Infection-induced glucose starvation triggers NINJ1-dependent macrophage lysis and pathogen escape

Infection-induced glucose starvation triggers NINJ1-dependent macrophage lysis and pathogen escape | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Infection-induced glucose starvation triggers NINJ1-dependent macrophage lysis and pathogen escape Harshini Weerasinghe, Orawan Tulyaprawat, Helen Stölting, Johannes Sonnberger, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7015602/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Pathogens compete for glucose with macrophages, which disrupts host glycolysis, modulates antimicrobial responses and causes macrophage death. We show that, upon glucose starvation caused by major fungal pathogens Candida albicans and Candida auris , macrophages lyse by activating NINJ1, the executioner of membrane rupture during cell death. In glucose-starved macrophages NINJ1 ruptures membranes independently of known cell death programs. Moreover, among cell death factors, NINJ1 is the dominant effector of fungal-induced macrophage damage. Supplementation of a single amino acid, alanine, rescues macrophages by inhibiting NINJ1 oligomerization, and C. albicans infection disrupts amino acid metabolism in mice and reduces serum alanine. Finally, NINJ1-mediated membrane rupture enables C. albicans egress from macrophage together with the toxin candidalysin. We establish the mechanism of glucose starvation-induced macrophage damage by activation of NINJ1, discover an approach to protect macrophages using a key mammalian metabolite, and demonstrate that NINJ1-dependent pathways are hijacked as an immune escape route. Biological sciences/Microbiology/Fungi/Fungal immune evasion Biological sciences/Immunology/Immune cell death Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Infection with fungal, bacterial, parasitic and viral pathogens changes glucose homeostasis in the host 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 . Consequently, proper regulation of glucose metabolism is important for host survival during infection 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 . An important aspect of glucose metabolism is the requirement for glycolysis in promoting antimicrobial responses by macrophages. Upon detection of microbes, macrophages rapidly adapt their metabolic pathways, with enhanced glycolysis being a defining feature 1 , 8 , 10 , 11 , 12 , 13 , 14 , 15 , 16 . Due to enhanced glycolysis, infected macrophages have an increased need for glucose, both as an energy source and for mounting effective antimicrobial responses, including production of antimicrobial cytokines, reactive oxygen species (ROS) and phagosomal acidification 1 , 6 , 8 , 11 , 12 , 17 , 18 , 19 , 20 , 21 . However, the demand for glucose can pose a challenge for macrophages because several pathogens are avid glucose consumers, leading to glucose-depleted conditions within infection microenvironments 1 , 2 , 22 , 23 , 24 , 25 , 26 . Moreover, pathogens can disrupt glucose metabolism in macrophages with immunological consequences. We have shown that Candida albicans and Candida auris , two fungal pathogens categorised as “ critical priority ” by the World Health Organization, deplete glucose and cause glucose starvation in macrophages that culminates in wide-spread immune cell death 1 , 24 . Similarly, disruption of macrophage glycolysis has been reported after infection with bacterial pathogens such as Salmonella typhimurium , Yersinia pseudotuberculosis and upon challenge with peptidoglycan 2 , 20 , 23 , 27 . Additionally, Staphylococcus aureus causes glucose stress in keratinocytes during skin infections 25 . Given its importance, modulating glucose homeostasis holds promise for promoting protective immune response and improving disease outcomes. However, the mechanisms that enable macrophages to respond to glucose starvation by inducing immune cell death and inflammation are incompletely defined. We have recently demonstrated that glucose-starved macrophages infected with C. albicans or C. auris undergo lytic cell death 1 , 24 . The lytic cell death is thought reduce the containment of fungi by macrophages and promote dissemination, since Candida species proliferate robustly in extracellular environments using a range of host nutrients. As such, reducing glucose starvation-induced macrophage lysis could increase fungal immune containment, but the mechanism linking metabolic dysfunction with macrophage lysis is unclear. Although glucose-starved macrophages activate the NLRP3 inflammasome in response to C. albicans 28 , and an activated NLRP3 inflammasome causes lytic macrophage pyroptosis under many conditions 29 , NLRP3 was surprisingly dispensable for macrophage lysis following C. albicans -induced glucose starvation despite inducing the secretion of the antifungal cytokine IL-1ß 28 . NLRP3 was also dispensable for macrophage lysis upon glucose starvation caused by C. auris 24 . Other mechanisms known to kill glucose-starved cancer cells, such as apoptosis or excessive accumulation of reactive oxygen species (ROS) 30 , 31 , 32 , also do not account for the lysis of glucose-starved macrophages upon Candida infection 1 , 24 , 28 . In this study we employed a nutritional approach to discover the trigger and mechanism of immune cell lysis in glucose-starved macrophages responding to fungal pathogens. We demonstrate that infection-induced glucose starvation is a trigger for activation of membrane damage that depends on NINJ1, the recently identified factor that induces plasma membrane rupture (PMR) and cell lysis upon immune cell death 33 . Our findings demonstrate that glucose starvation is a previously unidentified trigger of NINJ1-dependent PMR in infected macrophages, show that NINJ1 operates independently of major cell death programs in response to metabolic dysfunction, and demonstrate that NINJ1-dependent PMR can be inhibited by an important mammalian metabolite, the amino acid alanine, thereby suggesting a nutritional strategy to protect macrophages. Our comprehensive analyses of cell death pathways established that NINJ1 has the most dominant role in mediating C. albicans -induced macrophage damage and is the only cell death factor identified so far to respond to infection of macrophages by the emerging drug-resistant pathogen C. auris . Finally, we show a key role for NINJ1-induced macrophage PMR in facilitating fungal immune escape. RESULTS Alanine rescues macrophages from cell damage caused by glucose starvation In infected animals, C. albicans causes lowering of blood glucose levels, in addition to depleting available glucose for macrophages 1 , 34 . Supplementing glucose rescues C. albicans -infected macrophages by promoting their viability and cellular integrity, but it also potently promotes fungal growth and thus provides an advantage to C. albicans 1 . We therefore considered whether a nutritional rescue of macrophages without advantaging C. albicans is feasible. Amino acids metabolism may be a possible avenue to achieve this goal, since amino acids can support macrophage immunometabolism 35 , 36 , 37 . Furthermore, while C. albicans grows on amino acids, they are not its preferred carbon source. The effects of fungal infection on global host amino acid homeostasis are poorly understood. We found that mice systemically infected with C. albicans display profoundly perturbed amino acid homeostasis. There was a reduction in serum levels for arginine, aspartate, serine, glycine, alanine and several other amino acids (Fig. 1 A, Fig. S1 ). In contrast, glutamine, glutamate and others remained unchanged, while the levels of valine increased upon infection (Fig. 1 A, Fig. S1 ). We have previously shown that supplementation of arginine, serine, glutamine and leucine has no or very minor effects on rescuing C. albicans -infected macrophages 1 . Further inspection of affected amino acids showed that alanine displays a large, 2-fold reduction in serum levels in C. albicans -infected mice (Fig. 1 A). Alanine is an important metabolic substrate in mammals, both as a gluconeogenic substrate and feeding into the Krebs cycle for ATP production. Since alanine metabolism plays a role in low nutrient conditions and under metabolic stress 38 , we decided to test its effects on C. albicans -infected macrophages. To do so, we utilized our live-cell imaging platform with bone marrow-derived mouse macrophages (BMDMs) 1 . Here, macrophage plasma membrane permeabilization is quantified over time using the membrane-impermeable DNA dye DRAQ7. In this assay, C. albicans depletes glucose, resulting in glucose starvation of macrophages and cell death several hours after infection 1 and (Fig. 1 B). Supplementation of alanine rescued macrophages from glucose starvation-induced plasma membrane permeabilization, and the rescue was more potent than supplementation of glucose at the same concentrations (Fig. 1 B). As expected, glucose provided an advantage to C. albicans as seen by enhanced hyphal growth (Fig. 1 C ) . In contrast, alanine rescued macrophages without providing an advantage to C. albicans . C. albicans growth was the same in the presence or absence of alanine ( Fig. S2A ). Moreover, hyphal growth, which contributes to macrophage damage, was also the same (i.e. neither enhanced nor inhibited) in the presence of alanine (Fig. 1 C and Fig. S2B ). In addition to reducing membrane permeabilization (measured by DRAQ7-staining), supplementation of alanine also reduced lactate dehydrogenase (LDH) release into the extracellular medium, a measure of PMR and cell lysis (Fig. 1 D). Furthermore, alanine caused a reduction in secreted pro-inflammatory cytokine IL-1ß at 3 and 14 h post-infection (Fig. 1 E). At 16 h after infection alanine had no effect on IL-1ß secretion, which was high in the presence or absence of alanine (Fig. 1 E). The multiplicity of infection (ratio of C. albicans to macrophages) influences the kinetics of macrophage cell death and activation of glucose starvation 1 , 28 . Alanine rescued macrophages infected at both high (6:1) and low (1:1) multiplicity of infection (Fig. 1 F), indicating that it has a broadly protective effect. Additionally, C. albicans clinical isolates show distinct abilities to damage macrophages 28 , 39 . Thus, in addition to the prototype isolate SC5314 (Fig. 1 A), we established that alanine also rescues macrophages during infection with other C. albicans isolates (Fig. 1 G). Finally, rescue by alanine was recapitulated in primary monocyte-derived human macrophages (Fig. 1 H), and alanine also rescued C. auris -infected macrophages (Fig. 1 I). Collectively, these results show that alanine rescues macrophages infected with two different fungal pathogens, in response to multiple clinical isolates, and the rescue is recapitulated in mouse and human immune cells. C. albicans competes with macrophages for alanine using Alt1-dependent metabolism C. albicans is metabolically versatile and could compete with macrophages for alanine. To test this, we generated a C. albicans alt1D/D mutant that lacks alanine amino transferase needed for alanine utilization. The alt1D/D mutant grew as well as the wildtype in macrophage culture medium, in the presence or absence of alanine ( Fig. S2A ). Moreover, the mutant formed hyphae in culture medium and within macrophages (Fig. 2 A and S2 B), and infected macrophages normally (Fig. 2 B ) . Consistent with these phenotypes, under standard conditions (no additional alanine) the alt1D/D mutant triggered macrophage plasma membrane permeabilization with the same kinetics as the parental C. albicans strain (Fig. 2 C). Therefore, the alt1D/D mutant is not generally defective in infecting macrophages or triggering macrophage damage. However, in the presence of alanine, infections with alt1D/D led to prolonged rescue relative to parental C. albicans (Fig. 2 C, compare red and pink graphs). The prolonged rescue could be partially reversed by complementation of the mutant with ALT1 (Fig. 2 C, pink versus salmon graphs). Collectively, these data show that C. albicans competes with macrophages for alanine by using Alt1-dependent metabolism. Therefore, rescue by alanine is enhanced when competition from C. albicans is reduced in the alt1D/D mutant. Macrophage rescue does not require metabolic utilization of alanine Alanine rescued C. albicans- infected macrophages better than glucose (Fig. 1 B). This finding prompted us to consider if the rescue may be non-metabolic. To test this, we used L- and D-isomers of alanine. L-alanine is metabolized effectively by fungal and mammalian cells (and was used in our experiments so far). D-alanine is not metabolized well. Yet, D-alanine rescued C. albicans -infected macrophages as well as L-alanine (Fig. 3 A), without affecting the growth or filamentation of C. albicans (Fig. 3 B, Fig. S2A and S2B ). Moreover, consistent with D-alanine not being metabolised, infection with the C. albicans alt1D/D mutant did not lead to prolonged rescue in the presence of D-alanine (Fig. 3 C). As a control, we show that D-alanine did not have any effects on the growth or filamentation of the alt1D/D mutant ( Fig. S2A and S2B ). We have previously shown that C. albicans -induced glucose starvation triggers a hyperpolarization event in macrophage mitochondria, followed by depolarization 1 . These changes in mitochondrial membrane potential result from metabolic dysfunction and can be delayed by glucose supplementation 1 . Unlike glucose, supplementation of alanine did not delay mitochondrial hyperpolarization/depolarization, despite delaying macrophage plasma membrane permeabilization (Fig. 3 D and 3 E). Collectively, these results show that alanine does not correct the metabolic dysfunction caused by glucose starvation. Instead, it acts downstream of metabolic dysfunction to prevent macrophage plasma membrane damage. Alanine does not act on pyroptosis, necroptosis or apoptosis Next, we tested for a potential involvement of immune cell death programs, since C. albicans triggers NLRP3-caspase-1 inflammasome-dependent pyroptosis in the first few hours post infection 40 , 41 . In pyroptosis-negative Gsdmd −/− macrophages (lacking the pore-forming protein Gasdermin D) the initial membrane permeabilization was delayed as expected, but alanine caused a further delay showing that it does not need GSDMD to rescue macrophages (Fig. 4 A). Similarly, alanine rescued C. albicans -infected Nlrp3 −/− macrophages (Fig. 4 B). Of note, we have previously shown that in C. albicans -infected macrophages GSDMD processing to its pore-forming fragment depends on NLRP3; however, in our imaging assay, Nlrp3 −/− macrophages show a reduced initial delay in DRAQ7-positive macrophages relative to Gsdmd −/− due to increased formation of macrophage extracellular nets that are also stained by DRAQ7 42 . Consistent with the effect of alanine being independent of inflammasome-induced cell lysis, supplementation of alanine did not prevent the processing of caspase 1, IL-1ß or GSDMD in response to C. albicans , although it reduced their release into the supernatant due to reduced plasma membrane rupture (Fig. 4 C, Fig. S3 ). Alanine also rescued macrophages when all apoptotic caspase-dependent pathways were chemically inhibited with Q-VD-OPh (QVD) (Fig. 4 D) and when pyroptosis, necroptosis and extrinsic apoptosis were simultaneously disrupted using genetic approaches 43 (Fig. 4 E). NINJ1 is the target of alanine’s rescue and the effector of glucose starvation-induced macrophage plasma membrane damage To investigate other cell death factors potentially involved in alanine’s rescue, we re-analyzed our published RNAseq dataset from C. albicans -infected BMDMs 1 . Five genes encoding cell death factors showed increased expression upon C. albicans infection: Nlrp3 and Casp11 (pyroptosis), Mlkl (necroptosis), Zbp1 (apoptosis and necroptosis) and Ninj1 (performs macrophage plasma membrane rupture (PMR) downstream of several immune cell death) (Fig. 5 A). We ruled out pyroptosis and necroptosis in mediating the lysis of glucose-starved macrophages and being involved in macrophage rescue by alanine (Fig. 4 ). Zbp1 is also unlikely because it signals for caspase-8-driven apoptosis and MLKL-mediated necroptosis 44 , which we excluded (Fig. 4 ). We thus focused on the recently identified executioner of PMR, NINJ1 33 . Indeed, we show that Ninj1 expression is upregulated by C. albicans infection not only in mouse macrophages (Fig. 5 A), but also in human macrophages (Fig. 5 B and S4 A). Deletion of Ninj1 rescued C. albicans -infected macrophages from damage caused by glucose starvation, inhibiting both plasma membrane permeabilization (Fig. 5 C and Movie S1 ) and PMR (Fig. 5 D). Ninj1 loss conferred the strongest rescue of C. albicans -infected macrophages, when compared with all other host cell death factors that we have tested to date (compare Fig. 5 C with data in Fig. 4 and our previous work 1 , 28 , 40 , 45 ). Confirming its broad role in response to fungi, deletion of Ninj1 also rescued macrophages infected with C. auris (Fig. 5 E). Alanine did not further rescue Ninj1 −/− macrophages (neither membrane permeabilization nor PMR) (Fig. 5 C and 5 D ) , showing that alanine needs NINJ1 to exert its protective effects. Glycine, an amino acid that is structurally very similar to alanine, is reported to inhibit PMR by preventing NINJ1 oligomerization that is required for rupturing membranes 46 . Similarly to glycine, alanine also prevented NINJ1 oligomerization ( Fig. S4B ). Collectively, these results show that glucose starvation triggers NINJ1-dependent macrophage membrane damage in C. albicans and C. auris -infected macrophages. Moreover, alanine rescues glucose-starved macrophages by inhibiting NINJ1. To further test the involvement of NINJ1 in glucose starvation and fungal infections, we asked if glycine acts similarly to alanine in rescuing glucose-starved macrophages via NINJ1. Indeed, glycine rescued C. albicans -infected macrophages in a NINJ1-dependent manner (Fig. 5 F). Furthermore, like alanine glycine rescued Gsdmd −/− macrophages, both mouse (Fig. 5 G) and human (Fig. 5 H). This is consistent with no role for GSDMD-dependent pyroptosis in response to NINJ1-dependent membrane damage caused by glucose stress. IL-1ß secretion was reduced in C. albicans -infected Ninj1 −/− macrophages, with no further reduction upon supplementation with alanine or glycine (Fig. 5 I ) This is in line with NINJ1-dependent PMR causing some cytokine release, and glycine and alanine acting on NINJ1 to reduce cytokines. Despite lower levels of released IL-1ß, its proteolysis was normal in Ninj1 −/− macrophages, suggesting that reduced release of IL-1ß is due to lower PMR rather than inhibition of inflammasome signalling. Similarly, caspase-1 and GSDMD were proteolytically processed in C. albicans -infected Ninj1 −/− macrophages, although less cleaved GSDMD was found in supernatants consistent with reduced PMR (Fig. 5 J). NINJ1 is a macrophage escape factor for C. albicans working together with candidalysin Inactivation of NINJ1 caused a large reduction in membrane permeabilization following C. albicans infection, but the rescue was not complete (Fig. 5 C). We therefore decide to determine the mechanism causing the additional damage. The fungal pore-forming peptide candidalysin is secreted at high levels by C. albicans hyphae 47 , whereby it damages the macrophage plasma membrane during hyphal escape 42 , 48 , 49 . Therefore, candidalysin was a candidate factor to cause the residual membrane damage in the absence of NINJ1. Following infection with a mutant lacking the candidalysin-encoding gene ECE1 ( ece1D/D ), there was an initial delay in macrophage plasma membrane permeabilization (Fig. 6 A). This is consistent with the roles of candidalysin in hyphal escape 42 , 49 . Compared to wild type C. albicans , infection with ece1D/D further reduced plasma membrane permeabilization in Ninj1 -/- macrophages (Fig. 6 A). The additional effect of candidalysin was also seen in human macrophages when NINJ1 was inactivated by glycine (Fig. 6 B, Fig. S4C ). These data show that candidalysin is responsible for the residual damage in the absence of NINJ1 in mouse and human macrophages. NINJ1 oligomerizes in the membrane as prerequisite for PMR 33 , 50 . Consistently, C. albicans infection caused NINJ1 oligomerization, which was reduced in infections with ece1D/D (Fig. 6 C, Fig. S5 ). This shows that, in addition to working in parallel to