TFEB activation ameliorates listeriolysin O-mediated subversion of lysosomal and LC3 function during Listeria monocytogenes infection

TFEB activation ameliorates listeriolysin O-mediated subversion of lysosomal and LC3 function during Listeria monocytogenes infection | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results TFEB activation ameliorates listeriolysin O-mediated subversion of lysosomal and LC3 function during Listeria monocytogenes infection Aarti Pant , Sneha Bhatt , Parul Sen , Deepam Bhattacharya , Kuldeep Singh Rautela , Mamta Negi , Ravi Manjithaya doi: https://doi.org/10.1101/2025.11.30.691302 Aarti Pant 1 Autophagy Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research , Jakkur, Bangalore 560064, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sneha Bhatt 1 Autophagy Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research , Jakkur, Bangalore 560064, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Parul Sen 1 Autophagy Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research , Jakkur, Bangalore 560064, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Deepam Bhattacharya 1 Autophagy Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research , Jakkur, Bangalore 560064, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kuldeep Singh Rautela 1 Autophagy Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research , Jakkur, Bangalore 560064, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mamta Negi 1 Autophagy Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research , Jakkur, Bangalore 560064, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ravi Manjithaya 1 Autophagy Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research , Jakkur, Bangalore 560064, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: ravim{at}jncasr.ac.in Abstract Full Text Info/History Metrics Supplementary material Preview PDF Summary Xenophagy is a crucial innate subcellular defense mechanism, functioning as a selective autophagy pathway that captures intracellular pathogens within xenophagosomes for subsequent lysosomal degradation. However, many pathogens have evolved strategies to subvert this process, ensuring their intracellular replication and survival within the host cell. Despite its significance, the precise mechanisms by which intracellularly motile xenophagy cargo like Listeria monocytogenes ( Lm ) circumvent this selective autophagy pathway are not yet completely understood. In this study, we identify a previously unrecognized function of the secreted virulence factor listeriolysin (LLO) in orchestrating a multi-layered disruption of host autophagy-lysosomal machinery. While LLO is classically known for its role in mediating vacuolar escape, our findings reveal that it also contains a functional LIR motif which enables LLO to directly bind LC3 in the cytosol, rendering it unavailable for xenophagosome formation. Concurrently, LLO impairs lysosomal function by reducing its hydrolytic capacity and suppresses nuclear translocation of TFEB, a key transcription factor that governs autophagy and lysosome biogenesis, further compromising host degradative pathways. Using various Lm mutant strains, we show that actin-based motility is dispensable for xenophagy evasion. Instead, the intracellular persistence of Lm primarily stems from two factors: LLO-mediated LC3 binding, which diverts LC3 from its xenophagic functions, and the inhibition of TFEB nuclear translocation. Importantly, pharmacological activation of TFEB or genetic disruption of the LLO-LC3 interaction restores xenophagy and substantially reduces bacterial load. Collectively, our findings position LLO as a multifaceted virulence determinant that employs previously uncharacterized mechanisms to subvert host cell-autonomous defense. This study deepens mechanistic insight into the pathogenesis of Lm and highlights LC3 and TFEB as promising therapeutic targets for host-directed therapies against intracellular infections. Highlights ▪ Listeria monocytogenes ( Lm ) evades xenophagy independently of its host-actin polymerization ability ▪ Listeriolysin O (LLO) impairs lysosomal degradative potential and suppresses TFEB nuclear translocation ▪ LLO binds LC3 through its LC3-interacting region (LIR) motif, effectively limiting LC3 availability for xenophagosome formation ▪ Restoring LC3 and TFEB function enables intracellular clearance of Lm Graphical abstract The graphical abstract illustrates how vacuolar (Δ hlyA ), and cytosolic (WT and Δ actA ) Listeria monocytogenes differentially engage with host-degradative pathways. Listeriolysin O (LLO), a critical virulence factor, is highlighted as the linchpin determining whether these bacteria get targeted for degradation or successfully evade the host response. In Δ hlyA strain infected cells, where LLO is absent, (1) the bacteria remain confined within the phagosome. (2 - 4) The phagosomal compartment then fuse with lysosome, to form phagolysosome that is both acidified and hydrolytically active, enabling effective bacterial degradation while sustaining autophagy flux. (5) This process coincides with nuclear translocation of the master transcription regulator, TFEB, which enhances lysosomal biogenesis and function, boosting bacterial clearance. On the other hand, both WT and Δ actA strains produce LLO, (1, 2) which allows them to escape from the phagosome into the host cytosol. (3) WT strain induces polymerization of host cell actin, leading to the formation of actin clouds that mature into actin tails, facilitating bacterial mobility and cell to cell spread. (4) The Δ actA strain, despite lacking the ability to interact with host actin, still replicates in the cytosol. LLO plays a multifaceted role in evading host defenses. (5 - 6) It limits LC3 availability for nascent xenophagosome formation by binding to LC3, reducing bacterial capture by the xenophagy machinery. (7 - 8) Furthermore, even when a few bacteria are successfully sequestered within phagosome or xenophagosome, LLO-mediated lysosomal dysfunction impair their maturation into degradative phagolysosome and xenolysosome. (9) Compounding this, LLO suppresses nuclear translocation of TFEB, thereby dampening transcriptional activation of autophagy-lysosomal pathways. Collectively, these mechanisms compromise the host degradative capacity, allowing cytosolic bacteria to replicate and evade clearance. Several unanswered questions remain regarding the structural and mechanistic aspects of the interaction of LLO with LC3. It is still unclear whether LC3 conjugation occurs on single or double membrane vesicles, and whether these vesicular structures are fully sealed. Additionally, it is not yet determined if LLO binds LC3 specifically at sites of omegasome or phagophore formation. The aggregation behavior of LLO also raises intriguing possibilities: could these aggregates play a protective role in shielding LLO from its degradation? Furthermore, it remains to be clarified whether LLO exists freely in the cytosol or is associated with specific membrane-bound compartments. Finally, the precise mechanism by which LLO interferes with TFEB nuclear translocation is another critical open question requiring further investigation. Download figure Open in new tab Introduction Our cells maintain homeostasis by intricate quality control mechanisms, chief among them is the autophagy-lysosomal pathway 1 . Macroautophagy, hereafter referred to as autophagy, a fundamental catabolic process responsible for dismantling and recycling damaged organelles and macromolecules 2 , 3 . A specialized form of autophagy, xenophagy, plays a critical role in our innate immune response by specifically targeting and degrading intracellular pathogens within autophagy-lysosomal compartment 3 – 13 . Multiple studies suggest that xenophagy is regulated by a master transcriptional regulator, Transcription Factor EB (TFEB), which governs the expression of genes involved in lysosomal biogenesis, autophagy initiation, and autophagosome-lysosome fusion 14 – 16 . In certain intracellular infections, TFEB has been shown to translocate to the nucleus, thereby inducing a transcriptional program that reinforces xenophagic capacity 16 – 19 . Despite this powerful host defense system, successful intracellular pathogens have evolved strategies to circumvent host xenophagy and its regulation by TFEB. These evasion mechanisms typically involve interfering with pathogen recognition 20 , 21 , blocking xenophagosomes formation 22 , 23 and its maturation into xenolysosomes 24 , 25 , or impairing TFEB function by preventing its nuclear translocation 14 , 26 , 27 , downregulating its expression 28 , inhibiting its activation 29 , or facilitating its degradation 30 . A clearer understanding of these microbial tactics is essential for decoding host-pathogen dynamics and developing effective host-targeted therapies. To unravel the complexity of host-pathogen interactions and microbial immune evasion strategies, well-characterized infection model systems are routinely used as experimental frameworks. Among these, Lm stands out as an effective and versatile model for dissecting xenophagy, owing to its distinct and well-characterized intracellular lifestyle. Lm is a Gram-positive, facultative intracellular bacterium that rapidly transitions from a vacuolar compartment to the host cytosol, a principal site of xenophagic surveillance. This transition is mediated by its major virulence determinants, LLO (encoded by hlyA gene) 31 – 33 , a cholesterol-dependent pore-forming toxin, in coordination with accessory factors phospholipases C (PLCs), PI-PLC (encoded by plcA gene) and PC-PLC (encoded by plcB gene) 34 – 36 , which together facilitate efficient vacuolar membrane disruption. Once in the cytosol, Lm not only survives but thrives by exploiting host actin dynamics. Its surface protein actin assembly-inducing protein A (ActA), encoded by actA gene, nucleates actin polymerization, initially forming dense actin clouds and subsequently elongating into polarized actin comet tails 37 – 41 . These structures propel the bacterium through the cytosol and drive its spread into adjacent cells via protrusion and internalization, enabling tissue-level dissemination 42 – 44 . Beyond intracellular motility, studies indicate that ActA also functions as a molecular shield, helping the bacterium evade detection by the xenophagic machinery, by preventing ubiquitination 20 , 21 , 45 . This precisely coordinated transition from initial vacuolar entry to its subsequent evasion within the cytosol makes Lm an invaluable system for investigating host responses to both vacuole-contained and cytosol-exposed bacteria. Despite significant insights into the role of LLO in promoting vacuolar escape, its broader impact on host xenophagy remains incompletely understood. While LLO-mediated membrane damage is known to trigger host autophagy 46 , 47 , the precise mechanism by which Lm subsequently evades xenophagic degradation potentially through LLO remains unclear 48 . Although the recruitment of the autophagy marker LC3 to intracellular bacteria has been documented, the critical distinction between xenophagy initiation and effective bacterial degradation is often underappreciated. This distinction is essential since LC3 recruitment alone may indicate an activated autophagic response, but without corresponding bacterial clearance, it may reflect an incomplete or abortive host defense. To address this important gap, our study investigates the role of LLO in xenophagy evasion. We show that intracellular LLO binds LC3 in the host cytosol via a specific LIR motif, thereby preventing it from participating in xenophagy. In parallel, LLO disrupts lysosomal functionality and suppresses TFEB nuclear translocation, functionally crippling the autophagy-lysosomal pathway. Importantly, our findings demonstrate that pharmacological reactivation of TFEB or targeted disruption of the LLO-LC3 interaction effectively restores xenophagic clearance, resulting in enhanced elimination of the pathogen within host cells. Altogether, our study delineates a novel mechanism by which Lm subverts xenophagy, establishing LLO as a dual-function virulence factor that orchestrates both vacuolar escape and suppression of host degradative pathways. Results Lm evades xenophagy across intestinal epithelial and macrophage cells To investigate the interplay between host xenophagy and survival strategies adopted by Lm , an in vitro infection model was established using cell lines of different tissue origin. These include human intestinal epithelial cells (HCT116), human monocytic cells (THP-1) and murine macrophage cells (RAW 264.7) ( Fig. S1a ). The intracellular replication kinetics of Lm , along with its ability to exploit host actin dynamics were analyzed. Lm demonstrated a strikingly accelerated exponential replication across different host cell types - HCT116 ( Fig. S1b ), THP1 ( Fig. S1c ) and RAW 264.7 ( Fig. S1d ) between 1- and 6-hours post infection (hpi), with a pronounced surge observed during 3 to 6h window. Simultaneously, analysis of host actin recruitment ( Fig. S1, a and e ) revealed a progressive rise in the proportion of Lm displaying actin clouds and actin tails over time. By 6hpi, more than 40% of intracellular Lm were associated with host actin structures across all tested cell lines. Live cell imaging at 6hpi captured the dynamic actin-based motility of Lm in HeLa cells (Supplementary movie 1). Bacterial movement occurred at a mean speed of ∼ 0.2µm/s. Individual bacteria were initially surrounded by rounded actin “clouds,” which then rapidly polarized into characteristic actin “comet tails,” propelling intracellular movement and enabling direct cell-to-cell spread. These observations underscore the remarkable adaptability of Lm across diverse host cell types and highlight the critical role of actin-mediated motility in facilitating its dissemination, especially during the later stages of infection. Building on these observations, we next investigated whether Lm undergoes xenophagy-mediated degradation by evaluating