A Conserved Metabolic Network Regulates Titan Cell Formation in Cryptococcus neoformans

A Conserved Metabolic Network Orchestrates Melanin-Dependent Titan Cell Formation in Cryptococcus neoformans | 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 A Conserved Metabolic Network Orchestrates Melanin-Dependent Titan Cell Formation in Cryptococcus neoformans Pallavi S Phatak , Sudharsan Mathivathanan , Dhrumi Shah , Ishvarya Suresh , Mary Shejo , Santosh Kumar Das , Sriram Varahan doi: https://doi.org/10.1101/2025.09.02.673682 Pallavi S Phatak 1 CSIR-Centre for Cellular and Molecular Biology , Uppal Road, Hyderabad 500007, India 2 Academy of Scientific and Innovative Research (AcSIR) , Ghaziabad- 201002, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sudharsan Mathivathanan 1 CSIR-Centre for Cellular and Molecular Biology , Uppal Road, Hyderabad 500007, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dhrumi Shah 1 CSIR-Centre for Cellular and Molecular Biology , Uppal Road, Hyderabad 500007, India 2 Academy of Scientific and Innovative Research (AcSIR) , Ghaziabad- 201002, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ishvarya Suresh 1 CSIR-Centre for Cellular and Molecular Biology , Uppal Road, Hyderabad 500007, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mary Shejo 1 CSIR-Centre for Cellular and Molecular Biology , Uppal Road, Hyderabad 500007, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Santosh Kumar Das 1 CSIR-Centre for Cellular and Molecular Biology , Uppal Road, Hyderabad 500007, India 2 Academy of Scientific and Innovative Research (AcSIR) , Ghaziabad- 201002, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sriram Varahan 1 CSIR-Centre for Cellular and Molecular Biology , Uppal Road, Hyderabad 500007, India 2 Academy of Scientific and Innovative Research (AcSIR) , Ghaziabad- 201002, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: sriram.ccmb{at}csir.res.in Abstract Full Text Info/History Metrics Preview PDF Abstract Cryptococcus neoformans is an opportunistic fungal pathogen that primarily causes pulmonary infections, with the potential to cause life-threatening infections including meningoencephalitis in immunocompromised individuals. Key virulence factors including polysaccharide capsule and melanin, facilitate immune evasion and tissue invasion in the host. Recently, titan cell formation has been defined as another important virulence factor of C. neoformans and plays a pivotal role in disease progression. The cyclic AMP–protein kinase A (cAMP-PKA) pathway in C. neoformans has been shown to be an important regulator of virulence-associated processes, including capsule formation, melanin biosynthesis and titan cell formation. However, the upstream signals, critical for the activation of cAMP-PKA pathway in the context of titan cell formation remain poorly understood. In this study, we demonstrate that the central carbon metabolic pathway, glycolysis, is critical for cAMP-dependent titan cell formation. Pharmacological and genetic perturbation of glycolysis significantly attenuated titan cell formation. Remarkably, exogenous addition of cAMP completely reversed the titan cell defects, observed during glycolysis perturbation. Interestingly, melanin deficient strains exhibited a significant attenuation in titan cell formation establishing a novel link between dimorphic switching and melanin biosynthesis in C. neoformans . These findings establish a novel regulatory axis wherein central carbon metabolism orchestrates morphogenetic switching in C. neoformans by regulating the activity of the well-conserved cAMP-PKA pathway. We also demonstrate, for the first time, that melanin biosynthesis which is under the regulatory control of cAMP-PKA pathway, is critical for titan cell formation, providing new insights into the metabolic control of C. neoformans dimorphism. Summary A leading opportunistic fungal pathogen, Cryptococcus neoformans possess a significant threat to human health. Titan cell formation is one of the established virulence factors of C. neoformans . However, the mechanisms underlying the regulation of titan cell formation remains largely uncharacterized. Our study demonstrates that glycolysis regulates titan cell formation in a cAMP-PKA pathway dependent manner and melanin biosynthesis, which is under the direct regulatory control of cAMP-PKA pathway, is critical for titan cell formation. This study offers crucial insights into the metabolic regulation of dimorphic transitions in the human fungal pathogen, C. neoformans . Introduction Cryptococcus neoformans is an opportunistic fungal pathogen that significantly impacts global public health, and causes life-threatening infections especially in immunocompromised individuals ( Zhao et al. 2023 ). This pathogen is responsible for causing cryptococcosis that primarily manifests as a pulmonary infection but can disseminate to cause severe meningoencephalitis ( Rajasingham et al. 2017 ; Chen et al. 2018 ). With an estimated 220,000 cases of cryptococcal meningitis occurring annually worldwide, resulting in approximately 180,000 deaths, C. neoformans has been designated as a critical priority fungal pathogen by the World Health Organization (WHO) ( Rajasingham et al. 2017 ; Zhao et al. 2023 ). The pathogenicity of C. neoformans is attributed to several well-characterized virulence factors that facilitate immune evasion and tissue invasion in the host ( Zaragoza 2019 ). The polysaccharide capsule, composed primarily of glucuronoxylomannan and galactoxylomannan, serves as a major virulence determinant by inhibiting phagocytosis and modulating the host immune response in favour of C. neoformans survival ( Zaragoza et al. 2009 ; O’Meara and Andrew Alspaugh 2012 ). Additionally, melanin production through laccase-mediated oxidation of phenolic compounds, provides protection against oxidative stress and antimicrobial peptides, contributing to C. neoformans survival within the host ( Nosanchuk and Casadevall 2003 ; Eisenman and Casadevall 2012 ). In recent years, morphological plasticity has emerged as another critical virulence mechanism in C. neoformans ( Okagaki et al. 2010 ). While the organism typically exists as a budding yeast with cells ranging from 5-7μm in diameter, several studies have shown that, upon host entry, C. neoformans undergoes a dramatic morphological transition resulting in the formation of unusually large cells known as ‘Titan cells’ ( Cruickshank et al. 1973 ; Crabtree et al. 2012 ; Zhou and Ballou 2018 ). Titan cells are defined as enlarged cells with a diameter of ≥10μm (with some cells reaching sizes up to ∼100μm) ( Zhou and Ballou 2018 ). Several studies have shown that titan cells are resistant to phagocytosis as well as oxidative and nitrosative stresses within the host ( Crabtree et al. 2012 ; Okagaki and Nielsen 2012 ; García-Rodas et al. 2019 ). Similarly, in vivo studies suggest that titan cells play a pivotal role in cryptococcal dissemination and disease progression within the host ( Okagaki et al. 2010 ; García-Barbazán et al. 2016 ). Apart from increased cell size, titan cells exhibit a high degree of polyploidy, a significantly thickened cell wall and capsule with an altered compositions ( Mukaremera et al. 2018 ; Zhou and Ballou 2018 ; García-Rodas et al. 2019 ). Titan cells also exhibit significant variations in homeostatic cellular processes compared to typical yeast cells, including alterations in cell cycle progression with an extended G2 phase and G2/M arrest ( Zaragoza and Nielsen 2013 ; Altamirano et al. 2021 ). Cyclic adenosine-3’,5’-monophosphate-protein kinase A (cAMP-PKA) pathway is a highly conserved eukaryotic signalling cascade that regulates diverse cellular processes, including stress responses, metabolic adaptation, and morphogenetic transitions in various fungal species ( D’Souza and Heitman 2001 ; Pukkila-Worley and Alspaugh 2004 ; Caza and Kronstad 2019 ). cAMP-PKA pathway is highly responsive to nutrient availability, specifically glucose levels and glycolytic flux and regulates various cellular processes in response to these metabolic cues ( Busti et al. 2010 ; Caza and Kronstad 2019 ). In C. neoformans , cAMP-PKA pathway has emerged as a central regulator of key virulence factors including melanin, capsule and titan cell formation ( Alspaugh et al. 1997 ; D’Souza et al. 2001 ; Caza and Kronstad 2019 ; Yu et al. 2025 ). Deletion strains that lack the genes encoding various cAMP-PKA pathway components, such as Cac1 (adenylate cyclase) or Pka1 (catalytic subunit of PKA), are unable to undergo titanization both in vitro and in vivo ( D’Souza et al. 2001 ; Zaragoza et al. 2010 ; Hommel et al. 2018 ; Trevijano-Contador et al. 2018 ), while deletion strains that lack the Pka regulatory subunit, Pkr1 exhibit increased titanization ( Choi et al. 2012 ; Hommel et al. 2018 ). Recently, a series of landmark studies have established that in vitro titanization assays results in the reproducible generation of titan cells that faithfully recapitulate the various biological signatures of titan cells that are generated in vivo ( Dambuza et al. 2018 ; Hommel et al. 2018 ; Trevijano-Contador et al. 2018 ). Interestingly, one of these studies showed that several glycolytic genes were upregulated under in vitro titan cell inducing conditions suggesting a possible link between central carbon metabolism and titan cell formation ( Trevijano-Contador et al. 2018 ). However, the precise role of glycolysis in the context of titan cell formation remains unexplored. Interestingly, the link between glycolysis and cAMP-PKA signalling has been well-established in the model fungi Saccharomyces cerevisiae ( Busti et al. 2010 ; Peeters et al. 2017 ). The conservation of this regulatory link in other fungi, specifically the human pathogen C. neoformans , has not been completely explored. Given the fact that cAMP-PKA pathway is an important regulator of titan cell formation ( Pukkila-Worley and Alspaugh 2004 ; Okagaki et al. 2011 ; Caza and Kronstad 2019 ), we hypothesized that glycolysis flux could be one of the upstream signals critical for the activation cAMP-PKA pathway, in the context of titan cell formation. Our findings reveal a critical role for glycolysis, in regulating titan cell formation in C. neoformans , in a cAMP-PKA dependent manner. We demonstrate that perturbation of glycolysis using pharmacological inhibitors or pertinent knockout strains, significantly attenuated titan cell formation. Furthermore, we observed that perturbation of glycolysis negatively affects the intracellular levels of cAMP and subsequently, the expression of cAMP-PKA pathway regulated genes involved in capsule formation and melanin biosynthesis specifically under titan cell inducing conditions ( Pukkila-Worley et al. 2005 ; Cramer et al. 2006 ). Remarkably, the addition of exogenous cAMP reversed the defects in titan cell formation caused by glycolysis perturbation, thereby establishing a novel regulatory link between glycolysis and the cAMP–PKA pathway in C. neoformans . Additionally, for the first time, our study demonstrates a direct role of melanin biosynthesis during titan cell formation suggesting a possible interdependency between these two well-established virulence factors. Taken together, these findings elucidate the role of a conserved metabolic pathway in coordinating fungal morphogenesis via a well-conserved signaling module in the fungal pathogen C. neoformans . Results Perturbation of Glycolysis Attenuates Titan Cell Formation in C. neoformans Among a range of different morphotypes exhibited by C. neoformans ( Stempinski et al. 2023 ; Brown and Ballou 2024 ), titan cells, defined as cells with a diameter of ≥10μm, represent an important virulence lifestyle critical for host pathogenesis ( Okagaki et al. 2010 ; Crabtree et al. 2012 ). Previous studies have shown that despite their increased size, titan cells are metabolically active and exhibit significant variations in a variety of homeostatic cellular processes compared to the yeast cells of C. neoformans ( García-Barbazán et al. 2024 ). Titan cell formation is associated with altered cell cycle wherein cells are observed to have extended G2 phase and G2/M arrest ( Altamirano et al. 2021 ). Titan cells also possess a thicker cell wall and capsule with a significantly altered composition, compared to the yeast cells (cells with a diameter of 5-7μm) ( Hommel et al. 2018 ; Mukaremera et al. 2018 ). Several studies have suggested a strong interdependency between cellular metabolism and changes in cell size, ploidy or cell envelope composition ( Kalucka et al. 2015 ; Sachla and Helmann 2021 ; Cadart and Heald 2022 ; Diehl et al. 2024 ). Importantly, glycolysis plays a central role in the generation of precursors needed for the biosynthesis of various cell wall sugars (glucans, mannan, glucosamine, xylose, galactose etc.), nucleic acids, and lipids ( Kierans and Taylor 2024 ). Similarly, various studies have demonstrated a critical role for glycolysis in the regulation of cell cycle progression across different species ( Kalucka et al. 2015 ; Diehl et al. 2024 ). Despite the fact that titan cell formation in C. neoformans exhibits all the aforesaid changes including increase in cell size, alteration in cell cycle progression and variation in cell envelope architecture, the role of central carbon metabolic pathways including glycolysis has not been explored in this context. To investigate the functional requirement of glycolysis in titan cell formation, we tested the ability of C. neoformans to undergo titanization, using the previously established in vitro titan cell assay ( Trevijano-Contador et al. 2018 ). Briefly, wild-type C. neoformans strain H99 was cultured in Titan Cell Medium (TCM) in the presence and absence of sub-inhibitory concentrations of well-established glycolysis inhibitors, 2-Deoxy-D-glucose (2DG) and sodium citrate (NaCi), which target the early and rate-limiting steps of the glycolysis pathway and titan cell formation was subsequently assessed ( Trevijano-Contador et al. 2018 ) ( Fig. 1A ). 2DG, a glucose analogue, acts as a competitive inhibitor of hexokinase (Hxk) and phosphoglucose isomerase (Pgi), thereby perturbing glycolysis at its initial steps ( Cramer and Woodward 1952 ). Similarly, NaCi, interferes with the activity of phosphofructokinase (Pfk), which is a key rate-limiting enzyme in the glycolysis pathway ( Yoshino and Murakami 1982 ). Previous studies have shown that when C. neoformans grown in titan cell inducing medium, a heterogeneous population of cells emerge wherein the cell size ranges from 2μm to ≥10μm, and the cells that 2-3μm in diameter have been reported as titanides ( Feldmesser et al. 2001 ; Dambuza et al. 2018 ). Consistent with the literature, we also observed a heterogeneous population of cells including previously defined titanides (2-3μm), regular size yeast cells (5-7μm), cells between 7μm to 10μm and cells ≥10μm. In our study, we focused on the cells that are ≥10μm, designated as titan cells and the percentage of titan cells were quantified as the ratio of titan cells (≥10μm) to the total number of cells. Our data clearly demonstrates that sub-inhibitory concentrations of 2DG and NaCi significantly attenuate the ability of C. neoformans to undergo titan cell formation ( Fig. 1B and C ; Fig. S1A ). To rule out the possibility that the phenotype we observed is due to a general growth defect, we assessed the viability of C. neoformans , under titan cell inducing conditions in the presence of glycolysis inhibitors using CFU (Colony Forming Units) quantification. Our results clearly demonstrate that the reduction in titan cell formation is not due to a general growth defect, as presence of sub-inhibitory concentrations of 2DG and NaCi did not affect the viability of the wild-type strain ( Fig. 1D ). Titan cells are known to have a thicker and more complex capsule structure compared to the capsule of yeast cells. Additionally, in the field, titan cells have been defined by their cell size either including or excluding the capsule ( García-Rodas et al. 2019 ). Increased capsule expansion is thought to be an important morphological hallmark of titan cells, and given this, we wanted to assess whether the perturbation of glycolysis affects titan cell associated capsule thickness, and consequently, overall cell size including the capsule. In order to do this, wild-type cells were grown in TCM in the presence and absence of sub-inhibitory concentrations of glycolysis inhibitors and capsule was visualized using India ink staining and capsule thickness was quantified. Our data shows that the addition of 2DG or NaCi resulted in a significant reduction in overall capsule thickness and total