NINJ1-dependent pathways, candidalysin-induced membrane damage contributes to activation of NINJ1 following C. albicans infection. To further test the contributions of NINJ1 and candidalysin in Candida infections, we used C. albicans clinical isolates that display substantial diversity in candidalysin expression levels. The reference strain SC5314 (used in most of our experiments) readily forms hyphae under in vitro conditions and secrets high levels of candidalysin 47 . We selected four additional bloodstream infection isolates with reduced candidalysin expression 51 , 52 , 53 . These isolates also display lower hyphal growth within macrophages 28 . Deletion of Ninj1 strongly rescued macrophages in infections with all low-candidalysin isolates, and the rescue was stronger than with the candidalysin-high strain SC5314 (Fig. 6 D ) . Ninj1 −/− macrophages were protected from plasma membrane permeabilization for over 20 h upon infection with strain P78042, whereas for P57055, P57072 and P76067 plasma membrane permeabilization started late in Ninj1 −/− macrophages and progressed more slowly than with SC5314 (Fig. 6 D). For instance, at 20 h post-infection, plasma membrane permeabilization was seen for 30% of Ninj1 −/− macrophages for SC5314, 15% for P57055, 17% for P57072, 8% for P76067 and 6% for P78042. Collectively these results show that NINJ1 has a conserved role in macrophage plasma membrane damage across diverse C. albicans strains and an increasingly important role with strains that expresses lower candidalysin levels. Invasive hyphal growth and secreted candidalysin together damage macrophage membranes to promote immune escape of C. albicans 42 , 49 , 54 . This could promote dissemination, since C. albicans proliferates robustly in extracellular environments using a range of host nutrients. Based on data in Fig. 6 D we predicted that C. albicans strains with reduced hyphal growth and candidalysin expression might use NINJ1-dependent plasma membrane damage as their major mechanism for escape. Indeed, this was the case. Strain P78042 showed a large reduction in escape from Ninj1 −/− macrophages, with few externalized hyphae observable (Fig. 6 E and 6 F, Movie S2 ). In contrast, extensive escape of this strain was seen from wildtype macrophages, followed by proliferation of the externalized hyphae (Fig. 6 E and 6 F, Movie S2). Strains P57055, P57072 and P76067 strains were likewise delayed in their egress from Ninj1 −/− macrophages ( Fig. S6, Movie S2 ). These findings determine that NINJ1 is a major fungal escape factor. DISCUSSION In this study, we establish the mechanism by which glucose-starved macrophages lyse in response to fungal pathogens. We show that the mechanism is mediated by NINJ1, which causes macrophage plasma membrane permeabilization and rupture. This mechanism is conserved in both mouse and human macrophages, and establishes glucose starvation as a trigger for NINJ1 activation in infected macrophages. Our findings further show nutritional supplementation of alanine, an important mammalian metabolite, inhibits NINJ1-dependent damage without providing a growth advantage to C. albicans . Moreover, NINJ1 is the dominant host effector of macrophage damage in response to C. albicans , since inactivation of NINJ1 has by far the biggest effect in rescuing macrophages compared to all other cell death programs studied to date. For C. auris , NINJ1 is the only host factor discovered so far driving macrophage damage. Finally, C. albicans uses NINJ1-dependent plasma membrane damage to egress from macrophages, showing that NINJ1-dependent mechanisms contribute to immune evasion. Mediating pathogen escape is a yet unappreciated role for NINJ1 in infection. As we show here and previously, glucose starvation causes changes in mitochondrial membrane polarization, whereby hyperpolarization is followed by depolarization coinciding with plasma membrane permeabilization and damage (Fig. 3 ) and 1 . Like our results with C. albicans , the ATP synthase inhibitor oligomycin causes mitochondrial hyperpolarization and triggers NINJ1-dependent macrophage membrane damage 33 . Therefore, changes to mitochondrial function are a probable trigger for NINJ1 activation in response to glucose starvation and metabolic dysfunction caused by C. albicans . Furthermore, we show that the fungal pore-forming toxin candidalysin plays a role in activating NINJ1. Our results are supported by data showing that bacterial pore-forming toxins streptolycin and listeriolysin activate NINJ1 33 . The precise activating mechanisms for NINJ1 oligomerization and membrane rupture are still to be resolved and may differ depending on the stimulus. However, the current consensus is that changes to membrane structure, such as movement of phospholipids (scrambling), swelling or mechanical stress, are involved 50 , 55 , 56 , 57 . Candidalysin damages mitochondrial membranes and the plasma membrane of host cells 47 , 48 , 58 , providing the likely mechanism by which it activates NINJ1. Interestingly, a recent study showed that in addition to membrane damage caused by pore-forming toxins, mechanical damage is needed for full rupture mediated by NINJ1 57 . In line with our results with C. albicans , NINJ1-dependent membrane rupture triggered by mechanical challenge does not require the characterised cell death programs 57 . In the case of C. albicans , the mechanical damage maybe be provided by the growing fungal hyphae exerting force on the membrane 54 . Therefore, our data indicates that, during infection a combination of immunometabolic dysfunction, membrane-damaging toxins secreted by pathogens and mechanical stress on immune cell membranes from microbial growth are key stimuli for triggering NINJ1-mediated damage and PMR. Our findings further characterise a nutritional approach to protect macrophages by showing that supplementation of alanine rescues glucose-starved macrophages in a NINJ1-dependent manner. Alanine is an important metabolite under metabolic stress 38 , and our data in infected mice is consistent with major disruption of amino acid homeostasis in C. albicans -infected animals and a large reduction in serum levels of alanine. We show that alanine rescues macrophages without correcting their metabolic dysfunction, in line with studies showing that NINJ1-induced membrane damage occurs independently of metabolic dysfunction during cell death 33 , 50 , 59 . Moreover, supplementation of alanine or genetic deletion of Ninj1 cause a reduction in both membrane permeabilization and PMR in C. albicans -infected macrophages, in a manner that is independent of pyroptosis, apoptosis or necroptosis. Accordingly, the proteolytic processing of caspase-1, GSDMD and IL-1ß in response to C. albicans is not prevented by NINJ1 inhibition, although the amount of IL-1ß released by infected macrophages is reduced. Alanine is the third metabolite with NINJ1-inhibitory activity, in addition to glycine and the GABA receptor agonist muscimol 46 , 59 . These metabolites inhibit NINJ1 oligomer formation, although the biochemical mechanism remains to be understood 46 , 59 . Alanine and glycine are structurally similar, and muscimol also shares features of its chemical structure with glycine 59 . Therefore, the mechanism of NINJ1 inhibition by these molecules may be conserved. The requirement for NINJ1 for both membrane permeabilization and PMR of C. albicans -infected macrophages is similar to ferroptosis, infection with Yersinia or Salmonella , treatment with the lipopolysaccharide (LPS) in the presence of heat shock and mechanical membrane challenge as a trigger for NINJ1 57, 60, 61, 62 ; however, unlike our data with C. albicans , inactivation of NINJ1 does not impair release of IL-1ß in response to these bacterial pathogens and LPS plus heat shock 60 , 61 , 62 . Similar to our data with C. albicans , Ninj1 mutant mice had lower levels of plasma IL-1ß in response to infection with Escherichia coli 63 , showing that NINJ1-dependent damage drives the release of this major proinflammatory cytokine across highly diverse infections. Downstream of pyroptosis NINJ1 is required for terminal PMR but has no role in the earlier GSDMD-mediated membrane permeabilization event or secretion of IL-1ß 33 . The explanation is that, during pyroptosis, membrane permeabilization and IL-1ß secretion depend on GSDMD pores, which form independently on NINJ1 33 . Given that, as we and others have shown, C. albicans triggers NLRP3-dependent pyroptosis 40 , 41 , the question is why NINJ1 mediates membrane permeabilization in our experiments. The answer is that C. albicans triggers pyroptosis early after macrophage infection 40 , 41 , while glucose starvation occurs later and triggers NINJ1-dependent damage independently of GSDMD or pyroptosis, as we show here. Consistently, we show that alanine only needs NINJ1 but not any of the pyroptotic factors to rescue macrophages, and the same is true for glycine. Therefore, downstream of glucose starvation GSDMD has no role in macrophage membrane damage, explaining why NINJ1 is involved in both membrane permeabilization and PMR under these conditions. While GSDMD pores mediate the release of small molecules including IL-1ß, NINJ1-dependent PMR releases large pro-inflammatory danger-associated molecular patterns (DAMPs), such as LDH and the High Mobility Group Box 1 (HMGB1) protein 33 . Using diverse clinical isolates of C. albicans , we show that NINJ1-dependent PMR also promotes the release of the pathogen itself, thereby driving immune escape. Previous work by us and others has shown that C. albicans uses several mechanisms to egress from macrophages and disseminate. These include early escape via GSDMD-driven pyroptosis, as well as hyphae- and candidalysin-induced macrophage membrane damage 40 , 41 , 42 , 49 , 54 . However, these established mechanisms do not account for several findings. Firstly, C. albicans clinical strains are diverse, with several unable to form robust hyphae in vitro , activate pyroptosis or express high levels of candidalysin 28 , 51 , 52 , 53 . Secondly, only a proportion of macrophages in the population trigger pyroptosis upon C. albicans challenge 28 , 40 . We now show that NINJ1 plays an increasingly important role in macrophage membrane damage and egress for C. albicans isolates with reduced hyphal formation and candidalysin expression. These findings identify NINJ1 as a central host effector of fungal escape from macrophages. Our data with high – and low-candidalysin isolates and candidalysin mutant strains show that NINJ1 works together with candidalysin to damage macrophage membranes and promote C. albicans escape. In conclusion, our study identifies NINJ1 as the host effector of membrane damage following glucose starvation of Candida -infected macrophages and indicates that NINJ1 is a central control point for regulating inflammatory cytokine release, while also increasing immune containment of fungal pathogens to reduce dissemination. We propose that NINJ1 might be a general effector of PMR upon glycolytic dysfunction caused by a broad range of pathogens, such as Salmonella 23 , 59 . Moreover, given the central role of NINJ1 in performing PMR downstream of cell death pathways used by diverse pathogens to egress from macrophages 64 , we posit that NINJ1 may be commonly hijacked as a pathogen egress factor. MATERIALS AND METHODS Ethics statement All procedures involving mice for the isolation of BMDMs were conducted in compliance with protocols approved by the Monash University Animal Ethics Committee (approval ID ERM37724). Experiments involving the collection of human blood samples received ethical clearance from the Monash University Human Research Ethics Committee (ERM 44451). Experiments involving C. albicans infection of mice and blood collection were approved by the Monash University Animal Ethics Committee (approval ID ERM36304). Isolation of hMDMs in Fig S4 was conducted according to the principles of the Declaration of Helsinki. The blood donation protocol and use of blood for isolation of human monocyte-derived macrophages were approved by the institutional ethics committee of the University Hospital Jena (permission number 2207–01/08). Candida strains and media The strains used in this study are listed in Table S 1. Unless stated otherwise the clinical isolate SC5314 was the main strain used in this study. All clinical strains of C. albicans were obtained from BEI Resources, NAID, NIH ( https://www.niaid.nih.gov/research/bei-resources-repository ). Candida strains were routinely cultured on YPD medium composed of 1% yeast extract, 2% peptone, 2% glucose, and solidified with 2% agar, for 2 days. For experiments requiring overnight liquid cultures, such as growth curve kinetics, a single colony was picked from a freshly streaked YPD agar plate and incubated in liquid YPD at 30°C for 18 h. Cultures were then diluted to an initial OD 600 of 0.1 using prewarmed bone marrow-derived macrophage (BMDM) medium before use in assays. BMDM medium consisted of RPMI 1640 supplemented with 12.5 mM HEPES, 10% fetal bovine serum (FBS), 20% L-cell conditioned medium providing macrophage colony-stimulating factor, and 100 U/ml penicillin-streptomycin. RPMI 1640 (Thermo Fisher, catalog no. 11875093) was used as a base and supplemented with either glucose, alanine or glycine at 10 or 40 mM. For filamentation assays, macrophage infection studies and phagocytosis assays, a single colony was selected from fresh YPD solid medium and patched onto new plates and incubated at 30°C overnight. The patches were suspended in PBS the following day, and diluted to the desired concentration for the experiments, as determined by counting using a hemocytometer. Strain construction Primers used to construct the mutant strains in this study are listed in Table S2 . The ECE1 open reading frame (ORF) in C. albicans SC5314 was deleted using a CRISPR-Cas9 protocol 65 . The deletion of the ECE1 -ORF was confirmed via colony PCR, and the Cas9-expression vector was excised by Flp-recombination. The ECE1 -complemented strain was generated using the SAT1-Flipper method 66 . The cloned sequences were verified by Sanger-sequencing. The ECE1 complementation cassette was excised using Sac I/ Apa I and introduced into the C. albicans ece1 ∆/∆ strain (M2974) Correct integration was confirmed by colony PCR at the cassette junctions and the SAT1 -marker was excised by Flp-recombination. The homozygous deletion of ALT1 and the complemented strain were constructed in the SN152 strain ( his1 − leu2 − arg4 −) 67 , using the HIS1 and LEU2 markers for deletion and the ARG4 marker for complementation. The complemented strain has one copy of ALT1 reintroduced at the LEU2 locus. Transformants were confirmed by PCR. The reference strain SN425 ( HIS1 + LEU2 + ARG4 + ) was used as the control. Cell culture experiments Isolation of murine bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages (hMDMs) To isolate murine BMDMs, bone marrow cells were extracted from 6-8-week-old mouse femurs and tibias of either C57BL/6 mice obtained from the Monash Animal Research Platform or Nlrp3 −/− , Gsdmd −/− and Ninj1 −/− mice obtained from the Walter and Elisa Hall Institute and the Hudson Institute respectively. Following a previously described protocol 1 bone marrow containing monocytes was flushed out using 10 ml of BMDM medium. The cells were then incubated for 18–24 h at 37°C with 5% CO 2 . Following incubation, non-adherent cells were transferred to fresh BMDM medium and were allowed to differentiate into BMDMs for 5–7 days. To isolate hMDMs (Fig. 1 ), whole blood was obtained from healthy donors using Histopaque-1077 (Sigma-Aldrich) via density gradient centrifugation. Monocytes expressing CD14 and CD16 were subsequently enriched from peripheral blood mononuclear cells (PBMC) fraction using the Pan Monocyte Isolation Kit and magnetic labelling system (Miltenyi Biotec). The isolated monocytes were then seeded in 25 cm² tissue culture flasks and differentiated into human monocyte-derived macrophages (hMDMs) by culturing for 7 days at 37°C with 5% CO₂ in RPMI 1640 medium supplemented with 15 mM HEPES, 10% fetal bovine serum (Serana, Fisher Biotech), 100 U/ml penicillin-streptomycin (Sigma-Aldrich), and 50 ng/ml macrophage colony-stimulating factor (M-CSF, R&D Systems). For hMDM isolation in Fig. S4A , PBMCs were isolated from buffy coats obtained from healthy donors using Histopaque-1077 (Sigma-Aldrich) density gradient centrifugation. CD14-positive monocytes were subsequently purified by magnetic-activated cell sorting (MACS) using the autoMACS® system (Miltenyi Biotec), according to the manufacturer’s instructions. A total of 1 × 10⁷ CD14⁺ monocytes were seeded into 175 cm 2 flasks and cultured in RPMI 1640 (Thermo Fisher Scientific) and 10% FBS. Differentiation into macrophages was induced by adding 50 ng/mL rhM-CSF (ImmunoTools), and cells were incubated for seven days at 37°C, 5% CO₂. After differentiation, macrophages were detached using 50 mM EDTA in phosphate-buffered saline (PBS), and reseeded at a density of 2 × 10 5 cells/ml. Then 200 µl per well of a 96-well plate (TPP, 4 × 10 4 cells/well) were added for subsequent experiments. Live cell imaging Macrophages were harvested, seeded and stained following protocols detailed in our previous studies 1 , 24 . Following staining, macrophages were infected with Candida at a yeast-to-macrophage ratio of either 1:1, 3:1 or 6:1. After the phagocytosis step (1 h), extracellular yeast cells were removed by gentle washing with phosphate-buffered saline (PBS). Where specified, BMDM culture medium was supplemented with metabolites including glucose, alanine and glycine at concentrations detailed in the figure legends. These metabolites were added after the 1 h phagocytosis and wash steps. For experiments assessing the role of caspase inhibition, macrophages were pre-incubated with 10 µM Quinoline-Val-Asp-Difluorophenoxymethylketone (QVD) for 1 h. Following this, cells were washed and maintained in fresh BMDM medium prior to infection with C. albicans . Control (uninfected) macrophages were subjected to the same treatment conditions. After infection, culture media in both experimental and control wells was replaced were with fresh medium either with or without 10 µM QVD, which was maintained for the duration of the assay. All conditions included 0.6 mM DRAQ7 (Abcam) to monitor plasma membrane permeabilization in macrophages. Live-cell imaging was carried out using a Leica AF6000 LX epifluorescence microscope equipped with a 20× objective lens. Image analysis and quantification were performed using CellProfiler v2.1.1, following protocols previously established in our laboratory 42 , 68 . Time-lapse image sequences were compiled into movies using ImageJ software, and all quantitative data were visualized using Prism 10 (GraphPad Software, San Diego, CA). Human macrophage experiments were seeded as described previously in 24 . For visualization in live-cell assays, hMDMs were labelled with 1 µM CellTracker Green CMFDA (Thermo Fisher, C7025) for 25 min, following the same staining protocol used for BMDMs. Subsequently, cells were primed by incubation in RPMI 1640 supplemented with 15 mM HEPES, 10% FBS, and 50 ng/ml lipopolysaccharide (LPS, Sigma-Aldrich) for 2 h at 37°C and 5% CO₂. Post-priming, hMDMs were infected with Candida at a multiplicity of infection (MOI) of 3. After a 1 h infection period, the subsequent steps for live-cell imaging, including washing and analysis, were performed identically to the BMDM protocol described above. For immortalized bone marrow-derived macrophage (iBMDMs) experiments derived from C57BL/6 Cre-J2 mice 43 , including wild-type and genetically modified lines lacking caspase-1, caspase-11, caspase-12, caspase-8, or Receptor-Interacting Protein Kinase 3 (RIPK3) deficient in pyroptotic, necroptotic, and extrinsic apoptotic pathways or control and Ninj1 −/− macrophages described in 69 and Table S1 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin. Cells were maintained at 37°C in a humidified incubator with 5% CO₂. For live-cell imaging assays, iBMDMs were harvested by gentle scraping (BD Falcon cell scraper) and seeded into tissue culture-treated 96-well plates at a density of 8 × 10 4 cells per well. After overnight incubation to allow cell attachment recovery and multiplication (to 1 × 10 5 ), macrophages were infected with C. albicans at MOI of 3. Imaging and subsequent data acquisition and analysis were performed following the