the involvement of autophagy and lysosomal machinery at two distinct time points: an early stage (3hpi) and a late stage (6hpi). These time points were chosen based on the observed rapid intracellular proliferation occurring between 3 and 6hpi. The association of autophagy markers LC3 and GABARAP, along with the lysosomal marker LAMP1, with the bacterial surface was assessed using fluorescence microscopy. This approach enabled visualization of xenophagosomes, indicated by LC3 or GABARAP recruitment to Lm , and xenolysosomes, marked by the co-recruitment of LC3 and LAMP1 to Lm ( Fig. S1f ). Infection in HCT116 ( Fig. 1, a and b ), THP-1 ( Fig. 1, c and d ), and RAW 264.7 ( Fig. 1, e and f ) cells revealed that Lm is targeted by xenophagy, as evidenced by its sequestration into LC3-positive vesicles at 3hpi. These xenophagic vesicles comprise both xenophagosomes and xenolysosomes, indicating progression toward lysosomal fusion. Quantification showed that ∼ 15-20% of Lm in HCT116 cells and 20-30% in macrophage cells were associated with xenophagic vesicles at this time point. By 6hpi, a clear decline in xenophagic targeting was observed across all cell types, reflected by a reduction in the percentage of xenophagosomes to ∼ 10-12%. This decrease was even more pronounced for xenolysosomes, which dropped to around 5-7%. These findings point to a progressive decline in both formation of xenophagosomes and their subsequent maturation into xenolysosomes over time. Similarly, the recruitment of GABARAP, an isoform of the mammalian autophagy-related protein 8 family, to Lm in THP-1 and RAW 264.7 cells showed marked decline over time. At 3hpi, ∼ 25-35% of bacteria were associated with GABARAP-positive structures. By 6hpi, this proportion dropped significantly to 10-15%, as shown in Fig. 1, g and h . Interestingly, we observed notable cell line-specific differences in autophagy and lysosomal marker recruitment. GABARAP recruitment to Lm remained undetected in HCT116 cells throughout the infection period ( Fig. S2a ), as opposed to the clear recruitment observed in THP-1 and RAW 264.7 cells. This suggests that a predominantly LC3-driven xenophagic response operates in HCT116 cells. While LAMP1 was clearly recruited to Lm in both HCT116 and THP-1 cells, no such recruitment was evident in RAW 264.7 cells at either time point ( Fig. S2b ), indicating a complete blockade in xenophagosomes maturation into degradative xenolysosomes in this macrophage model. Overall, the observed decline in the percentage of xenophagosomes and xenolysosomes across different host cell types suggests that Lm employs evasion strategies to counteract xenophagy-mediated clearance. By restricting xenophagosomes formation and potentially preventing their maturation into xenolysosomes, Lm effectively circumvents its degradation within the host. Download figure Open in new tab Figure 1. Lm evades xenophagy across intestinal epithelial and macrophage cells. The human intestinal cell line HCT116, differentiated human macrophage cell line THP-1 and mouse macrophage cell line RAW 264.7 were infected with mCherry expressing WT strain of Lm 10403S. At 3 and 6hpi, cells were immunostained using LC3, GABARAP, and LAMP1 antibodies to assess xenophagy. a) Fluorescence micrographs of HCT116 cells show xenophagosomes (recruitment of LC3 to Lm ) and xenolysosomes (recruitment of both LC3 and LAMP1 to Lm ) indicated by blue color arrowheads. Plot profiles along the blue line traced within the insets display intensity overlap of Lm , LC3, and LAMP1. b) Percentage of xenophagosomes and xenolysosomes at 3 and 6hpi in HCT116 cells. c) Fluorescence micrographs and plot profile of infected THP-1 cells show xenophagosomes and xenolysosomes. d) Percentage of xenophagosomes and xenolysosomes at 3 and 6hpi in THP-1 cells. e) Fluorescence micrographs show xenophagosomes in RAW 264.7 cells. f) Percentage of xenophagosomes at 3 and 6hpi in RAW 264.7 cells. g) Fluorescence micrographs of infected THP-1 and RAW 264.7 show recruitment of GABARAP to Lm . h) Percentage of xenophagosomes (recruitment of GABARAP to Lm) in THP-1 and RAW 264.7 cells at 3 and 6hpi. Scale bars: 5µm; zoom panel: 2µm. b, d, f, h) Data represents mean ± SEM ( N = 3 independent experiments, n ≥ 100 bacteria per sample for each experiment). *P < 0.05, ***P < 0.001, ****P < 0.0001 using unpaired two-tailed t-test. Cytosolic (WT and Δ actA ) and vacuolar Lm (Δ hlyA ) show distinct fates and degradation patterns in intestinal epithelial cells ActA and LLO, encoded by actA and hlyA genes respectively, are key Lm virulence factors crucial for its pathogenesis 49 . Several studies suggest their involvement in modulating autophagy. For instance, ActA has been reported to potentially help Lm evade autophagic recognition by promoting actin polymerization on the bacterial surface 20 . Conversely, LLO promotes phagosomal damage, allowing bacterial escape from the host cell vacuole, an event known to trigger autophagy activation 47 , 50 . Despite these proposed roles, the precise mechanisms by which ActA and LLO influence autophagy are still not fully understood. To delve deeper into how autophagy differentiates between cytosolic and vacuolar Lm , we examined wild type (WT), Δ actA (lacking the actA gene), and Δ hlyA (lacking the hlyA gene) strains. Our experimental strategy was guided by prior studies showing that WT and Δ actA strains typically escape into the host cytosol within 30-60 minutes post-entry, whereas the Δ hlyA strain remains confined within the vacuole due to its inability to disrupt the vacuolar membrane 51 . Leveraging these differences in intracellular localization, we assessed xenophagy by monitoring LC3 and LAMP1 recruitment to WT, Δ actA , and Δ hlyA strains in HCT116 at 3 and 6hpi using fluorescence microscopy. Gene deletions in the Δ actA and Δ hlyA strains were confirmed by colony PCR ( Fig. S2d ), and their extracellular growth kinetics, assessed via growth assays ( Fig. S2e ), showed no significant differences compared to the wild-type strain. Our results indicate that at 3hpi, both WT and Δ actA were targeted by xenophagy, with xenophagosomes ( Fig. 2, a and b ) and xenolysosomes ( Fig. 2, a and c ) each comprising ∼15-20% of the bacterial population. However, by 6hpi, xenophagy-associated compartments declined significantly in both strains, with Δ actA displaying a more pronounced reduction than WT. Since Δ hlyA lacks LC3 engagement, this negates the possibility of xenophagic or LC3-associated phagocytosis (LAP) degradation. However, the potential for Δ hlyA to be sequestered within LAMP1-positive compartments remains. To further characterize this mode of bacterial containment, we quantified LAMP1-only recruitment, defined as LAMP1-positive compartments lacking LC3 association. At 3hpi, ∼ 58% of Δ hlyA resided within LAMP1-positive compartments, whereas the percentages were notably lower for WT (∼20%) and Δ actA (∼5%). Over time, LAMP1-positive bacterial localization progressively declined in WT and Δ actA but remained stable in Δ hlyA ( Fig. 2d ). Additionally, Δ hlyA infected cells exhibited a substantially greater number of LAMP1 puncta relative to WT and Δ actA infected cells at 3 ( Fig. S3a ) and 6hpi ( Fig. S3b ). Due to observed differences in LAMP1 puncta number in WT, Δ actA , and Δ hlyA infected cells, we proceeded to analyze LC3 and LAMP1 colocalization events, which are indicative of autolysosomal number ( Fig. 2e ). Consistent with LAMP1 puncta quantification, Δ hlyA displayed significantly increased colocalization compared to WT and Δ actA at 3 ( Fig. 2f ) and 6hpi ( Fig. 2g ). The elevated number of LAMP1 puncta and LC3-LAMP1 colocalization events in Δ hlyA infected cells suggests a heightened lysosomal and autophagy response compared to WT, Δ actA infected and uninfected cells. Given the temporal decline in xenophagic vesicles in WT and Δ actA strains, we investigated the recruitment of the autophagy receptor p62 to Lm ( Fig. S3c ) to assess cargo recognition. At 3hpi, ∼ 15% of WT and ∼ 18% of Δ actA exhibited p62 recruitment; however, the percentage of p62-positive Lm decreased significantly at 6hpi to ∼ 4 to 7% for both strains ( Fig. S3d ). In contrast, Δ hlyA showed minimal p62 recruitment, consistent with its LC3 recruitment profile, suggesting diminished recognition by the xenophagy machinery due to its retention within vacuolar compartments ( Fig. S3d ). To explore the functional consequence of this differential host response to Lm strains, we compared intracellular bacterial load among WT, Δ actA , and Δ hlyA at 6hpi in infected HCT116, THP-1, and RAW 264.7 cells. In line with its extensive sequestration within LAMP1-positive compartments, Δ hlyA burden was significantly reduced in all cell types, indicating impaired intracellular survival, potentially due to phagolysosomal-mediated degradation. Compared to the WT strain, Δ actA showed similar intracellular load within THP-1 ( Fig. S3f ) and RAW 264.7 ( Fig. S3g ) cells but exhibited enhanced proliferation in HCT116 cells ( Fig. S3e ). This cell type-dependent disparity suggests that innate immune mechanisms such as inflammation and reactive oxygen species production 52 , 53 may contribute to limiting Δ actA proliferation in macrophages than in epithelial cells. Collectively, these findings highlight distinct autophagy-related outcomes for cytosolic and vacuolar populations of Lm . Although, WT and Δ actA strains escape into the cytosol and are initially targeted by xenophagy, this response wanes over time. Conversely, Δ hlyA strain appears to be primarily routed through the phagolysosomal pathway rather than being cleared via xenophagy or LAP. The pronounced autophagy and lysosomal response observed in Δ hlyA infected cells, coupled with its consistently reduced intracellular burden across multiple cell types, support a potential role for LLO in evading autophagy-lysosomal defenses and promoting Lm intracellular survival. Download figure Open in new tab Figure 2. Cytosolic (WT and Δ actA ) and vacuolar Lm (Δ hlyA ) show distinct fates and degradation patterns in intestinal epithelial cells. HCT116 cells were infected with mCherry expressing WT, Δ actA , and Δ hlyA strains of Lm and immunostained with LC3 (green) and LAMP1 (grey) antibodies at 3 and 6hpi. a) Fluorescence micrographs of infected with WT, Δ actA , and Δ hlyA show xenophagosomes (recruitment of LC3 to Lm ), xenolysosomes (recruitment of both LC3 and LAMP1 to Lm ) and LAMP1-only recruitment to Lm (LAMP1 positive, LC3 negative Lm ), indicated by blue color arrowheads. Percentage of xenophagosomes b) , xenolysosomes c) , and LAMP1-only recruitment to Lm in cells infected with WT, Δ actA , and Δ hlyA strains d) . e) Fluorescence micrographs and puncta colocalization between LC3 and LAMP1 in cells infected with WT, Δ actA , and Δ hlyA at 3 f) and 6hpi g) ; uninfected cells serve as a control. Scale bars: 5µm. b-d) Data represents mean ± SEM ( N = 3 independent experiments, n ≥ 100 bacteria per sample for each experiment). **P < 0.01, ***P < 0.001, ****P < 0.0001 using two-way ANOVA with Tukey’s multiple comparison test. f, g) Violin plots represent the density distribution of the number of autolysosomes at 3 and 6hpi in infected cells ( N = 3 independent experiments, n ≥ 30 infected cells per sample for each experiment). The black bar indicates the interquartile range (IQR). Individual data points are shown as dots. *P < 0.05, **P < 0.01 using one-way ANOVA with Tukey’s multiple comparison test. Lm perturbs lysosomal acidification and hydrolytic activity to evade lysosomal-dependent degradation pathways in an LLO-dependent manner Based on the observed differences in LAMP1 puncta abundance, LC3-LAMP1 colocalization, and LAMP1 recruitment to Lm in HCT116 cells infected with WT, Δ actA , and Δ hlyA strains, we sought to determine whether these variations reflect underlying differences in lysosomal function. Considering that lysosomal dysfunction impairs autophagy and disrupts other cellular processes dependent on lysosomal function, potentially leading to defects in intracellular recycling and homeostasis 54 , we proceeded to evaluate lysosomal functionality. We assessed lysosomal acidity and proteolytic capacity in infected HCT116 cells. Lysosomal acidity was determined using LysoTracker staining 55 ( Fig. 3a ), a method that relies on selective accumulation of this dye in acidic organelles to serve as an indicator of lysosomal pH. To evaluate lysosomal proteolytic activity, we performed the DQ-BSA assay ( Fig. 3c ), wherein fluorescence is emitted upon enzymatic degradation of dye-quenched bovine serum albumin by lysosomal hydrolases 56 . Quantification of LAMP1 and LysoTracker-positive Lm revealed that a significantly higher proportion (∼55%) of Δ hlyA bacteria localized within acidic, LAMP1-positive compartments compared to WT and Δ actA at 6hpi ( Fig. 3b ). Consistently, a greater fraction of Δ hlyA bacteria colocalized with both DQ-BSA and LysoTracker signals, indicating that ∼ 40% of Δ hlyA resided in hydrolytically active lysosomal compartments by 6hpi, whereas WT and Δ actA were largely negative for these markers ( Fig. 3d ). To assess overall lysosomal functionality, we quantified LysoTracker ( Fig. 3e ) and DQ-BSA ( Fig. 3f ) fluorescence intensities in infected HCT116 cells at 3 and 6hpi. Both indicators were significantly elevated in Δ hlyA infected cells compared to those infected with WT and Δ actA , reflecting increased lysosomal acidification and proteolytic activity. Interestingly, Δ actA infected cells displayed a marked reduction in both signals, especially at 6hpi, when compared to WT infected cells. Collectively, these results indicate that LLO expression dampens lysosomal acidification and enzymatic activity, thereby facilitating evasion of lysosomal-mediated degradation by WT and Δ actA strains. Download figure Open in new tab Figure 3. Lm perturbs lysosomal acidification and hydrolytic activity to evade lysosomal-dependent degradation pathways in an LLO-dependent manner. HCT116 cells were infected with mCherry expressing WT, Δ actA , and Δ hlyA strains of Lm and immunostained with LAMP1 antibody at 3 and 6hpi. Lysosomal acidification and degradative capacity were quantified using LysoTracker and DQ-BSA dyes. a) Fluorescence micrographs, and b) quantification of percentage LAMP1 and LysoTracker positive Lm in cells infected with WT, Δ actA , and Δ hlyA at 3 and 6hpi. c) Fluorescence micrographs, and d) quantification of percentage DQ-BSA and LysoTracker positive Lm in HCT116 cells infected with WT, Δ actA and Δ hlyA at 3 and 6hpi. Quantification of fluorescence intensity of LysoTracker e) and DQ-BSA f) at 3 and 6hpi in cells infected with WT, Δ actA , and Δ hlyA . Scale bars: a) 5µm, c) 10µm. b, d) Data represents mean ± S.E.M ( N = 3 independent experiments, n ≥ 100 bacteria per sample for each experiment). ****P < 0.0001 using two-way ANOVA with Tukey’s multiple comparison test. e, f) Violin plots represent the density distribution of fluorescence intensity of LysoTracker and DQ-BSA at 3 and 6hpi in infected HCT116 cells ( N = 3 independent experiments, n ≥ 30 infected cells per sample for each experiment). The black bar indicates the interquartile range (IQR). Individual data points are shown as dots. **P < 0.01, ***P < 0.001, ****P 0.9999 using one-way ANOVA with Tukey’s multiple comparison test. Lm suppresses TFEB nuclear translocation through the expression of LLO Since Lm strains expressing LLO display impairment in both autophagy and lysosomal pathways, we hypothesized that these effects may be mediated through modulation of TFEB, a master transcriptional regulator of genes governing autophagy and lysosomal pathways. Under basal conditions, TFEB is phosphorylated and retained in the cytoplasm. Upon exposure to stress signals such as nutrient deprivation or intracellular infection, it becomes dephosphorylated and translocates to the nucleus to activate transcriptional programs linked to autophagy and lysosomal biogenesis 57 . To determine whether TFEB is modulated in response to Lm infection, we assessed its activation by monitoring its dephosphorylation status and nuclear localization. Dephosphorylation was evaluated by immunoblotting, utilizing the phosphorylation-dependent mobility shift, whereby phosphorylated TFEB migrates more slowly and appears as a higher molecular weight “supershifted” band on the immunoblot 58 . As a positive control, cells treated with Earle’s Balanced Salt Solution (EBSS), a known inducer of TFEB activation via starvation 59 , exhibited complete conversion of phosphorylated TFEB to its dephosphorylated form, clearly visualized as a mobility shift on the immunoblot. WT and Δ actA infected cells displayed significantly reduced levels of dephosphorylated TFEB. In contrast, Δ hlyA infected cells showed significantly elevated levels of dephosphorylated TFEB relative to WT, Δ actA , and uninfected controls ( Fig. 4, a and b ), indicating enhanced TFEB activation in the absence of LLO. Consistent with TFEB results, LAMP1 level was significantly diminished in WT and Δ actA infected cells at 6hpi, whereas Δ hlyA infected cells exhibited increased LAMP1 levels ( Fig. 4, a and c ). The mCherry band in the immunoblot ( Fig. 4a ) reflects the presence of mCherry-labeled Lm in infected cells, with its intensity serving as a proxy for intracellular bacterial load. These findings reinforce a mechanistic link between TFEB suppression and compromised lysosomal function during infection with LLO expressing Lm strains. Additionally, we assessed TFEB nuclear translocation by quantifying nuclear TFEB intensity in infected HCT116 cells at 6hpi ( Fig. 4d ). Notably, Δ hlyA infected cells exhibited significantly higher TFEB nuclear localization compared to WT and Δ actA strains ( Fig. 4e ), indicating that in the absence of LLO, TFEB activation is not suppressed, thereby enabling robust autophagic and lysosomal response. To validate the direct effect of LLO on autophagy regulation, HCT116 cells were treated with 20µM purified recombinant LLO (rLLO), a concentration chosen based on assays demonstrating hemolytic activity ( Fig. S4b ) while maintaining host cell viability ( Fig. S4a ). The impact of rLLO on TFEB dephosphorylation ( Fig. 5f ) and LC3 I to LC3 II conversion ( Fig. 5h ) was examined by immunoblotting. In the presence of Torin1, an mTOR inhibitor that typically induces TFEB activation, rLLO treatment significantly reduced levels of dephosphorylated TFEB ( Fig. 5g ). Furthermore, rLLO exposure led to an increased LC3 II to I ratio following chloroquine (CQ) treatment ( Fig. 5i ) consistent with autophagosome accumulation due to impaired lysosomal fusion. Notably, LLO protein levels remained constant across Torin1- and CQ-treated samples ( Fig. 5j and S4c ), suggesting that LLO may be relatively resistant to autophagic degradation rather than being efficiently targeted as a cargo. Intriguingly, fluorescence microscopy revealed that intracellular LLO in infected cells appeared as dense punctate signals, persisting even at later time point ( Fig. S4d ). This was particularly evident in Δ actA infected cells, where bacteria escape from the primary vacuole but remain restricted to the initial host cell due to impaired actin-based motility, thereby preventing cell-to-cell spread and secondary vacuole formation 60 . Despite this confinement, the sustained presence of dense LLO signal in Δ actA mutants implies its cytosolic relevance, even in the absence of its classic phagosomal function. Notably, Δ actA infected cells exhibited higher LLO levels per cell compared to WT ( Fig. S4e ), which coincided with a greater bacterial burden and a more pronounced suppression of autophagy, lysosomal function, and TFEB nuclear translocation. However, quantification of intracellular LLO levels in bacterial lysates revealed comparable expression between WT and Δ actA strains ( Fig. S4, f and g ), suggesting that the elevated cellular LLO levels observed in Δ actA infected cells likely result from more intracellular bacterial load rather than inherent differences in LLO production. Collectively, these findings highlight the pivotal role of LLO in intracellular survival of Lm by suppressing TFEB nuclear translocation and consequently blocking xenophagy. Download figure Open in new tab Figure 4. Lm suppresses TFEB nuclear translocation through the expression of LLO. a) Western blots depicting phospho- and dephosphorylated (faster-migrating TFEB band corresponds to the dephosphorylated form of the protein levels of TFEB), along with LAMP1 and mCherry levels in HCT116 cells following infection with WT, Δ actA , or Δ hlyA strains at 3 and 6hpi. Uninfected and EBSS-treated samples serve as controls. b) Dephosphorylated TFEB, and c) LAMP1 levels were quantified. d) Fluorescence micrograph, and e) quantification of nuclear fluorescence intensity of TFEB in HCT116 cells upon infection with WT, Δ actA , and Δ hlyA strains at 6hpi. HCT116 cells were treated with 20µM rLLO for 2h, with or without Torin1. Untreated cells and those treated only with Torin1 serve as controls. f) Western blots show phosphorylated and dephosphorylated TFEB. g) Dephosphorylated TFEB levels were quantified. HCT116 cells were treated with 20µM rLLO for 2h, with or without CQ. Untreated cells and CQ-only treated cells serve as controls. h) Western blots show lipidated (LC3II) and non-lipidated (LC3I) forms of LC3. i) LC3II/I ratio was quantified. j) Western blots display LLO levels in untreated cells and in cells treated with LLO alone, LLO combined with Torin 1, or LLO combined with CQ. Scale bar: 5µm. b, c) Data represents mean ± S.E.M ( N = 3 independent experiments). *P < 0.05, **P < 0.01 using one-way ANOVA with Tukey’s multiple comparison test. e) Violin plot represents the density distribution of nuclear fluorescence intensity of TFEB per infected cell at 6hpi ( N = 3 independent experiments, n ≥ 50 infected cells per sample for each experiment). The black bar indicates the interquartile range (IQR). Individual data points are shown as dots. *P < 0.05, **P < 0.01 using one-way ANOVA with Tukey’s multiple comparison test. g, i) Data represents mean ± S.E.M (N = 3 independent experiments). *P < 0.05, **P < 0.01 using one-way ANOVA with Tukey’s multiple comparison test. Download figure Open in new tab Figure 5. LLO binds to LC3 via an LIR motif and suppresses autophagy. a) The structure of monomeric LLO with two labeled putative LIR motifs, 98 YIVV 101 (located in the D2 domain) and 435 WDEV 438 (located in the D4 domain). Highlighted hydrophobic amino acids Y (tyrosine) and W (tryptophan) were mutated to A (alanine) using a site-directed mutagenesis approach to assess the role of the putative LIR motifs in LC3 interaction. b) LLO was obtained from the supernatant of bacterial cultures, (Δ hlyA , Δ hlyA ::pH- hlyA- PL3, Δ hlyA ::pH- hlyA (Y98A)-PL3, Δ hlyA ::pH- hlyA (W435A)-PL3 and Δ hlyA ::pH- hlyA (Y98A,W435A)-PL3) and is tested for its interaction with LC3 in HCT116 cell lysate. This interaction is examined using co-immunoprecipitation (Co-IP), via an antibody specific to LC3. c) Fluorescence micrograph and d) quantification of colocalization between LLO and LC3 in HCT116 cells infected with different strains as mentioned in b . e) Fluorescence micrographs of HCT116 cells infected with Δ hlyA ::pH- hlyA- PL3 and Δ hlyA ::pH- hlyA (Y98A)-PL3 strains show LLO and LC3 colocalization at 6hpi. Plot profiles reveal intensity overlap between LLO and LC3 along a defined blue line. f) Quantification of percentage xenophagosomes in HCT116 infected with Δ hlyA ::pH- hlyA- PL3, Δ hlyA ::pH- hlyA (Y98A)-PL3, Δ hlyA ::pH- hlyA (W435A)-PL3 and Δ hlyA ::pH- hlyA (Y98A,W435A)-PL3 strains. Scale bar: 5µm. d) Data represents mean ± S.E.M ( N = 3 independent experiments, n ≥ 25 infected cells per sample for each experiment). ***P 0.9999 using one-way ANOVA with Tukey’s multiple comparison test. f) Data represents mean ± S.E.M (N = 3 independent experiments, n ≥ 50 bacteria per sample for each experiment) *P < 0.05, **P 0.9999 using one-way ANOVA with Tukey’s multiple comparison test. LLO binds to LC3 via an LIR motif and suppresses autophagy Persisting in the host cytosol as a dense punctate signal, LLO coincides with suppression of TFEB nuclear translocation, along with impaired autophagy and lysosomal function. These findings led to the hypothesis that LLO, in addition to mediating vacuolar escape, may exert a direct role in subverting host degradative pathways. To explore the potential interaction between LLO and LC3, we analyzed the LLO protein sequence using the Eukaryotic Linear Motif (ELM) 61 and iLIR prediction 62 tools. This in silico analysis identified two putative LIR motifs: one located at residues 98-101 ( 98 YIVV 101 ) and another at residues 435-438 ( 435 WDEV 4 38 ) ( Fig. 5a ). To determine the functional importance of these motifs, site-directed mutagenesis was carried out to replace the conserved hydrophobic residue with alanine, resulting in the generation of three mutant Lm strains: Δ hlyA ::pH- hlyA (Y98A)-PL3, Δ hlyA ::pH- hlyA (W435A)-PL3 and Δ hlyA ::pH- hlyA (Y98A,W435A)-PL3. No significant differences were observed in the extracellular growth kinetics ( Fig. S5a ) or in the levels of intracellular ( Fig. S5, b and c ) and secreted ( Fig. S5, b and d ) LLO between the mutant strains and the complemented control strain Δ hlyA ::pH- hlyA -PL3 expressing WT LLO. To determine whether these mutations affect the interaction between LLO and LC3, co-immunoprecipitation (Co-IP) was performed. Culture supernatants from Lm strains expressing either WT or mutant LLO were incubated with lysates from HCT116 cells, followed by immunoprecipitation using an anti-LC3 antibody to capture LC3-bound LLO. Co-IP results revealed that LLO secreted from the Δ hlyA ::pH- hlyA (Y98A)-PL3 mutant retained its ability to interact with LC3, comparable to the complemented Δ hlyA ::pH- hlyA -PL3 strain. In contrast, LLO from both Δ hlyA ::pH- hlyA (W435A)-PL3 and the double mutant Δ hlyA ::pH- hlyA (Y98A,W435A)-PL3 failed to co-immunoprecipitate with LC3, indicating that the 435 WDEV 4 38 motif is critical for mediating this interaction ( Fig. 5b ). To corroborate the Co-IP results in a cellular context, we performed immunofluorescence microscopy on HCT116 cells infected with the respective mutants and complemented Lm strains to quantify colocalization between LLO and LC3 ( Fig. 5c and Fig. 5e ). Both WT LLO and LLOY98A exhibited clear colocalization with LC3, whereas the LLOW435A and the double mutant LLOY98A,W435A showed minimal overlap with LC3 signals ( Fig. 5d ). Interestingly, in strains harboring the 435 WDEV 4 38 motif mutation, LC3 signal was found to colocalize with the bacteria ( Fig. 5e ), a pattern not observed in the complemented strain or the Δ hlyA ::pH- hlyA (Y98A)-PL3 strain. Quantification of LC3 recruitment confirmed that both Δ hlyA ::pH- hlyA (W435A)-PL3 and the double mutant Δ hlyA ::pH- hlyA (Y98A,W435A)-PL3 exhibited significantly enhanced LC3 recruitment compared to the Y98A mutant and the strain expressing wild-type LLO ( Fig. 5f ). Consistent with this increased LC3 recruitment, lysosomal marker analysis revealed that LLO mutants lacking the 435 WDEV 4 38 motif, exhibited significantly higher percentage of LAMP1 and LysoTracker positive bacteria ( Fig. S5, f and g ). Supporting these observations, intracellular CFU assays demonstrated a marked reduction in bacterial survival for both Δ hlyA ::pH- hlyA (W435A)-PL3 and Δ hlyA ::pH- hlyA (Y98A,W435A)-PL3 strains compared to the wild-type LLO and Y98A mutant strains ( Fig. S5e ). These findings identify the 435 WDEV 4 38 motif as the main contributor for LLO-LC3 interaction, contributing significantly to xenophagy evasion and enhancing intracellular survival of Lm . Overall, these findings reveal that LLO executes multiple strategies to disrupt the host autophagy-lysosomal pathway. It binds to LC3 in the cytosol through a defined LIR motif, 435 WDEV 4 38 , limiting its availability from engaging in xenophagy. Restoring TFEB nuclear translocation enhances intracellular degradation of Lm Since cytosolic Lm