cell diameter ( Fig. 1E and F ; Fig. S1B ). This demonstrates that the perturbation of glycolysis disrupts titan cell associated capsule expansion. Download figure Open in new tab Fig. S1. Wider Field Images of TCM Grown C. neoformans Treated with Glycolysis Inhibitors. (A) Representative wider field images of TCM grown wild-type cultures, in presence and absence of sub-inhibitory concentration of glycolysis inhibitors (2DG and NaCi). Scale bar represents 10μm. (B) Representative wider field images of India ink staining for capsule visualization of TCM grown wild-type cultures, in presence and absence of sub-inhibitory concentration of glycolysis inhibitors (2DG and NaCi). Scale bar represents 10μm. This figure was created using Biorender. Download figure Open in new tab Fig. 1. Effect of Glycolysis Inhibitors on Titan Cell Formation in C. neoformans . (A) Schematic overview of glycolysis and its inhibition by glycolysis inhibitors (2-Deoxy-D-Glucose (2DG) and sodium citrate (NaCi)). (B) Wild-type strain was cultured in Titan Cell Medium (TCM) with and without inhibitor (2DG (0.025%)) at 37 °C with 5% CO2 for 72 hours. Cells were fixed and imaged using Zeiss Apotome microscope. Percentage of titan cells was quantified as a ratio of cells with diameter ≥10μm to total number of cells. Cell diameter was measured using Zen 2.3 software. Threshold for cell body diameter of titan cells is indicated by the red line. More than 400 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001) and **(p<0.01). Error bars represent SEM Scale bars represents 2μm. (C) Wild-type strain was cultured in TCM with and without inhibitor (NaCi (0.5%)) at 37 °C with 5% CO2 for 72 hours. Cells were fixed and imaged using Zeiss Apotome microscope. Percentage of titan cells was quantified as a ratio of cells with diameter ≥10μm to total number of cells. Cell diameter was measured using Zen 2.3 software. Threshold for cell body diameter of titan cells is indicated by the red line. More than 400 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001) and **(p<0.01). Error bars represent SEM Scale bars represents 2μm. (D) Colony Forming Units (CFU) analysis was performed to monitor overall viability of wild-type strain in TCM, +2DG and +NaCi conditions. Wild-type cultures were grown under TCM conditions with and without glycolysis inhibitors for 72 hours and plated on YPD plates and incubated at 30 °C for 48 hours. CFU quantification was done post incubation. Statistical analysis was done using unpaired t-test, ns (non-significant). Error bars represent SEM. (E) For capsule visualization, wild-type strain was cultured in TCM with and without 2DG at 37 °C with 5% CO2 for 72 hours, cells were stained with India ink and imaging was done using Zeiss Apotome microscope. Capsule thickness was measured using Zen 2.3 software. Approximately 150 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001). Error bars represent SEM Scale bars represents 2μm. (F) For capsule visualization, wild-type strain was cultured in TCM with and without NaCi at 37 °C with 5% CO2 for 72 hours, cells were stained with India ink and imaging was done using Zeiss Apotome microscope. Capsule thickness was measured using Zen 2.3 software. Approximately 150 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001). Error bars represent SEM. Scale bars represents 2μm. This figure was created using Biorender. Given that, 2DG and NaCi exhibited a strong inhibitory effect on titan cell formation, we wanted to corroborate these observations by using pertinent genetic knockout strains that are compromised in their ability to metabolize glucose via glycolysis ( Price et al. 2011 ). In order to do this, we tested the ability of knockout strains which lack the genes that encode for enzymes that are involved in the glycolysis pathway including, Hxk2 (Hexokinase 2) and Pyk1 (Pyruvate kinase 1) ( Price et al. 2011 ) ( Fig. 2A ). In C. neoformans, hxk2 encodes for the enzyme that catalyses the phosphorylation of glucose to glucose-6-phosphate ( Idnurm et al. 2007 ), while pyk1 encodes for the enzyme responsible for converting phosphoenolpyruvate to pyruvate ( Price et al. 2011 ). Briefly, hxk2 Δ and pyk1 Δ ( hxk2 Δ refers to the hxk2 knockout strain and pyk1 Δ refers to the pyk1 knockout strain) along with the wild-type were cultured in TCM and incubated at 37 °C under 5% CO2 for 72 hours. Cells were imaged using bright field microscopy, and percentage of titan cells was quantified as the ratio of titan cells (≥10μm) to the total number of cells. Our result demonstrates that the deletion of hxk2 and pyk1 resulted in a significant reduction in titan cell formation, compared to the wild-type strain ( Fig. 2B and C ; Fig. S2A ). To rule out the possibility that the phenotype we observed is due to a general growth defect, we performed CFU assay using the wild-type and knockout strains ( hxk2 Δ and pyk1 Δ ) grown in titan cell inducing conditions. Our data suggests that the reduction in titan cell formation is not due to a general growth defect as there was no significant difference in the viability of the wild-type and mutants grown under titan cell inducing conditions. ( Fig. 2D ). Given that the perturbation of glycolysis using 2DG or NaCi leads to reduced titan cell associated capsule thickness in the wild-type, we wanted to corroborate these observations by using pertinent genetic knockout strains ( hxk2 Δ and pyk1 Δ ) that are compromised in their ability to undergo glycolysis. In order to do this, glycolysis knockout strains including, hxk2 Δ and pyk1 Δ along with the wild-type were cultured in TCM and capsule was visualized using India ink staining and capsule thickness was measured. Bright field microscopy revealed that glycolysis knockout strains exhibited a significant decrease in capsule thickness compared to the wild-type strain ( Fig. 2E and F ; Fig. S2B ). Download figure Open in new tab Fig. S2. Wider Field Images of TCM Grown Wild-type and Glycolysis Mutants of C. neoformans . (A) Representative wider field images of wild-type, and glycolysis mutants including hxk2 Δ, pyk1 Δ grown under titan cell inducing conditions. Scale bar represents 10μm. (B) Representative wider field images of India ink staining for capsule visualization of wild-type, and glycolysis mutants including hxk2 Δ, pyk1 Δ grown under titan cell inducing conditions. Scale bar represents 10μm. (C) Representative wider field images of wild-type, hxk2 Δ, pyk1 Δ, hxk2Δ + HXK2 and pyk1Δ + PYK1 strains grown under titan cell inducing conditions. Scale bar represents 10μm. (D) Representative wider field images of India ink staining for capsule visualization of wild-type, hxk2 Δ, pyk1 Δ, hxk2Δ + HXK2 and pyk1Δ + PYK1 strains grown under titan cell inducing conditions. Scale bar represents 10μm. This figure was created using Biorender. Download figure Open in new tab Fig. 2. Genetic Perturbation of Glycolysis Attenuates Titan Cell Formation in C. neoformans . Overview of glycolysis pathway showing targeted gene deletions. (B) Wild-type and hxk2Δ strains were cultured in Titan Cell Medium (TCM) at 37 °C with 5% CO2 for 72 hours. Cells were fixed and imaged using Zeiss Apotome microscope. Percentage of titan cells was quantified as a ratio of cells with diameter ≥10μm to total number of cells. Cell diameter was measured using Zen 2.3 software. Threshold for cell body diameter of titan cells is indicated by the red line. More than 400 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001). Error bars represent SEM. Scale bars represents 2μm. (C) Wild-type and pyk1Δ strains were cultured in TCM at 37 °C with 5% CO2 for 72 hours. Cells were fixed and imaged using Zeiss Apotome microscope. Percentage of titan cells was quantified as a ratio of cells with diameter ≥10μm to total number of cells. Cell diameter was measured using Zen 2.3 software. Threshold for cell body diameter of titan cells is indicated by the red line. More than 400 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001) **(p<0.01). Error bars represent SEM. Scale bars represents 2μm. (D) CFU analysis was performed to monitor overall viability of wild-type and knockout strains including hxk2Δ , and pyk1Δ in TCM. Wild-type, hxk2Δ , and pyk1Δ strains were grown under TCM conditions for 72 hours and plated on YPD plates and incubated at 30 °C for 48 hours. CFU quantification was done post