same protocol established for primary BMDM experiments. BlaER1 cell culture and transdifferentiation The B-cell leukemia cell line C/EBPαER clone 1 (BLaER1) was previously generated 70 and cultured and transdifferentiated as described in their work with minor adjustments regarding cell numbers. In short, BLaER1 cells were cultured in RPMI 1640 medium supplemented with 10 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin (all Thermo Fisher Scientific) and 10% fetal bovine serum (FBS) (BLaER1 medium) at 37°C, 5% CO 2 . Cells were passaged at a sub-cultivation ratio of 1:3–1:5 every 2 to 3 days to a maximum of 30 passages. For transdifferentiation, BLaER1 cells were adjusted to 3.3 x 10 5 cells/ml in BLaER1 medium supplemented additionally with 10 ng/ml recombinant human macrophage colony-stimulating factor (rhM-CSF, ImmunoTools), 10 ng/ml recombinant human interleukin 3 (rhIL-3, ImmunoTools) and 200 nM β-estradiol (Sigma-Aldrich) (Transdifferentiation medium). Three ml per well of this suspension were seeded in 6-well plates (TPP) and incubated at 37°C, 5% CO 2 for seven days. Every two to three days, 1.5 ml of the medium was replaced with fresh, transdifferentiation medium. At day seven, the undifferentiated suspension cells were removed by aspirating the supernatant, while the transdifferentiated, adherent cells (tdBLaER1) were carefully harvested by thorough washing of the wells with medium. The concentration of the tdBLaER1 was adjusted to 4 x 10 5 cells/ml in transdifferentiation medium. Of this suspension, 200 µl/well were seeded into 96-well plates (TPP). The cells were left at room temperature for 20–30 min to allow attachment and incubated at 37°C, 5% CO 2 overnight for experiments on the following day. The cells were checked microscopically at the days of the medium exchange. Transdifferentiated cells appeared as GFP-positive, attached, and elongated with a spindle-shaped, macrophage-like morphology. Preparation of supernatants and cell lysates for IL-1β ELISA, LDH release, and immunoblotting The collection procedure for supernatant and cell lysate collection was described previously 24 and where relevant were performed in BMDM or iBMDM (control and Ninj1 −/− ) cells. Macrophages were seeded at a density of 5 × 10⁵ cells per well in 24-well plates using BMDM medium, following the protocol described under “Live cell imaging” methods. Cells were primed with 50 ng/ml lipopolysaccharide (LPS) for 3 h at 37°C in 5% CO₂, after which they were either left uninfected or challenged with Candida albicans (strain SC5314, MOI 3) for 1 h. Following infection, wells were washed three times with PBS to remove non-phagocytosed fungal cells and replaced with 250 µl of fresh BMDM medium. Where relevant media was supplemented with 10mM alanine or glycine at this point. Infected and control cells were then incubated for an additional 3, 14, 16 h. As a positive control for inflammasome activation, macrophages were treated with 10 µM nigericin or 0.01% TritonX for 3 h. For both tdBLaER1 cells and hMDMs LDH release assays, cells were primed with 50 ng/ml LPS from Escherichia coli O55:B5 (Sigma Aldrich) for 2 h at 37°C, 5% CO 2 . During priming, glycine from a sterile-filtered 1 M stock solution in double-distilled water, was added to RPMI 1640 to a final concentration of 50 mM. After priming, the LPS-containing medium was aspirated and 100 µl fresh RPMI 1640 containing either 50 mM L-glycine or no additional supplement was added and incubated for 10 min prior to infection to block NINJ1 oligomerization according to the procedure previously described for human primary macrophages by 46 . C. albicans strains were adjusted to MOI 1 for tdBLaER1 and MOI 3 for hMDMs as described above, either in 100 µl RPMI 1640 or 100µl RPMI 1640 + 50 mM L-glycine depending on the condition. For infection, 100 µl of the adjusted strains were added to the cells and incubated for 24 h at 37°C, 5% CO 2 . IL-1β quantification ELISA Levels of IL-1β in culture supernatants were quantified using the Mouse IL-1β/IL-1F2 DuoSet ELISA kit (R&D Systems, DY401), following the manufacturer’s protocol. Absorbance was read at 450 nm with a correction wavelength of 540 nm using a Tecan Infinite M200 microplate reader. A standard curve was generated using the recombinant IL-1β standards supplied in the kit, and cytokine concentrations in experimental samples were calculated by interpolation from this curve using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA). Lactate dehydrogenase (LDH) release assay Cell death was assessed by measuring lactate dehydrogenase (LDH) release into cell culture supernatants using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, G1780), following the protocol provided by the manufacturer. Absorbance was recorded at 492 nm using a Tecan Spark 10M microplate reader. LDH activity in each sample was expressed as fold change relative to the positive control treated with 10 µM nigericin. For both tdBLaER1 cells and hMDMs LDH release assays, infection plates were centrifuged at 250 g for 10 min, the supernatants were collected and diluted 1:5 in PBS. LDH concentrations were measured using the Cytotoxicity detection kit (Roche) according to manufacturer’s instructions. Western blot analysis For detection of IL-1β, caspase-1, and Gasdermin D (GSDMD), cell lysates and/or supernatants were denatured by boiling at 95°C for 10 min prior to electrophoresis. Samples were then separated on 4–12% Criterion™ XT Bis-Tris Protein Gel (Biorad) and transferred overnight onto Immobilon-P Membrane, PVDF, 0.45 uM (Millipore). Membranes were briefly stained with Ponceau S to confirm equal loading and transfer. Following blocking with 5% (v/v) skim milk in TBS containing 0.1% (v/v) Tween-20 (TBS-T) for 1 h at room temperature, membranes were incubated overnight at 4°C on a rotating platform with the following primary antibodies (1:1000 dilution): anti-IL-1β (R&D Systems, #AF-401-NA), anti-caspase-1 (Adipogen, AG-20B-0042-C100), and anti-GSDMD (Abcam, ab209845). The next day, membranes were washed and probed with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000 dilution) for 1 h at room temperature. Signal was developed using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher 34094) and imaged using FUJI medical X-ray films. Figures were prepared using Adobe Illustrator. Infection and RNA isolation of tdBLaER1 cells and hMDMs for analysis of Ninj1 expression For host RNA extraction upon C. albicans infection, 1 × 10 6 and 2 × 10 6 of hMDMs and tdBlaER1 cells respectively, were seeded in 6-wells at the concentrations described above. On the day of infection, the medium in each well was replaced with 2 ml RPMI without FBS, infected with C. albicans at an MOI 2, and incubated at 37°C, 5% CO2. Samples for RNA isolation were collected at 1 h, 3 h, and 6 h post infection. The well content was removed and replaced with 1 ml of RNeasy Lysis (RLT) buffer (Qiagen) supplemented with 1% β-mercaptoethanol (Roth). Cells were detached using a cell scraper (< 3 min) and centrifuged for 10 min (20,000 g, 4°C). The host RNA-containing supernatant was transferred to a new tube, immediately shock-frozen in liquid nitrogen, and stored at -80°C until further processing. Once thawed, the supernatant was mixed with an equal volume of 70% ethanol (prepared in diethyl pyrocarbonate [DEPC]-treated water), and total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Reverse transcription-quantitative PCR (RT-qPCR) The RNA (500 ng) was treated with Baseline-ZERO™ DNase (Biozym) according to the manufacturer's guidelines and subsequently transcribed into cDNA using 0.5 µg Oligo(dT)12–18 Primer, 200 U Superscript™ III Reverse Transcriptase, and 40 U RNaseOUT™ Recombinant RNase Inhibitor (Thermo Fischer Scientific). Obtained cDNA was diluted 1:5 and used for RT-qPCR with GoTaq® qPCR Master Mix (Promega) in a CFX96 thermocycler (Bio-Rad). Expression levels were normalized to beta-actin. All primer sequences are listed in Table S2 . Blue Native PAGE For native gel analysis, samples were prepared following the protocol provided by the manufacturer (Thermo Fisher Scientific). In short, cells were lysed and then subjected to centrifugation at 100 × g for 2 minutes. The resulting pellet was resuspended in 1× Sample Buffer supplemented with 1% digitonin. After mixing the suspension gently by pipetting five times, it was spun down at 20,000 × g for 30 min at 4°C. The supernatant was carefully collected for analysis. Proteins were separated using 3–12% NativePAGE™ Bis-Tris Mini Protein Gels (Thermo Fisher Scientific, BN1002BOX Gels were then transferred onto PVDF membranes and subjected to immunoblotting.). Detection of the NINJ1 protein was carried out using anti-human NINJ1 antibody (R&D Systems, AF5105) at a concentration of 1 µg/ml. Detection of NINJ1 was performed using a primary Rabbit anti-NINJ1 Invitrogen (Cat#: BS-11105R (for blots in Figure S4 ) and PA5-95755; RRID: AB_2807557(for blots in Figure S5 ) antibody at 1:1000, while secondary Donkey anti-Rabbit IgG (H + L) (Cat#: A-31573; RRID: AB_2536183) ( Figure S4 ) and Goat anti-Rabbit IgG (H + L) (Cat#: 31460; RRID: AB_228341) ( Figure S5 ) antibodies was used at a 1:5000 dilution. Phagocytosis assay BMDMs were seeded at a density of 5 × 10⁵ cells in 500 µl of a 24-well plate and incubated overnight at 37°C with 5% CO₂. The following day, macrophages were infected with C. albicans strains at a MOI of 2, and co-incubated for 1 h in BMDM medium. Non-phagocytosed C. albicans cells were removed by washing the wells three times with PBS. Five images per well were captured using Olympus BX60 microscope at 40x magnification. The number of internalized C. albicans cells and macrophages was manually counted for at least 100 macrophages using the Cell Counter plugin in ImageJ. The phagocytosis index was calculated as the ratio of engulfed C. albicans cells to the total number of macrophages per field. Two biological replicates, each with two technical replicates were carried out for the experiment. Growth curves analysis C. albicans strains were adjusted to an optical density (OD₆₀₀) of 0.1 and suspended in BMDM medium, either without supplementation or supplemented with 10 mM L-alanine or D-alanine (BMDM medium: RPMI 1640 with 12.5 mM HEPES, 20% L-cell conditioned medium, 15% heat-inactivated and filtered fetal bovine serum, and 100 U/ml of penicillin-streptomycin). The growth of the strains was monitored at 37°C using a Tecan Spark 10M microplate reader, with OD₆₀₀ measurements taken every 30 min for 24 h. Three biological replicates, each with three technical replicates were performed for the experiment. Filamentation assay Candida strain suspensions were prepared at a concentration of 1 × 10⁷ cells/ml, as determined by hemocytometer. A volume of 10µl of this suspension was added to 140 µl of BMDM medium, supplemented with either 10 mM L-alanine or D-alanine, in a 96-well plate. The plate was placed under live-cell imaging conditions at 37°C with 5% CO₂. Images were captured every 15 minutes for a total duration of 24 h. Confocal microscopy Immortalized bone marrow-derived macrophage (iBMDMs) derived from control and Ninj1 +/+ macrophages in the C57BL/6J Cas9 + background described in 69 were used. Cells were seeded at 1 × 10⁵ cells per well in a cell culture chamber slide (8-well, catalogue no. 94.6170.802) in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin. Cells were maintained at 37°C in a humidified incubator with 5% CO₂. Cells were allowed to adhere and recover overnight at 37°C in a humidified incubator with 5% CO₂. Macrophages were infected with C. albicans at a yeast-to-macrophage ratio of 3:1 and phagocytosis (1 h), extracellular yeast cells were removed by gentle washing with PBS. At the 7 h post infection media was spiked with 10 µg/ml of calcofluor white (CFW) and allowed to proceed for (8 h total) before fixation with 2% paraformaldehyde for 20 min protected from light. Samples were covered in foil and stored overnight in PBS. The following day, fixed cells were stained with Phalloidin-iFluor 555 (1:1000 dilution, Abcam, ab176756) in PBS with 0.1% Triton X-100 (Sigma, T9284) for 40 min at room temperature. The chambers were then rinsed with PBS three times before confocal imaging on the Zeiss 980 LSM microscope. 16-bit, 1024x1024 frame px frame size images were captured using a Plan-Apochromat 40x/1.3 NA objective lens. Images were processed using Image J 1.54f 71 . Serum amino acid extraction and analysis Female C57BL/6J mice (Monash Animal Research Platform), aged 10–11 weeks and weighing approximately 18–22 g, were individually housed as part of a pair-feeding study requiring accurate monitoring of food intake. These mice had been previously allocated to an unrelated, aborted experiment and were therefore slightly older than typically used. Systemic C. albicans infection was induced via intravenous injection into the lateral tail vein using strain SC5314 at a dose of 1 × 10⁶ CFU per 20 g of body weight (equivalent to 5 × 10⁷ CFU/kg), administered in 100 µl sterile PBS. Mice were euthanized 24 h post-infection by cervical dislocation. Mice were decapitated immediately following euthanasia and trunk blood was collected and centrifuged at 8,000 × g for 10 min at 4°C to separate serum. Serum samples were aliquoted and stored at − 80°C for downstream amino acid profiling. For sample preparation, 25 µl of serum was mixed with 360 µl of ice-cold 100% methanol containing 5 µM of stable isotope-labelled amino acids. Samples were vortexed for 2 min, followed by the addition of 200 µl chloroform and incubation at 37°C for 5 min with shaking. Subsequently, 400 µl of double-distilled water was added, and the mixture was vortexed again for 2 min. Samples were then centrifuged at maximum speed (≥ 12,000 × g) for 10 min at room temperature. A volume of 360 µl from the resulting aqueous phase was transferred to a new tube, evaporated with nitrogen (N2) gas until no liquid remained, and stored at − 80°C until analysis. Statistical analysis All statistical analyses were performed using GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA). Specific statistical tests applied are indicated in the corresponding figure legends. For comparisons involving multiple groups, one-way or two-way ANOVA followed by Tukey’s post hoc test was used for analysis of LDH release, IL-1β levels (ELISA), gene expression by qPCR and mouse CFU enumeration. Non-parametric data sets, including hMDM LDH assay, were analysed using a Mann-Whitney test with Welsh’s correction. For mouse serum nutrient experiments a student’s t-test with multiple comparison analysis was used. A p-value of less than 0.05 was considered statistically significant throughout. Declarations ACKNOWLEDGMENTS We acknowledge expert support provided by Monash University Research Platforms, particularly the Monash MicroImaging Facility and the Monash Animal Research Platform. We are grateful to Marco Herold, Yexuan Deng, and Marcel Doerflinger (Walter and Eliza Hall Institute) for providing mutant macrophages, Maria Almira S. Cleofe for preparing the serum samples for amino acid analyses, and Rhys Dunstan, Chris Stubenrauch, Ivan Poon and Bo Shi for advice and assistance with BN-PAGE. We thank Tim Tucey for initial observations related to the study. We further thank BEI Resources for the C. albicans clinical isolates and the Australian Mycology Reference Centre (Sarah Kidd and Sharon Chen) for sharing the C. auris strain used in this study. This work was supported by grants from the National Health and Medical Research Council of Australia: Investigator grant 2033452 (AT), Investigator grant 2008692 (JEV), Ideas grant 2019765 (AT, AJR), Ideas grant APP2002520 (AT), Project grant APP158678 (AT), Ideas grant 2020757 (AJR, SB), Ideas grant APP1181089 (KL), the Australian Research Council: Future Fellowship FT190100733 (AT), Future Fellowship FT190100266 (KEL), the German Research Foundation (Deutsche Forschungsgemeinschaft) (DFG) within the Priority Program SPP2225 “Exit strategies of intracellular pathogens” (Project 446404928) (BH, JS, TL) and within the Cluster of Excellence ‘Balance of the Microverse’, under Germany’s Excellence Strategy, EXC 2051, Project ID 390713860 (BH, TBS). AUTHOR CONTRIBUTIONS Conceptualization: HW, OT, HS, JS, BvD, BH, AJR, AT Investigation: HW, OT, HS, JS, JN, TL, BvD, TLL, FABO, SB Validation: BvD Formal analysis: HW, OT, HS, JS, TL, SB Resources: CH, JS, JV, KEL, TBS, BH Visualization: HW Funding acquisition: AT, AJR, BH, JS, TL, TBS, JV, KEL Project administration: AT Supervision: TN, BH, AJR, AT Writing – original draft: AT Writing – review & editing: HW, AT with contribution from the other authors. COMPETING INTERESTS: Authors declare that they have no competing interests. DATA AVAILABILITY: All data is available within the manuscript and supplementary files. The iBMDM mutant macrophages obtained from the Walter and Eliza Hall Institute are subject to an MTA. References Tucey TM et al (2018) Glucose Homeostasis Is Important for Immune Cell Viability during Candida Challenge and Host Survival of Systemic Fungal Infection. 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Cell Death Dis 14:755 Borges JP et al (2024) NINJ1 is activated by calcium-driven plasma membrane lipid scrambling during lytic cell death. bioRxiv , 2024.2010.2023.619800 Zhu Y et al (2025) NINJ1 regulates plasma membrane fragility under mechanical strain. Nature Blagojevic M et al (2021) Candidalysin triggers epithelial cellular stresses that induce necrotic death. Cell Microbiol 23:e13371 den Hartigh AB, Loomis WP, Anderson MJ, Frølund B, Fink SL (2023) Muscimol inhibits plasma membrane rupture and ninjurin-1 oligomerization during pyroptosis. Commun Biol 6:1010 Ramos S, Hartenian E, Santos JC, Walch P, Broz P (2024) NINJ1 induces plasma membrane rupture and release of damage-associated molecular pattern molecules during ferroptosis. Embo j 43:1164–1186 Bjanes E et al (2021) Genetic targeting of Card19 is linked to disrupted NINJ1 expression, impaired cell lysis, and increased susceptibility to Yersinia infection. PLoS Pathog 17:e1009967 Han JH et al (2024) NINJ1 mediates inflammatory cell death, PANoptosis, and lethality during infection conditions and heat stress. Nat Commun 15:1739 Cui J et al (2025) Inhibiting NINJ1-dependent plasma membrane rupture protects against inflammasome-induced blood coagulation and inflammation. Elife 12 Traven A, Naderer T (2014) Microbial egress: a hitchhiker's guide to freedom. PLoS Pathog 10:e1004201 Vyas VK, Barrasa MI, Fink GR (2015) A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families. Sci Adv 1:e1500248 Reuss O, Vik A, Kolter R, Morschhäuser J (2004) The SAT1 flipper, an optimized tool for gene disruption in Candida albicans. Gene 341:119–127 Noble SM, Johnson AD (2005) Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans. Eukaryot Cell 4:298–309 Olivier FAB, Traven A (2023) Quantitative live-cell imaging of Candida albicans escape from immune phagocytes. STAR Protoc 4:102737 Simpson DS et al (2022) Interferon-γ primes macrophages for pathogen ligand-induced killing via a caspase-8 and mitochondrial cell death pathway. Immunity 55:423–441e429 Rapino F et al (2013) C/EBPα induces highly efficient macrophage transdifferentiation of B lymphoma and leukemia cell lines and impairs their tumorigenicity. Cell Rep 3:1153–1163 Schindelin J et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682 Additional Declarations There is NO Competing Interest. Supplementary Files WeerasingheetalDatasetS1.xlsx Dataset S1 WeerasingheetalSupplementaryfile30062025.pdf Supplementary Figures and Tables MovieS1.mp4 Ninj1 is the effector of glucose starvation-induced macrophage cell lysis during C. albicans infection MovieS2.mp4 Ninj1 is a macrophage escape factor for C. albicans clinical isolates SUPPLEMENTARYFIGURELEGENDS.