uses multiple strategies to evade autophagy-lysosomal pathway, we sought to counteract these evasion mechanisms through targeted chemical modulation. We made use of small molecules known to modulate autophagy. Torin1 (2µM) and rapamycin (4µM), both mTOR inhibitors that promote TFEB nuclear translocation 63 – 65 , were used to restore transcriptional regulation of autophagy-related genes. To evaluate the contribution of autophagy at different stages, we also included an early-stage autophagy inhibitor SBI-0206965, an ULK1 inhibitor (2µM) 66 and a late-stage inhibitor, CQ (40µM) which blocks autophagosome–lysosome fusion 67 and assessed their impact on Lm intracellular proliferation ( Fig. 6a ). To rule out any direct antibacterial effects of these compounds, we first performed extracellular growth curve, which showed no significant differences in growth rates compared to untreated controls ( Fig. 6b ), indicating that the compounds do not affect Lm replication directly. Subsequent intracellular CFU assays in HCT116, THP-1, and RAW 264.7 cells ( Fig. 6c ) revealed distinct outcomes based on the type of autophagy modulation. Treatment with torin1 or rapamycin significantly reduced bacterial burden relative to untreated controls, while CQ treatment resulted in a marked increase in intracellular CFU. SBI-0206965 also showed an upward trend of Lm proliferation, although the change was not statistically significant across all cell types. These findings demonstrate that manipulation of the autophagy-lysosomal axis strongly influences Lm intracellular survival. To further validate the role of TFEB in this process, we silenced TFEB in HCT116 cells and measured bacterial burden at 6hpi ( Fig. 6d ). Both WT ( Fig. 6e ) and Δ actA Lm ( Fig. 6f ) strains showed significantly enhanced intracellular survival upon TFEB knockdown, reinforcing the critical role of TFEB in restricting Lm replication. Altogether, these results emphasize the importance of TFEB and the autophagy-lysosomal pathway as key regulators of host defense system against cytosolic Lm . Download figure Open in new tab Figure 6. Restoring TFEB nuclear translocation enhances intracellular degradation of cytosolic Lm . a) Schematic illustrates the function of TFEB in autophagy and lysosomal biogenesis, highlighting its regulation via mTOR and the influence of small molecules including torin1, rapamycin, SBI-0206965, and CQ at various stages of these pathways. b) Growth curve of Lm following treatment with the small molecules mentioned in a) . Untreated samples and ampicillin-treated controls are included as controls. c, d) Quantification of intracellular bacterial load of WT and Δ actA in HCT116, THP-1, and RAW 264.7 cells at 6hpi. Infected cells were lysed, and colony-forming units (CFUs) were counted. CFU values from treated samples were normalized to those from untreated infected cells to determine fold change in bacterial burden. e) Western blot validation of TFEB siRNA knockdown efficiency. Intracellular load of f ) WT and g) Δ actA Lm upon TFEB silencing. CFU data from siRNA-treated samples were normalized to CFU values from untreated infected cells to quantify fold change in bacterial burden. c, d) Data represents mean ± S.E.M (N = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P 0.9999 using one-way ANOVA with Dunnett’s multiple comparison test. f, g) Data represents mean ± S.E.M (N = 3 independent experiments). *P < 0.05, **P < 0.01 using unpaired two-tailed t-test. Discussion Lm is known to deploy multiple strategies to evade xenophagic clearance, allowing it to persist within host cells. Among its virulence factors, ActA and Internalin K (InlK) have been shown to subvert xenophagy by hijacking host proteins. ActA promotes actin polymerization to shield the bacterium 45 , while InlK recruits major vault protein (MVP) to mask its surface 68 , 69 . Through these distinct yet convergent mechanisms, both ActA and InlK prevent xenophagic recognition and targeting. Additionally, one study reports that Lm modulates host lipid signaling through phospholipases C (PlcA and PlcB), reducing phosphatidylinositol 3-phosphate (PI3P) levels and stalling pre-autophagosomal structures, thereby interfering with autophagosome maturation 70 , 71 . Building upon this established framework, our study reveals that Lm leverages a multipronged xenophagy disruptive function of its principal virulence factor, LLO. Beyond its canonical role in vacuolar escape, LLO simultaneously interferes with multiple components of the host autophagy-lysosomal axis. We show that intracellular LLO interacts with LC3 via a defined LIR motif, in the cytosol to reduce its availability to participate in the formation of xenophagosome. Simultaneously, LLO impairs lysosomal function and represses activation of the master regulator TFEB by inhibiting its nuclear translocation, further dampening the host xenophagy machinery. Crucially, our data reveals that pharmacological activation of TFEB or targeted disruption of the LLO-LC3 interaction is sufficient to restore xenophagy, leading to enhanced intracellular bacterial clearance. Taken together, our findings redefine the role of LLO as a critical effector that antagonizes xenophagic clearance post-vacuolar escape, expanding our understanding of immune evasion tactics of Lm . Our comparative analysis of xenophagic targeting across distinct Lm strains differing in intracellular localization and motility: WT (cytosolic and spreading), Δ actA (cytosolic but non-spreading), and Δ hlyA (vacuolar) provides new insights into bacterial survival strategies. Both WT and Δ actA strains, successfully evade xenophagy at later stages, indicating that actin-based motility is not essential for avoiding xenophagic degradation. In contrast, the vacuole-confined Δ hlyA strain fails to engage with LC3 yet strongly associate with LAMP1, indicating its retention within phagolysosomes rather than diversion to either xenophagy or LAP pathway. These findings reinforce the centrality of LLO-mediated phagosomal escape and suggest that ActA is dispensable for intracellular survival. Our results, along with some of previous studies, demonstrate that ActA is not strictly required for intracellular survival 50 , 68 , 72 , defined as the ability of Lm to remain viable and replicate within a host cell in the absence of ActA. In line with this, our finding and reports from others show comparable intracellular burden for both WT and Δ actA strains across multiple host cell types and tissues, including the colon and ileum 43 , 73 , 74 , highlighting that actin-based motility is not universally required for intracellular survival. Nonetheless, ActA remains critical when considering bacterial dissemination beyond the initial site of infection. Its role in polymerization of actin enables Lm to traverse cellular barriers and colonize distal tissues like the brain and placenta 43 , 75 – 77 . Thus, while dispensable in some contexts, ActA confers a selective advantage to Lm during systemic infection by enabling efficient dissemination to distant tissues, balancing the metabolic cost of actin polymerisation with the benefit of enhanced tissue tropism and pathogenesis. In addition to evading xenophagic targeting, cytosolic Lm disrupts the abundance and functionality of degradative autophagosomal and lysosomal compartments, as evidenced by reduced autolysosomal population, altered lysosomal pH and activity. This impairment suggests that cytosolic Lm , independent of actin-based motility, also interferes with downstream autophagic maturation and lysosomal function. These observations are in line with previous reports implicating cholesterol-dependent cytolysins (CDCs) such as perfringolysin O (PFO), pneumolysin (PLY), and streptolysin O (SLO) to impair lysosomal integrity 78 – 80 . The shared ability of these toxins underscores a conserved immune evasion strategy adapted by Gram-positive pathogens to enhance intracellular survival. In parallel, our findings reveal that LLO suppresses activation of TFEB, a critical transcription factor responsible for autophagic and lysosomal biogenesis at 6hpi. While one study has demonstrated that TFEB translocates to the nucleus during the initial phase of Lm infection, this nuclear translocation diminishes by 4hpi, suggesting a tightly regulated temporal response. Although LLO-induced membrane damage has been identified as the upstream trigger for TFEB activation, the experimental assessments of TFEB nuclear translocation were limited to an early timepoint (1hpi) 17 . This constraint precludes a full understanding of the temporal dynamics and regulatory mechanisms governing TFEB localization beyond the initial phase of infection. Indeed, multiple bacterial and viral pathogens have been reported to target TFEB as part of their host immune evasion tactics 16 , further emphasizing its role as a key regulator of antimicrobial responses. Central to our study is the mechanistic role of LLO in subverting host autophagy. While previous reports have suggested that LLO induces autophagy 47 , this response likely represents a host-driven mechanism aimed at eliminating damaged vacuoles post-escape, rather than a bacterial survival tactic. A key observation from our data is the sustained secretion of LLO into the cytosol. Notably, we detect LLO as dense, punctate signals within the host cytosol at later stages of infection, prompting an investigation into its post-escape role. Mechanistically, we demonstrate that secreted LLO directly binds to LC3 via a functional LIR motif, 435 WDEV 4 38 , with a preferential interaction likely toward the lipidated LC3 which is LC3II form. This interaction is supported by our findings of LC3 puncta formation and LC3II accumulation upon Lm infection or LLO treatment. However, the conjugation of LC3 to vesicles remains ambiguous, especially regarding the nature of these vesicles whether they exhibit single or double membranes, whether they are fully sealed during the LLO-LC3 interaction. Furthermore, it is unclear whether LLO exists freely within the cytosol or is associated with specific membrane-bound compartments. Defying the autophagic degradation of Cry5B toxin of Bacillus thuringiensis in Caenorhabditis elegans 81 , LLO exhibits an extraordinary resilience, bypassing degradation even when autophagy is pharmacologically activated by Torin1. This persistent cytosolic accumulation suggests that the LLO-LC3 complex forms a stable, non-productive structure that evades degradative pathway. We propose that LLO acts as a molecular decoy, sequestering LC3II away from its role in xenophagosome formation. Reported evidence of LLO forming aggregates further supports this hypothesis 82 . One report suggests these cytosolic LLO structures resemble neurodegeneration-associated aggregates 83 and another show LLO colocalizing with ubiquitin and p62 though whether these are subject to autophagic degradation remains unclear 84 . Additionally, while degradation of LLO monomers and aggregates via the ubiquitin-proteasome system (N-end rule pathway) is well established 31 , 85 , 86 , our data raise the possibility that some LLO aggregates may escape this route by entering autophagic vesicles and blocking fusion with lysosomes. Similar autophagy-evasion strategies have also been observed in other pathogens. For example, the non-structural small (NSs) protein from Rift Valley fever virus binds LC3 through a LIR motif, leading to nuclear retention of LC3 and xenophagy inhibition 87 . Other examples include IcsB effector of Shigella flexneri binds ATG5 to prevent xenophagosome formation 88 , RavZ protease of Legionella pneumophila that cleaves LC3 from autophagosomal membranes to prevent its autophagic degradation 23 , and M2 protein of Influenza A virus hijacks LC3 to facilitate viral egress 89 . Together, these examples underscore a shared evolutionary strategy among intracellular pathogens to mimic host short linear motifs (SLiMs) 90 , 91 , particularly LIR motif to manipulate autophagy and ensure intracellular persistence. Understanding this molecular mimicry will reveal foundational principles in host-pathogen interactions and could accelerate the development of novel antimicrobial therapies 92 . Importantly, our study demonstrates that restoring TFEB activation using pharmacological activators or disrupting LLO-LC3 interaction via targeted mutagenesis, significantly reduces the intracellular burden of Lm . TFEB activation not only restores lysosomal function but also boosts LC3 and other autophagy proteins levels 93 , 94 , producing sufficient LC3 and other factors to outcompete LLO for binding and thereby counteract its sequestration effect. Furthermore, the reduced intracellular burden of Lm following disruption of the LLO-LC3 interaction substantiates our conclusion that ActA is not necessary for xenophagy evasion. Overall, these findings establish LLO not only as a pore-forming effector essential for vacuolar escape but also as a potent modulator of the host autophagy-lysosomal pathway. The dual targeting of LC3 and TFEB reveals parallel axes of immune subversion, reinforcing the idea that Lm has evolved a highly integrated approach to manipulating host cellular defenses. While the critical role of LLO in xenophagy inhibition is clearly established, questions remain regarding the spatial and temporal regulation of its activity and aggregation propensity within host compartments. Though its pore-forming function is pH-dependent 95 , it is plausible that additional regulatory layers and differential expression 96 modulate its dual functionality, balancing immune evasion with host cell viability. Of particular interest is the mechanism by which LLO inhibits TFEB nuclear translocation, which remains undefined. Emerging studies have identified LC3 lipidation and GABARAP conjugation to selective autophagic membranes as key triggers of TFEB activation during lysophagy and xenophagy, respectively 97 , 98 . The sequestration of LC3 by LLO may therefore interfere with this signaling axis thereby suppressing TFEB activation. Unravelling this