The NLRP3 inflammasome has emerged as a central immune regulator that senses virulence factors expressed by microbial pathogens for triggering inflammation. Inflammation can be harmful and therefore ...this response must be tightly controlled. The mechanisms by which immune cells, such as macrophages, discriminate benign from pathogenic microbes to control the NLRP3 inflammasome remain poorly defined. Here we used live cell imaging coupled with a compendium of diverse clinical isolates to define how macrophages respond and activate NLRP3 when faced with the human yeast commensal and pathogen Candida albicans. We show that metabolic competition by C. albicans, rather than virulence traits such as hyphal formation, activates NLRP3 in macrophages. Inflammasome activation is triggered by glucose starvation in macrophages, which occurs when fungal load increases sufficiently to outcompete macrophages for glucose. Consistently, reducing Candida's ability to compete for glucose and increasing glucose availability for macrophages tames inflammatory responses. We define the mechanistic requirements for glucose starvation-dependent inflammasome activation by Candida and show that it leads to inflammatory cytokine production, but it does not trigger pyroptotic macrophage death. Pyroptosis occurs only with some Candida isolates and only under specific experimental conditions, whereas inflammasome activation by glucose starvation is broadly relevant. In conclusion, macrophages use their metabolic status, specifically glucose metabolism, to sense fungal metabolic activity and activate NLRP3 when microbial load increases. Therefore, a major consequence of Candida-induced glucose starvation in macrophages is activation of inflammatory responses, with implications for understanding how metabolism modulates inflammation in fungal infections.
Regulation of gene expression programs is crucial for the survival of microbial pathogens in host environments and for their ability to cause disease. Here we investigated the epigenetic regulator ...RSC (Remodels the Structure of Chromatin) in the most prevalent human fungal pathogen Candida albicans. Biochemical analysis showed that CaRSC comprises 13 subunits and contains two novel non-essential members, which we named Nri1 and Nri2 (Novel RSC Interactors) that are exclusive to the CTG clade of Saccharomycotina. Genetic analysis showed distinct essentiality of C. albicans RSC subunits compared to model fungal species suggesting functional and structural divergence of RSC functions in this fungal pathogen. Transcriptomic and proteomic profiling of a conditional mutant of the essential catalytic subunit gene STH1 demonstrated global roles of RSC in C. albicans biology, with the majority of growth-related processes affected, as well as mis-regulation of genes involved in morphotype switching, host-pathogen interaction and adaptive fitness. We further assessed the functions of non-essential CaRSC subunits, showing that the novel subunit Nri1 and the bromodomain subunit Rsc4 play roles in filamentation and stress responses; and also interacted at the genetic level to regulate cell viability. Consistent with these roles, Rsc4 is required for full virulence of C. albicans in the murine model of systemic infection. Taken together, our data builds the first comprehensive study of the composition and roles of RSC in C. albicans, showing both conserved and distinct features compared to model fungal systems. The study illuminates how C. albicans uses RSC-dependent transcriptional regulation to respond to environmental signals and drive survival fitness and virulence in mammals.
To fight infections, macrophages undergo a metabolic shift whereby increased glycolysis fuels antimicrobial inflammation and killing of pathogens. Here we demonstrate that the pathogen Candida ...albicans turns this metabolic reprogramming into an Achilles' heel for macrophages. During Candida-macrophage interactions intertwined metabolic shifts occur, with concomitant upregulation of glycolysis in both host and pathogen setting up glucose competition. Candida thrives on multiple carbon sources, but infected macrophages are metabolically trapped in glycolysis and depend on glucose for viability: Candida exploits this limitation by depleting glucose, triggering rapid macrophage death. Using pharmacological or genetic means to modulate glucose metabolism of host and/or pathogen, we show that Candida infection perturbs host glucose homeostasis in the murine candidemia model and demonstrate that glucose supplementation improves host outcomes. Our results support the importance of maintaining glucose homeostasis for immune cell survival during Candida challenge and for host survival in systemic infection.
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•The macrophage Warburg effect becomes a liability during microbial challenge•In vitro, Candida competes for glucose, triggering massive macrophage death•In vivo, Candida infection promotes disruption of host glucose homeostasis•A glucose-rich diet improves host outcomes in systemic fungal infection
Glucose is essential for innate immune cells to mount effective anti-fungal responses. Tucey et al. show that, for infected macrophages, dependence on glucose becomes their downfall: the human fungal pathogen Candida albicans rapidly consumes glucose, causing macrophages to die. In mice, maintaining host glucose homeostasis is important to prevent life-threatening fungal infection.