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7015602","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":482847965,"identity":"9d3ee686-2119-48b2-be87-cfbcd03051d3","order_by":0,"name":"Harshini Weerasinghe","email":"","orcid":"","institution":"Department of Biochemistry and Molecular Biology, Infection Program, Biomedicine Discovery Institute, Monash University, Clayton 3800, VICTORIA, AUSTRALIA","correspondingAuthor":false,"prefix":"","firstName":"Harshini","middleName":"","lastName":"Weerasinghe","suffix":""},{"id":482847966,"identity":"69641cf9-6417-445e-bcbb-088733d35669","order_by":1,"name":"Orawan Tulyaprawat","email":"","orcid":"","institution":"Department of Biochemistry and Molecular Biology, Infection Program, Biomedicine Discovery Institute, Monash University, Clayton 3800, VICTORIA, AUSTRALIA","correspondingAuthor":false,"prefix":"","firstName":"Orawan","middleName":"","lastName":"Tulyaprawat","suffix":""},{"id":482847967,"identity":"431c07bb-2c66-4fa2-9705-1ae225245974","order_by":2,"name":"Helen Stölting","email":"","orcid":"https://orcid.org/0000-0002-7830-2776","institution":"Department of Biochemistry and Molecular Biology, Infection Program, Biomedicine Discovery Institute, Monash University, Clayton 3800, VICTORIA, AUSTRALIA; Department of Biochemistry and Molecular Biology, Metabolism, Diabetes and Obesity Program, Biomedicine Discovery Institute, Monash University, Clayton 3800, VICTORIA, AUSTRALIA","correspondingAuthor":false,"prefix":"","firstName":"Helen","middleName":"","lastName":"Stölting","suffix":""},{"id":482847968,"identity":"1ac76b65-3f7d-4bd5-b0e6-f9535530b6db","order_by":3,"name":"Johannes Sonnberger","email":"","orcid":"","institution":"Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, GERMANY","correspondingAuthor":false,"prefix":"","firstName":"Johannes","middleName":"","lastName":"Sonnberger","suffix":""},{"id":482847969,"identity":"a3f5988d-62be-434a-8b84-85b69b88e28c","order_by":4,"name":"Joshua Nickson","email":"","orcid":"","institution":"Department of Biochemistry and Molecular Biology, Infection Program, Biomedicine Discovery Institute, Monash University, Clayton 3800, VICTORIA, AUSTRALIA","correspondingAuthor":false,"prefix":"","firstName":"Joshua","middleName":"","lastName":"Nickson","suffix":""},{"id":482847970,"identity":"626f5e7a-7f10-4b50-a53a-a5112677f056","order_by":5,"name":"Theresa Lange","email":"","orcid":"","institution":"Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, GERMANY","correspondingAuthor":false,"prefix":"","firstName":"Theresa","middleName":"","lastName":"Lange","suffix":""},{"id":482847971,"identity":"41f080b1-7f26-405d-bc03-321aba91c956","order_by":6,"name":"Bryce van Denderen","email":"","orcid":"","institution":"Department of Biochemistry and Molecular Biology, Infection Program, Biomedicine Discovery Institute, Monash University, Clayton 3800, VICTORIA, AUSTRALIA","correspondingAuthor":false,"prefix":"","firstName":"Bryce","middleName":"van","lastName":"Denderen","suffix":""},{"id":482847972,"identity":"6f3d9c7e-9032-4a38-a378-e160e86b3c79","order_by":7,"name":"Tricia L. 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Lawlor","email":"","orcid":"https://orcid.org/0000-0003-0471-6842","institution":"The Walter and Eliza Hall Institute of Medical Research, University of Melbourne, Parkville 3052, VICTORIA, AUSTRALIA; Centre for Innate Immunity and Infectious Diseases at the Hudson Institute of Medical Research, Clayton 3168, VICTORIA, AUSTRALIA; Department of Molecular and Translational Science, Monash University; Clayton 3168, VICTORIA, AUSTRALIA","correspondingAuthor":false,"prefix":"","firstName":"Kate","middleName":"E.","lastName":"Lawlor","suffix":""},{"id":482847979,"identity":"943e929e-1341-4c49-bcaa-66b3845e56da","order_by":14,"name":"Stefan Bröer","email":"","orcid":"https://orcid.org/0000-0002-8040-1634","institution":"Research School of Biology, Australian National University, Canberra 0200, AUSTRALIAN CAPITAL TERRITORY, AUSTRALIA","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Bröer","suffix":""},{"id":482847980,"identity":"8d2b8c40-89af-4ace-8edc-ecea84570b85","order_by":15,"name":"Bernhard Hube","email":"","orcid":"https://orcid.org/0000-0002-6028-0425","institution":"Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, GERMANY; Cluster of Excellence Balance of the Microverse, Friedrich Schiller University Jena, Jena, GERMANY; Institute of Microbiology, Friedrich Schiller University, Jena, GERMANY","correspondingAuthor":false,"prefix":"","firstName":"Bernhard","middleName":"","lastName":"Hube","suffix":""},{"id":482847981,"identity":"2de4ad08-7533-465e-9811-85148d8d93a9","order_by":16,"name":"Thomas Naderer","email":"","orcid":"https://orcid.org/0000-0003-2691-0283","institution":"Department of Biochemistry and Molecular Biology, Infection Program, Biomedicine Discovery Institute, Monash University, Clayton 3800, VICTORIA, AUSTRALIA","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Naderer","suffix":""},{"id":482847982,"identity":"29f3ca0c-d667-42cb-8196-9e30f9ce7e7c","order_by":17,"name":"Adam J. 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Quantitative data for alanine levels is shown in the graph. p. i.= post-infection\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eMacrophages (BMDMs) were challenged with \u003cem\u003eC. albicans \u003c/em\u003e(strain SC5314, MOI 3) in base medium with 10 mM glucose (blue graph) or with supplementation of additional glucose or alanine as indicated. Membrane permeabilization was quantified as DRAQ7-positive nuclei. Shown are means and SEM for 3 independent experiments. At least 3000 macrophages were quantified per experiment and experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eMicroscopy images from \u003cem\u003eC. albicans\u003c/em\u003e infection of BMDMs (MOI 3, strain SC5314) at 5 and 13 h post infection in based media (which contains 10 mM glucose), or with supplementation of of 10 mM glucose or alanine. Glucose, but not alanine provides an advantage to \u003cem\u003eC. albicans\u003c/em\u003e, as shown by the escaped hyphae growing thicker at the 13 h time point. Fungal growth curves showing no effect of alanine supplementation on \u003cem\u003eC. albicans\u003c/em\u003egrowth are shown in \u003cstrong\u003eFig. S2A\u003c/strong\u003e. Scale bar= 50µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003eLDH release at 3, 14 and 16 h post BMDM challenge with\u003cem\u003e C. albicans\u003c/em\u003e. Shown are means and SEM from 4 independent experiments (2-way ANOVA Tukey’s multiple comparison test, ** p≤0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. \u003c/strong\u003eIL-1ß ELISA with and without 10 mM L-alanine. Following priming with 50 ng/ml LPS for 3 h, BMDMs were challenged with \u003cem\u003eC. albicans\u003c/em\u003e and samples taken at the indicated time points. Shown are means and SEM for 4 independent experiments (2-way ANOVA Tukey’s multiple comparison test, * p≤0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. \u003c/strong\u003eBMDMs were challenged with \u003cem\u003eC. albicans \u003c/em\u003eSC5314 at MOI 1 or 6. Shown are means and SEM for 2 independent experiments. At least 3500 macrophages were imaged in each experiment per experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG. \u003c/strong\u003eAs in A, but infection with the indicated \u003cem\u003eC. albicans\u003c/em\u003e isolates (MOI 3). Shown are means and SEM for 2 independent experiments. At least 3000 macrophages were quantified per experiment and each experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH. \u003c/strong\u003eAs in A, but with human monocyte-derived macrophages (hMDMs). Shown are means and SEM for 2 independent donors. At least 1400 macrophages were quantified per experiment, for each experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI. \u003c/strong\u003eAs in A, but BMDMs were challenged with \u003cem\u003eC. auris \u003c/em\u003e(MOI 6). Shown are means and SEM for 3 independent experiments. At least 3000 macrophages were quantified per experiment and each experimental group.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7015602/v1/23298ab69ee017d09e0bd0ce.png"},{"id":86385982,"identity":"2a846167-14c2-4b63-ae11-bd068e408daf","added_by":"auto","created_at":"2025-07-10 05:51:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":379554,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eC. albicans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ecompetes with macrophages for alanine using Alt1-dependent metabolism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eImages of macrophages infected with \u003cem\u003eC. albicans\u003c/em\u003e \u003cem\u003ealt1\u003c/em\u003eΔ/Δ and the complemented \u003cem\u003ealt1\u003c/em\u003eΔ/Δ +\u003cem\u003eALT1\u003c/em\u003e strain, with or without 10 mM L-alanine. Images were taken 8 h post-infection. Scale bar=50 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003ePhagocytosis index calculated as the ratio of engulfed \u003cem\u003eC. albicans\u003c/em\u003e to the total number of macrophages (BMDMs) (MOI 2). After 1 h of infection, images were captured, and the number of phagocytosed \u003cem\u003eC. albicans\u003c/em\u003e and macrophages counted. Data were calculated from 2 independent replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eMacrophages (BMDMs) infected with wild type \u003cem\u003eC. albicans\u003c/em\u003e, \u003cem\u003ealt1Δ/Δ\u003c/em\u003e and the complemented strain \u003cem\u003ealt1Δ/Δ\u003c/em\u003e+\u003cem\u003eALT1\u003c/em\u003e (MOI 1), with or without 10 mM L-alanine. Membrane permeabilization was quantified using DRAQ7. Shown are means and SEM from 3 independent experiments. At least 3000 macrophages were imaged per experiment and each experimental group.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7015602/v1/f2fe3c5aa286fe3f2655bafe.png"},{"id":86384938,"identity":"64d3b5f5-ac5d-470b-9214-c57cb7ebb0d1","added_by":"auto","created_at":"2025-07-10 05:35:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":712644,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlanine rescues macrophages independently of its metabolism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eMacrophage membrane permeabilization during \u003cem\u003eC. albicans \u003c/em\u003einfection (strain SC5314, BMDMs) in base medium (blue) or with supplementation of 10 mM L- or D-alanine. Shown are means and SEM for 3 independent experiments. At least 3000 macrophages were quantified per experiment for each of the experimental groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eImages of \u003cem\u003eC. albicans\u003c/em\u003e hyphae during infections of BMDMs, with or without 10 mM L- or D-alanine. Images were taken 8 h post-infection. Scale bar=50 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eMacrophage membrane permeabilization during infection\u003cem\u003e \u003c/em\u003ewith wild type \u003cem\u003eC. albicans\u003c/em\u003e, or the \u003cem\u003ealt1Δ/Δ\u003c/em\u003e mutant, in the presence or absence of D-alanine. Shown are the means and SEM for 2 independent experiments. At least 2500 macrophages were quantified per experiment for each of the experimental groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003eImages showing mitochondrial hyperpolarisation (TMRM staining) at the indicated time points following \u003cem\u003eC. albicans\u003c/em\u003e infection. In control conditions, hyperpolarization at 11 h is followed by de-polarization at 13 h. Supplementation of glucose corrects metabolic dysfunction in macrophages, delaying mitochondrial hyperpolarisation. In contrast, L-alanine does not delay mitochondrial hyperpolarisation. The images are from experiments shown in panel E. Scale bar= 50µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. \u003c/strong\u003eLeft panel: quantification of \u003cem\u003eC. albicans\u003c/em\u003e-infected BMDMs displaying hyperpolarized mitochondria using TMRM fluorescence. Base medium was supplemented with glucose or L-alanine as indicated. Right panel – macrophage membrane permeabilization from the same experiments quantified using DRAQ7 staining. Although alanine delays membrane permeabilization (DRAQ7-staining), it does not delay mitochondrial hyperpolarization (TMRM staining). Shown are means and SEM from 3 independent experiments. At least 3000 macrophages were quantified per experiment and each experimental group.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7015602/v1/34bdb2a5f58c2301f2fb7ebb.png"},{"id":86384941,"identity":"31134563-0aae-4c1d-a3ff-3584a5fe4477","added_by":"auto","created_at":"2025-07-10 05:35:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":576312,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlanine rescues macrophages independently of pyroptosis, necroptosis and apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u0026nbsp; \u003c/strong\u003eWild type (WT) or \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e BMDMs challenged with \u003cem\u003eC. albicans \u003c/em\u003eSC5314 (MOI 6) with or without supplementation of 10 mM L-alanine. DRAQ7-positive macrophages were quantified using live-cell imaging. Shown are means and SEM for 2 independent experiments. At least 3000 macrophages were quantified per experiment and experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u0026nbsp; \u003c/strong\u003eAs in A but wild type (WT) and \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e BMDMs (MOI 3). Shown are means and SEM for 2 independent experiments. At least 3000 macrophages were quantified per experiment and experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u0026nbsp; \u003c/strong\u003eImmunoblots from \u003cem\u003eC. albicans\u003c/em\u003e-infected BMDMs with or without 10 mM L-alanine. Samples were pre-treated with 50 ng/ml LPS. Active, proteolytically processed fragments are p17 for IL-1b, p20 for caspase-1 and p30 for GSDMD. Uncropped blots are shown in \u003cstrong\u003eFig. S3A\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u0026nbsp; \u003c/strong\u003eBMDMs were treated with the pan-apoptotic caspase inhibitor QVD (10 mM) and 10 mM L-alanine. Shown are means and SEM for 3 independent experiments. At least 3500 macrophages were quantified per experiment and experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u0026nbsp; \u003c/strong\u003eAs in D but using immortalized BMDMs (iBMDMs), wild type (WT) or mutant lacking \u003cem\u003eCasp1\u003c/em\u003e, \u003cem\u003eCasp4\u003c/em\u003e, \u003cem\u003eCasp12\u003c/em\u003e, \u003cem\u003eCasp8\u003c/em\u003e and \u003cem\u003eRipk3\u003c/em\u003e. Shown are means and SEM for 3 independent experiments. At least 3500 macrophages were quantified per experiment and experimental group.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7015602/v1/806288d0ab2345be7f4e75bb.png"},{"id":86384946,"identity":"a2e75294-d51c-4b88-b1fd-b893fdb986f8","added_by":"auto","created_at":"2025-07-10 05:35:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":608300,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNINJ1 drives membrane damage of glucose-starved macrophages and is required for alanine’s rescue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u0026nbsp; \u003c/strong\u003eHeatmap of differential gene expression in \u003cem\u003eC. albicans\u003c/em\u003e-infected vs uninfected macrophages. Shown is log2 fold change, FDR \u0026lt;0.05.\u0026nbsp; Data is from our published manuscript \u003csup\u003e1\u003c/sup\u003e, and is accessible at http://rnasystems.erc.monash.edu/2017/papers/glucose_comp/. On the right we show \u003cem\u003eNinj1 \u003c/em\u003eexpression (average of the normalized counts) in infected and uninfected macrophages over time.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u0026nbsp; \u003c/strong\u003eQuantitative PCR of \u003cem\u003eNinj1\u003c/em\u003e expression normalized to actin (\u003cem\u003eActb\u003c/em\u003e), in \u003cem\u003eC. albicans\u003c/em\u003e-infected tdBLaER1 human macrophage-like cell line. Shown are means and SEM for 3 independent experiments, two technical replicates each (2-way ANOVA Tukey’s multiple comparison test, * p≤0.05). Data for activation of \u003cem\u003eNINJ1 \u003c/em\u003eexpression in human monocyte-derived macrophages (hMDMs) is shown in \u003cstrong\u003eFig S4A\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u0026nbsp; \u003c/strong\u003eWild type (WT) and \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e BMDMs infected with \u003cem\u003eC. albicans \u003c/em\u003eat MOI 6 in base medium (dark and light blue curves) or with supplementation of 10 mM L-alanine (red and orange curves). Membrane permeabilization was quantified using DRAQ7. Shown are means and SEM for 2 independent experiments. At least 3000 macrophages were quantified per experiment and experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u0026nbsp; \u003c/strong\u003eLDH release from \u003cem\u003eC. albicans-\u003c/em\u003einfected control or \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e iBMDMs at 14 h post infection, relative to Triton X-lysed macrophages. Shown are means and SEM from 4 independent experiments (2-way ANOVA Tukey’s multiple comparison test, * p≤0.05, **** p≤0.0001)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u0026nbsp; \u003c/strong\u003eAs in C, but wild type (WT) and \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e BMDMs challenged with \u003cem\u003eC. auris \u003c/em\u003e(MOI 6). Shown are means and SEM for 2 independent experiments. At least 3000 macrophages were quantified per experiment and experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.\u0026nbsp;\u0026nbsp; \u003c/strong\u003eAs in C but with 10 mM glycine. Shown are means and SEM for 2 independent experiments. Infections here and in panel C were performed within the same experiments, and therefore the control data (WT and \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e macrophages without supplements) is the same as in panel C. At least 3000 macrophages were quantified per experiment and experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG.\u0026nbsp; \u003c/strong\u003eAs in \u003cstrong\u003eFig. 4A\u003c/strong\u003e but with 10 mM glycine. Shown are means and SEM for 2 independent experiments. At least 3000 macrophages were imaged per experiment, for each experimental group. Infections here and in \u003cstrong\u003eFig. 4A\u003c/strong\u003e were performed together, and therefore the control data (WT and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e macrophages without supplements) is the same.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH.\u0026nbsp; \u003c/strong\u003etdBLaER1 human macrophage-like cell line WT and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003echallenged with \u003cem\u003eC. albicans \u003c/em\u003eand treated with or without glycine. LDH release was determined at 24 h post-infection. Shown are the means and SEM of 3 independent experiments. Measurements for all of WT groups (infected and uninfected) were performed and analyzed within the same experimental setup as strains used in \u003cstrong\u003eFig. S4C\u003c/strong\u003e, using a shared control group across the figures. (Welsh’s t-test correction * p≤0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI.\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003eIL-1b ELISA from control and \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e iBMDMs upon challenge with \u003cem\u003eC. albicans \u003c/em\u003e+/- 10 mM L-alanine. Triton-X and uninfected macrophages were the positive and negative control respectively. Shown are means and SEM for 4 independent experiments (2-way ANOVA Tukey’s multiple comparison test, ** p≤0.01, *** p≤0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ.\u0026nbsp;\u0026nbsp; \u003c/strong\u003eImmunoblot for IL-1ß (p17), caspase-1 (p20), GSDMD (p30) processing from \u003cem\u003eC. albicans\u003c/em\u003e infected wild type (WT) and \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e BMDMs +/- 10 mM L-alanine or glycine, 16 h post-infection. All samples were pre-treated with 50 ng/ml LPS. Uncropped blots are shown in \u003cstrong\u003eFig. S3B\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7015602/v1/8b78286ebbc73db792c91f32.png"},{"id":86384945,"identity":"eae4ff19-f049-44ae-883c-c0af9d7d12de","added_by":"auto","created_at":"2025-07-10 05:35:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":694725,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNINJ1 is a macrophage escape factor for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. albicans \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eworking in conjunction with candidalysin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u0026nbsp; \u003c/strong\u003eControl\u003cem\u003e Ninj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/ +\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e iBMDMs were infected with wild type \u003cem\u003eC. albicans\u003c/em\u003e (BWP17) and the isogenic candidalysin mutant \u003cem\u003eece1Δ/Δ\u003c/em\u003e (MOI 3). Macrophage plasma membrane permeabilization was quantified by DRAQ7 in live-cell imaging. Shown are means and SEM from 3 independent replicates. At least 3500 macrophages were quantified per experiment, for each experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u0026nbsp; \u003c/strong\u003eLDH release from hMDMs infected with \u003cem\u003eC. albicans \u003c/em\u003ewild type (WT) or \u003cem\u003eece1Δ/Δ\u003c/em\u003e at 24 h. NINJ1 was inhibited with 50 mM glycine. Shown are means and SEM from 4 independent donors (t-test with Welsh’s correction * p≤0.05)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u0026nbsp; \u003c/strong\u003eQuantification of band intensity for oligomerised NINJ1 from BN-PAGE of BMDMs infected with wild type (WT) or \u003cem\u003eece1Δ/Δ\u003c/em\u003e \u003cem\u003eC. albicans\u003c/em\u003e or treated with nigericin +/- 10 mM glycine. Uninfected and \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e macrophages were used as controls. The blot and quantification are shown in \u003cstrong\u003eFig. S5\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u0026nbsp; \u003c/strong\u003eControl \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/ +\u003c/em\u003e\u003c/sup\u003eand \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e iBMDMs challenged with the indicated \u003cem\u003eC. albicans\u003c/em\u003e isolate and imaged to quantify DRAQ7-positive macrophages (MOI 3). The hyphal index (hyphal length divided by width) within macrophages is indicated on each graph (h.i.) \u003csup\u003e28\u003c/sup\u003e. Shown are means and SEM for 3 independent experiments. At least 3000 macrophages were quantified per experiment, for each experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u0026nbsp; \u003c/strong\u003eTime-lapse images of \u003cem\u003eC. albicans \u003c/em\u003estrain P78042 escaping from control\u003cem\u003e Ninj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/ +\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eor \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e iBMDMs. The white dashed line demarcates intact macrophages containing \u003cem\u003eC. albicans. \u003c/em\u003eYellow arrows indicate escaped hyphae and their extracellular growth over time. The DRAQ7 signal (indicating permeabilized macrophages) is overlayed in red. Scale bar=100 µm. See also \u003cstrong\u003eMovie S2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.\u0026nbsp;\u0026nbsp; \u003c/strong\u003eConfocal imaging of escaped \u003cem\u003eC. albicans \u003c/em\u003e(strain P78042), stained with the fungal cell wall dye calcofluor white (blue). Calcofluor white differentially labels escaped fungi \u003csup\u003e42\u003c/sup\u003e. Control\u003cem\u003e Ninj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/ +\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e iBMDMs were stained with phalloidin (red). Images were taken at 8 h post-infection. Scale bar=100 µm\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7015602/v1/6c6184ff7dfbae011cf34a9a.png"},{"id":86524121,"identity":"e0f9711f-271b-4c92-aadb-d6f3a050226f","added_by":"auto","created_at":"2025-07-11 15:37:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5380873,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7015602/v1/360fd495-b6db-4d4f-b31c-11a998532a85.pdf"},{"id":86384932,"identity":"19d6291c-ac43-4400-827d-596756f5c4ef","added_by":"auto","created_at":"2025-07-10 05:35:11","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":371138,"visible":true,"origin":"","legend":"Dataset S1","description":"","filename":"WeerasingheetalDatasetS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7015602/v1/86f2f832fb9afa318b55ff9b.xlsx"},{"id":86384933,"identity":"bd2a3b8f-d401-4a19-9b52-07d70048cff8","added_by":"auto","created_at":"2025-07-10 05:35:11","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4150693,"visible":true,"origin":"","legend":"Supplementary Figures and Tables","description":"","filename":"WeerasingheetalSupplementaryfile30062025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7015602/v1/f001390a059db80657c2ba2d.pdf"},{"id":86384944,"identity":"8b7d4409-6661-4b25-99c3-73d0c9fe60fe","added_by":"auto","created_at":"2025-07-10 05:35:11","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":17530152,"visible":true,"origin":"","legend":"Ninj1 is the effector of glucose starvation-induced macrophage cell lysis during C. albicans infection","description":"","filename":"MovieS1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7015602/v1/02c1fb55ea3100c138827a98.mp4"},{"id":86384952,"identity":"019cdad6-690f-4f4b-ade1-4e5820594dd3","added_by":"auto","created_at":"2025-07-10 05:35:12","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":74960788,"visible":true,"origin":"","legend":"Ninj1 is a macrophage escape factor for C. albicans clinical isolates","description":"","filename":"MovieS2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7015602/v1/cd018b3c840c379d02033daf.mp4"},{"id":86385648,"identity":"a1dff276-a802-4a78-b4b8-6115a0acfb91","added_by":"auto","created_at":"2025-07-10 05:43:11","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":22989,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYFIGURELEGENDS.docx","url":"https://assets-eu.researchsquare.com/files/rs-7015602/v1/dffae0622975fb03f2cbb41b.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Infection-induced glucose starvation triggers NINJ1-dependent macrophage lysis and pathogen escape","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eInfection with fungal, bacterial, parasitic and viral pathogens changes glucose homeostasis in the host \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Consequently, proper regulation of glucose metabolism is important for host survival during infection \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. An important aspect of glucose metabolism is the requirement for glycolysis in promoting antimicrobial responses by macrophages. Upon detection of microbes, macrophages rapidly adapt their metabolic pathways, with enhanced glycolysis being a defining feature \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Due to enhanced glycolysis, infected macrophages have an increased need for glucose, both as an energy source and for mounting effective antimicrobial responses, including production of antimicrobial cytokines, reactive oxygen species (ROS) and phagosomal acidification \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, the demand for glucose can pose a challenge for macrophages because several pathogens are avid glucose consumers, leading to glucose-depleted conditions within infection microenvironments \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Moreover, pathogens can disrupt glucose metabolism in macrophages with immunological consequences. We have shown that \u003cem\u003eCandida albicans\u003c/em\u003e and \u003cem\u003eCandida auris\u003c/em\u003e, two fungal pathogens categorised as \u0026ldquo;\u003cem\u003ecritical priority\u003c/em\u003e\u0026rdquo; by the World Health Organization, deplete glucose and cause glucose starvation in macrophages that culminates in wide-spread immune cell death \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Similarly, disruption of macrophage glycolysis has been reported after infection with bacterial pathogens such as \u003cem\u003eSalmonella typhimurium\u003c/em\u003e, \u003cem\u003eYersinia pseudotuberculosis\u003c/em\u003e and upon challenge with peptidoglycan \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Additionally, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e causes glucose stress in keratinocytes during skin infections \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eGiven its importance, modulating glucose homeostasis holds promise for promoting protective immune response and improving disease outcomes. However, the mechanisms that enable macrophages to respond to glucose starvation by inducing immune cell death and inflammation are incompletely defined. We have recently demonstrated that glucose-starved macrophages infected with \u003cem\u003eC. albicans\u003c/em\u003e or \u003cem\u003eC. auris\u003c/em\u003e undergo lytic cell death\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The lytic cell death is thought reduce the containment of fungi by macrophages and promote dissemination, since \u003cem\u003eCandida\u003c/em\u003e species proliferate robustly in extracellular environments using a range of host nutrients. As such, reducing glucose starvation-induced macrophage lysis could increase fungal immune containment, but the mechanism linking metabolic dysfunction with macrophage lysis is unclear. Although glucose-starved macrophages activate the NLRP3 inflammasome in response to \u003cem\u003eC. albicans\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, and an activated NLRP3 inflammasome causes lytic macrophage pyroptosis under many conditions\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, NLRP3 was surprisingly dispensable for macrophage lysis following \u003cem\u003eC. albicans\u003c/em\u003e-induced glucose starvation despite inducing the secretion of the antifungal cytokine IL-1\u0026szlig; \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. NLRP3 was also dispensable for macrophage lysis upon glucose starvation caused by \u003cem\u003eC. auris\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Other mechanisms known to kill glucose-starved cancer cells, such as apoptosis or excessive accumulation of reactive oxygen species (ROS) \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, also do not account for the lysis of glucose-starved macrophages upon \u003cem\u003eCandida\u003c/em\u003e infection \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study we employed a nutritional approach to discover the trigger and mechanism of immune cell lysis in glucose-starved macrophages responding to fungal pathogens. We demonstrate that infection-induced glucose starvation is a trigger for activation of membrane damage that depends on NINJ1, the recently identified factor that induces plasma membrane rupture (PMR) and cell lysis upon immune cell death \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Our findings demonstrate that glucose starvation is a previously unidentified trigger of NINJ1-dependent PMR in infected macrophages, show that NINJ1 operates independently of major cell death programs in response to metabolic dysfunction, and demonstrate that NINJ1-dependent PMR can be inhibited by an important mammalian metabolite, the amino acid alanine, thereby suggesting a nutritional strategy to protect macrophages. Our comprehensive analyses of cell death pathways established that NINJ1 has the most dominant role in mediating \u003cem\u003eC. albicans\u003c/em\u003e-induced macrophage damage and is the only cell death factor identified so far to respond to infection of macrophages by the emerging drug-resistant pathogen \u003cem\u003eC. auris\u003c/em\u003e. Finally, we show a key role for NINJ1-induced macrophage PMR in facilitating fungal immune escape.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eAlanine rescues macrophages from cell damage caused by glucose starvation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn infected animals, \u003cem\u003eC. albicans\u003c/em\u003e causes lowering of blood glucose levels, in addition to depleting available glucose for macrophages \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Supplementing glucose rescues \u003cem\u003eC. albicans\u003c/em\u003e-infected macrophages by promoting their viability and cellular integrity, but it also potently promotes fungal growth and thus provides an advantage to \u003cem\u003eC. albicans\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. We therefore considered whether a nutritional rescue of macrophages without advantaging \u003cem\u003eC. albicans\u003c/em\u003e is feasible. Amino acids metabolism may be a possible avenue to achieve this goal, since amino acids can support macrophage immunometabolism \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Furthermore, while \u003cem\u003eC. albicans\u003c/em\u003e grows on amino acids, they are not its preferred carbon source.\u003c/p\u003e\u003cp\u003eThe effects of fungal infection on global host amino acid homeostasis are poorly understood. We found that mice systemically infected with \u003cem\u003eC. albicans\u003c/em\u003e display profoundly perturbed amino acid homeostasis. There was a reduction in serum levels for arginine, aspartate, serine, glycine, alanine and several other amino acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). In contrast, glutamine, glutamate and others remained unchanged, while the levels of valine increased upon infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). We have previously shown that supplementation of arginine, serine, glutamine and leucine has no or very minor effects on rescuing \u003cem\u003eC. albicans\u003c/em\u003e-infected macrophages \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Further inspection of affected amino acids showed that alanine displays a large, 2-fold reduction in serum levels in \u003cem\u003eC. albicans\u003c/em\u003e-infected mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Alanine is an important metabolic substrate in mammals, both as a gluconeogenic substrate and feeding into the Krebs cycle for ATP production. Since alanine metabolism plays a role in low nutrient conditions and under metabolic stress \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, we decided to test its effects on \u003cem\u003eC. albicans\u003c/em\u003e-infected macrophages.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo do so, we utilized our live-cell imaging platform with bone marrow-derived mouse macrophages (BMDMs) \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Here, macrophage plasma membrane permeabilization is quantified over time using the membrane-impermeable DNA dye DRAQ7. In this assay, \u003cem\u003eC. albicans\u003c/em\u003e depletes glucose, resulting in glucose starvation of macrophages and cell death several hours after infection \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Supplementation of alanine rescued macrophages from glucose starvation-induced plasma membrane permeabilization, and the rescue was more potent than supplementation of glucose at the same concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). As expected, glucose provided an advantage to \u003cem\u003eC. albicans\u003c/em\u003e as seen by enhanced hyphal growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. In contrast, alanine rescued macrophages without providing an advantage to \u003cem\u003eC. albicans\u003c/em\u003e. \u003cem\u003eC. albicans\u003c/em\u003e growth was the same in the presence or absence of alanine (\u003cb\u003eFig. S2A\u003c/b\u003e). Moreover, hyphal growth, which contributes to macrophage damage, was also the same (i.e. neither enhanced nor inhibited) in the presence of alanine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cb\u003eFig. S2B\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eIn addition to reducing membrane permeabilization (measured by DRAQ7-staining), supplementation of alanine also reduced lactate dehydrogenase (LDH) release into the extracellular medium, a measure of PMR and cell lysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Furthermore, alanine caused a reduction in secreted pro-inflammatory cytokine IL-1\u0026szlig; at 3 and 14 h post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). At 16 h after infection alanine had no effect on IL-1\u0026szlig; secretion, which was high in the presence or absence of alanine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The multiplicity of infection (ratio of \u003cem\u003eC. albicans\u003c/em\u003e to macrophages) influences the kinetics of macrophage cell death and activation of glucose starvation \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Alanine rescued macrophages infected at both high (6:1) and low (1:1) multiplicity of infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), indicating that it has a broadly protective effect. Additionally, \u003cem\u003eC. albicans\u003c/em\u003e clinical isolates show distinct abilities to damage macrophages \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Thus, in addition to the prototype isolate SC5314 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), we established that alanine also rescues macrophages during infection with other \u003cem\u003eC. albicans\u003c/em\u003e isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Finally, rescue by alanine was recapitulated in primary monocyte-derived human macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), and alanine also rescued \u003cem\u003eC. auris\u003c/em\u003e-infected macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Collectively, these results show that alanine rescues macrophages infected with two different fungal pathogens, in response to multiple clinical isolates, and the rescue is recapitulated in mouse and human immune cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eC. albicans\u003c/b\u003e \u003cb\u003ecompetes with macrophages for alanine using Alt1-dependent metabolism\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e is metabolically versatile and could compete with macrophages for alanine. To test this, we generated a \u003cem\u003eC. albicans alt1D/D\u003c/em\u003e mutant that lacks alanine amino transferase needed for alanine utilization. The \u003cem\u003ealt1D/D\u003c/em\u003e mutant grew as well as the wildtype in macrophage culture medium, in the presence or absence of alanine (\u003cb\u003eFig. S2A\u003c/b\u003e). Moreover, the mutant formed hyphae in culture medium and within macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB), and infected macrophages normally (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Consistent with these phenotypes, under standard conditions (no additional alanine) the \u003cem\u003ealt1D/D\u003c/em\u003e mutant triggered macrophage plasma membrane permeabilization with the same kinetics as the parental \u003cem\u003eC. albicans\u003c/em\u003e strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Therefore, the \u003cem\u003ealt1D/D\u003c/em\u003e mutant is not generally defective in infecting macrophages or triggering macrophage damage. However, in the presence of alanine, infections with \u003cem\u003ealt1D/D\u003c/em\u003e led to prolonged rescue relative to parental \u003cem\u003eC. albicans\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, compare red and pink graphs). The prolonged rescue could be partially reversed by complementation of the mutant with \u003cem\u003eALT1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, pink versus salmon graphs). Collectively, these data show that \u003cem\u003eC. albicans\u003c/em\u003e competes with macrophages for alanine by using Alt1-dependent metabolism. Therefore, rescue by alanine is enhanced when competition from \u003cem\u003eC. albicans\u003c/em\u003e is reduced in the \u003cem\u003ealt1D/D\u003c/em\u003e mutant.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMacrophage rescue does not require metabolic utilization of alanine\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAlanine rescued \u003cem\u003eC. albicans-\u003c/em\u003einfected macrophages better than glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This finding prompted us to consider if the rescue may be non-metabolic. To test this, we used L- and D-isomers of alanine. L-alanine is metabolized effectively by fungal and mammalian cells (and was used in our experiments so far). D-alanine is not metabolized well. Yet, D-alanine rescued \u003cem\u003eC. albicans\u003c/em\u003e-infected macrophages as well as L-alanine (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), without affecting the growth or filamentation of \u003cem\u003eC. albicans\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cb\u003eFig. S2A and S2B\u003c/b\u003e). Moreover, consistent with D-alanine not being metabolised, infection with the \u003cem\u003eC. albicans alt1D/D\u003c/em\u003e mutant did not lead to prolonged rescue in the presence of D-alanine (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). As a control, we show that D-alanine did not have any effects on the growth or filamentation of the \u003cem\u003ealt1D/D\u003c/em\u003e mutant (\u003cb\u003eFig. S2A and S2B\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe have previously shown that \u003cem\u003eC. albicans\u003c/em\u003e-induced glucose starvation triggers a hyperpolarization event in macrophage mitochondria, followed by depolarization \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. These changes in mitochondrial membrane potential result from metabolic dysfunction and can be delayed by glucose supplementation \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Unlike glucose, supplementation of alanine did not delay mitochondrial hyperpolarization/depolarization, despite delaying macrophage plasma membrane permeabilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Collectively, these results show that alanine does not correct the metabolic dysfunction caused by glucose starvation. Instead, it acts downstream of metabolic dysfunction to prevent macrophage plasma membrane damage.