regulatory crosstalk will be essential for fully characterizing multifunctionality of LLO and developing host-targeted therapeutic strategies to strengthen autophagic surveillance. While the early phase (∼ 1hpi) of Lm infection is defined by the responses to listeriolysin O (LLO), including release of cathepsins from lysosomes 79 and TFEB translocation 17 . However, the progressive decline of these responses despite the continued presence of LLO raises a critical question on the role of LLO at the later stages of infection. Our study addresses this question by investigating how sustained LLO exposure influences intracellular survival beyond the acute cellular response window. We demonstrate that Lm leverages LLO not only to breach vacuolar confinement but also to subvert host xenophagic defenses through LC3 binding, inhibition of TFEB nuclear translocation, and lysosomal disruption. This late-stage immune interference allows the pathogen to persist within host cells, revealing an intricately coordinated strategy for long-term survival. Given its central role, LLO stands out as a compelling therapeutic target, with multiple studies supporting its inhibition to effectively control Lm infection both in vitro and in vivo 99 – 101 . These insights underscore the multifaceted impact of LLO on host-pathogen dynamics and reinforce the therapeutic potential of modulating autophagy-related regulators. Supplementary figures Download figure Open in new tab Figure S1. Intracellular Lm exhibits exponential growth and host actin-based motility at later time points of infection across different cells. HCT116, THP-1, and RAW 264.7 cells were infected with GFP expressing WT strain of Lm . Cells were stained with phalloidin dye to visualize host actin network at 1, 3, and 6hpi. a) Fluorescence micrographs depict intracellular proliferation of Lm and host actin-based clouds and tail formation by Lm across different time points. b - d ) Intracellular growth kinetics of Lm in HCT116, THP-1, and RAW 264.7 cells. e) Quantification of Lm -mediated host actin cloud and actin tail formation at indicated timepoints in HCT116, RAW 264.7, and THP-1 cells. f) Schematic shows measurement of xenophagy using fluorescence intensity plot profile. Scale bars: 5µm; zoom panel: 5µm. b, c, d) Data represents mean ± S.E.M ( N = 3 independent experiments). e) Data represents mean ± S.E.M ( N = 3 independent experiments, n ≥ 100 bacteria per sample for each experiment). Download figure Open in new tab Figure S2. a) Representative fluorescence micrographs of infected HCT116 cells demonstrate the absence of GABARAP recruitment to Lm . b) Representative fluorescence micrographs of HCT116, THP-1, and RAW 264.7 cells infected with Lm show the recruitment of actin and LAMP1 to the bacteria. c) Quantitation of percentage LAMP1 recruitment to Lm at 6hpi. d) PCR confirmation of hlyA and actA gene deletions in putative Δ hlyA and Δ actA strains of Lm . Complementary strains Δ hlyA ::pH- hlyA -PL3 and Δ actA ::pPL2t. P hyper - actA served as controls. e) Growth curves of WT, Δ actA , and Δ hlyA strains in BHI broth at 37°C, assessing their proliferation over time. Scale bar: 5µm. Download figure Open in new tab Figure S3. Quantification of the number of LAMP1 puncta in HCT116 cells infected with WT, Δ actA , and Δ hlyA at 3 a) and 6hpi b) , fluorescence micrographs are shown in Figure 2 e . HCT116 cells were infected with mCherry expressing WT, Δ actA , and Δ hlyA strains of Lm and immunostained with p62 (green) at 3 and 6hpi. Host actin network was labeled using phalloidin dye (grey). c) Fluorescence micrographs and d) quantification of percentage p62 recruitment to Lm at 3 and 6hpi in HCT116 cells. Quantification of intracellular CFU of WT, Δ actA , and Δ hlyA in HCT116 e) , THP-1 f) , and RAW 264.7 cells g) at 6hpi. Intracellular CFU was determined by lysing the infected cells, plating on BHI agar plate, and counting the CFUs. Scale bar: 5µm. a, b) Violin plots represent the density distribution of the number of lysosomes at 3 and 6hpi in infected HCT116 cells ( N = 3 independent experiments, n ≥ 30 infected cells per sample for each experiment). The black bar indicates the interquartile range (IQR). Individual data points are shown as dots. *P < 0.05, ****P < 0.0001 using one-way ANOVA with Tukey’s multiple comparison test. d) Data represents mean ± S.E.M ( N = 3 independent experiments, n ≥ 100 bacteria per sample for each experiment). **P < 0.01, ****P < 0.0001 using two-way ANOVA with Tukey’s multiple comparison test. e-g) Data represents mean ± S.E.M (N = 3 independent experiments). *P < 0.05, **P 0.9999 using one-way ANOVA with Tukey’s multiple comparison test. Download figure Open in new tab Figure S4. a) Cell viability assessment in HCT116 cells following rLLO treatment, quantified as relative luminescence units (RLU). b) Percentage hemolysis observed at varying concentrations of rLLO. c) Western blot-based quantification of LLO levels in HCT116 cells treated with 20µM rLLO for 2h, with and without Torin1 or CQ treatment (data corresponding to Figure 4j ). d) Fluorescence micrographs and e) quantification of LLO fluorescence intensity in HCT116 cells infected with WT, Δ actA , and Δ hlyA strains at 6hpi. f) Western blot analysis and j) quantification of LLO protein levels in WT, Δ actA , and Δ hlyA strains of Lm . Scale bar: 5µm. c, g) Data represents mean ± S.E.M ( N = 3 independent experiments), ns >0.9999, using one-way ANOVA with Tukey’s multiple comparison test. e) Data represents mean ± S.E.M ( N = 3 independent experiments, n = 25 infected cells per sample for each experiment), **P < 0.01, using one-way ANOVA with Tukey’s multiple comparison test. Download figure Open in new tab Figure S5. Disruption of LLO-LC3 interaction reinstates host xenophagy against Lm . a) Growth curve of WT, Δ hlyA , Δ hlyA ::pH- hlyA- PL3, Δ hlyA ::pH- hlyA (Y98A)-PL3, Δ hlyA ::pH- hlyA (W435A)-PL3 and Δ hlyA ::pH- hlyA (Y98A,W435A)-PL3 strains. b-d) Western blot analysis of intracellular and secreted LLO levels across the same strains. e) Intracellular CFU quantification of WT, Δ hlyA , Δ hlyA ::pH- hlyA- PL3, Δ hlyA ::pH- hlyA (Y98A)-PL3, Δ hlyA ::pH- hlyA (W435A)-PL3 and Δ hlyA ::pH- hlyA (Y98A,W435A)-PL3 strains in HCT116 cells at 6hpi. CFU values from WT Lm infected strains were normalized to those from cells infected with mutant and complemented strains to calculate the fold change in bacterial burden. f, g) Representative fluorescence micrographs, and quantification of percentage LAMP1 and LysoTracker positive Lm in HCT116 cells infected with Δ hlyA ::pH- hlyA -PL3, Δ hlyA ::pH- hlyA (Y98A)-PL3, Δ hlyA ::pH- hlyA (W435A)-PL3 and Δ hlyA ::pH- hlyA (Y98A,W435A)-PL3 strains at 6hpi. b-d) Data shown as mean ± S.E.M (N = 3 independent experiments), ns > 0.9999 using one-way ANOVA with Tukey’s multiple comparison test. e) Data represents mean ± S.E.M (N = 3 independent experiments). *P < 0.05, **P 0.9999 using one-way ANOVA with Dunnett’s multiple comparison test. g) Data represents mean ± S.E.M (N = 3 independent experiments, n ≥ 50 bacteria per sample for each experiment) *P < 0.05, **P 0.9999 using one-way ANOVA with Tukey’s multiple comparison test. Supplementary movie 1: Lm live cell.avi Intracellular and intercellular host actin-mediated movement of Lm . Materials and Methods Bacterial strains and plasmids All Lm strains used in this study (listed in Table 1 ) are derived from 10403S, a streptomycin-resistant isolate of strain 10403S (kind gift from Prof. Daniel A Portnoy, UCB, USA). Lm was cultured in Brain Heart Infusion (BHI) (BD Difco, 237500) broth to perform infection assays as follows: a single colony was inoculated into 2 mL of filter-sterilized (0.22µm) BHI broth and incubated overnight at 37°C in a tube lying flat. The resulting primary culture was diluted 1:10 into fresh BHI broth and grown at 37°C at 200 rpm until the optical density at 600 nm (OD 600 ) reached 0.8–1.0, typically within 3-5h. This secondary culture was then used for infection assays in host cells. E. coli strains (listed in Table 2 ) were grown in Luria-Bertani (LB) (HiMedia, M1245) broth at 37°C. The following antibiotic concentrations were used: streptomycin (G-Biosciences, RC-197) (200μg/mL), chloramphenicol (Sigma-Aldrich, C0378) (7.5μg/mL for Lm and 10μg/mL for E. coli ) and tetracycline (Sigma-Aldrich, 64-75-5) (2μg/mL). All plasmids used in this study are listed in Table 3 . To perform bacterial imaging, plasmids pPL2-mCherry and pPL2-GFP were transconjugated from E. coli SM10 (kind gift from Prof. Daniel A Portnoy, UCB, USA) into Lm to generate mCherry and GFP-expressing Lm strains. pPL2t. P hyper - actA and pH- hlyA- PL3 plasmids were transconjugated from E. coli SM10 into Δ actA and Δ hlyA Lm strains respectively to generate complemented strains Δ actA ::pPL2t. P hyper - actA and Δ hlyA ::pH- hlyA- PL3 Lm . Colony PCR was used to verify these complemented strains with primers detailed in Table 3 . Point mutations were introduced into hlyA gene within pH- hlyA- PL3 plasmid using SPRIP (Single-Primer Reactions IN Parallel) mutagenesis method 102 . The primers used to create the specific nucleotide change are listed in Table 3 . The mutations were confirmed through DNA sequencing. Further, pH- hlyA- PL3 plasmids carrying point mutation were transformed into competent E. coli SM10 and selected on a chloramphenicol containing LB plate. Donor E. coli SM10 containing the mutant plasmid and recipient Δ hlyA Lm were streaked in an overlapping manner on a non-selective BHI plate and incubated for 24h at 37°C. Transconjugants were then selected by picking overlapping colonies and plating them on a BHI plate containing streptomycin and chloramphenicol. View this table: View inline View popup Table 1. Details of bacterial strains used. View this table: View inline View popup Table 2. Details of plasmids used. View this table: View inline View popup Table 3. Details of oligonucleotides used. Cell culture HCT116 cells were maintained in McCoy’s 5A medium (Sigma-Aldrich, D5648) supplemented with 2.2g/L sodium bicarbonate (Sigma-Aldrich, S5761), 100U/mL penicillin-streptomycin (Pen-Strep) (Gibco, 15140122), and 10% fetal bovine serum (FBS) (Gibco, 10270106). THP-1 cells were cultured in RPMI medium (Sigma-Aldrich, R6504) supplemented with 2.2g/L sodium bicarbonate, 100 U/mL Pen-Strep, and 10% FBS. RAW 264.7 and HeLa cells were grown in DMEM (Sigma-Aldrich, D5648) supplemented with 3.7g/L sodium bicarbonate, 100 U/mL Pen-Strep, and 10% FBS. All cell lines were incubated at 37°C in a 5% CO₂ humidified atmosphere and were procured from NCCS, Pune, India (Cell Repository). For infection assays, THP-1 cells were differentiated into macrophages by pre-treatment with 25ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, P1585) for 24h. Following differentiation, cells were washed once with 1X DPBS (Sigma-Aldrich, D5773) and cultured in fresh RPMI medium for an additional 24h prior to use. Intracellular Colony Forming Unit (CFU) infection Assay HCT116 cells were infected at a multiplicity of infection (MOI) of 50 for 1h. Differentiated THP-1 and RAW 264.7 cells were infected at an MOI of 20 and 5, respectively, for 30min. HeLa cells were infected at an MOI of 100 for 1h. Infections were performed in incomplete media (without FBS). Following bacterial incubation, cells were washed three times with 1X DPBS. To eliminate extracellular bacteria, gentamicin (50μg/mL) (Genticyn) was added to incomplete media for 20min. Cells were then washed with 1X DPBS and incubated in complete media containing 25μg/mL gentamicin to prevent extracellular bacterial growth, with or without the compound (listed in Table 4 ), until the time of collection. To lyse host cells and release intracellular bacteria, cells were treated with 0.1% ice-cold Triton X-100 (Millipore, 648466) in 1X DPBS. Lysates were plated on BHI agar and incubated at 37°C for 24h. Intracellular bacterial replication or survival was quantified by counting CFU. View this table: View inline View popup Table 4. Details of antibodies, reagents and other resources used. Immunofluorescence microscopy HCT116 and RAW 264.7 cells were seeded on coverslips and allowed to attach overnight. THP-1 cells were seeded on coverslips and treated with 20 ng/mL PMA for 48h to promote attachment. After attachment, cells were infected and/or treated with the indicated compound (Table 5) for 6h. Cells were then fixed using a fixation buffer (4% paraformaldehyde in 1X PBS). For antibody staining, cells were permeabilized with a permeabilization buffer (0.1% Triton X-100 in 1X PBS) for 10 min, followed by blocking with a blocking buffer (2% BSA in 1X PBS) for 1h at room temperature. Cells were incubated with the primary antibodies (listed in Table 4 ) overnight at 4°C, followed by a 1h incubation with the secondary antibodies (listed in Table 4 ) at room temperature in the dark. Phalloidin (Sigma-Aldrich, 68825) dye was then added at a 1:60 dilution and incubated for 1h at room temperature in the dark. When combined with immunostaining, phalloidin dye was co-incubated with the secondary antibody under the same conditions. Coverslips were mounted using Vectashield antifade reagent with DAPI (VectorLabs, H-1200-10), and fluorescence was observed using a DeltaVision Elite Deconvolution Microscope. Measurement of lysosomal acidity and hydrolytic activity For fixed-cell microscopy, cells were stained with 100nM LysoTracker Deep Red (Invitrogen, 12492) for 30 min at 37°C in 5% CO₂. After staining, cells were washed with 1X DPBS, fixed with fixation buffer, and imaged. DQ-BSA (Abcam, ab286868) was added at a concentration of 16µg/mL along with 50nM LysoTracker in DMEM supplemented with 0.5% FBS and incubated for 1h. Cells were then washed twice with 1X DPBS, and a live-cell imaging solution (Invitrogen, A59688DJ) was added. Live-cell snapshots were acquired. Immunoblotting HCT116 cells were seeded in 12-well plates and incubated overnight for attachment. Subsequently, cells were either infected for 3 and 6h or treated with rLLO (Abcam, ab83345) for 2h, in the presence or absence of Torin1 (Tocris Bioscience, 4247) or CQ (Sigma-Aldrich, C6628). After the specified treatment durations, cells were washed with ice-cold PBS and lysed in 1X SDS protein loading dye using a cell scraper on ice. The resulting cell lysates were then boiled at 99°C for 15min. Subsequently, the samples were separated by SDS-PAGE and transferred to PVDF membranes (PALL Life Sciences, 79548A) via wet transfer method. The membranes were incubated with primary antibodies (listed in Table 4 ) overnight at 4°C, followed by a 1h incubation with HRP-conjugated secondary antibodies (listed in Table 4 ) at room temperature. Protein bands were visualized using an enhanced chemiluminescence substrate (Clarity Bio-Rad, 170-5061), and images were captured using a gel documentation system. Finally, band intensities were quantified using ImageJ software. Bacterial growth curve in BHI broth A single colony was inoculated in BHI broth and incubated overnight at 37°C without shaking. The following day, a secondary culture was prepared by diluting the overnight culture 1:10 in fresh BHI broth. Once the culture reached an OD 600 of 0.8 to 1.0, a total of 100µl of culture was added to each well of a 96-well plate, ensuring that the final OD 600 of the added culture was ∼ 0.1 to 0.3 for all bacterial strains. Absorbance at 600 nm was then measured every hour for 12 or 24h using a Varioskan Flash plate reader (Thermo Fisher Scientific). Measurement of LLO-mediated hemolytic activity Lm strains were grown in 2.5 mL BHI broth at 37°C overnight without shaking. Stationary-phase cultures were diluted 1:10 in 5 mL fresh BHI broth and incubated at 37°C with shaking until OD 600 reached 0.8 to 1.0. 