incubation. Statistical analysis was done using unpaired t-test, ns (non-significant). Error bars represent SEM. (E) For capsule visualization, wild-type, and hxk2Δ strains were cultured in TCM at 37 °C with 5% CO2 for 72 hours, cells were stained with India ink and imaging was done using Zeiss Apotome microscope. Capsule thickness was measured using Zen 2.3 software. Approximately 150 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001). Error bars represent SEM. Scale bars represents 2μm. (F) For capsule visualization, wild-type and pyk1Δ strains were cultured in TCM at 37 °C with 5% CO2 for 72 hours, cells were stained with India ink and imaging was done using Zeiss Apotome microscope. Capsule thickness was measured using Zen 2.3 software. Approximately 150 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001). Error bars represent SEM. Scale bars represents 2μm. (G) Wild-type, hxk2Δ, pyk1Δ, hxk2Δ + HXK2 and pyk1Δ + PYK1 strains were cultured in TCM at 37 °C with 5% CO2 for 72 hours. Cells were fixed and imaged using Zeiss Apotome microscope. Percentage of titan cells was quantified as a ratio of cells with diameter ≥10μm to total number of cells. Cell diameter was measured using Zen 2.3 software. Threshold for cell body diameter of titan cells is indicated by the red line. More than 400 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001) **(p<0.01). Error bars represent SEM Scale bars represents 2μm. (H) For capsule visualization, wild-type, hxk2Δ, pyk1Δ, hxk2Δ + HXK2 and pyk1Δ + PYK1 strains were cultured in TCM at 37 °C with 5% CO2 for 72 hours, cells were stained with India Ink and imaging was done using Zeiss Apotome microscope. Capsule thickness was measured using Zen 2.3 software. Approximately 150 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001). Error bars represent SEM. Scale bars represents 2μm. This figure was created using Biorender. To confirm that the defects observed in titan cell formation, in the glycolysis mutants ( hxk2 Δ and pyk1 Δ), were not due to any off-target integrations, chromosomal deletions/duplications, and tandem integrations caused by the transformation procedure, we validated our results using complemented strains for these mutants. Our results demonstrated that the complemented strains, hxk2 Δ + HXK2 and pyk1 Δ + PYK1 exhibited complete rescue of the titanization defects exhibited by the mutants, hxk2 Δ and pyk1 Δ ( Fig. 2G ; Fig. S2C ). These complementation strains were also tested for titan cell associated capsule formation and total cell size, using India ink staining. As shown in the Fig. 2H , complemented strains hxk2 Δ + HXK2 and pyk1 Δ + PYK1 exhibited complete rescue of the defects observed in titan cell associated capsule formation in the glycolysis mutants hxk2 Δ and pyk1 Δ ( Fig. S2D ). Taken together, our results clearly demonstrate that the ability of C. neoformans to efficiently metabolize glucose via glycolysis is critical for titan cell formation. Active Glycolysis is Critical for cAMP-PKA Signalling in Titan Cell Inducing Conditions Our data clearly demonstrates that the perturbation of glycolysis using inhibitors or pertinent knockout strains attenuates titan cell formation in C. neoformans . Several studies have established that fungi use different pathways to sense nutrient availability and modulate their metabolic state in order to regulate growth and proliferation ( Bahn et al. 2007 ; Yuan et al. 2013 ). For example, glucose availability and the glycolytic flux is sensed by the conserved signalling pathway, cyclic-AMP-Protein Kinase A (cAMP-PKA) pathway in S. cerevisiae and it has been shown that active glycolysis is critical for the full activation of this pathway ( Busti et al. 2010 ; Peeters et al. 2017 ). In C. neoformans , cAMP-PKA pathway is known to play an important role in host pathogenesis by regulating three critical virulence factors including capsule formation, melanin biosynthesis and titan cell formation ( Hicks et al. 2004 ; Caza and Kronstad 2019 ; Mahmood et al. 2024 ). Given the fundamental and highly conserved nature of both glycolysis and the cAMP-PKA pathway across various fungal species ( Fuller and Rhodes 2012 ; Caza and Kronstad 2019 ; Yu and Rollins 2022 ), we hypothesized that the glycolysis-dependent regulation of titan cell formation is mediated through the cAMP-PKA pathway. To test this hypothesis, we sought to evaluate the activity of cAMP-PKA pathway at two distinct levels, during glycolysis perturbation in titan cell inducing conditions. First, we quantified the intracellular levels of cAMP, in C. neoformans wild-type and glycolysis mutants. Second, we analysed the gene expression of known downstream effectors of cAMP-PKA pathway in C. neoformans ( Pukkila-Worley et al. 2005 ; Cramer et al. 2006 ), in glycolysis mutants, compared to the wild-type, under titan cell inducing conditions. In order to quantify the intracellular levels of cAMP in the wild-type and glycolysis mutants, these strains were grown in TCM for 24 hours, and intracellular cAMP levels were measured using the cAMP-Glo assay kit (#V1501 - Promega). Our results demonstrate that intracellular cAMP levels were consistently higher in the wild-type strain as compared to the glycolysis mutants, including hxk2 Δ and pyk1 Δ under the tested conditions. This suggests that perturbation of glycolysis negatively influences the intracellular levels of cAMP, leading to the reduced activation of cAMP-PKA cascade ( Fig. 3C ). In order to confirm that the perturbation of glycolysis leads to downregulation of the cAMP-PKA pathway, in titan cell inducing conditions, we measured the expression of a known downstream target, cap10 . In C. neoformans , Cap10 has been shown to be involved in capsule biosynthesis ( Fig. 3A ) ( Pukkila-Worley et al. 2005 ). To determine the relative expression of cap10 glycolysis knockout strains including hxk2 Δ and pyk1 Δ along with the wild-type strain were cultured in titan cell inducing conditions for 72 hours, following which total RNA was extracted and gene expression levels were quantified using RT-qPCR. Our data shows that the expression of cap10 was significantly downregulated in hxk2 Δ and pyk1 Δ as compared to wild-type strain ( Fig 3B ). Similarly, we monitored the expression of other cAMP-PKA downstream targets, lac1 and lac2 ( Pukkila-Worley et al. 2005 ; Yu et al. 2025 ) in glycolysis knockout strains including, hxk2 Δ and pyk1 Δ along with the wild-type strain in titan cell inducing conditions. In C. neoformans , both lac1 and lac2 encodes for the laccase enzyme which catalyses an important step in melanin biosynthesis ( Pukkila-Worley et al. 2005 ) ( Fig. 3A ). Briefly, glycolysis knockout strains including hxk2 Δ and pyk1 Δ, along with the wild-type strain were cultured in titan cell inducing conditions for 72 hours, following which total RNA was extracted and gene expression levels were quantified using RT-qPCR. Our results indicate that both lac1 and lac2 were significantly downregulated in hxk2 Δ and pyk1 Δ compared to wild-type strain ( Fig. 3B ). Download figure Open in new tab Fig. 3. Active Glycolysis is Critical for cAMP-PKA Signalling in Titan Cell Inducing Conditions. (A) Overview of cAMP-PKA pathway regulating the expression of key genes involved in capsule formation and melanin biosynthesis. (B) Wild-type, hxk2 Δ and pyk1 Δ strains were cultured in Titan Cell Medium (TCM) at 37 °C with 5% CO2 for 72 hours. Total RNA was extracted after 72 hours and comparative RT-qPCR was performed. Statistical analysis was done using Two-Way ANOVA, ***(p<0.001), **(p<0.01), *(p<0.05). Error bars represent SEM. (C) Schematic for intracellular cAMP measurement assay. Wild-type strain, hxk2 Δ, and pyk1 Δ strains were cultured in TCM at 37 °C in shaking conditions for 24 hours. cAMP measurement assay was performed using cAMP-Glo assay kit. Statistical analysis was done using unpaired t-test, **(p<0.01). Error bars represent SEM, (D) Schematic for intracellular cAMP measurement assay and RT-qPCR analysis for YPD grown cultures. Wild-type strain, hxk2 Δ, and pyk1 Δ strains were cultured in YPD medium at 37 °C in shaking conditions and cells were collected at mid-log phase. cAMP measurement assay was performed using cAMP-Glo assay kit. Statistical analysis was done using unpaired t-test, ns (non-significant). Error bars represent SEM, (E) Wild-type