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAlanine does not act on pyroptosis, necroptosis or apoptosis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNext, we tested for a potential involvement of immune cell death programs, since \u003cem\u003eC. albicans\u003c/em\u003e triggers NLRP3-caspase-1 inflammasome-dependent pyroptosis in the first few hours post infection \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In pyroptosis-negative \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e macrophages (lacking the pore-forming protein Gasdermin D) the initial membrane permeabilization was delayed as expected, but alanine caused a further delay showing that it does not need GSDMD to rescue macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Similarly, alanine rescued \u003cem\u003eC. albicans\u003c/em\u003e-infected \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Of note, we have previously shown that in \u003cem\u003eC. albicans\u003c/em\u003e-infected macrophages GSDMD processing to its pore-forming fragment depends on NLRP3; however, in our imaging assay, \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e macrophages show a reduced initial delay in DRAQ7-positive macrophages relative to \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e due to increased formation of macrophage extracellular nets that are also stained by DRAQ7 \u003csup\u003e42\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eConsistent with the effect of alanine being independent of inflammasome-induced cell lysis, supplementation of alanine did not prevent the processing of caspase 1, IL-1\u0026szlig; or GSDMD in response to \u003cem\u003eC. albicans\u003c/em\u003e, although it reduced their release into the supernatant due to reduced plasma membrane rupture (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cb\u003eFig. S3\u003c/b\u003e). Alanine also rescued macrophages when all apoptotic caspase-dependent pathways were chemically inhibited with Q-VD-OPh (QVD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) and when pyroptosis, necroptosis and extrinsic apoptosis were simultaneously disrupted using genetic approaches \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eNINJ1 is the target of alanine\u0026rsquo;s rescue and the effector of glucose starvation-induced macrophage plasma membrane damage\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate other cell death factors potentially involved in alanine\u0026rsquo;s rescue, we re-analyzed our published RNAseq dataset from \u003cem\u003eC. albicans\u003c/em\u003e-infected BMDMs \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Five genes encoding cell death factors showed increased expression upon \u003cem\u003eC. albicans\u003c/em\u003e infection: \u003cem\u003eNlrp3\u003c/em\u003e and \u003cem\u003eCasp11\u003c/em\u003e (pyroptosis), \u003cem\u003eMlkl\u003c/em\u003e (necroptosis), \u003cem\u003eZbp1\u003c/em\u003e (apoptosis and necroptosis) and \u003cem\u003eNinj1\u003c/em\u003e (performs macrophage plasma membrane rupture (PMR) downstream of several immune cell death) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We ruled out pyroptosis and necroptosis in mediating the lysis of glucose-starved macrophages and being involved in macrophage rescue by alanine (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e Zbp1 is also unlikely because it signals for caspase-8-driven apoptosis and MLKL-mediated necroptosis \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, which we excluded (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We thus focused on the recently identified executioner of PMR, NINJ1\u003csup\u003e33\u003c/sup\u003e. Indeed, we show that \u003cem\u003eNinj1\u003c/em\u003e expression is upregulated by \u003cem\u003eC. albicans\u003c/em\u003e infection not only in mouse macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), but also in human macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDeletion of \u003cem\u003eNinj1\u003c/em\u003e rescued \u003cem\u003eC. albicans\u003c/em\u003e-infected macrophages from damage caused by glucose starvation, inhibiting both plasma membrane permeabilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eC \u003cb\u003eand Movie S1\u003c/b\u003e) and PMR (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). \u003cem\u003eNinj1\u003c/em\u003e loss conferred the strongest rescue of \u003cem\u003eC. albicans\u003c/em\u003e-infected macrophages, when compared with all other host cell death factors that we have tested to date (compare Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eC with data in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e and our previous work \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e). Confirming its broad role in response to fungi, deletion of \u003cem\u003eNinj1\u003c/em\u003e also rescued macrophages infected with \u003cem\u003eC. auris\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Alanine did not further rescue \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e macrophages (neither membrane permeabilization nor PMR) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e, showing that alanine needs NINJ1 to exert its protective effects. Glycine, an amino acid that is structurally very similar to alanine, is reported to inhibit PMR by preventing NINJ1 oligomerization that is required for rupturing membranes \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Similarly to glycine, alanine also prevented NINJ1 oligomerization (\u003cb\u003eFig. S4B\u003c/b\u003e). Collectively, these results show that glucose starvation triggers NINJ1-dependent macrophage membrane damage in \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eC. auris\u003c/em\u003e-infected macrophages. Moreover, alanine rescues glucose-starved macrophages by inhibiting NINJ1.\u003c/p\u003e\u003cp\u003eTo further test the involvement of NINJ1 in glucose starvation and fungal infections, we asked if glycine acts similarly to alanine in rescuing glucose-starved macrophages \u003cem\u003evia\u003c/em\u003e NINJ1. Indeed, glycine rescued \u003cem\u003eC. albicans\u003c/em\u003e-infected macrophages in a NINJ1-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Furthermore, like alanine glycine rescued \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e macrophages, both mouse (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eG) and human (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). This is consistent with no role for GSDMD-dependent pyroptosis in response to NINJ1-dependent membrane damage caused by glucose stress.\u003c/p\u003e\u003cp\u003eIL-1\u0026szlig; secretion was reduced in \u003cem\u003eC. albicans\u003c/em\u003e-infected \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e macrophages, with no further reduction upon supplementation with alanine or glycine (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eI\u003cb\u003e)\u003c/b\u003e This is in line with NINJ1-dependent PMR causing some cytokine release, and glycine and alanine acting on NINJ1 to reduce cytokines. Despite lower levels of released IL-1\u0026szlig;, its proteolysis was normal in \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e macrophages, suggesting that reduced release of IL-1\u0026szlig; is due to lower PMR rather than inhibition of inflammasome signalling. Similarly, caspase-1 and GSDMD were proteolytically processed in \u003cem\u003eC. albicans\u003c/em\u003e-infected \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e macrophages, although less cleaved GSDMD was found in supernatants consistent with reduced PMR (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ).\u003c/p\u003e\u003cp\u003e\u003cb\u003eNINJ1 is a macrophage escape factor for\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e \u003cb\u003eworking together with candidalysin\u003c/b\u003e\u003c/p\u003e\u003cp\u003eInactivation of NINJ1 caused a large reduction in membrane permeabilization following \u003cem\u003eC. albicans\u003c/em\u003e infection, but the rescue was not complete (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). We therefore decide to determine the mechanism causing the additional damage. The fungal pore-forming peptide candidalysin is secreted at high levels by \u003cem\u003eC. albicans\u003c/em\u003e hyphae \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, whereby it damages the macrophage plasma membrane during hyphal escape \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Therefore, candidalysin was a candidate factor to cause the residual membrane damage in the absence of NINJ1.\u003c/p\u003e\u003cp\u003eFollowing infection with a mutant lacking the candidalysin-encoding gene \u003cem\u003eECE1\u003c/em\u003e (\u003cem\u003eece1D/D\u003c/em\u003e), there was an initial delay in macrophage plasma membrane permeabilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). This is consistent with the roles of candidalysin in hyphal escape \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Compared to wild type \u003cem\u003eC. albicans\u003c/em\u003e, infection with \u003cem\u003eece1D/D\u003c/em\u003e further reduced plasma membrane permeabilization in \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The additional effect of candidalysin was also seen in human macrophages when NINJ1 was inactivated by glycine (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, \u003cb\u003eFig. S4C\u003c/b\u003e). These data show that candidalysin is responsible for the residual damage in the absence of NINJ1 in mouse and human macrophages. NINJ1 oligomerizes in the membrane as prerequisite for PMR \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Consistently, \u003cem\u003eC. albicans\u003c/em\u003e infection caused NINJ1 oligomerization, which was reduced in infections with \u003cem\u003eece1D/D\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, \u003cb\u003eFig. S5\u003c/b\u003e). This shows that, in addition to working in parallel to NINJ1-dependent pathways, candidalysin-induced membrane damage contributes to activation of NINJ1 following \u003cem\u003eC. albicans\u003c/em\u003e infection.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further test the contributions of NINJ1 and candidalysin in \u003cem\u003eCandida\u003c/em\u003e infections, we used \u003cem\u003eC. albicans\u003c/em\u003e clinical isolates that display substantial diversity in candidalysin expression levels. The reference strain SC5314 (used in most of our experiments) readily forms hyphae under \u003cem\u003ein vitro\u003c/em\u003e conditions and secrets high levels of candidalysin \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. We selected four additional bloodstream infection isolates with reduced candidalysin expression \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. These isolates also display lower hyphal growth within macrophages \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Deletion of \u003cem\u003eNinj1\u003c/em\u003e strongly rescued macrophages in infections with all low-candidalysin isolates, and the rescue was stronger than with the candidalysin-high strain SC5314 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e macrophages were protected from plasma membrane permeabilization for over 20 h upon infection with strain P78042, whereas for P57055, P57072 and P76067 plasma membrane permeabilization started late in \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e macrophages and progressed more slowly than with SC5314 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). For instance, at 20 h post-infection, plasma membrane permeabilization was seen for 30% of \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e macrophages for SC5314, 15% for P57055, 17% for P57072, 8% for P76067 and 6% for P78042. Collectively these results show that NINJ1 has a conserved role in macrophage plasma membrane damage across diverse \u003cem\u003eC. albicans\u003c/em\u003e strains and an increasingly important role with strains that expresses lower candidalysin levels.\u003c/p\u003e\u003cp\u003eInvasive hyphal growth and secreted candidalysin together damage macrophage membranes to promote immune escape of \u003cem\u003eC. albicans\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. This could promote dissemination, since \u003cem\u003eC. albicans\u003c/em\u003e proliferates robustly in extracellular environments using a range of host nutrients. Based on data in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eD we predicted that \u003cem\u003eC. albicans\u003c/em\u003e strains with reduced hyphal growth and candidalysin expression might use NINJ1-dependent plasma membrane damage as their major mechanism for escape. Indeed, this was the case. Strain P78042 showed a large reduction in escape from \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e macrophages, with few externalized hyphae observable (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, \u003cb\u003eMovie S2\u003c/b\u003e). In contrast, extensive escape of this strain was seen from wildtype macrophages, followed by proliferation of the externalized hyphae (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, \u003cb\u003eMovie S2).\u003c/b\u003e Strains P57055, P57072 and P76067 strains were likewise delayed in their egress from \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e macrophages (\u003cb\u003eFig. S6, Movie S2\u003c/b\u003e). These findings determine that NINJ1 is a major fungal escape factor.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we establish the mechanism by which glucose-starved macrophages lyse in response to fungal pathogens. We show that the mechanism is mediated by NINJ1, which causes macrophage plasma membrane permeabilization and rupture. This mechanism is conserved in both mouse and human macrophages, and establishes glucose starvation as a trigger for NINJ1 activation in infected macrophages. Our findings further show nutritional supplementation of alanine, an important mammalian metabolite, inhibits NINJ1-dependent damage without providing a growth advantage to \u003cem\u003eC. albicans\u003c/em\u003e. Moreover, NINJ1 is the dominant host effector of macrophage damage in response to \u003cem\u003eC. albicans\u003c/em\u003e, since inactivation of NINJ1 has by far the biggest effect in rescuing macrophages compared to all other cell death programs studied to date. For \u003cem\u003eC. auris\u003c/em\u003e, NINJ1 is the only host factor discovered so far driving macrophage damage. Finally, \u003cem\u003eC. albicans\u003c/em\u003e uses NINJ1-dependent plasma membrane damage to egress from macrophages, showing that NINJ1-dependent mechanisms contribute to immune evasion. Mediating pathogen escape is a yet unappreciated role for NINJ1 in infection.\u003c/p\u003e\u003cp\u003eAs we show here and previously, glucose starvation causes changes in mitochondrial membrane polarization, whereby hyperpolarization is followed by depolarization coinciding with plasma membrane permeabilization and damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Like our results with \u003cem\u003eC. albicans\u003c/em\u003e, the ATP synthase inhibitor oligomycin causes mitochondrial hyperpolarization and triggers NINJ1-dependent macrophage membrane damage \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Therefore, changes to mitochondrial function are a probable trigger for NINJ1 activation in response to glucose starvation and metabolic dysfunction caused by \u003cem\u003eC. albicans\u003c/em\u003e. Furthermore, we show that the fungal pore-forming toxin candidalysin plays a role in activating NINJ1. Our results are supported by data showing that bacterial pore-forming toxins streptolycin and listeriolysin activate NINJ1 \u003csup\u003e33\u003c/sup\u003e. The precise activating mechanisms for NINJ1 oligomerization and membrane rupture are still to be resolved and may differ depending on the stimulus. However, the current consensus is that changes to membrane structure, such as movement of phospholipids (scrambling), swelling or mechanical stress, are involved \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Candidalysin damages mitochondrial membranes and the plasma membrane of host cells \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, providing the likely mechanism by which it activates NINJ1. Interestingly, a recent study showed that in addition to membrane damage caused by pore-forming toxins, mechanical damage is needed for full rupture mediated by NINJ1 \u003csup\u003e57\u003c/sup\u003e. In line with our results with \u003cem\u003eC. albicans\u003c/em\u003e, NINJ1-dependent membrane rupture triggered by mechanical challenge does not require the characterised cell death programs \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. In the case of \u003cem\u003eC. albicans\u003c/em\u003e, the mechanical damage maybe be provided by the growing fungal hyphae exerting force on the membrane \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Therefore, our data indicates that, during infection a combination of immunometabolic dysfunction, membrane-damaging toxins secreted by pathogens and mechanical stress on immune cell membranes from microbial growth are key stimuli for triggering NINJ1-mediated damage and PMR.\u003c/p\u003e\u003cp\u003eOur findings further characterise a nutritional approach to protect macrophages by showing that supplementation of alanine rescues glucose-starved macrophages in a NINJ1-dependent manner. Alanine is an important metabolite under metabolic stress \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, and our data in infected mice is consistent with major disruption of amino acid homeostasis in \u003cem\u003eC. albicans\u003c/em\u003e-infected animals and a large reduction in serum levels of alanine. We show that alanine rescues macrophages without correcting their metabolic dysfunction, in line with studies showing that NINJ1-induced membrane damage occurs independently of metabolic dysfunction during cell death \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Moreover, supplementation of alanine or genetic deletion of Ninj1 cause a reduction in both membrane permeabilization and PMR in \u003cem\u003eC. albicans\u003c/em\u003e-infected macrophages, in a manner that is independent of pyroptosis, apoptosis or necroptosis. Accordingly, the proteolytic processing of caspase-1, GSDMD and IL-1\u0026szlig; in response to \u003cem\u003eC. albicans\u003c/em\u003e is not prevented by NINJ1 inhibition, although the amount of IL-1\u0026szlig; released by infected macrophages is reduced. Alanine is the third metabolite with NINJ1-inhibitory activity, in addition to glycine and the GABA receptor agonist muscimol \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. These metabolites inhibit NINJ1 oligomer formation, although the biochemical mechanism remains to be understood \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Alanine and glycine are structurally similar, and muscimol also shares features of its chemical structure with glycine \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Therefore, the mechanism of NINJ1 inhibition by these molecules may be conserved.