1 mL of each culture (normalized to 0.8 to 1.0 OD 600 ) was centrifuged at 13,000 x g for 10 min. The human red blood cells (RBCs) (voluntary donation with informed consent through a signed consent form) were washed twice with 1X PBS (pH 7.4) and resuspended to a final concentration of 5% (v/v) in 1X PBS. Supernatants from bacterial cultures were collected and supplemented with 1 mM dithiothreitol (DTT) (Thermo Fischer Scientific, R0862) for 10min at room temperature. In a 96-well plate, 100µL of the bacterial supernatant and 100µL of the 5% RBC suspension were combined (1:1 ratio, 200µL total volume). The mixtures were incubated at 37°C for 45min without shaking. Following incubation, plates were centrifuged, and hemoglobin release in the supernatant was quantified by measuring absorbance at 550nm. Hemolytic activity was determined by quantifying the lysis of RBCs induced by LLO present in culture supernatants. CellTiter-Glo (CTG) luminescent cell viability assay HCT116 cells were counted using the Countess 3 Automated Cell Counter, and equal numbers (10,000 cells per well) were plated on a 96-well plate. A dose-response assay for rLLO (ranging from 2.5µM to 20µM) was performed for 2, 4, and 6h. At each incubation time point, 10µL of CellTiter-Glo reagent (Promega, G7570) was added to each well, and luminescence was measured using the Varioskan Flash plate reader (Thermo Fisher Scientific). esiRNA transient transfection HCT116 were plated in a 24-well plate at a confluency of ∼ 30%. Cells were transfected with 400ng of TFEB esiRNA (Sigma-Aldrich, EHU059261) using Lipofectamine 2000 (Invitrogen, 11668019) transfection reagent for 48h, universal negative control siRNA (Sigma-Aldrich, SIC001) was used as control. After 48h, cells were subjected to intracellular CFU infection assay. Trichloroacetic acid (TCA) precipitation To quantify the secretory levels of LLO from the supernatant of Lm cultures grown in BHI broth, TCA (Thermo Fischer Scientific, 76-03-9) precipitation was performed. Lm strains were first grown to stationary phase in BHI broth overnight. The cultures were then diluted 10-fold in fresh BHI broth and incubated at 37°C with shaking until they reached an OD600 of 0.8-1.0. For sample preparation, 1 mL of culture was transferred into a microcentrifuge tube and centrifuged at 13,000 x g for 15 min. The bacterial pellet was resuspended in 1 X SDS loading dye to prepare samples for intracellular LLO analysis, while the supernatant was mixed with 100µL of 100% TCA and incubated on ice for 1h to precipitate secreted proteins. After incubation, the mixture was centrifuged at 13,000 x g for 10 min, and the resulting protein pellet was washed twice with ice-cold acetone. The final pellet was resuspended in 1 X SDS loading dye containing 0.1N NaOH. Co-immunoprecipitation HCT116 cells were cultured in 100 mm dishes. Monolayers were trypsinized, washed twice with 1 X DPBS, and centrifuged at 600 x g for 5 min. The resulting cell pellets were lysed in ice-cold RIPA buffer (20 mM Tris-Cl, pH 7.4; 150 mM NaCl; 2% glycerol; 0.5% Triton-X 100; 1.6 mM EDTA) supplemented with a protease inhibitor cocktail (Roche, COEDTAF-RO) and 1 mM PMSF (Sigma-Aldrich, P7626). Lysates were vortexed and sonicated twice for 30 seconds each, with 1 min incubation on ice between sonication steps. Following centrifugation at 12,000 x g for 30 min at 4°C, supernatants were collected. Bacterial supernatant was obtained from a secondary culture grown to an OD 600 of 1. Bacterial cells were removed by centrifugation at 14,000 x g for 10 min, and the supernatant was filtered through a 0.22µm pore-size filter for further use. For co-immunoprecipitation, mammalian protein lysate and bacterial supernatant were incubated with anti-LC3 antibodies (MBL Life Sciences, M152-3), following the manufacturer’s recommendations. Protein-antibody complexes were captured using Protein G Dynabeads (Invitrogen, 10004D) and incubated overnight at 4°C with rotation. After incubation, beads were washed three times with washing buffer (20 mM Tris-Cl (pH 7.4), 150 mM NaCl). Finally, the beads were then resuspended in 1X SDS protein loading dye and subsequently analyzed by SDS-PAGE and western blotting. Live cell imaging HeLa cells (ATCC) were cultured and transfected with Lifeact-mCherry (Addgene plasmid #193300) using Lipofectamine 2000 transfection reagent (Invitrogen, 11668019). After 24h of transfection, cells were infected with GFP-tagged WT Lm as per the previously described infection protocol. Live cell imaging was carried out at 6hpi using a DeltaVision Elite Deconvolution Microscope at a frame rate of 1 frame every 1s for total duration of 1.3min (eight Z sections of 0.2µm thickness). Image analysis Intracellular motility dynamics of Lm were quantified using the TrackMate plugin in ImageJ 103 . Xenophagosomes (identified by the colocalization of LC3 and Lm ) and xenolysosomes (identified by the triple colocalization of LC3, LAMP1, and Lm ) were manually quantified using the Arbitrary line profile tool in Softworx software on individual z-stack images. The same approach was used to analyze the recruitment of p62 to Lm , as well as the colocalization of LAMP1 and LysoTracker or DQ-BSA with Lm . Actin tail formation and actin cloud association with Lm were quantified using the Cell Counter plugin in ImageJ. LAMP1-positive puncta were quantified using the Analyze Particles tool in ImageJ. To assess LC3-LAMP1 and LLO-LC3 colocalization, the Colocalization plugin in ImageJ was utilized, followed by particle analysis on the merged signal to estimate the number of colocalized puncta. The fluorescence intensity of LysoTracker and DQ-BSA was determined by quantifying the Integrated Density (RawIntDen) in ImageJ. Similarly, nuclear translocation of TFEB was quantified by measuring RawIntDen within nuclear ROIs. Statistical analysis All statistical analyses and graphical representations were performed using GraphPad Prism 10.0.0. Data are presented as the mean ± SEM. P values were determined using the specific methods outlined in the respective figure legends. P value less than 0.05 (P < 0.05) was considered statistically significant. Antibodies, reagents and other resources Details of all antibodies, reagents and other resources are provided in Table 4 . Declaration of interests The authors declare no competing interests. Author contributions A.P. and R.M. conceptualized the study. A.P. and R.M. developed the methodology. A.P., S.B., P.S., and D.B. conducted the investigation. K.S.R., S.B., P.S., A.P., and M.N. analyzed the data. A.P. and R.M. drafted, edited and reviewed the manuscript, while rest of the authors reviewed it. R.M. supervised the project and acquired funding. Declaration of generative AI and AI-assisted technologies in the writing process During the preparation of this work the authors used ChatGPT to improve the language and readability. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication. Acknowledgments This work was supported by the Life Sciences Research Board-DRDO (LSRB-310/BTB/2017), the Science and Engineering Research Board (SERB/MN/4471), the Indian Council of Medical Research (ICMR/RM/4846) and the intramural funds from JNCASR (JNCASR/MBGU/RM) to RM. We extend our sincere thanks to all members of the Autophagy Laboratory for their valuable support and insightful discussions. We are deeply grateful to Prof. Daniel Portnoy and Andrea Anaya Sanchez for their continual support with the plasmid constructs and Lm strains. We also thank Dr. Veena Ammanathan, Dr. Keerti, Dr. Kushagra Bansal and Dr. Kesavardana Sannula for their thoughtful input and guidance. We acknowledge the research facilities provided by JNCASR. AP received support through a doctoral fellowship from JNCASR. SB and KSR are thankful to JNCASR for its continued support. PS acknowledges IISER Mohali for funding, and DB is grateful to JNCASR for her Integrated Ph.D. fellowship. MN is thankful to JNCASR for post-doctoral fellowship. All the figures were made using Adobe Illustrator with changes applied uniformly across all tests and controls unless mentioned otherwise in the methods. Schematic representations were made using BioRender through a paid subscription. References 1. ↵ Deretic , V. , and Kroemer , G . ( 2022 ). Autophagy in metabolism and quality control: opposing, complementary or interlinked functions? Autophagy 18 , 283 – 292 . doi: 10.1080/15548627.2021.1933742 . OpenUrl CrossRef 2. ↵ Klionsky , D.J. , Petroni , G. , Amaravadi , R.K. , Baehrecke , E.H. , Ballabio , A. , Boya , P. , Bravo-San Pedro , J.M. , Cadwell , K. , Cecconi , F. , Choi , A.M.K. , et al. ( 2021 ). Autophagy in major human diseases . EMBO J 40 , e108863 . doi: 10.15252/embj.2021108863 . OpenUrl CrossRef PubMed 3. ↵ Lu , S.-L. , Omori , H. , Zhou , Y. , Lin , Y.-S. , Liu , C.-C. , Wu , J.-J. , and Noda , T . ( 2022 ). VEGF-Mediated Augmentation of Autophagic and Lysosomal Activity in Endothelial Cells Defends against Intracellular Streptococcus pyogenes . mBio 13 , e01233 – 01222 . doi: 10.1128/mbio.01233-22 . OpenUrl CrossRef PubMed 4. Mao , K. , and Klionsky , D.J . ( 2017 ). Xenophagy: A battlefield between host and microbe, and a possible avenue for cancer treatment . Autophagy 13 , 223 – 224 . doi: 10.1080/15548627.2016.1267075 . OpenUrl CrossRef PubMed 5. Wang , Z. , and Li , C . ( 2020 ). Xenophagy in innate immunity: A battle between host and pathogen . Dev Comp Immunol 109 , 103693 . doi: 10.1016/j.dci.2020.103693 . OpenUrl CrossRef PubMed 6. Mehto , S. , Jena , K.K. , Yadav , R. , Priyadarsini , S. , Samal , P. , Krishna , S. , Dhar , K. , Jain , A. , Chauhan , N.R. , Murmu , K.C. , et al. ( 2022 ). Selective autophagy of RIPosomes maintains innate immune homeostasis during bacterial infection . The EMBO Journal 41 , e111289 . doi: 10.15252/embj.2022111289 . OpenUrl CrossRef PubMed 7. Pant , A. , Yao , X. , Lavedrine , A. , Viret , C. , Dockterman , J. , Chauhan , S. , Shi , C.-S. , Manjithaya , R. , Cadwell , K. , A. Kufer , T. , et al. ( 2022 ). Interactions of Autophagy and the Immune System in Health and Diseases . Autophagy Reports 1 , 438 – 515 . doi: 10.1080/27694127.2022.2119743 . OpenUrl CrossRef PubMed 8. Gomes , Ligia C. , and Dikic , I . ( 2014 ). Autophagy in Antimicrobial Immunity . Molecular Cell 54 , 224 – 233 . doi: 10.1016/j.molcel.2014.03.009 . OpenUrl CrossRef PubMed 9. Cong , Y. , Dinesh Kumar , N. , Mauthe , M. , Verlhac , P. , and Reggiori , F . ( 2020 ). Manipulation of selective macroautophagy by pathogens at a glance . J Cell Sci 133 . doi: 10.1242/jcs.240440 . OpenUrl Abstract / FREE Full Text 10. Aguilera , M.O. , Delgui , L.R. , Reggiori , F. , Romano , P.S. , and Colombo , M.I . ( 2024 ). Autophagy as an innate immunity response against pathogens: a Tango dance . FEBS Lett 598 , 140 – 166 . doi: 10.1002/1873-3468.14788 . OpenUrl CrossRef PubMed 11. Fujita , N. , Morita , E. , Itoh , T. , Tanaka , A. , Nakaoka , M. , Osada , Y. , Umemoto , T. , Saitoh , T. , Nakatogawa , H. , Kobayashi , S. , et al. ( 2013 ). Recruitment of the autophagic machinery to endosomes during infection is mediated by ubiquitin . J Cell Biol 203 , 115 – 128 . doi: 10.1083/jcb.201304188 . OpenUrl Abstract / FREE Full Text 12. Xu , Y. , Zhou , P. , Cheng , S. , Lu , Q. , Nowak , K. , Hopp , A.K. , Li , L. , Shi , X. , Zhou , Z. , Gao , W. , et al. ( 2019 ). A Bacterial Effector Reveals the V-ATPase-ATG16L1 Axis that Initiates Xenophagy . Cell 178 , 552 – 566 .e520. doi: 10.1016/j.cell.2019.06.007 . OpenUrl CrossRef PubMed 13. ↵ Nakagawa , I. , Amano , A. , Mizushima , N. , Yamamoto , A. , Yamaguchi , H. , Kamimoto , T. , Nara , A. , Funao , J. , Nakata , M. , Tsuda , K. , et al. ( 2004 ). Autophagy Defends Cells Against Invading Group A Streptococcus . Science 306 , 1037 – 1040 . doi: 10.1126/science.1103966 . OpenUrl Abstract / FREE Full Text 14. ↵ Ammanathan , V. , Mishra , P. , Chavalmane , A.K. , Muthusamy , S. , Jadhav , V. , Siddamadappa , C. , and Manjithaya , R . ( 2020 ). Restriction of intracellular Salmonella replication by restoring TFEB-mediated xenophagy . Autophagy 16 , 1584 – 1584 . doi: 10.1080/15548627.2019.1689770 . OpenUrl CrossRef PubMed 15. Capurro , M.I. , Prashar , A. , Gao , X. , and Jones , N.L . ( 2023 ). Survival of intracellular pathogens in response to mTORC1- or TRPML1-TFEB-induced xenophagy . Autophagy Rep 2 , 2191918 . doi: 10.1080/27694127.2023.2191918 . OpenUrl