Human Fungal Pathogen Candida Glabrata Research Articles

The Hog1 MAPK substrate governs Candida glabrata-epithelial cell adhesion via the histone H2A variant.

CgHog1, terminal kinase of the high-osmolarity glycerol signalling pathway, orchestrates cellular response to multiple external stimuli including surplus-environmental iron in the human fungal pathogen Candida glabrata (Cg). However, CgHog1 substrates remain unidentified. Here, we show that CgHog1 adversely affects Cg adherence to host stomach and kidney epithelial cells in vitro, but promotes Cg survival in the iron-rich gastrointestinal tract niche. Further, CgHog1 interactome and in vitro phosphorylation analysis revealed CgSub2 (putative RNA helicase) to be a CgHog1 substrate, with CgSub2 also governing iron homeostasis and host adhesion. CgSub2 positively regulated EPA1 (encodes a major adhesin) expression and host adherence via its interactor CgHtz1 (histone H2A variant). Notably, both CgHog1 and surplus environmental iron had a negative impact on CgSub2-CgHtz1 interaction, with CgHTZ1 or CgSUB2 deletion reversing the elevated adherence of Cghog1Δ to epithelial cells. Finally, the surplus-extracellular iron led to CgHog1 activation, increased CgSub2 phosphorylation, elevated CgSub2-CgHta (canonical histone H2A) interaction, and EPA1 transcriptional activation, thereby underscoring the iron-responsive, CgHog1-induced exchange of histone partners of CgSub2. Altogether, our work mechanistically defines how CgHog1 couples Epa1 adhesin expression with iron abundance, and point towards specific chromatin composition modification programs that probably aid fungal pathogens align their adherence to iron-rich (gut) and iron-poor (blood) host niches.

Phosphatidylinositol 3-phosphate regulates iron transport via PI3P-binding CgPil1 protein

Iron homeostasis, which is pivotal to virulence, is regulated by the phosphatidylinositol 3-kinase CgVps34 inthe human fungal pathogen Candida glabrata. Here, we identify CgPil1 as a phosphatidylinositol 3-phosphate (PI3P)-binding protein and unveil its role in retaining the high-affinity iron transporter CgFtr1 at the plasma membrane (PM), with PI3P negatively regulating CgFtr1-CgPil1 interaction. PI3P production and its PM localization are elevated in the high-iron environment. Surplus iron also leads to intracellular distribution and vacuolar delivery of CgPil1 and CgFtr1, respectively, from the PM. Loss of CgPil1 or CgFtr1 ubiquitination at lysines 391 and 401 results in CgFtr1 trafficking to the endoplasmic reticulum and a decrease in vacuole-localized CgFtr1. The E3-ubiquitin ligase CgRsp5 interacts with CgFtr1 and forms distinct CgRsp5-CgFtr1 puncta at the PM, with high iron resulting in their internalization. Finally, PI3P controls retrograde transport of many PM proteins. Altogether, we establish PI3P as a key regulator of membrane transport in C.glabrata.

Functional reprogramming of Candida glabrata epithelial adhesins: the role of conserved and variable structural motifs in ligand binding

For host-cell interaction, the human fungal pathogen Candida glabrata harbors a large family of more than 20 cell wall-attached epithelial adhesins (Epas). Epa family members are lectins with binding pockets containing several conserved and variable structural hot spots, which were implicated in mediating functional diversity. In this study, we have performed an elaborate structure-based mutational analysis of numerous Epa paralogs to generally determine the role of diverse structural hot spots in conferring host cell binding and ligand binding specificity. Our study reveals that several conserved structural motifs contribute to efficient host cell binding. Moreover, our directed motif exchange experiments reveal that the variable loop CBL2 is key for programming ligand binding specificity, albeit with limited predictability. In contrast, we find that the variable loop L1 affects host cell binding without significantly influencing the specificity of ligand binding. Our data strongly suggest that variation of numerous structural hot spots in the ligand binding pocket of Epa proteins is a main driver of their functional diversification and evolution.