\u003c/p\u003e\u003cp\u003eThe requirement for NINJ1 for both membrane permeabilization and PMR of \u003cem\u003eC. albicans\u003c/em\u003e-infected macrophages is similar to ferroptosis, infection with \u003cem\u003eYersinia\u003c/em\u003e or \u003cem\u003eSalmonella\u003c/em\u003e, treatment with the lipopolysaccharide (LPS) in the presence of heat shock and mechanical membrane challenge as a trigger for NINJ1 \u003csup\u003e57, 60, 61, 62\u003c/sup\u003e; however, unlike our data with \u003cem\u003eC. albicans\u003c/em\u003e, inactivation of NINJ1 does not impair release of IL-1\u0026szlig; in response to these bacterial pathogens and LPS plus heat shock \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Similar to our data with \u003cem\u003eC. albicans\u003c/em\u003e, Ninj1 mutant mice had lower levels of plasma IL-1\u0026szlig; in response to infection with \u003cem\u003eEscherichia coli\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e, showing that NINJ1-dependent damage drives the release of this major proinflammatory cytokine across highly diverse infections. Downstream of pyroptosis NINJ1 is required for terminal PMR but has no role in the earlier GSDMD-mediated membrane permeabilization event or secretion of IL-1\u0026szlig; \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The explanation is that, during pyroptosis, membrane permeabilization and IL-1\u0026szlig; secretion depend on GSDMD pores, which form independently on NINJ1 \u003csup\u003e33\u003c/sup\u003e. Given that, as we and others have shown, \u003cem\u003eC. albicans\u003c/em\u003e triggers NLRP3-dependent pyroptosis \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, the question is why NINJ1 mediates membrane permeabilization in our experiments. The answer is that \u003cem\u003eC. albicans\u003c/em\u003e triggers pyroptosis early after macrophage infection \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, while glucose starvation occurs later and triggers NINJ1-dependent damage independently of GSDMD or pyroptosis, as we show here. Consistently, we show that alanine only needs NINJ1 but not any of the pyroptotic factors to rescue macrophages, and the same is true for glycine. Therefore, downstream of glucose starvation GSDMD has no role in macrophage membrane damage, explaining why NINJ1 is involved in both membrane permeabilization and PMR under these conditions.\u003c/p\u003e\u003cp\u003eWhile GSDMD pores mediate the release of small molecules including IL-1\u0026szlig;, NINJ1-dependent PMR releases large pro-inflammatory danger-associated molecular patterns (DAMPs), such as LDH and the High Mobility Group Box 1 (HMGB1) protein \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Using diverse clinical isolates of \u003cem\u003eC. albicans\u003c/em\u003e, we show that NINJ1-dependent PMR also promotes the release of the pathogen itself, thereby driving immune escape. Previous work by us and others has shown that \u003cem\u003eC. albicans\u003c/em\u003e uses several mechanisms to egress from macrophages and disseminate. These include early escape via GSDMD-driven pyroptosis, as well as hyphae- and candidalysin-induced macrophage membrane damage \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. However, these established mechanisms do not account for several findings. Firstly, \u003cem\u003eC. albicans\u003c/em\u003e clinical strains are diverse, with several unable to form robust hyphae \u003cem\u003ein vitro\u003c/em\u003e, activate pyroptosis or express high levels of candidalysin \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Secondly, only a proportion of macrophages in the population trigger pyroptosis upon \u003cem\u003eC. albicans\u003c/em\u003e challenge \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. We now show that NINJ1 plays an increasingly important role in macrophage membrane damage and egress for \u003cem\u003eC. albicans\u003c/em\u003e isolates with reduced hyphal formation and candidalysin expression. These findings identify NINJ1 as a central host effector of fungal escape from macrophages. Our data with high \u0026ndash; and low-candidalysin isolates and candidalysin mutant strains show that NINJ1 works together with candidalysin to damage macrophage membranes and promote \u003cem\u003eC. albicans\u003c/em\u003e escape.\u003c/p\u003e\u003cp\u003eIn conclusion, our study identifies NINJ1 as the host effector of membrane damage following glucose starvation of \u003cem\u003eCandida\u003c/em\u003e-infected macrophages and indicates that NINJ1 is a central control point for regulating inflammatory cytokine release, while also increasing immune containment of fungal pathogens to reduce dissemination. We propose that NINJ1 might be a general effector of PMR upon glycolytic dysfunction caused by a broad range of pathogens, such as \u003cem\u003eSalmonella\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Moreover, given the central role of NINJ1 in performing PMR downstream of cell death pathways used by diverse pathogens to egress from macrophages \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e, we posit that NINJ1 may be commonly hijacked as a pathogen egress factor.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cb\u003eEthics statement\u003c/b\u003e\u003c/p\u003e\u003cp\u003e All procedures involving mice for the isolation of BMDMs were conducted in compliance with protocols approved by the Monash University Animal Ethics Committee (approval ID ERM37724). Experiments involving the collection of human blood samples received ethical clearance from the Monash University Human Research Ethics Committee (ERM 44451). Experiments involving \u003cem\u003eC. albicans\u003c/em\u003e infection of mice and blood collection were approved by the Monash University Animal Ethics Committee (approval ID ERM36304). Isolation of hMDMs in \u003cb\u003eFig S4\u003c/b\u003e was conducted according to the principles of the Declaration of Helsinki. The blood donation protocol and use of blood for isolation of human monocyte-derived macrophages were approved by the institutional ethics committee of the University Hospital Jena (permission number 2207\u0026ndash;01/08).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCandida\u003c/b\u003e \u003cb\u003estrains and media\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe strains used in this study are listed in \u003cb\u003eTable S\u003c/b\u003e1. Unless stated otherwise the clinical isolate SC5314 was the main strain used in this study. All clinical strains of \u003cem\u003eC. albicans\u003c/em\u003e were obtained from BEI Resources, NAID, NIH (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.niaid.nih.gov/research/bei-resources-repository\u003c/span\u003e\u003cspan address=\"https://www.niaid.nih.gov/research/bei-resources-repository\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). \u003cem\u003eCandida\u003c/em\u003e strains were routinely cultured on YPD medium composed of 1% yeast extract, 2% peptone, 2% glucose, and solidified with 2% agar, for 2 days. For experiments requiring overnight liquid cultures, such as growth curve kinetics, a single colony was picked from a freshly streaked YPD agar plate and incubated in liquid YPD at 30\u0026deg;C for 18 h. Cultures were then diluted to an initial OD\u003csub\u003e600\u003c/sub\u003e of 0.1 using prewarmed bone marrow-derived macrophage (BMDM) medium before use in assays. BMDM medium consisted of RPMI 1640 supplemented with 12.5 mM HEPES, 10% fetal bovine serum (FBS), 20% L-cell conditioned medium providing macrophage colony-stimulating factor, and 100 U/ml penicillin-streptomycin. RPMI 1640 (Thermo Fisher, catalog no. 11875093) was used as a base and supplemented with either glucose, alanine or glycine at 10 or 40 mM. For filamentation assays, macrophage infection studies and phagocytosis assays, a single colony was selected from fresh YPD solid medium and patched onto new plates and incubated at 30\u0026deg;C overnight. The patches were suspended in PBS the following day, and diluted to the desired concentration for the experiments, as determined by counting using a hemocytometer.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStrain construction\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePrimers used to construct the mutant strains in this study are listed in \u003cb\u003eTable S2\u003c/b\u003e. The \u003cem\u003eECE1\u003c/em\u003e open reading frame (ORF) in \u003cem\u003eC. albicans\u003c/em\u003e SC5314 was deleted using a CRISPR-Cas9 protocol \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. The deletion of the \u003cem\u003eECE1\u003c/em\u003e-ORF was confirmed via colony PCR, and the Cas9-expression vector was excised by Flp-recombination. The \u003cem\u003eECE1\u003c/em\u003e-complemented strain was generated using the SAT1-Flipper method \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. The cloned sequences were verified by Sanger-sequencing. The \u003cem\u003eECE1\u003c/em\u003e complementation cassette was excised using \u003cem\u003eSac\u003c/em\u003eI/\u003cem\u003eApa\u003c/em\u003eI and introduced into the \u003cem\u003eC. albicans ece1\u003c/em\u003e∆/∆ strain (M2974) Correct integration was confirmed by colony PCR at the cassette junctions and the \u003cem\u003eSAT1\u003c/em\u003e-marker was excised by Flp-recombination. The homozygous deletion of \u003cem\u003eALT1\u003c/em\u003e and the complemented strain were constructed in the SN152 strain (\u003cem\u003ehis1\u003c/em\u003e\u0026thinsp;\u0026minus;\u0026thinsp;\u003cem\u003eleu2\u003c/em\u003e\u0026thinsp;\u0026minus;\u0026thinsp;\u003cem\u003earg4\u003c/em\u003e\u0026minus;) \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, using the \u003cem\u003eHIS1\u003c/em\u003e and \u003cem\u003eLEU2\u003c/em\u003e markers for deletion and the \u003cem\u003eARG4\u003c/em\u003e marker for complementation. The complemented strain has one copy of \u003cem\u003eALT1\u003c/em\u003e reintroduced at the \u003cem\u003eLEU2\u003c/em\u003e locus. Transformants were confirmed by PCR. The reference strain SN425 (\u003cem\u003eHIS1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e \u003cem\u003eLEU2\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e \u003cem\u003eARG4\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e) was used as the control.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell culture experiments\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIsolation of murine bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages (hMDMs)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo isolate murine BMDMs, bone marrow cells were extracted from 6-8-week-old mouse femurs and tibias of either C57BL/6 mice obtained from the Monash Animal Research Platform or \u003cem\u003eNlrp3\u003c/em\u003e \u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eGsdmd\u003c/em\u003e \u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNinj1\u003c/em\u003e \u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice obtained from the Walter and Elisa Hall Institute and the Hudson Institute respectively. Following a previously described protocol \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e bone marrow containing monocytes was flushed out using 10 ml of BMDM medium. The cells were then incubated for 18\u0026ndash;24 h at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Following incubation, non-adherent cells were transferred to fresh BMDM medium and were allowed to differentiate into BMDMs for 5\u0026ndash;7 days.\u003c/p\u003e\u003cp\u003eTo isolate hMDMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), whole blood was obtained from healthy donors using Histopaque-1077 (Sigma-Aldrich) via density gradient centrifugation. Monocytes expressing CD14 and CD16 were subsequently enriched from peripheral blood mononuclear cells (PBMC) fraction using the Pan Monocyte Isolation Kit and magnetic labelling system (Miltenyi Biotec). The isolated monocytes were then seeded in 25 cm\u0026sup2; tissue culture flasks and differentiated into human monocyte-derived macrophages (hMDMs) by culturing for 7 days at 37\u0026deg;C with 5% CO₂ in RPMI 1640 medium supplemented with 15 mM HEPES, 10% fetal bovine serum (Serana, Fisher Biotech), 100 U/ml penicillin-streptomycin (Sigma-Aldrich), and 50 ng/ml macrophage colony-stimulating factor (M-CSF, R\u0026amp;D Systems).\u003c/p\u003e\u003cp\u003eFor hMDM isolation in \u003cb\u003eFig. S4A\u003c/b\u003e, PBMCs were isolated from buffy coats obtained from healthy donors using Histopaque-1077 (Sigma-Aldrich) density gradient centrifugation. CD14-positive monocytes were subsequently purified by magnetic-activated cell sorting (MACS) using the autoMACS\u0026reg; system (Miltenyi Biotec), according to the manufacturer\u0026rsquo;s instructions. A total of 1 \u0026times; 10⁷ CD14⁺ monocytes were seeded into 175 cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e flasks and cultured in RPMI 1640 (Thermo Fisher Scientific) and 10% FBS. Differentiation into macrophages was induced by adding 50 ng/mL rhM-CSF (ImmunoTools), and cells were incubated for seven days at 37\u0026deg;C, 5% CO₂. After differentiation, macrophages were detached using 50 mM EDTA in phosphate-buffered saline (PBS), and reseeded at a density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/ml. Then 200 \u0026micro;l per well of a 96-well plate (TPP, 4 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) were added for subsequent experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLive cell imaging\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMacrophages were harvested, seeded and stained following protocols detailed in our previous studies \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Following staining, macrophages were infected with \u003cem\u003eCandida\u003c/em\u003e at a yeast-to-macrophage ratio of either 1:1, 3:1 or 6:1. After the phagocytosis step (1 h), extracellular yeast cells were removed by gentle washing with phosphate-buffered saline (PBS). Where specified, BMDM culture medium was supplemented with metabolites including glucose, alanine and glycine at concentrations detailed in the figure legends. These metabolites were added after the 1 h phagocytosis and wash steps. For experiments assessing the role of caspase inhibition, macrophages were pre-incubated with 10 \u0026micro;M Quinoline-Val-Asp-Difluorophenoxymethylketone (QVD) for 1 h. Following this, cells were washed and maintained in fresh BMDM medium prior to infection with \u003cem\u003eC. albicans\u003c/em\u003e. Control (uninfected) macrophages were subjected to the same treatment conditions. After infection, culture media in both experimental and control wells was replaced were with fresh medium either with or without 10 \u0026micro;M QVD, which was maintained for the duration of the assay. All conditions included 0.6 mM DRAQ7 (Abcam) to monitor plasma membrane permeabilization in macrophages.\u003c/p\u003e\u003cp\u003eLive-cell imaging was carried out using a Leica AF6000 LX epifluorescence microscope equipped with a 20\u0026times; objective lens. Image analysis and quantification were performed using CellProfiler v2.1.1, following protocols previously established in our laboratory \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Time-lapse image sequences were compiled into movies using ImageJ software, and all quantitative data were visualized using Prism 10 (GraphPad Software, San Diego, CA).\u003c/p\u003e\u003cp\u003eHuman macrophage experiments were seeded as described previously in \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. For visualization in live-cell assays, hMDMs were labelled with 1 \u0026micro;M CellTracker Green CMFDA (Thermo Fisher, C7025) for 25 min, following the same staining protocol used for BMDMs. Subsequently, cells were primed by incubation in RPMI 1640 supplemented with 15 mM HEPES, 10% FBS, and 50 ng/ml lipopolysaccharide (LPS, Sigma-Aldrich) for 2 h at 37\u0026deg;C and 5% CO₂. Post-priming, hMDMs were infected with \u003cem\u003eCandida\u003c/em\u003e at a multiplicity of infection (MOI) of 3. After a 1 h infection period, the subsequent steps for live-cell imaging, including washing and analysis, were performed identically to the BMDM protocol described above.\u003c/p\u003e\u003cp\u003eFor immortalized bone marrow-derived macrophage (iBMDMs) experiments derived from C57BL/6 Cre-J2 mice \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, including wild-type and genetically modified lines lacking caspase-1, caspase-11, caspase-12, caspase-8, or Receptor-Interacting Protein Kinase 3 (RIPK3) deficient in pyroptotic, necroptotic, and extrinsic apoptotic pathways or control and \u003cem\u003eNinj1\u003c/em\u003e \u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e macrophages described in \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e and \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin. Cells were maintained at 37\u0026deg;C in a humidified incubator with 5% CO₂.\u003c/p\u003e\u003cp\u003eFor live-cell imaging assays, iBMDMs were harvested by gentle scraping (BD Falcon cell scraper) and seeded into tissue culture-treated 96-well plates at a density of 8 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well. After overnight incubation to allow cell attachment recovery and multiplication (to 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e), macrophages were infected with \u003cem\u003eC. albicans\u003c/em\u003e at MOI of 3. Imaging and subsequent data acquisition and analysis were performed following the same protocol established for primary BMDM experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBlaER1 cell culture and transdifferentiation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe B-cell leukemia cell line C/EBPαER clone 1 (BLaER1) was previously generated \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e and cultured and transdifferentiated as described in their work with minor adjustments regarding cell numbers. In short, BLaER1 cells were cultured in RPMI 1640 medium supplemented with 10 mM HEPES, 100 U/ml penicillin, and 100 \u0026micro;g/ml streptomycin (all Thermo Fisher Scientific) and 10% fetal bovine serum (FBS) (BLaER1 medium) at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were passaged at a sub-cultivation ratio of 1:3\u0026ndash;1:5 every 2 to 3 days to a maximum of 30 passages.\u003c/p\u003e\u003cp\u003eFor transdifferentiation, BLaER1 cells were adjusted to 3.3 x 10\u003csup\u003e5\u003c/sup\u003e cells/ml in BLaER1 medium supplemented additionally with 10 ng/ml recombinant human macrophage colony-stimulating factor (rhM-CSF, ImmunoTools), 10 ng/ml recombinant human interleukin 3 (rhIL-3, ImmunoTools) and 200 nM β-estradiol (Sigma-Aldrich) (Transdifferentiation medium). Three ml per well of this suspension were seeded in 6-well plates (TPP) and incubated at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e for seven days. Every two to three days, 1.5 ml of the medium was replaced with fresh, transdifferentiation medium. At day seven, the undifferentiated suspension cells were removed by aspirating the supernatant, while the transdifferentiated, adherent cells (tdBLaER1) were carefully harvested by thorough washing of the wells with medium. The concentration of the tdBLaER1 was adjusted to 4 x 10\u003csup\u003e5\u003c/sup\u003e cells/ml in transdifferentiation medium. Of this suspension, 200 \u0026micro;l/well were seeded into 96-well plates (TPP). The cells were left at room temperature for 20\u0026ndash;30 min to allow attachment and incubated at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e overnight for experiments on the following day. The cells were checked microscopically at the days of the medium exchange. Transdifferentiated cells appeared as GFP-positive, attached, and elongated with a spindle-shaped, macrophage-like morphology.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of supernatants and cell lysates for IL-1β ELISA, LDH release, and immunoblotting\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe collection procedure for supernatant and cell lysate collection was described previously \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and where relevant were performed in BMDM or iBMDM (control and \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) cells. Macrophages were seeded at a density of 5 \u0026times; 10⁵ cells per well in 24-well plates using BMDM medium, following the protocol described under \u0026ldquo;Live cell imaging\u0026rdquo; methods. Cells were primed with 50 ng/ml lipopolysaccharide (LPS) for 3 h at 37\u0026deg;C in 5% CO₂, after which they were either left uninfected or challenged with \u003cem\u003eCandida albicans\u003c/em\u003e (strain SC5314, MOI 3) for 1 h. Following infection, wells were washed three times with PBS to remove non-phagocytosed fungal cells and replaced with 250 \u0026micro;l of fresh BMDM medium. Where relevant media was supplemented with 10mM alanine or glycine at this point. Infected and control cells were then incubated for an additional 3, 14, 16 h. As a positive control for inflammasome activation, macrophages were treated with 10 \u0026micro;M nigericin or 0.01% TritonX for 3 h.