Microbes are exposed to nutritional and stress challenges in their environmental and host niches. To rise to these challenges, they regulate transcriptional programs that enable cellular adaptation. ...For instance, metabolite concentrations regulate post-translational modifications of chromatin, such as histone acetylation. In this way, metabolic signals are integrated with transcription. Over the last decade, several histone acylations have been discovered, including histone crotonylation. Their roles in microbial biology, environmental adaptation, and microbe-host interactions are incompletely defined. Here we show that the short-chain fatty acid crotonate, which is used to study histone crotonylation, changes cell morphology and immune interactions of
. Crotonate reduces invasive hyphal morphogenesis of
within macrophages, thereby delaying macrophage killing and pathogen escape, as well as reducing inflammatory cytokine maturation. Crotonate's ability to reduce hyphal growth is environmentally contingent and pronounced within macrophages. Moreover, crotonate is a stronger hyphal inhibitor than butyrate under the conditions that we tested. Crotonate causes increased histone crotonylation in
under hyphal growth conditions and reduces transcription of hyphae-induced genes in a manner that involves the Nrg1 repressor pathway. Increasing histone acetylation by histone deacetylase inhibition partially rescues hyphal growth and gene transcription in the presence of crotonate. These results indicate that histone crotonylation might compete with acetylation in the regulation of hyphal morphogenesis. Based on our findings, we propose that diverse acylations of histones (and likely also non-histone proteins) enable
to respond to environmental signals, which in turn regulate its cell morphology and host-pathogen interactions.IMPORTANCEMacrophages curtail the proliferation of the pathogen
within human body niches. Within macrophages,
adapts its metabolism and switches to invasive hyphal morphology. These adaptations enable fungal growth and immune escape by triggering macrophage lysis. Transcriptional programs regulate these metabolic and morphogenetic adaptations. Here we studied the roles of chromatin in these processes and implicate lysine crotonylation, a histone mark regulated by metabolism, in hyphal morphogenesis and macrophage interactions by
. We show that the short-chain fatty acid crotonate increases histone crotonylation, reduces hyphal formation within macrophages, and slows macrophage lysis and immune escape of
. Crotonate represses hyphal gene expression, and we propose that
uses diverse acylation marks to regulate its cell morphology in host environments. Hyphal formation is a virulence property of
. Therefore, a further importance of our study stems from identifying crotonate as a hyphal inhibitor.
A leading objective in biology is to identify the complete set of activities that each gene performs
In this study, we have asked whether a genetic approach can provide an efficient means of ...achieving this goal, through the identification and analysis of a comprehensive set of separation-of-function (
) mutations in a gene. Toward this goal, we have subjected the
gene, which encodes a regulatory subunit of telomerase, to intensive mutagenesis (with an average coverage of one mutation for every 4.5 residues), using strategies that eliminated those mutations that disrupted protein folding/stability. The resulting set of
mutations defined four biochemically distinct activities for the Est1 telomerase protein: two temporally separable steps in telomerase holoenzyme assembly, a telomerase recruitment activity, and a fourth newly discovered regulatory function. Although biochemically distinct, impairment of each of these four different activities nevertheless conferred a common phenotype (critically short telomeres) comparable to that of an
-∆ null strain. This highlights the limitations of gene deletions, even for nonessential genes; we suggest that employing a representative set of
mutations for each gene in future high- and low-throughput investigations will provide deeper insights into how proteins interact inside the cell.
Identification of multiple histone acylations diversifies transcriptional control by metabolism, but their functions are incompletely defined. Here we report evidence of histone crotonylation in the ...human fungal pathogen Candida albicans. We define the enzymes that regulate crotonylation and show its dynamic control by environmental signals: carbon sources, the short-chain fatty acids butyrate and crotonate, and cell wall stress. Crotonate regulates stress-responsive transcription and rescues C. albicans from cell wall stress, indicating broad impact on cell biology. The YEATS domain crotonylation readers Taf14 and Yaf9 are required for C. albicans virulence, and Taf14 controls gene expression, stress resistance, and invasive growth via its chromatin reader function. Blocking the Taf14 C terminus with a tag reduced virulence, suggesting that inhibiting Taf14 interactions with chromatin regulators impairs function. Our findings shed light on the regulation of histone crotonylation and the functions of the YEATS proteins in eukaryotic pathogen biology and fungal infections.