Glyoxylate cycle gene ICL1 is essential for the metabolic flexibility and virulence of Candida glabrata

The human fungal pathogen Candida glabrata appears to utilise unique stealth, evasion and persistence strategies in subverting the onslaught of host immune response during systemic infection. However, macrophages actively deprive the intracellular fungal pathogen of glucose, and therefore alternative carbon sources probably support the growth and survival of engulfed C. glabrata. The present study aimed to investigate the role of the glyoxylate cycle gene ICL1 in alternative carbon utilisation and its importance for the virulence of C. glabrata. The data showed that disruption of ICL1 rendered C. glabrata unable to utilise acetate, ethanol or oleic acid. In addition, C. glabrata icl1∆ cells displayed significantly reduced biofilm growth in the presence of several alternative carbon sources. It was also found that ICL1 is crucial for the survival of C. glabrata in response to macrophage engulfment. Disruption of ICL1 also conferred a severe attenuation in the virulence of C. glabrata in the mouse model of invasive candidiasis. In conclusion, a functional glyoxylate cycle is essential for C. glabrata to utilise certain alternative carbon sources in vitro and to display full virulence in vivo. This reinforces the view that antifungal drugs that target fungal Icl1 have potential for future therapeutic intervention.

Adhesins in Candida glabrata.

The human fungal pathogen Candida glabrata is causing more and more problems in hospitals, as this species shows an intrinsic antifungal drug resistance or rapidly becomes resistant when challenged with antifungals. C. glabrata only grows in the yeast form, so it is lacking a yeast-to-hyphae switch, which is one of the main virulence factors of C. albicans. An important virulence factor of C. glabrata is its capacity to strongly adhere to many different substrates. To achieve this, C. glabrata expresses a large number of adhesin-encoding genes and genome comparisons with closely related species, including the non-pathogenic S. cerevisiae, which revealed a correlation between the number of adhesin-encoding genes and pathogenicity. The adhesins are involved in the first steps during an infection; they are the first point of contact with the host. For several of these adhesins, their importance in adherence to different substrates and subsequent biofilm formation was demonstrated in vitro or in vivo. In this review, we provide an overview of the role of C. glabrata adhesins during adhesion and biofilm formation both, under in vitro and in vivo conditions.

Evolution of regulatory networks in Candida glabrata: learning to live with the human host.

The opportunistic human fungal pathogen Candida glabrata is second only to C. albicans as the cause of Candida infections and yet is more closely related to Saccharomyces cerevisiae. Recent advances in functional genomics technologies and computational approaches to decipher regulatory networks, and the comparison of these networks among these and other Ascomycete species, have revealed both unique and shared strategies in adaptation to a human commensal/opportunistic pathogen lifestyle and antifungal drug resistance in C. glabrata. Recently, several C. glabrata sister species in the Nakeseomyces clade representing both human associated (commensal) and environmental isolates have had their genomes sequenced and analyzed. This has paved the way for comparative functional genomics studies to characterize the regulatory networks in these species to identify informative patterns of conservation and divergence linked to phenotypic evolution in the Nakaseomyces lineage.

Structural Hot Spots Determine Functional Diversity of the Candida glabrata Epithelial Adhesin Family

For host colonization, the human fungal pathogen Candida glabrata is known to utilize a large family of highly related surface-exposed cell wall proteins, the lectin-like epithelial adhesins (Epas). To reveal the structure-function relationships within the entire Epa family, we have performed a large scale functional analysis of the adhesion (A) domains of 17 Epa paralogs in combination with three-dimensional structural studies of selected members with cognate ligands. Our study shows that most EpaA domains exert lectin-like functions and together recognize a wide variety of glycans with terminal galactosides for conferring epithelial cell adhesion. We further identify several conserved and variable structural features within the diverse Epa ligand binding pockets, which affect affinity and specificity. These features rationalize why mere phylogenetic relationships within the Epa family are weak indicators for functional classification and explain how Epa-like adhesins have evolved in C. glabrata and related fungal species.

Genetic Transformation of Candida glabrata by Heat Shock.

Here, we report a method for the transformation of Candida glabrata using a heat shock method. The protocol can be used for transformations in single well or in 96-well scale. It has been employed as an alternative method to the electroporation protocol to construct a genome-scale gene deletion collection in the human fungal pathogen Candida glabrata ATCC2001 and related strains. Furthermore, the protocol can be used to generate gene deletions in clinical isolates of Candida glabrata (C. glabrata).

Genetic Transformation of Candida glabrata by Electroporation.

Here, we report a method for the transformation by electroporation of the human fungal pathogen Candida glabrata (C. glabrata). The protocol can be used for transformations in single well or in 96-well microtiter plates. It has been extensively used to generate a genome-scale gene deletion library using the C. glabrata background recipient strain ATCC2001 (Schwarzmüller et al., 2014).