CrossRef PubMed 16. ↵ Keerti , Pant , A. , and Manjithaya , R. ( 2021 ). TFEB - at the crossroads of host-pathogen interactions . J Cell Sci 134 . doi: 10.1242/jcs.252981 . OpenUrl CrossRef 17. ↵ Cabral-Fernandes , L. , Goyal , S. , Farahvash , A. , Tsalikis , J. , Philpott , D.J. , and Girardin , S.E . ( 2022 ). Invading Bacterial Pathogens Activate Transcription Factor EB in Epithelial Cells through the Amino Acid Starvation Pathway of mTORC1 Inhibition . Mol Cell Biol 42 , e0024122 . doi: 10.1128/mcb.00241-22 . OpenUrl CrossRef PubMed 18. Visvikis , O. , Ihuegbu , N. , Labed , S.A. , Luhachack , L.G. , Alves , A.F. , Wollenberg , A.C. , Stuart , L.M. , Stormo , G.D. , and Irazoqui , J.E . ( 2014 ). Innate host defense requires TFEB-mediated transcription of cytoprotective and antimicrobial genes . Immunity 40 , 896 – 909 . doi: 10.1016/j.immuni.2014.05.002 . OpenUrl CrossRef PubMed 19. ↵ El-Houjeiri , L. , Possik , E. , Vijayaraghavan , T. , Paquette , M. , Martina , J.A. , Kazan , J.M. , Ma , E.H. , Jones , R. , Blanchette , P. , Puertollano , R. , and Pause , A . ( 2019 ). The Transcription Factors TFEB and TFE3 Link the FLCN-AMPK Signaling Axis to Innate Immune Response and Pathogen Resistance . Cell Reports 26 , 3613 – 3628 .e3616. doi: 10.1016/j.celrep.2019.02.102 . OpenUrl CrossRef PubMed 20. ↵ Yoshikawa , Y. , Ogawa , M. , Hain , T. , Yoshida , M. , Fukumatsu , M. , Kim , M. , Mimuro , H. , Nakagawa , I. , Yanagawa , T. , Ishii , T. , et al. ( 2009 ). Listeria monocytogenes ActA-mediated escape from autophagic recognition . Nat Cell Biol 11 , 1233 – 1240 . doi: 10.1038/ncb1967 . OpenUrl CrossRef PubMed Web of Science 21. ↵ Cheng , M.I. , Chen , C. , Engström , P. , Portnoy , D.A. , and Mitchell , G . ( 2018 ). Actin-based motility allows Listeria monocytogenes to avoid autophagy in the macrophage cytosol . Cell Microbiol 20 , e12854 . doi: 10.1111/cmi.12854 . OpenUrl CrossRef PubMed 22. ↵ Seto , S. , Tsujimura , K. , and Koide , Y. Coronin-1a inhibits autophagosome formation around Mycobacterium tuberculosis-containing phagosomes and assists mycobacterial survival in macrophages . 23. ↵ Choy , A. , Dancourt , J. , Mugo , B. , O’Connor , T.J. , Isberg , R.R. , Melia , T.J. , and Roy , C.R . ( 2012 ). The Legionella effector RavZ inhibits host autophagy through irreversible Atg8 deconjugation . Science 338 , 1072 – 1076 . doi: 10.1126/science.1227026 . OpenUrl Abstract / FREE Full Text 24. ↵ Pujol , C. , Klein , K.A. , Romanov , G.A. , Palmer , L.E. , Cirota , C. , Zhao , Z. , and Bliska , J.B . ( 2009 ). Yersinia pestis can reside in autophagosomes and avoid xenophagy in murine macrophages by preventing vacuole acidification . Infect Immun 77 , 2251 – 2261 . doi: 10.1128/iai.00068-09 . OpenUrl Abstract / FREE Full Text 25. ↵ Liu , P.F. , Cheng , J.S. , Sy , C.L. , Huang , W.C. , Yang , H.C. , Gallo , R.L. , Huang , C.M. , and Shu , C.W . ( 2015 ). IsaB Inhibits Autophagic Flux to Promote Host Transmission of Methicillin-Resistant Staphylococcus aureus . J Invest Dermatol 135 , 2714 – 2722 . doi: 10.1038/jid.2015.254 . OpenUrl CrossRef PubMed 26. ↵ Zhao , D. , Qiang , L. , Lei , Z. , Ge , P. , Lu , Z. , Wang , Y. , Zhang , X. , Qiang , Y. , Li , B. , Pang , Y. , et al. ( 2024 ). TRIM27 elicits protective immunity against tuberculosis by activating TFEB-mediated autophagy flux . Autophagy 20 , 1483 – 1504 . doi: 10.1080/15548627.2024.2321831 . OpenUrl CrossRef PubMed 27. ↵ Killips , B. , Heaton , E.J.B. , Augusto , L. , Omsland , A. , and Gilk , S.D . ( 2024 ). Coxiella burnetii inhibits nuclear translocation of TFEB, the master transcription factor for lysosomal biogenesis . J Bacteriol 206 , e0015024 . doi: 10.1128/jb.00150-24 . OpenUrl CrossRef PubMed 28. ↵ Rao , S. , Xu , T. , Xia , Y. , and Zhang , H . ( 2020 ). Salmonella and S. aureus Escape From the Clearance of Macrophages via Controlling TFEB . Front Microbiol 11 , 573844 . doi: 10.3389/fmicb.2020.573844 . OpenUrl CrossRef PubMed 29. ↵ Paik , S. , Kim , K.T. , Kim , I.S. , Kim , Y.J. , Kim , H.J. , Choi , S. , and Jo , E.K . ( 2022 ). Mycobacterial acyl carrier protein suppresses TFEB activation and upregulates miR-155 to inhibit host defense . Front Immunol 13 , 946929 . doi: 10.3389/fimmu.2022.946929 . OpenUrl CrossRef PubMed 30. ↵ Lear , T.B. , Larsen , M.B. , Lin , B. , Treat , B.R. , Cao , Q. , Boudreau , Á. , Lockwood , K.C. , Alfaras , I. , Kennerdell , J.R. , Salminen , L. , et al. ( 2025 ). Inhibition of virally induced TFEB proteasomal degradation as a host-centric therapeutic approach for coronaviral infection . Sci Adv 11 , eadv4033 . doi: 10.1126/sciadv.adv4033 . OpenUrl CrossRef PubMed 31. ↵ Nguyen , B.N. , Peterson , B.N. , and Portnoy , D.A . ( 2019 ). Listeriolysin O: A phagosome-specific cytolysin revisited . Cell Microbiol 21 , e12988 . doi: 10.1111/cmi.12988 . OpenUrl CrossRef 32. Hamon , M.A. , Ribet , D. , Stavru , F. , and Cossart , P . ( 2012 ). Listeriolysin O: the Swiss army knife of Listeria . Trends Microbiol 20 , 360 – 368 . doi: 10.1016/j.tim.2012.04.006 . OpenUrl CrossRef PubMed Web of Science 33. ↵ Osborne , S.E. , and Brumell , J.H . ( 2017 ). Listeriolysin O: from bazooka to Swiss army knife . Philos Trans R Soc Lond B Biol Sci 372 . doi: 10.1098/rstb.2016.0222 . OpenUrl CrossRef PubMed 34. ↵ Smith , G.A. , Marquis , H. , Jones , S. , Johnston , N.C. , Portnoy , D.A. , and Goldfine , H . ( 1995 ). The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread . Infect Immun 63 , 4231 – 4237 . doi: 10.1128/iai.63.11.4231-4237.1995 . OpenUrl Abstract / FREE Full Text 35. Portnoy , D.A. , Chakraborty , T. , Goebel , W. , and Cossart , P . ( 1992 ). Molecular determinants of Listeria monocytogenes pathogenesis . Infect Immun 60 , 1263 – 1267 . doi: 10.1128/iai.60.4.1263-1267.1992 . OpenUrl FREE Full Text 36. ↵ Alberti-Segui , C. , Goeden , K.R. , and Higgins , D.E . ( 2007 ). Differential function of Listeria monocytogenes listeriolysin O and phospholipases C in vacuolar dissolution following cell-to-cell spread . Cell Microbiol 9 , 179 – 195 . doi: 10.1111/j.1462-5822.2006.00780.x . OpenUrl CrossRef PubMed 37. ↵ Skoble , J. , Portnoy , D.A. , and Welch , M.D . ( 2000 ). Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and Listeria monocytogenes motility . J Cell Biol 150 , 527 – 538 . doi: 10.1083/jcb.150.3.527 . OpenUrl Abstract / FREE Full Text 38. ↵ Ireton , K. , and Cossart , P . ( 1997 ). Host-pathogen interactions during entry and actin-based movement of Listeria monocytogenes . Annu Rev Genet 31 , 113 – 138 . doi: 10.1146/annurev.genet.31.1.113 . OpenUrl CrossRef PubMed Web of Science 39. Rafelski , S.M. , and Theriot , J.A . ( 2005 ). Bacterial shape and ActA distribution affect initiation of Listeria monocytogenes actin-based motility . Biophys J 89 , 2146 – 2158 . doi: 10.1529/biophysj.105.061168 . OpenUrl CrossRef PubMed 40. Cossart , P . ( 1995 ). Actin-based bacterial motility . Curr Opin Cell Biol 7 , 94 – 101 . doi: 10.1016/0955-0674(95)80050-6 . OpenUrl CrossRef PubMed Web of Science 41. ↵ Pillich , H. , Puri , M. , and Chakraborty , T . ( 2017 ). ActA of Listeria monocytogenes and Its Manifold Activities as an Important Listerial Virulence Factor . Curr Top Microbiol Immunol 399 , 113 – 132 . doi: 10.1007/82_2016_30 . OpenUrl CrossRef PubMed 42. ↵ Dowd , G.C. , Mortuza , R. , and Ireton , K . ( 2021 ). Molecular Mechanisms of Intercellular Dissemination of Bacterial Pathogens . Trends Microbiol 29 , 127 – 141 . doi: 10.1016/j.tim.2020.06.008 . OpenUrl CrossRef 43. ↵ Tucker , J.S. , Cho , J. , Albrecht , T.M. , Ferrell , J.L. , and D’Orazio , S.E.F . ( 2023 ). Egress of Listeria monocytogenes from Mesenteric Lymph Nodes Depends on Intracellular Replication and Cell-to-Cell Spread . Infect Immun 91 , e0006423 . doi: 10.1128/iai.00064-23 . OpenUrl CrossRef 44. ↵ Czuczman , M.A. , Fattouh , R. , van Rijn , J.M. , Canadien , V. , Osborne , S. , Muise , A.M. , Kuchroo , V.K. , Higgins , D.E. , and Brumell , J.H. Listeria monocytogenes exploits efferocytosis to promote cell-to-cell spread . 45. ↵ Yoshikawa , Y. , Ogawa , M. , Hain , T. , Chakraborty , T. , and Sasakawa , C . ( 2009 ). Listeria monocytogenes ActA is a key player in evading autophagic recognition . Autophagy 5 , 1220 – 1221 . doi: 10.4161/auto.5.8.10177 . OpenUrl CrossRef PubMed Web of Science 46. ↵ Puri , M. , La Pietra , L. , Mraheil , M.A. , Lucas , R. , Chakraborty , T. , and Pillich , H. ( 2017 ). Listeriolysin O Regulates the Expression of Optineurin, an Autophagy Adaptor That Inhibits the Growth of Listeria monocytogenes . Toxins (Basel) 9 . doi: 10.3390/toxins9090273 . OpenUrl CrossRef 47. ↵ Meyer-Morse , N. , Robbins , J.R. , Rae , C.S. , Mochegova , S.N. , Swanson , M.S. , Zhao , Z. , Virgin , H.W. , and Portnoy , D . ( 2010 ). Listeriolysin O is necessary and sufficient to induce autophagy during Listeria monocytogenes infection . PLoS One 5 , e8610 . doi: 10.1371/journal.pone.0008610 . OpenUrl CrossRef PubMed 48. ↵ Mitchell , G. , Cheng , M.I. , Chen , C. , Nguyen , B.N. , Whiteley , A.T. , Kianian , S. , Cox , J.S. , Green , D.R. , McDonald , K.L. , and Portnoy , D.A . ( 2018 ). Listeria monocytogenes triggers noncanonical autophagy upon phagocytosis, but avoids subsequent growth-restricting xenophagy . Proc Natl Acad Sci U S A 115 , E210 – e217 . doi: 10.1073/pnas.1716055115 . OpenUrl Abstract / FREE Full Text 49. ↵ Asano , K. , Sashinami , H. , Osanai , A. , Hirose , S. , Ono , H.K. , Narita , K. , Hu , D.L. , and Nakane , A . ( 2016 ). Passive immunization with anti-ActA and anti-listeriolysin O antibodies protects against Listeria monocytogenes infection in mice . Sci Rep 6 , 39628 . doi: 10.1038/srep39628 . OpenUrl CrossRef PubMed 50. ↵ Birmingham , C.L. , Canadien , V. , Gouin , E. , Troy , E.B. , Yoshimori , T. , Cossart , P. , Higgins , D.E. , and Brumell , J.H . ( 2007 ). Listeria monocytogenes evades killing by autophagy during colonization of host cells . Autophagy 3 , 442 – 451 . doi: 10.4161/auto.4450 . OpenUrl CrossRef PubMed 51. ↵ Peron-Cane , C. , Fernandez , J.C. , Leblanc , J. , Wingertsmann , L. , Gautier , A. , Desprat , N. , and Lebreton , A . ( 2020 ). Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells . PLoS Pathog 16 , e1009001 . doi: 10.1371/journal.ppat.1009001 . OpenUrl CrossRef PubMed 52. ↵ Herb , M. , and Schramm , M . ( 2021 ). Functions of ROS in Macrophages and Antimicrobial Immunity . Antioxidants (Basel) 10 . doi: 10.3390/antiox10020313 . OpenUrl CrossRef PubMed 53. ↵ Zhang , L. , and Wang , C.C . ( 2014 ). Inflammatory response of macrophages in infection . Hepatobiliary Pancreat Dis Int 13 , 138 – 152 . doi: 10.1016/s1499-3872(14)60024-2 . OpenUrl CrossRef PubMed 54. ↵ Ballabio , A. , and Bonifacino , J.S. Lysosomes as dynamic regulators of cell and organismal homeostasis . 55. ↵ Chazotte , B . ( 2011 ). Labeling lysosomes in live cells with LysoTracker . Cold Spring Harb Protoc 2011 , pdb.prot5571. doi: 10.1101/pdb.prot5571 . OpenUrl Abstract / FREE Full Text 56. ↵ Frost , L.S. , Dhingra , A. , Reyes-Reveles , J. , and Boesze-Battaglia , K . ( 2017 ). The Use of DQ-BSA to Monitor the Turnover of Autophagy-Associated Cargo . Methods Enzymol 587 , 43 – 54 . doi: 10.1016/bs.mie.2016.09.052 . OpenUrl CrossRef PubMed 57. ↵ Settembre , C. , Di Malta , C. , Polito , V.A. , Garcia Arencibia , M. , Vetrini , F. , Erdin , S. , Erdin , S.U. , Huynh , T. , Medina , D. , Colella , P. , et al. ( 2011 ). TFEB links autophagy to lysosomal biogenesis . Science 332 , 1429 – 1433 . doi: 10.1126/science.1204592 . OpenUrl Abstract / FREE Full Text 58. ↵ Lee , C.-R. , Park , Y.-H. , Min , H. , Kim , Y.-R. , and Seok , Y.-J . ( 2019 ). Determination of protein phosphorylation by polyacrylamide gel electrophoresis . Journal of Microbiology 57 , 93 – 100 . doi: 10.1007/s12275-019-9021-y . OpenUrl CrossRef PubMed 59. ↵ Paquette , M. , El-Houjeiri , L. , L, C.Z., Puustinen , P. , Blanchette , P. , Jeong , H. , Dejgaard , K. , Siegel , P.M. , and Pause , A. AMPK-dependent phosphorylation is required for transcriptional activation of TFEB and TFE3 . 60. ↵ Gedde , M.M. , Higgins , D.E. , Tilney , L.G. , and Portnoy , D.A . ( 2000 ). Role of listeriolysin O in cell-to-cell spread of Listeria monocytogenes . Infect Immun 68 , 999 – 1003 . doi: 10.1128/iai.68.2.999-1003.2000 . OpenUrl Abstract / FREE Full Text 61. ↵ Kumar , M. , Michael , S. , Alvarado-Valverde , J. , Zeke , A. , Lazar , T. , Glavina , J. , Nagy-Kanta , E. , Donagh , J.M. , Kalman , Z.E. , Pascarelli , S. , et al. ( 2024 ). ELM-the Eukaryotic Linear Motif resource-2024 update . Nucleic Acids Res 52 , D442 – d455 . doi: 10.1093/nar/gkad1058 . OpenUrl CrossRef PubMed 62. ↵ Jacomin , A.C. , Samavedam , S. , Promponas , V. , and Nezis , I.P . ( 2016 ). iLIR database: A web resource for LIR motif-containing proteins in eukaryotes . Autophagy 12 , 1945 – 1953 . doi: 10.1080/15548627.2016.1207016 . OpenUrl CrossRef PubMed 63. ↵ Chao , X. , Wang , S. , Zhao , K. , Li , Y. , Williams , J.A. , Li , T. , Chavan , H. , Krishnamurthy , P. , He , X.C. , Li , L. , et al. ( 2018 ). Impaired TFEB-Mediated Lysosome Biogenesis and Autophagy Promote Chronic Ethanol-Induced Liver Injury and Steatosis in Mice . Gastroenterology 155 , 865 – 879 .e812. doi: 10.1053/j.gastro.2018.05.027 . OpenUrl CrossRef PubMed 64. Martina , J.A. , Chen , Y. , Gucek , M. , and Puertollano , R . ( 2012 ). MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB . Autophagy 8 , 903 – 914 . doi: 10.4161/auto.19653 . OpenUrl CrossRef PubMed Web of Science 65. ↵ Zhitomirsky , B. , Yunaev , A. , Kreiserman , R. , Kaplan , A. , Stark , M. , and Assaraf , Y.G . ( 2018 ). Lysosomotropic drugs activate TFEB via lysosomal membrane fluidization and consequent inhibition of mTORC1 activity . Cell Death Dis 9 , 1191 . doi: 10.1038/s41419-018-1227-0 . OpenUrl CrossRef PubMed 66. ↵ Tang , F. , Hu , P. , Yang , Z. , Xue , C. , Gong , J. , Sun , S. , Shi , L. , Zhang , S. , Li , Z. , Yang , C. , et al. ( 2017 ). SBI0206965, a novel inhibitor of Ulk1, suppresses non-small cell lung cancer cell growth by modulating both autophagy and apoptosis pathways . Oncol Rep 37 , 3449 – 3458 . doi: 10.3892/or.2017.5635 . OpenUrl CrossRef PubMed 67. ↵ Mauthe , M. , Orhon , I. , Rocchi , C. , Zhou , X. , Luhr , M. , Hijlkema , K.J. , Coppes , R.P. , Engedal , N. , Mari , M. , and Reggiori , F . ( 2018 ). Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion . Autophagy 14 , 1435 – 1455 . doi: 10.1080/15548627.2018.1474314 . OpenUrl CrossRef PubMed 68. ↵ Dortet , L. , Mostowy , S. , Samba-Louaka , A. , Gouin , E. , Nahori , M.A. , Wiemer , E.A. , Dussurget , O. , and Cossart , P . ( 2011 ). Recruitment of the major vault protein by InlK: a Listeria monocytogenes strategy to avoid autophagy . PLoS Pathog 7 , e1002168 . doi: 10.1371/journal.ppat.1002168 . OpenUrl CrossRef PubMed 69. ↵ Dortet , L. , Mostowy , S. , and Cossart , P . ( 2012 ). Listeria and autophagy escape: involvement of InlK, an internalin-like protein . Autophagy 8 , 132 – 134 . doi: 10.4161/auto.8.1.18218 . OpenUrl CrossRef PubMed 70. ↵ Tattoli , I. , Sorbara , M.T. , Yang , C. , Tooze , S.A. , Philpott , D.J. , and Girardin , S.E . ( 2013 ). Listeria phospholipases subvert host autophagic defenses by stalling pre-autophagosomal structures . EMBO J 32 , 3066 – 3078 . doi: 10.1038/emboj.2013.234 . OpenUrl CrossRef PubMed 71. ↵ Mitchell , G. , Ge , L. , Huang , Q. , Chen , C. , Kianian , S. , Roberts , M.F. , Schekman , R. , and Portnoy , D.A . ( 2015 ). Avoidance of autophagy mediated by PlcA or ActA is required for Listeria monocytogenes growth in macrophages . Infect Immun 83 , 2175 – 2184 . doi: 10.1128/IAI.00110-15 . OpenUrl Abstract / FREE Full Text 72. ↵ Angelakopoulos , H. , Loock , K. , Sisul , D.M. , Jensen , E.R. , Miller , J.F. , and Hohmann , E.L . ( 2002 ). Safety and shedding of an attenuated strain of Listeria monocytogenes with a deletion of actA/plcB in adult volunteers: a dose escalation study of oral inoculation . Infect Immun 70 , 3592 – 3601 . doi: 10.1128/IAI.70.7.3592-3601.2002 . OpenUrl Abstract / FREE Full Text 73. ↵ Kortebi , M. , Milohanic , E. , Mitchell , G. , Péchoux , C. , Prevost , M.C. , Cossart , P. , and Bierne , H . ( 2017 ). Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells . PLoS Pathog 13 , e1006734 . doi: 10.1371/journal.ppat.1006734 . OpenUrl CrossRef PubMed 74. ↵ Tavares-Gomes , L. , Polidori , M. , Monney , C. , Neuhaus , G. , Vidondo , B. , Witz , G. , Hemphill , A. , and Oevermann , A . ( 2025 ). Divergent host-pathogen interactions in neurolisteriosis: cytosolic replication vs. phagosomal dormancy of Listeria monocytogenes in CNS macrophages . Acta Neuropathol 149 , 63 . doi: 10.1007/s00401-025-02900-8 . OpenUrl CrossRef PubMed 75. ↵ Maudet , C. , Kheloufi , M. , Levallois , S. , Gaillard , J. , Huang , L. , Gaultier , C. , Tsai , Y.H. , Disson , O. , and Lecuit , M . ( 2022 ). Bacterial inhibition of Fas-mediated killing promotes neuroinvasion and persistence . Nature 603 , 900 – 906 . doi: 10.1038/s41586-022-04505-7 . OpenUrl CrossRef PubMed 76. Bakardjiev , A.I. , Stacy , B.A. , and Portnoy , D.A . ( 2005 ). Growth of Listeria monocytogenes in the guinea pig placenta and role of cell-to-cell spread in fetal infection . J Infect Dis 191 , 1889 – 1897 . doi: 10.1086/430090 . OpenUrl CrossRef PubMed Web of Science 77. ↵ Le Monnier , A. , Autret , N. , Join-Lambert , O.F. , Jaubert , F. , Charbit , A. , Berche , P. , and Kayal , S. ( 2007 ). ActA is required for crossing of the fetoplacental barrier by Listeria monocytogenes . Infect Immun 75 , 950 – 957 . doi: 10.1128/iai.01570-06 . OpenUrl Abstract / FREE Full Text 78. ↵ Shaughnessy , L.M. , Hoppe , A.D. , Christensen , K.A. , and Swanson , J.A . ( 2006 ). Membrane perforations inhibit lysosome fusion by altering pH and calcium in Listeria monocytogenes vacuoles . Cell Microbiol 8 , 781 – 792 . doi: 10.1111/j.1462-5822.2005.00665.x . OpenUrl CrossRef PubMed Web of Science 79. ↵ Malet , J.K. , Cossart , P. , and Ribet , D . ( 2017 ). Alteration of epithelial cell lysosomal integrity induced by bacterial cholesterol-dependent cytolysins . Cell Microbiol 19 . doi: 10.1111/cmi.12682 . OpenUrl CrossRef 80. ↵ Bastiat-Sempe , B. , Love , J.F. , Lomayesva , N. , and Wessels , M.R . ( 2014 ). Streptolysin O and NAD-glycohydrolase prevent phagolysosome acidification and promote group A Streptococcus survival in macrophages . mBio 5 , e01690 – 01614 . doi: 10.1128/mBio.01690-14 . OpenUrl CrossRef PubMed 81. ↵ Chen , H.D. , Kao , C.Y. , Liu , B.Y. , Huang , S.W. , Kuo , C.J. , Ruan , J.W. , Lin , Y.H. , Huang , C.R. , Chen , Y.H. , Wang , H.D. , et al. ( 2017 ). HLH-30/TFEB-mediated autophagy functions in a cell-autonomous manner for epithelium intrinsic cellular defense against bacterial pore-forming toxin in C. elegans . Autophagy 13 , 371 – 385 . doi: 10.1080/15548627.2016.1256933 . OpenUrl CrossRef PubMed 82. ↵ Bavdek , A. , Kostanjšek , R. , Antonini , V. , Lakey , J.H. , Dalla Serra , M. , Gilbert , R.J.C. , and Anderluh , G . ( 2012 ). pH dependence of listeriolysin O aggregation and pore-forming ability . The FEBS Journal 279 , 126 – 141 . doi: 10.1111/j.1742-4658.2011.08405.x . OpenUrl CrossRef PubMed 83. ↵ Viala , J.P.M. , Mochegova , S.N. , Meyer-Morse , N. , and Portnoy , D.A . ( 2008 ). A bacterial pore-forming toxin forms aggregates in cells that resemble those associated with neurodegenerative diseases . Cellular Microbiology 10 , 985 – 993 . doi: 10.1111/j.1462-5822.2007.01100.x . OpenUrl CrossRef PubMed 84. ↵ Chen , C. , Nguyen , B.N. , Mitchell , G. , Margolis , S.R. , Ma , D. , and Portnoy , D.A . ( 2018 ). The Listeriolysin O PEST-like Sequence Co-opts AP-2-Mediated Endocytosis to Prevent Plasma Membrane Damage during Listeria Infection . Cell Host Microbe 23 , 786 – 795 .e785. doi: 10.1016/j.chom.2018.05.006 . OpenUrl CrossRef PubMed 85. ↵ Schnupf , P. , Zhou , J. , Varshavsky , A. , and Portnoy , D.A . ( 2007 ). Listeriolysin O secreted by Listeria monocytogenes into the host cell cytosol is degraded by the N-end rule pathway . Infect Immun 75 , 5135 – 5147 . doi: 10.1128/iai.00164-07 . OpenUrl Abstract / FREE Full Text 86. ↵ Schnupf , P. , Portnoy , D.A. , and Decatur , A.L . ( 2006 ). Phosphorylation, ubiquitination and degradation of listeriolysin O in mammalian cells: role of the PEST-like sequence . Cellular Microbiology 8 , 353 – 364 . doi: 10.1111/j.1462-5822.2005.00631.x . OpenUrl CrossRef PubMed 87. ↵ Petraccione , K. , Ali , M.G.H. , Cyr , N. , Wahba , H.M. , Stocker , T. , Akhrymuk , M. , Akhrymuk , I. , Panny , L. , Bracci , N. , Cafaro , R. , et al. ( 2024 ). An LIR motif in the Rift Valley fever virus NSs protein is critical for the interaction with LC3 family members and inhibition of autophagy . PLOS Pathogens 20 , e1012093 . doi: 10.1371/journal.ppat.1012093 . OpenUrl CrossRef PubMed 88. ↵ Kayath , C.A. , Hussey , S. , El hajjami , N. , Nagra , K. , Philpott , D. , and Allaoui , A . ( 2010 ). Escape of intracellular Shigella from autophagy requires binding to cholesterol through the type III effector, IcsB . Microbes Infect 12 , 956 – 966 . doi: 10.1016/j.micinf.2010.06.006 . OpenUrl CrossRef PubMed 89. ↵ Beale , R. , Wise , H. , Stuart , A. , Ravenhill , B.J. , Digard , P. , and Randow , F . ( 2014 ). A LC3-interacting motif in the influenza A virus M2 protein is required to subvert autophagy and maintain virion stability . Cell Host Microbe 15 , 239 – 247 . doi: 10.1016/j.chom.2014.01.006 . OpenUrl CrossRef PubMed 90. ↵ Goswami , S. , Samanta , D. , and Duraivelan , K . ( 2023 ). Molecular mimicry of host short linear motif-mediated interactions utilised by viruses for entry . Mol Biol Rep 50 , 4665 – 4673 . doi: 10.1007/s11033-023-08389-2 . OpenUrl CrossRef PubMed 91. ↵ Sámano-Sánchez , H. , and Gibson , T.J . ( 2020 ). Mimicry of Short Linear Motifs by Bacterial Pathogens: A Drugging Opportunity . Trends in Biochemical Sciences 45 , 526 – 544 . doi: 10.1016/j.tibs.2020.03.003 . OpenUrl CrossRef PubMed 92. ↵ Apte , S. , Bhutda , S. , Ghosh , S. , Sharma , K. , Barton , T.E. , Dibyachintan , S. , Sahay , O. , Roy , S. , Sinha , A.R. , Adicherla , H. , et al. An innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins . Science Advances 9 , eade1851 . doi: 10.1126/sciadv.ade1851 . OpenUrl CrossRef 93. ↵ Napolitano , G. , and Ballabio , A . ( 2016 ). TFEB at a glance . Journal of Cell Science 129 , 2475 – 2481 . doi: 10.1242/jcs.146365 . OpenUrl Abstract / FREE Full Text 94. ↵ Palmieri , M. , Impey , S. , Kang , H. , di Ronza , A. , Pelz , C. , Sardiello , M. , and Ballabio , A. ( 2011 ). Characterization of the CLEAR network reveals an integrated control of cellular clearance pathways . Human Molecular Genetics 20 , 3852 – 3866 . doi: 10.1093/hmg/ddr306 . OpenUrl CrossRef PubMed Web of Science 95. ↵ Schuerch , D.W. , Wilson-Kubalek , E.M. , and Tweten , R.K . ( 2005 ). Molecular basis of listeriolysin O pH dependence . Proceedings of the National Academy of Sciences 102 , 12537 – 12542 . doi: 10.1073/pnas.0500558102 . OpenUrl Abstract / FREE Full Text 96. ↵ Santra , S. , Nayak , I. , Paladhi , A. , Das , D. , and Banerjee , A . ( 2024 ). Estimates of differential toxin expression governing heterogeneous intracellular lifespans of Streptococcus pneumoniae . Journal of Cell Science 137 , jcs260891 . doi: 10.1242/jcs.260891 . OpenUrl CrossRef PubMed 97. ↵ Goodwin , J.M. , Walkup , W.G. , Hooper , K. , Li , T. , Kishi-Itakura , C. , Ng , A. , Lehmberg , T. , Jha , A. , Kommineni , S. , Fletcher , K. , et al. ( 2021 ). GABARAP sequesters the FLCN-FNIP tumor suppressor complex to couple autophagy with lysosomal biogenesis . Sci Adv 7 , eabj2485 . doi: 10.1126/sciadv.abj2485 . OpenUrl CrossRef PubMed 98. ↵ Nakamura , S. , Akayama , S. , and Yoshimori , T . ( 2021 ). Autophagy-independent function of lipidated LC3 essential for TFEB activation during the lysosomal damage responses . Autophagy 17 , 581 – 583 . doi: 10.1080/15548627.2020.1846292 . OpenUrl CrossRef PubMed 99. ↵ Li , S. , Xu , X. , Wei , L. , Wang , L. , and Lv , Q . ( 2022 ). Acacetin Alleviates Listeria monocytogenes Virulence Both In Vitro and In Vivo via the Inhibition of Listeriolysin O . Foodborne Pathog Dis 19 , 115 – 125 . doi: 10.1089/fpd.2021.0021 . OpenUrl CrossRef PubMed 100. Wang , T. , Liu , B. , Zhang , C. , Fang , T. , Ding , Y. , Song , G. , Zhang , S. , Shi , L. , Deng , X. , and Wang , J . ( 2022 ). Kaempferol-Driven Inhibition of Listeriolysin O Pore Formation and Inflammation Suppresses Listeria monocytogenes Infection . Microbiol Spectr 10 , e0181022 . doi: 10.1128/spectrum.01810-22 . OpenUrl CrossRef 101. ↵ Liu , M. , Lv , Q. , Xu , J. , Liu , B. , Zhou , Y. , Zhang , S. , Shen , X. , and Wang , L . ( 2023 ). Isoflavone glucoside genistin, an inhibitor targeting Sortase A and Listeriolysin O, attenuates the virulence of Listeria monocytogenes in vivo and in vitro . Biochem Pharmacol 209 , 115447 . doi: 10.1016/j.bcp.2023.115447 . OpenUrl CrossRef PubMed 102. ↵ Edelheit , O. , Hanukoglu , A. , and Hanukoglu , I . ( 2009 ). Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies . BMC Biotechnol 9 , 61 . doi: 10.1186/1472-6750-9-61 . OpenUrl CrossRef PubMed 103. ↵ Schindelin , J. , Arganda-Carreras , I. , Frise , E. , Kaynig , V. , Longair , M. , Pietzsch , T. , Preibisch , S. , Rueden , C. , Saalfeld , S. , Schmid , B. , et al. Fiji: an open-source platform for biological-image analysis . 104. Bishop , D.K. , and Hinrichs , D.J. Adoptive transfer of immunity to Listeria monocytogenes . The influence of in vitro stimulation on lymphocyte subset requirements . 105. Skoble , J. , Portnoy , D.A. , and Welch , M.D. Three Regions within Acta Promote Arp2/3 Complex-Mediated Actin Nucleation and Listeria monocytogenes Motility . 106. Jones , S. , and Portnoy , D.A. Characterization of Listeria monocytogenes pathogenesis in a strain expressing perfringolysin O in place of listeriolysin O . 107. Simon , R. , Priefer , U. , and Pühler , A. A Broad Host Range Mobilization System for In Vivo Genetic Engineering: Transposon Mutagenesis in Gram Negative Bacteria . 108. Balestrino , D. , Hamon , M.A. , Dortet , L. , Nahori , M.-A. , Pizarro-Cerda , J. , Alignani , D. , Dussurget , O. , Cossart , P. , and Toledo-Arana , A. Single-Cell Techniques Using Chromosomally Tagged Fluorescent Bacteria To Study . 109. Reniere , M.L. , Whiteley , A.T. , and Portnoy , D.A . ( 2016 ). An In Vivo Selection Identifies Listeria monocytogenes Genes Required to Sense the Intracellular Environment and Activate Virulence Factor Expression . PLoS Pathog 12 , e1005741 . doi: 10.1371/journal.ppat.1005741 . OpenUrl CrossRef PubMed 110. Thomas , E.J. , King , R.K. , Burchak , J. , and Gannon , V.P. Sensitive and specific detection of Listeria monocytogenes in milk and ground beef with the polymerase chain reaction . View the discussion thread. Back to top Previous Next Posted December 01, 2025. 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Share TFEB activation ameliorates listeriolysin O-mediated subversion of lysosomal and LC3 function during Listeria monocytogenes infection Aarti Pant , Sneha Bhatt , Parul Sen , Deepam Bhattacharya , Kuldeep Singh Rautela , Mamta Negi , Ravi Manjithaya bioRxiv 2025.11.30.691302; doi: https://doi.org/10.1101/2025.11.30.691302 Share This Article: Copy Citation Tools TFEB activation ameliorates listeriolysin O-mediated subversion of lysosomal and LC3 function during Listeria monocytogenes infection Aarti Pant , Sneha Bhatt , Parul Sen , Deepam Bhattacharya , Kuldeep Singh Rautela , Mamta Negi , Ravi Manjithaya bioRxiv 2025.11.30.691302; doi: https://doi.org/10.1101/2025.11.30.691302 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cell Biology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17697) Bioengineering (13895) Bioinformatics (41951) Biophysics (21456) Cancer Biology (18594) Cell Biology (25520) Clinical Trials (138) Developmental Biology (13381) Ecology (19903) Epidemiology (2067) Evolutionary Biology (24323) Genetics (15612) Genomics (22510) Immunology (17738) Microbiology (40401) Molecular Biology (17184) Neuroscience (88622) Paleontology (667) Pathology (2833) Pharmacology and Toxicology (4825) Physiology (7644) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4296) Systems Biology (9825) Zoology (2271)