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•Metabolism and stress responses control histone crotonylation in Candida albicans•The YEATS crotonylation readers Taf14 and Yaf9 are important for Candida virulence•Taf14 participates in stress responses, cell wall, and filamentation pathways•The SCFA crotonate affects transcription and stress susceptibility in Candida
Diverse histone acylations expand functional linkages among metabolism, gene regulation, and cellular responses. Wang et al. report that metabolic and stress cues dynamically control histone crotonylation in the human commensal and pathogen Candida albicans and show the requirement for crotonylation readers in pathogenesis-related pathways and mammalian virulence.
Actin, one of the most abundant proteins in the eukaryotic cell, also has an abundance of relatives in the eukaryotic proteome. To date though, only five families of actins have been characterized in ...bacteria. We have conducted a phylogenetic search and uncovered more than 35 highly divergent families of actin-like proteins (Alps) in bacteria. Their genes are found primarily on phage genomes, on plasmids and on integrating conjugative elements, and are likely to be involved in a variety of functions. We characterize three Alps and find that all form filaments in the cell. The filaments of Alp7A, a plasmid partitioning protein and one of the most divergent of the Alps, display dynamic instability and also treadmill. Alp7A requires other elements from the plasmid to assemble into dynamic polymers in the cell. Our findings suggest that most if not all of the Alps are indeed actin relatives, and that actin is very well represented in bacteria.
The interactions of mitochondria with the endoplasmic reticulum (ER) are crucial for maintaining proper mitochondrial morphology, function and dynamics. This enables cells to utilize their ...mitochondria optimally for energy production and anabolism, and it further provides for metabolic control over developmental decisions. In fungi, a key mechanism by which ER and mitochondria interact is via a membrane tether, the protein complex ERMES (ER-Mitochondria Encounter Structure). In the model yeast
, the mitochondrial GTPase Gem1 interacts with ERMES, and it has been proposed to regulate its activity. Here we report on the first characterization of Gem1 in a human fungal pathogen. We show that in
Gem1 has a dominant role in ensuring proper mitochondrial morphology, and our data is consistent with Gem1 working with ERMES in this role. Mitochondrial respiration and steady state cellular phospholipid homeostasis are not impacted by inactivation of
in
. There are two major virulence-related consequences of disrupting mitochondrial morphology by
inactivation:
becomes hypersusceptible to cell wall stress, and is unable to grow invasively. In the
Δ
Δ mutant, it is specifically the invasive capacity of hyphae that is compromised, not the ability to transition from yeast to hyphal morphology, and this phenotype is shared with ERMES mutants. As a consequence of the hyphal invasion defect, the
Δ
Δ mutant is drastically hypovirulent in the worm infection model. Activation of the mitogen activated protein (MAP) kinase Cek1 is reduced in the
Δ
Δ mutant, and this function could explain both the susceptibility to cell wall stress and lack of invasive growth. This result establishes a new, respiration-independent mechanism of mitochondrial control over stress signaling and hyphal functions in
. We propose that ER-mitochondria interactions and the ER-Mitochondria Organizing Network (ERMIONE) play important roles in adaptive responses in fungi, in particular cell surface-related mechanisms that drive invasive growth and stress responsive behaviors that support fungal pathogenicity.
Telomerase is essential for continuous cellular proliferation. Substantial insights have come from studies of budding yeast telomerase, which consists of a catalytic core in association with two ...regulatory proteins, ever shorter telomeres 1 and 3 (Est1 and Est3). We report here a high-resolution structure of the Est3 telomerase subunit determined using a recently developed strategy that combines minimal NMR experimental data with Rosetta de novo structure prediction algorithms. Est3 adopts an overall protein fold which is structurally similar to that adopted by the shelterin component TPP1. However, the characteristics of the surface of the experimentally determined Est3 structure are substantially different from those predicted by prior homology-based models of Est3. Structure-guided mutagenesis of the complete surface of the Est3 protein reveals two adjacent patches on a noncanonical face of the protein that differentially mediate telomere function. Mapping these two patches on the Est3 structure defines a set of shared features between Est3 and HsTPP1, suggesting an analogous multifunctional surface on TPP1.
In budding yeast, association of the Est1 regulatory protein with telomerase is thought to be limited to the late S phase, when telomere elongation occurs. By monitoring the stoichiometry of ...telomerase subunits, we show instead that a telomerase complex containing Est1 is assembled much earlier in the cell cycle. We also report a biochemical interaction between Est1 and the telomere binding protein Cdc13 that recapitulates the previously observed genetic relationship between EST1 and CDC13. This supports a model in which regulated binding of Cdc13 to chromosome termini dictates subsequent interaction of a recruitment-competent telomerase complex with telomeres.
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