strain, hxk2 Δ, and pyk1 Δ strains were cultured in YPD medium at 37 °C in shaking conditions and cells were collected at mid-log phase. Total RNA was extracted after 72 hours and comparative RT-qPCR was performed. Statistical analysis was done using Two-Way ANOVA, ns (non-significant). Error bars represent SEM. This figure was created using Biorender. These findings indicate a clear regulatory link between glycolysis and the cAMP-PKA pathway during titan cell formation in C. neoformans . While it is established that cAMP levels in C. neoformans is linked to glucose availability, the specific extracellular glucose receptors and underlying mechanistic details remain undiscovered ( Hommel et al. 2018 ). Our data demonstrate a positive correlation between glycolysis and the cAMP-PKA pathway specifically under titan cell inducing conditions. To determine if this connection is conserved under general nutrient-rich growth environments, we quantified cAMP levels and the expression of key cAMP-PKA target genes in both wild-type and glycolysis-deficient mutants grown in standard YPD medium at 37 °C ( Fig. 3D ). Interestingly, we observed no significant differences in cAMP levels or target gene expression between the mutants and the wild-type strain under these standard laboratory growth conditions ( Fig. 3D and E ). This suggests that the regulatory influence of glycolysis on the cAMP-PKA pathway exists specifically under titan cell inducing conditions. Overall, these results indicate that the perturbation of glycolysis results in reduced intracellular levels of cAMP which in turn negatively influences the expression of genes which are regulated by the cAMP-PKA pathway under titan cell inducing condition. This suggests that active glycolytic flux might be important for the robust activation of the cAMP-PKA pathway and its downstream regulon, only during titan cell formation and not during general growth in nutrient-rich medium. Glycolysis Orchestrates Titan Cell Formation in a cAMP-PKA Dependent Manner Our RT-qPCR results clearly demonstrated that the perturbation of glycolysis negatively affects several downstream targets of cAMP-PKA pathway under titan cell inducing conditions. Moreover, we observed significant reduction in intracellular cAMP levels in glycolysis mutants under titan cell inducing treatment. Given the central role of cAMP-PKA pathway in titan cell formation, the observed defects in titanization under glycolysis perturbation might be attributable to glycolysis-mediated reduction in cAMP levels and henceforth downregulation of cAMP-PKA pathway. To test this hypothesis, we performed cAMP-add back assays wherein glycolysis knockout strains including hxk2 Δ and pyk1 Δ along with wild-type strain were cultured in TCM with and without the exogenous supplementation of cAMP (10mM) and incubated under titan cell inducing conditions. Cells were imaged using bright field microscopy, and percentage of titan cells was quantified as the ratio of titan cells (≥10μm) to the total number of cells. Remarkably, exogenous supplementation of cAMP significantly rescued the titan cell formation defect observed in hxk2 Δ and pyk1 Δ strains ( Fig. 4A and C ; Fig. S3A ). The percentage of titan cells were significantly higher in the cAMP treated hxk2 Δ and pyk1 Δ strains compared to the untreated controls, reaching levels comparable to the wild-type strain ( Fig. 4A and C ; Fig. S3A ). Download figure Open in new tab Fig. S3. Wider Field Images of cAMP Add-back Assay for Glycolysis Mutants of C. neoformans in TCM. (A) Representative wider field images of wild-type, and glycolysis mutants including hxk2 Δ and pyk1 Δ grown under titan cell inducing conditions with and without exogenous supplementation of cAMP. Scale bar represents 10μm. (B) Representative wider field images of India ink staining for capsule visualization of wild-type, and glycolysis mutants including hxk2 Δ and pyk1 Δ grown under titan cell inducing conditions with and without exogenous supplementation of cAMP. Scale bar represents 10μm. This figure was created using Biorender. Download figure Open in new tab Fig. 4. Glycolysis Orchestrates Titan Cell Formation in a cAMP-PKA Dependent Manner. (A) Wild-type and hxk2 Δ strains were cultured in Titan Cell Medium (TCM) in the presence and absence of cAMP (10mM) at 37 °C with 5% CO2 for 72 hours. Cells were fixed and imaged using Zeiss Apotome microscope. Percentage of titan cells was quantified as a ratio of cells with diameter ≥10μm to total number of cells. Cell diameter was measured using Zen 2.3 software. Threshold for cell body diameter of titan cells is indicated by the red line. More than 400 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001), and ns (non-significant). Error bars represent SEM. Scale bar represents 2μm. (B) For capsule visualization, wild-type and hxk2 Δ strains were cultured in TCM in the presence and absence of cAMP (10mM) at 37 °C with 5% CO2 for 72 hours, cells were stained with India ink and imaging was done using Zeiss Apotome microscope. Capsule thickness was measured using Zen 2.3 software. Approximately 150 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001). Error bars represent SEM Scale bar represents 2μm. (C) Wild-type and pyk1 Δ strains were cultured in TCM in the presence and absence of cAMP (10mM) at 37 °C with 5% CO2 for 72 hours. Cells were fixed and imaged using Zeiss Apotome microscope. Percentage of titan cells was quantified as a ratio of cells with diameter ≥10μm to total number of cells. Cell diameter was measured using Zen 2.3 software. Threshold for cell body diameter of titan cells is indicated by the red line. More than 400 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001), **(p<0.01), *(p<0.05) and ns (non-significant). Error bars represent SEM. Scale bar represents 2μm. (D) For capsule visualization, wild-type and pyk1 Δ strains were cultured in TCM in the presence and absence of cAMP (10mM) at 37 °C with 5% CO2 for 72 hours, cells were stained with India Ink and imaging was done using Zeiss Apotome microscope. Capsule thickness was measured using Zen 2.3 software. Approximately 150 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001). Error bars represent SEM. Scale bar represents 2μm. This figure was created using Biorender. cAMP-PKA pathway is a known positive regulator of capsule formation in C. neoformans ( Leadsham and Gourlay 2010 ). Given that exogenous supplementation of cAMP significantly rescued titan cell formation defects observed in the glycolysis knockout strains, we wanted to assess whether cAMP add-back rescues titan cell associated capsule thickness, and consequently, overall cell size including the capsule. In order to do this, glycolysis knockout strains including hxk2 Δ and pyk1 Δ along with the wild-type strain were cultured in TCM with and without exogenous supplementation of cAMP (10mM) and incubated under titan cell inducing conditions. Capsule was visualized using India ink staining and capsule thickness was measured. Our data demonstrates that the exogenous supplementation of cAMP leads to significant increase in capsule thickness of hxk2 Δ and pyk1 Δ strains compared to the untreated cultures of hxk2 Δ and pyk1 Δ strains, reaching levels comparable to the wild-type strain ( Fig. 4B and D ; Fig. S3B ). Collectively, our results indicate that glycolysis regulates titan cell formation through the activation of the cAMP-PKA pathway. Melanin Biosynthesis is Critical for Titan Cell Formation in C. neoformans Our RT-qPCR results demonstrated that under titan cell inducing conditions, functional glycolysis is essential for the expression of genes involved in melanin biosynthesis including lac1 and lac2 , in a cAMP-PKA pathway dependent manner. Although in C. neoformans , melanin is a known virulence factor that contributes towards host pathogenesis, its direct involvement in titan cell formation remains unexplored ( Zaragoza 2019 ). In order to determine whether the downregulation of genes involved in melanin biosynthesis, directly contributes towards the attenuation of titan cell formation under glycolysis perturbation, we tested the ability of a knockout strain ( lac2 Δ) which is impaired in melanin biosynthesis ( Pukkila-Worley et al. 2005 ), to undergo titan cell formation. To confirm that the phenotype observed in the melanin deficient strain ( lac2 Δ), were