\u003c/p\u003e\u003cp\u003eFor both tdBLaER1 cells and hMDMs LDH release assays, cells were primed with 50 ng/ml LPS from Escherichia coli O55:B5 (Sigma Aldrich) for 2 h at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e. During priming, glycine from a sterile-filtered 1 M stock solution in double-distilled water, was added to RPMI 1640 to a final concentration of 50 mM. After priming, the LPS-containing medium was aspirated and 100 \u0026micro;l fresh RPMI 1640 containing either 50 mM L-glycine or no additional supplement was added and incubated for 10 min prior to infection to block NINJ1 oligomerization according to the procedure previously described for human primary macrophages by \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eC. albicans\u003c/em\u003e strains were adjusted to MOI 1 for tdBLaER1 and MOI 3 for hMDMs as described above, either in 100 \u0026micro;l RPMI 1640 or 100\u0026micro;l RPMI 1640\u0026thinsp;+\u0026thinsp;50 mM L-glycine depending on the condition. For infection, 100 \u0026micro;l of the adjusted strains were added to the cells and incubated for 24 h at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIL-1β quantification ELISA\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLevels of IL-1β in culture supernatants were quantified using the Mouse IL-1β/IL-1F2 DuoSet ELISA kit (R\u0026amp;D Systems, DY401), following the manufacturer\u0026rsquo;s protocol. Absorbance was read at 450 nm with a correction wavelength of 540 nm using a Tecan Infinite M200 microplate reader. A standard curve was generated using the recombinant IL-1β standards supplied in the kit, and cytokine concentrations in experimental samples were calculated by interpolation from this curve using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eLactate dehydrogenase (LDH) release assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCell death was assessed by measuring lactate dehydrogenase (LDH) release into cell culture supernatants using the CytoTox 96\u0026reg; Non-Radioactive Cytotoxicity Assay (Promega, G1780), following the protocol provided by the manufacturer. Absorbance was recorded at 492 nm using a Tecan Spark 10M microplate reader. LDH activity in each sample was expressed as fold change relative to the positive control treated with 10 \u0026micro;M nigericin. For both tdBLaER1 cells and hMDMs LDH release assays, infection plates were centrifuged at 250 g for 10 min, the supernatants were collected and diluted 1:5 in PBS. LDH concentrations were measured using the Cytotoxicity detection kit (Roche) according to manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blot analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor detection of IL-1β, caspase-1, and Gasdermin D (GSDMD), cell lysates and/or supernatants were denatured by boiling at 95\u0026deg;C for 10 min prior to electrophoresis. Samples were then separated on 4\u0026ndash;12% Criterion\u0026trade; XT Bis-Tris Protein Gel (Biorad) and transferred overnight onto Immobilon-P Membrane, PVDF, 0.45 uM (Millipore). Membranes were briefly stained with Ponceau S to confirm equal loading and transfer. Following blocking with 5% (v/v) skim milk in TBS containing 0.1% (v/v) Tween-20 (TBS-T) for 1 h at room temperature, membranes were incubated overnight at 4\u0026deg;C on a rotating platform with the following primary antibodies (1:1000 dilution): anti-IL-1β (R\u0026amp;D Systems, #AF-401-NA), anti-caspase-1 (Adipogen, AG-20B-0042-C100), and anti-GSDMD (Abcam, ab209845). The next day, membranes were washed and probed with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000 dilution) for 1 h at room temperature. Signal was developed using SuperSignal\u0026trade; West Femto Maximum Sensitivity Substrate (Thermo Fisher 34094) and imaged using FUJI medical X-ray films. Figures were prepared using Adobe Illustrator.\u003c/p\u003e\u003cp\u003e\u003cb\u003eInfection and RNA isolation of tdBLaER1 cells and hMDMs for analysis of\u003c/b\u003e \u003cb\u003eNinj1\u003c/b\u003e \u003cb\u003eexpression\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor host RNA extraction upon \u003cem\u003eC. albicans\u003c/em\u003e infection, 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e and 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e of hMDMs and tdBlaER1 cells respectively, were seeded in 6-wells at the concentrations described above. On the day of infection, the medium in each well was replaced with 2 ml RPMI without FBS, infected with C. albicans at an MOI 2, and incubated at 37\u0026deg;C, 5% CO2. Samples for RNA isolation were collected at 1 h, 3 h, and 6 h post infection. The well content was removed and replaced with 1 ml of RNeasy Lysis (RLT) buffer (Qiagen) supplemented with 1% β-mercaptoethanol (Roth). Cells were detached using a cell scraper (\u0026lt;\u0026thinsp;3 min) and centrifuged for 10 min (20,000 g, 4\u0026deg;C). The host RNA-containing supernatant was transferred to a new tube, immediately shock-frozen in liquid nitrogen, and stored at -80\u0026deg;C until further processing. Once thawed, the supernatant was mixed with an equal volume of 70% ethanol (prepared in diethyl pyrocarbonate [DEPC]-treated water), and total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eReverse transcription-quantitative PCR (RT-qPCR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe RNA (500 ng) was treated with Baseline-ZERO\u0026trade; DNase (Biozym) according to the manufacturer's guidelines and subsequently transcribed into cDNA using 0.5 \u0026micro;g Oligo(dT)12\u0026ndash;18 Primer, 200 U Superscript\u0026trade; III Reverse Transcriptase, and 40 U RNaseOUT\u0026trade; Recombinant RNase Inhibitor (Thermo Fischer Scientific). Obtained cDNA was diluted 1:5 and used for RT-qPCR with GoTaq\u0026reg; qPCR Master Mix (Promega) in a CFX96 thermocycler (Bio-Rad). Expression levels were normalized to beta-actin. All primer sequences are listed in \u003cb\u003eTable S2\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBlue Native PAGE\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor native gel analysis, samples were prepared following the protocol provided by the manufacturer (Thermo Fisher Scientific). In short, cells were lysed and then subjected to centrifugation at 100 \u0026times; g for 2 minutes. The resulting pellet was resuspended in 1\u0026times; Sample Buffer supplemented with 1% digitonin. After mixing the suspension gently by pipetting five times, it was spun down at 20,000 \u0026times; g for 30 min at 4\u0026deg;C. The supernatant was carefully collected for analysis. Proteins were separated using 3\u0026ndash;12% NativePAGE\u0026trade; Bis-Tris Mini Protein Gels (Thermo Fisher Scientific, BN1002BOX Gels were then transferred onto PVDF membranes and subjected to immunoblotting.). Detection of the NINJ1 protein was carried out using anti-human NINJ1 antibody (R\u0026amp;D Systems, AF5105) at a concentration of 1 \u0026micro;g/ml. Detection of NINJ1 was performed using a primary Rabbit anti-NINJ1 Invitrogen (Cat#: BS-11105R (for blots in \u003cb\u003eFigure S4\u003c/b\u003e) and PA5-95755; RRID: AB_2807557(for blots in \u003cb\u003eFigure S5\u003c/b\u003e) antibody at 1:1000, while secondary Donkey anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (Cat#: A-31573; RRID: AB_2536183) (\u003cb\u003eFigure S4\u003c/b\u003e) and Goat anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (Cat#: 31460; RRID: AB_228341) (\u003cb\u003eFigure S5\u003c/b\u003e) antibodies was used at a 1:5000 dilution.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhagocytosis assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBMDMs were seeded at a density of 5 \u0026times; 10⁵ cells in 500 \u0026micro;l of a 24-well plate and incubated overnight at 37\u0026deg;C with 5% CO₂. The following day, macrophages were infected with \u003cem\u003eC. albicans\u003c/em\u003e strains at a MOI of 2, and co-incubated for 1 h in BMDM medium. Non-phagocytosed \u003cem\u003eC. albicans\u003c/em\u003e cells were removed by washing the wells three times with PBS. Five images per well were captured using Olympus BX60 microscope at 40x magnification. The number of internalized \u003cem\u003eC. albicans\u003c/em\u003e cells and macrophages was manually counted for at least 100 macrophages using the Cell Counter plugin in ImageJ. The phagocytosis index was calculated as the ratio of engulfed \u003cem\u003eC. albicans\u003c/em\u003e cells to the total number of macrophages per field. Two biological replicates, each with two technical replicates were carried out for the experiment.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGrowth curves analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e strains were adjusted to an optical density (OD₆₀₀) of 0.1 and suspended in BMDM medium, either without supplementation or supplemented with 10 mM L-alanine or D-alanine (BMDM medium: RPMI 1640 with 12.5 mM HEPES, 20% L-cell conditioned medium, 15% heat-inactivated and filtered fetal bovine serum, and 100 U/ml of penicillin-streptomycin). The growth of the strains was monitored at 37\u0026deg;C using a Tecan Spark 10M microplate reader, with OD₆₀₀ measurements taken every 30 min for 24 h. Three biological replicates, each with three technical replicates were performed for the experiment.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFilamentation assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eCandida\u003c/em\u003e strain suspensions were prepared at a concentration of 1 \u0026times; 10⁷ cells/ml, as determined by hemocytometer. A volume of 10\u0026micro;l of this suspension was added to 140 \u0026micro;l of BMDM medium, supplemented with either 10 mM L-alanine or D-alanine, in a 96-well plate. The plate was placed under live-cell imaging conditions at 37\u0026deg;C with 5% CO₂. Images were captured every 15 minutes for a total duration of 24 h.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConfocal microscopy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eImmortalized bone marrow-derived macrophage (iBMDMs) derived from control and \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e macrophages in the C57BL/6J Cas9\u0026thinsp;+\u0026thinsp;background described in \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e were used. Cells were seeded at 1 \u0026times; 10⁵ cells per well in a cell culture chamber slide (8-well, catalogue no. 94.6170.802) in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin. Cells were maintained at 37\u0026deg;C in a humidified incubator with 5% CO₂. Cells were allowed to adhere and recover overnight at 37\u0026deg;C in a humidified incubator with 5% CO₂. Macrophages were infected with \u003cem\u003eC. albicans\u003c/em\u003e at a yeast-to-macrophage ratio of 3:1 and phagocytosis (1 h), extracellular yeast cells were removed by gentle washing with PBS. At the 7 h post infection media was spiked with 10 \u0026micro;g/ml of calcofluor white (CFW) and allowed to proceed for (8 h total) before fixation with 2% paraformaldehyde for 20 min protected from light. Samples were covered in foil and stored overnight in PBS. The following day, fixed cells were stained with Phalloidin-iFluor 555 (1:1000 dilution, Abcam, ab176756) in PBS with 0.1% Triton X-100 (Sigma, T9284) for 40 min at room temperature. The chambers were then rinsed with PBS three times before confocal imaging on the Zeiss 980 LSM microscope. 16-bit, 1024x1024 frame px frame size images were captured using a Plan-Apochromat 40x/1.3 NA objective lens. Images were processed using Image J 1.54f \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSerum amino acid extraction and analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFemale C57BL/6J mice (Monash Animal Research Platform), aged 10\u0026ndash;11 weeks and weighing approximately 18\u0026ndash;22 g, were individually housed as part of a pair-feeding study requiring accurate monitoring of food intake. These mice had been previously allocated to an unrelated, aborted experiment and were therefore slightly older than typically used. Systemic \u003cem\u003eC. albicans\u003c/em\u003e infection was induced via intravenous injection into the lateral tail vein using strain SC5314 at a dose of 1 \u0026times; 10⁶ CFU per 20 g of body weight (equivalent to 5 \u0026times; 10⁷ CFU/kg), administered in 100 \u0026micro;l sterile PBS. Mice were euthanized 24 h post-infection by cervical dislocation. Mice were decapitated immediately following euthanasia and trunk blood was collected and centrifuged at 8,000 \u0026times; g for 10 min at 4\u0026deg;C to separate serum. Serum samples were aliquoted and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for downstream amino acid profiling. For sample preparation, 25 \u0026micro;l of serum was mixed with 360 \u0026micro;l of ice-cold 100% methanol containing 5 \u0026micro;M of stable isotope-labelled amino acids. Samples were vortexed for 2 min, followed by the addition of 200 \u0026micro;l chloroform and incubation at 37\u0026deg;C for 5 min with shaking. Subsequently, 400 \u0026micro;l of double-distilled water was added, and the mixture was vortexed again for 2 min. Samples were then centrifuged at maximum speed (\u0026ge;\u0026thinsp;12,000 \u0026times; g) for 10 min at room temperature. A volume of 360 \u0026micro;l from the resulting aqueous phase was transferred to a new tube, evaporated with nitrogen (N2) gas until no liquid remained, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis.\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed using GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA). Specific statistical tests applied are indicated in the corresponding figure legends. For comparisons involving multiple groups, one-way or two-way ANOVA followed by Tukey\u0026rsquo;s post hoc test was used for analysis of LDH release, IL-1β levels (ELISA), gene expression by qPCR and mouse CFU enumeration. Non-parametric data sets, including hMDM LDH assay, were analysed using a Mann-Whitney test with Welsh\u0026rsquo;s correction. For mouse serum nutrient experiments a student\u0026rsquo;s t-test with multiple comparison analysis was used. A p-value of less than 0.05 was considered statistically significant throughout.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge expert support provided by Monash University Research Platforms, particularly the Monash MicroImaging Facility and the Monash Animal Research Platform. We are grateful to Marco Herold, Yexuan Deng, and Marcel Doerflinger (Walter and Eliza Hall Institute) for providing mutant macrophages,\u0026nbsp;Maria Almira S. Cleofe\u0026nbsp;for preparing the serum samples for amino acid analyses, and Rhys Dunstan, Chris Stubenrauch, Ivan Poon and Bo Shi for advice and assistance with BN-PAGE.\u0026nbsp;We thank Tim Tucey for initial observations related to the study. We further thank BEI Resources for the \u003cem\u003eC. albicans\u003c/em\u003e clinical isolates and the Australian Mycology Reference Centre (Sarah Kidd and Sharon Chen) for sharing the \u003cem\u003eC. auris\u003c/em\u003e strain used in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the\u0026nbsp;National Health and Medical Research Council of Australia: Investigator grant 2033452 (AT), Investigator grant 2008692 (JEV), Ideas grant 2019765 (AT, AJR), Ideas grant APP2002520 (AT), Project grant APP158678 (AT), Ideas grant 2020757 (AJR, SB), Ideas grant APP1181089 (KL), the Australian Research Council: Future Fellowship FT190100733 (AT),\u0026nbsp;Future Fellowship FT190100266 (KEL), the\u0026nbsp;German Research Foundation (Deutsche Forschungsgemeinschaft) (DFG) within the Priority Program SPP2225 \u0026ldquo;Exit strategies of intracellular pathogens\u0026rdquo;\u0026nbsp;(Project 446404928)\u0026nbsp;(BH, JS, TL) and within the Cluster of Excellence \u0026lsquo;Balance of the Microverse\u0026rsquo;, under Germany\u0026rsquo;s Excellence Strategy, EXC 2051, Project ID 390713860 (BH, TBS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: HW, OT, HS, JS, BvD, BH, AJR, AT\u003c/p\u003e\n\u003cp\u003eInvestigation: HW, OT, HS, JS, JN, TL, BvD, TLL, FABO, SB\u003c/p\u003e\n\u003cp\u003eValidation: BvD\u003c/p\u003e\n\u003cp\u003eFormal analysis: HW, OT, HS, JS, TL, SB\u003c/p\u003e\n\u003cp\u003eResources: CH, JS, JV, KEL, TBS, BH\u003c/p\u003e\n\u003cp\u003eVisualization: HW\u003c/p\u003e\n\u003cp\u003eFunding acquisition: AT, AJR, BH, JS, TL, TBS, JV, KEL\u003c/p\u003e\n\u003cp\u003eProject administration: AT\u003c/p\u003e\n\u003cp\u003eSupervision: TN, BH, AJR, AT\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft: AT\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; review \u0026amp; editing: HW, AT with contribution from the other authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS:\u003c/strong\u003e Authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY:\u0026nbsp;\u003c/strong\u003eAll data is available within the manuscript and supplementary files. The iBMDM mutant macrophages obtained from the Walter and Eliza Hall Institute are subject to an MTA.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTucey TM et al (2018) Glucose Homeostasis Is Important for Immune Cell Viability during Candida Challenge and Host Survival of Systemic Fungal Infection. Cell Metab 27:988\u0026ndash;1006e1007\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang Y et al (2024) Yersinia infection induces glucose depletion and AMPK-dependent inhibition of pyroptosis in mice. Nat Microbiol 9:2144\u0026ndash;2159\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi DD et al (2022) Fungal sensing enhances neutrophil metabolic fitness by regulating antifungal Glut1 activity. Cell Host Microbe 30:530\u0026ndash;544e536\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang A et al (2016) Opposing Effects of Fasting Metabolism on Tissue Tolerance in Bacterial and Viral Inflammation. 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Nat Methods 9:676\u0026ndash;682\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7015602/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7015602/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePathogens compete for glucose with macrophages, which disrupts host glycolysis, modulates antimicrobial responses and causes macrophage death. We show that, upon glucose starvation caused by major fungal pathogens \u003cem\u003eCandida albicans\u003c/em\u003e and \u003cem\u003eCandida auris\u003c/em\u003e, macrophages lyse by activating NINJ1, the executioner of membrane rupture during cell death. In glucose-starved macrophages NINJ1 ruptures membranes independently of known cell death programs. Moreover, among cell death factors, NINJ1 is the dominant effector of fungal-induced macrophage damage. Supplementation of a single amino acid, alanine, rescues macrophages by inhibiting NINJ1 oligomerization, and \u003cem\u003eC. albicans\u003c/em\u003e infection disrupts amino acid metabolism in mice and reduces serum alanine. Finally, NINJ1-mediated membrane rupture enables \u003cem\u003eC. albicans\u003c/em\u003e egress from macrophage together with the toxin candidalysin. We establish the mechanism of glucose starvation-induced macrophage damage by activation of NINJ1, discover an approach to protect macrophages using a key mammalian metabolite, and demonstrate that NINJ1-dependent pathways are hijacked as an immune escape route.\u003c/p\u003e","manuscriptTitle":"Infection-induced glucose starvation triggers NINJ1-dependent macrophage lysis and pathogen escape","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 05:35:06","doi":"10.21203/rs.3.rs-7015602/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bbc0c101-2910-4de4-9f7d-5a3ef6858172","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":51268165,"name":"Biological sciences/Microbiology/Fungi/Fungal immune evasion"},{"id":51268166,"name":"Biological sciences/Immunology/Immune cell death"}],"tags":[],"updatedAt":"2026-05-01T04:20:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-10 05:35:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7015602","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7015602","identity":"rs-7015602","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}