not due to any off-target integrations, chromosomal deletions/duplications, and tandem integrations caused by the transformation procedure, we included a complementation strain ( lac2 Δ + LAC2) in our assays. Briefly, lac2 Δ and lac2 Δ + LAC2 strains along with the wild-type strain were cultured in TCM and incubated at 37 °C under 5% CO2, for 72 hours. Cells were imaged using bright field microscopy, and percentage of titan cells was quantified as the ratio of titan cells (≥10μm) to the total number of cells. Our results demonstrate that the deletion of lac2 Δ resulted in a significant reduction in titan cell formation, compared to the wild-type strain ( Fig. 5A ; Fig. S4A ), while the complemented strain exhibited titanization comparable to the wild type strain. In C. neoformans , laccase activity is encoded by two paralogs, lac1 and lac2 ( Pukkila-Worley et al. 2005 ). Since lac1 remains functional in lac2Δ mutants, we further evaluated titan cell formation in a double-deletion strain ( lac1 Δ lac2 Δ) which is completely deficient in laccase production. The double deletion strain, lac1 Δ lac2 Δ resulted in significant reduction in titan cell formation ( Fig. 5A ; Fig. S4A ) similar to the lac2 Δ strain. As a confirmation, the aforementioned strains were tested for their ability to synthesize melanin, in melanin inducing medium. As shown in the Fig. 5B , deletion strains lac2 Δ and lac1 Δ lac2 Δ were defective in melanin biosynthesis after incubation for 36 hours in melanin inducing medium. Download figure Open in new tab Fig. S4. Wider Field Images of TCM Grown Wild-Type and Melanin Deficient Mutants of C. neoformans in TCM. (A) Representative wider field images of wild-type, lac2 Δ, lac2 Δ + LAC2 and lac1 Δ lac2 Δ strains grown under titan cell inducing conditions. Scale bar represents 10μm. (B) Representative wider field images of India ink staining for capsule visualization of wild-type, lac2 Δ, lac2 Δ + LAC2 and lac1 Δ lac2 Δ strains grown under titan cell inducing conditions. Scale bar represents 10μm. This figure was created using Biorender. Download figure Open in new tab Fig. 5. Melanin Biosynthesis is Critical for Titan Cell Formation in C. neoformans . (A) Wild-type, lac2 Δ, lac2 Δ + LAC2 and lac1 Δ lac2 Δ strains were cultured in Titan Cell Medium (TCM) at 37 °C with 5% CO2 for 72 hours. Cells were fixed and imaged using Zeiss Apotome microscope. Percentage of Titan cells was quantified as a ratio of cells with diameter ≥10μm to total number of cells. Cell diameter was measured using Zen 2.3 software. Threshold for cell body diameter of titan cells is indicated by red line. More than 200 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001), **(p<0.01) and ns (non-significant). Error bars represent SEM. Scale bar represents 2μm. (B) Wild-type, lac2 Δ, lac2 Δ + LAC2 and lac1 Δ lac2 Δ strains were spotted on melanin inducing medium and incubated at 37 °C for 36 hours. After incubation the colonies were imaged using SZX16 microscope. (C) For capsule visualization, wild-type, lac2 Δ and lac2 Δ + LAC2 strains were cultured in TCM at 37 °C with 5% CO2 for 72 hours, cells were stained with India ink and imaging was done using Zeiss Apotome microscope. Capsule thickness was measured using Zen 2.3 software. Approximately 150 cells were counted for each condition. Statistical analysis was done using unpaired t-test, ***(p<0.001). Scale bar represents 2μm. (D) For capsule visualization, wild-type and lac1 Δ lac2 Δ strains were cultured in TCM at 37 °C with 5% CO2 for 72 hours, cells were stained with India ink and imaging was done using Zeiss Apotome microscope. Capsule thickness was measured using Zen 2.3 software. Approximately 150 cells were counted for each condition. Statistical analysis was done using unpaired t-test, Error bars represent SEM, ***(p<0.001). Scale bar represents 2μm. This figure was created using Biorender. Additionally, we measured titan cell associated capsule formation for lac2 Δ, its complemented strain, lac2 Δ + LAC2 and double knockout strain lac1 Δ lac2 Δ, in titan cell inducing conditions, similar to our previous experiments. Strains, lac2 Δ, lac2 Δ + LAC2, lac1 Δ lac2 Δ along with the wild-type strain were cultured in TCM and incubated at 37 °C under 5% CO2 for 72 hours. Capsule was visualized using India ink staining and capsule thickness was measured. Bright field microscopy revealed that lac2 Δ exhibited a significant decrease in total cell diameter which includes both the cell body and the surrounding capsule relative to the wild-type strain ( Fig. 5C and D ; Fig. S4B ). These findings, for the first time, establish a direct role for melanin biosynthesis during titan cell formation in C. neoformans , highlighting the regulatory interdependencies of these virulence factors. Discussion Our findings provide compelling evidence for a conserved regulatory axis that links central carbon metabolism, specifically glycolysis and cAMP-PKA pathway in the regulation of titan cell formation in C. neoformans . Even though several studies have demonstrated that cAMP-PKA pathway is critical for titan cell formation ( Zaragoza et al. 2010 ; Okagaki et al. 2011 ; Zhou and Ballou 2018 ; Caza and Kronstad 2019 ), the upstream signals that are required for the activation of this pathway during titan cell formation remains poorly understood. Titan cells exhibit dramatically altered cellular and morphological changes compared to the yeast morphotype and these include increase in cell size, polyploidy resulting from endoreduplication and thicker cellular envelope (cell wall and polysaccharide capsule) with altered composition ( Zhou and Ballou 2018 ; García-Rodas et al. 2019 ). Importantly, glycolysis plays a critical role in generating various precursors required for cell wall and capsule biosynthesis, and the role of glycolysis in regulating cell size and cell cycle progression in various organisms including multiple fungal species is well established ( Cadart and Heald 2022 ; Diehl et al. 2024 ; Kierans and Taylor 2024 ). Furthermore, glycolysis has been recently implicated in the regulation of fungal morphogenesis, particularly in S. cerevisiae and Candida albicans ( Shah et al. 2026 ). A recent study that reported the successful generation of in vitro titan cells which are remarkably similar to the titan cells that are formed in vivo , during C. neoformans infections, demonstrated that in vitro titan cells have increased expression of several glycolytic genes ( Trevijano-Contador et al. 2018 ). Given these observations, we hypothesized that glycolysis might play an important role in the regulation of titan cell formation in C. neoformans . Our results clearly demonstrated that the inhibition of glycolysis with sub-inhibitory concentrations of 2DG or NaCi significantly reduced titan cell formation, providing strong evidence for the metabolic dependency of this morphogenetic process. These findings were further supported by the results from titan cell assays using pertinent glycolysis deletion strains, wherein hxk2 Δ ( hxk2 Δ refers to the hxk2 deletion strain) and pyk1 Δ ( pyk1 Δ refers to the pyk1 deletion strain) showed a substantial reduction in titan cell formation compared to the wild-type. The cAMP-PKA pathway is a well-established sensor of nutrient availability, particularly glucose, across various fungal species ( Pukkila-Worley and Alspaugh 2004 ; Busti et al. 2010 ). In Saccharomyces cerevisiae , this pathway integrates extracellular glucose levels and intracellular metabolic flux through distinct mechanisms. The membrane-bound G-protein coupled receptor (GPCR), Gpr1, responds to ambient glucose concentrations, to modulate cAMP synthesis. Concurrently, the glycolysis intermediate fructose-1,6-bisphosphate regulates cAMP synthesis in a Ras1-dependent manner. ( Xue et al. 1998 ; Pukkila-Worley and Alspaugh 2004 ; Busti et al. 2010 ; Peeters et al. 2017 ). In C. neoformans , intracellular cAMP levels increases in response to glucose availability, though the underlying mechanisms remain unknown ( Xue et al. 2006 ). While the G-protein coupled receptor Gpr4 shares significant homology with the S. cerevisiae glucose sensor Gpr1, it primarily responds to methionine levels to regulate the cAMP-PKA pathway ( Xue et al. 2006 ). Consequently, the specific extracellular receptors and the precise molecular mechanisms governing glucose mediated regulation of this pathway in C. neoformans are yet to be identified ( Pukkila-Worley and Alspaugh 2004 ). Given the nutrient responsive nature of cAMP-PKA pathway and the fact that this cascade is central for the regulation of titan cell formation, we hypothesised that the effect of glycolysis perturbation on titan cell formation could be mediated through the activation of cAMP-PKA pathway. Supporting our hypothesis, we observed significant reduction in the intracellular cAMP levels, in the glycolysis mutants, compared to the wild-type, in titan cell inducing conditions. The significantly reduced intracellular levels of cAMP, explains the reduced activity of the cAMP-PKA pathway in the glycolysis mutants ( hxk2 Δ and pyk1 Δ). This was demonstrated by the transcriptional downregulation of cAMP-PKA pathway target genes ( cap10, lac1 , and lac2 ) in pertinent glycolysis knockout strains including hxk2 Δ and pyk1 Δ compared to the wild-type strain, confirming our aforesaid hypothesis that perturbation of glycolysis leads to reduced intracellular levels of cAMP which in turn results in downregulation of the cAMP-PKA pathway during titan cell formation. Remarkably, exogenous supplementation of cAMP rescues titan cell formation defects exhibited by glycolysis knockout strains ( hxk2 Δ and pyk1 Δ ) , strongly suggesting that glycolysis functions upstream of cAMP-PKA activation, thereby establishing a metabolic checkpoint for this morphogenetic transition ( Fig. 6 ). Notably, this regulatory coupling appears to be context-dependent, as we observed no significant change in cAMP levels or the expression of downstream target genes regulated by the cAMP-PKA pathway, when mutants were cultured under standard nutrient-rich (YPD) conditions. These findings suggest that the metabolic crosstalk between glycolysis and the cAMP-PKA pathway in C. neoformans is uniquely required during titan cell formation. The cAMP-PKA pathway is a widely recognized central regulator of cell growth. It plays a key role in influencing essential cellular processes, including ribosome biogenesis, mitochondrial biogenesis, and the regulation of cell wall biosynthesis ( Hu et al. 2007 ; Yoboue et al. 2012 ; Guerra et al. 2022 ). Given that, titan cells exhibit characteristic features of increased cell size and altered cell wall compositions, our finding that glycolysis-mediated activation of cAMP-PKA pathway is critical for titan cell formation, is highly significant and further studies are required to delineate the underlying mechanism. Download figure Open in new tab Fig. 6. A Conserved Metabolic Network Regulates Titan Cell Formation in C. neoformans . Active glycolysis plays a critical role in the regulation of the cAMP-PKA signalling pathway which in turn affects the regulation of virulence factors of C. neoformans including capsule formation, melanin biosynthesis and titan cell formation. Perturbation of glycolysis either through glycolysis inhibitors (2DG and NaCi) or pertinent knockout strains ( hxk2 Δ and pyk1 Δ), leads to a downregulation of cAMP-PKA pathway. The suppression of cAMP-PKA pathway impairs capsule formation and melanin biosynthesis, ultimately resulting in attenuated titan cell formation. This figure was created using Biorender. The cAMP-PKA signaling pathway is a well-established central regulator of three primary virulence factors in C. neoformans , including capsule, melanin, and titan cell formation ( Zaragoza 2019 ). While previous research has established that capsule deficient mutants are defective in titanization ( Hommel et al. 2018 ), the direct involvement of melanin biosynthesis in titan cell formation has remained unexplored. To address this knowledge gap and determine if impaired melanization contributes to titan cell formation, we evaluated the titanization ability of melanin deficient strains ( lac2 Δ and lac1 Δ lac2 Δ) ( Pukkila-Worley et al. 2005 ). Interestingly, our results demonstrate that the deletion of lac genes results in a significant reduction in titan cell formation. These findings, for the first time, establish a direct role for melanin biosynthesis in titan cell formation in C. neoformans . Melanin could contribute towards titan cell formation via several integrated physiological mechanisms. Melanin accumulation is positively correlated with cell wall rigidity and structural integrity ( Langfelder et al. 2003 ; Moses et al. 2006 ). Thus, melanin accumulation might provide the mechanical support necessary for the overall cell size expansion, characteristic of titan cells. Furthermore, literature suggests a correlation between melanization and increased cell diameter, indicating a possible role for melanin in supporting cell size growth in C. neoformans . ( Baker et al. 2024 ). Previous studies have shown that melanin acts as an antioxidant when C. neoformans encounters the host environment, effectively scavenging excessive ROS to maintain cellular redox homeostasis ( Zaragoza 2019 ).. While, endogenous reactive oxygen species (ROS) are known to induce endoreduplication and polyploidy that are required for titanization ( Altamirano et al. 2021 ; García-Barbazán et al. 2024 ), excessive ROS accumulation that likely occurs in melanin deficient strains, can be cytotoxic or trigger pleiotropic inhibitory effects. Hence, maintenance of optimum ROS levels would be an important factor for titan cell formation. By scavenging excessive ROS, melanin may act as a redox buffer and maintain the ROS levels within the threshold that is optimal for titan cell formation. A recent study shows that deletion of gsh2 , a gene encoding for glutathione synthetase, a key enzyme involved in redox homeostasis of C. neoformans , attenuates titan cell formation further strengthening the aforesaid hypothesis ( Black et al. 2024 ). While the cAMP-PKA pathway is recognized as a master regulator of capsule expansion, melanin biosynthesis, and titan cell formation in C. neoformans , the functional interdependency among these three virulence factors remains poorly defined. Recent studies have indicated a competitive relationship between melanin and the capsule, in capsule-inducing media, wherein melanin deposition within the cell wall negatively impacts capsule expansion by sequestering shared cellular resources ( Baker et al. 2024 ). In contrast to this observation, our findings indicate a synergistic relationship between capsule and melanin during titanization. Specifically, melanin biosynthesis, along with capsule formation ( Hommel et al. 2018 ) appears to promote titan cell formation, in standard in vitro titanization conditions. This variation suggests that the regulation of these virulence factors differ depending on the environmental stimuli. Under titan cell-inducing conditions, the cells may metabolically rewire and reallocate its resources to simultaneously promote the biosynthesis of both capsule and melanin to support this dramatic morphological transition. Deciphering the precise molecular mechanisms underlying this complex crosstalk between capsule, melanin and titanization requires further investigation. In conclusion, our study establishes glycolysis as a critical metabolic pathway that modulates titan cell formation in C. neoformans through the regulation of the cAMP-PKA signalling ( Fig. 6 ). Additionally, we also demonstrate that, melanin, a well-characterized virulence factor of C. neoformans , is critical for titan cell formation, underscoring its important role in C. neoformans pathogenesis. These findings provide new insights into the metabolic basis of morphogenetic transitions in C. neoformans and highlight the connections between central carbon metabolism and pathogenesis. Understanding these metabolic requirements not only advances our fundamental knowledge of C. neoformans biology but also reveals potential targets for therapeutic intervention against this opportunistic pathogen. Materials and Methods Strains and Media All strains used in this study are listed in Table 1 . All experiments were performed using Cryptococcus neoformans var. grubii, serotype A, H99 wild-type, and its derivatives. All strains used in this study were generously provided by Dr. Joeseph Heitman, Duke University, USA. Yeast Peptone (YP) (1% (w/v) yeast extract and 2% (w/v) peptone) medium supplemented with glycerol was used for cryo-preservation of strains at -80 °C. All the strains were recovered at 30 °C on YPD agar plates (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose and 2% (w/v) agar). View this table: View inline View popup Download powerpoint Table 1: C. neoformans Strains Used in This Study YPD broth containing 1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) glucose was used for the overnight growth of the cultures. Overnight cultures of various strains were grown in YPD broth and incubated at 30 °C with continuous shaking at 200 Rotations Per Minute (RPM). Titan Cell Medium (TCM) containing 5% (w/v) SDA (Sabouraud), 5% (v/v) FBS (Fetal Bovine Serum), 15μM Sodium Azide and 50mM MOPS was used for all in vitro titan cell assays. In Vitro Titan Cell Assay For in vitro titan cell inducing assays, overnight grown cultures of different C. neoformans strains (listed in Table 1 ) were inoculated in TCM at a cell density of 10 6 CFU per ml. For titan cell inducing assays with glycolysis inhibitors, cultures were grown in TCM at cell density of 10 6 CFU per ml with 0.025% (w/v) of 2-Deoxy-D-Glucose (2DG) and 0.5% (w/v) of sodium citrate for 72 hours at 37 °C under 5% CO2. For cAMP add-back assays, wild-type and knockout strains of C. neoformans were cultured in TCM at cell density of 10 6 CFU per ml in the presence and absence of 10mM cAMP for 72 hours at 37 °C under 5% CO2. Following 72 hours of incubation, cells were fixed and imaged using Zeiss Apotome microscope. Cell diameter was measured using Zen 2.3 software and titan cell quantification was done (>400 cells per sample). Titan cell percentage was calculated as a ratio of cells with cell body diameter of ≥10μm to total number of cells. Statistical analysis was performed using unpaired t-test with GraphPad Prism (v9.0). Error bars represent the Standard Error of the Mean (SEM). All statistical comparison was based on a minimum of three independent biological replicates. Capsule Visualization and Analysis For visualization and analysis of capsule thickness, overnight grown cultures of different C. neoformans strains (listed in Table 1 ) were inoculated in TCM at cell density of 10 6 CFU per ml for 72 hours at 37 °C under 5% CO2. Following 72 hours of incubation, cells were fixed and stained using India ink. Samples were imaged using Zeiss Apotome microscope. Capsule thickness was measured using Zen 2.3 software (150 cells each sample). Statistical analysis was performed using unpaired t-test with GraphPad Prism (v9.0). Error bars represent the Standard Error of the Mean (SEM). All statistical comparison was based on a minimum of three independent biological replicates. Intracellular cAMP Measurement Assay Overnight grown cultures of wild-type and glycolysis mutants hxk2 Δ and pyk1 Δ were inoculated in TCM or YPD and grown at 37 °C under shaking conditions. Cells were harvested at mid-log phase (YPD) or after 24 hours (TCM) washed twice with sterile 1X PBS and 10 4 cells from each strain were used for cAMP measurement assay using cAMP-Glo assay kit (#V1501 - Promega), as per the manufacturer’s instructions. Statistical analysis was performed using unpaired t-test with GraphPad Prism (v9.0). Error bars represent the Standard Error of the Mean (SEM). All statistical comparison was based on a minimum of three independent biological replicates. Cell Viability Assay To study the effect of glycolysis inhibitors (2DG and NaCi) on general growth, the overnight culture of wild-type, grown in YPD, was inoculated into TCM at a cell density of 10 6 CFU per ml with and without 2DG (0.025%) or sodium citrate (0.5%) and incubated at 37 °C under 5% CO2 for 72 hours. After incubation, cells were washed twice with sterile 1X PBS and plated on YPD Agar plates (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose, 2% (w/v) agar) and incubated for 48 hours at 30 °C. For glycolysis mutants, the overnight cultures of glycolysis mutants ( hxk2 Δ and pyk1 Δ) along with the wild-type, was grown in YPD and inoculated into TCM at a cell density of 10 6 CFU per mL and incubated at 37 °C under 5% CO2 for 72 hours. After incubation, cells were washed twice with sterile 1X PBS and plated on YPD Agar plates and incubated for 48 hours at 30 °C. Post-incubation colonies were counted and calculated to determine the number of colonies per millilitre. Statistical analysis was performed using unpaired t-test with GraphPad Prism (v9.0). Error bars represent the Standard Error of the Mean (SEM). All statistical comparison was based on a minimum of three independent biological replicates. Reverse Transcriptase-Quantitative Polymerase Chain Reaction (RT-qPCR) Assay Wild-type and knockout strains (listed in Table 1 ) of C. neoformans were cultured in TCM at cell density of 10 6 CFU per ml for 72 hours at 37 °C under 5% CO2. Total RNA was extracted following 72 hours of incubation using the Ribopure RNA purification kit (yeast) (cat. #AM1926) following the manufacturers instruction. cDNA biosynthesis was done using PrimeScript cDNA biosynthesis kit (cat. #6110A). Primers used for RT-qPCR were designed using PrimeQuest tool from Integrated DNA Technologies (IDT) and the primers used in these experiments are listed in Table 2 . RT-qPCR was performed using iTaq Universal SYBR Green Master Mix (cat. #1725121). Actin was used as housekeeping gene for normalization. The results were analysed using the 2 -ΔΔCT method and Statistical analysis was performed using Two-way ANOVA test with GraphPad Prism (v9.0). Error bars represent the Standard Error of the Mean (SEM). All statistical comparison was based on a minimum of three independent biological replicates. View this table: View inline View popup Download powerpoint Table 2: Primers Used in This Study Melanin Biosynthesis Assay To assess melanin biosynthesis, wild-type and melanin defective strains (listed in Table 1 ) were cultured in liquid YPD and incubated at 30 °C overnight. Post incubation, cells were washed with 1X PBS and spotted onto melanin inducing medium (L-DOPA medium, 1g L-asparagine, 3g KH2PO4, 0.25g MgSO4, 1mg thiamine, 5μg biotin, and 100mg L-DOPA containing 0.1% glucose). Cells were incubated at 37 °C and were imaged using SZX16 after 36 hours. Illustrations Figure illustrations were created using Biorender ( https://app.biorender.com ). Author Contributions Pallavi S Phatak: Investigation; Formal analysis; Visualization; Writing-original draft. Sudharsan Mathivathanan: Investigation; Writing-original draft. Dhrumi Shah: Investigation; Writing-original draft. Ishvarya Suresh: Investigation; Writing-original draft. Mary Shejo: Investigation; Writing-original draft. Santosh Kumar Das : Investigation; Sriram Varahan: Conceptualization; Supervision; Funding acquisition; Investigation; Formal analysis; Visualization; Writing-original draft; Writing-review and editing. Conflict of Interest The authors declare that they have no conflict of interests. Acknowledgements We extend our immense gratitude to Dr. Joseph Heitman and Mrs. Anna Floyd-Averette for generously providing the strains used in this study. We would like to thank Dr. Sunil Laxman (BRIC-inStem) for critical reading of our manuscript. We gratefully acknowledge the invaluable support and resources offered by CSIR-Centre for Cellular and Molecular Biology (CCMB) central facilities including Advanced Microscopy and Imaging facility (AMIF). PSP thanks Council of Scientific and Industrial Research (CSIR), India for her research fellowship. SV acknowledges funding from Indian Council of Medical Research (ICMR) (IIRPSG-2024-01-02717), India; Anusandhan National Research Foundation (ANRF) (SRG/2023/000470), India; Council of Scientific & Industrial Research (CSIR) (FTT070505), India; and DBT-Wellcome Trust India Alliance (IA/E/16/1/502996), India. Funder Information Declared Indian Council of Medical Research (ICMR) , IIRPSG-2024-01-02717 Council of Scientific and Industrial Research (CSIR) , FTT070505 Anusandhan National Research Foundation (ANRF) , SRG/2023/000470 DBT-Wellcome Trust India Alliance , IA/E/16/1/502996 Footnotes We have revised multiple figures, included additional data and have made pertinent changes to the manuscript text. References 1. ↵ Alspaugh JA , Perfect JR , Heitman J. 1997 . Cryptococcus neoformans mating and virulence are regulated by the G-protein α subunit GPA1 and cAMP . Genes Dev . 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