Large-scale Phenotypic Profiling of Gene Deletion Mutants in Candida glabrata.

Here, we describe a method enabling the phenotypic profiling of genome-scale deletion collections of fungal mutants to detect phenotypes for various stress conditions. These stress conditions include among many others antifungal drug susceptibility, temperature-induced and osmotic as well as heavy metal or oxidative stress. The protocol was extensively used to phenotype a collection of gene deletion mutants in the human fungal pathogen Candida glabrata (C. glabrata) (Schwarzmüller et al., 2014).

A systematic analysis reveals an essential role for high-affinity iron uptake system, haemolysin and CFEM domain-containing protein in iron homoeostasis and virulence in Candida glabrata.

Iron is an essential nutrient for all living organisms and human pathogens employ a battery of factors to scavenge iron from the high-affinity iron-binding host proteins. In the present study, we have elucidated, via a candidate gene approach, major iron acquisition and homoeostatic mechanisms operational in an opportunistic human fungal pathogen Candida glabrata. Phenotypic, biochemical and molecular analysis of a set of 13 C. glabrata strains, deleted for proteins potentially implicated in iron metabolism, revealed that the high-affinity reductive iron uptake system is required for utilization of alternate carbon sources and for growth under both in vitro iron-limiting and in vivo conditions. Furthermore, we show for the first time that the cysteine-rich CFEM (common in fungal extracellular membranes) domain-containing cell wall structural protein, CgCcw14, and a putative haemolysin, CgMam3, are essential for maintenance of intracellular iron content, adherence to epithelial cells and virulence. Consistent with their roles in iron homoeostasis, mitochondrial aconitase activity was lower and higher in mutants disrupted for high-affinity iron transport, and haemolysin respectively. Additionally, we present evidence that the mitochondrial frataxin, CgYfh1, is pivotal to iron metabolism. Besides yielding insights into major in vitro and in vivo iron acquisition strategies, our findings establish high-affinity iron uptake mechanisms as critical virulence determinants in C. glabrata.

Dissection of Ire1 functions reveals stress response mechanisms uniquely evolved in Candida glabrata.

Proper protein folding in the endoplasmic reticulum (ER) is vital in all eukaryotes. When misfolded proteins accumulate in the ER lumen, the transmembrane kinase/endoribonuclease Ire1 initiates splicing of HAC1 mRNA to generate the bZIP transcription factor Hac1, which subsequently activates its target genes to increase the protein-folding capacity of the ER. This cellular machinery, called the unfolded protein response (UPR), is believed to be an evolutionarily conserved mechanism in eukaryotes. In this study, we comprehensively characterized mutant phenotypes of IRE1 and other related genes in the human fungal pathogen Candida glabrata. Unexpectedly, Ire1 was required for the ER stress response independently of Hac1 in this fungus. C. glabrata Ire1 did not cleave mRNAs encoding Hac1 and other bZIP transcription factors identified in the C. glabrata genome. Microarray analysis revealed that the transcriptional response to ER stress is not mediated by Ire1, but instead is dependent largely on calcineurin signaling and partially on the Slt2 MAPK pathway. The loss of Ire1 alone did not confer increased antifungal susceptibility in C. glabrata contrary to UPR-defective mutants in other fungi. Taken together, our results suggest that the canonical Ire1-Hac1 UPR is not conserved in C. glabrata. It is known in metazoans that active Ire1 nonspecifically cleaves and degrades a subset of ER-localized mRNAs to reduce the ER load. Intriguingly, this cellular response could occur in an Ire1 nuclease-dependent fashion in C. glabrata. We also uncovered the attenuated virulence of the C. glabrata Δire1 mutant in a mouse model of disseminated candidiasis. This study has unveiled the unique evolution of ER stress response mechanisms in C. glabrata.

Pathogenesis and Antifungal Drug Resistance of the Human Fungal Pathogen Candida glabrata

Candida glabrata is a major opportunistic human fungal pathogen causing superficial as well as systemic infections in immunocompromised individuals and several other patient cohorts. C. glabrata represents the second most prevalent cause of candidemia and a better understanding of its virulence and drug resistance mechanisms is thus of high medical relevance. In contrast to the diploid dimorphic pathogen C. albicans, whose ability to undergo filamentation is considered a major virulence trait, C. glabrata has a haploid genome and lacks the ability to switch to filamentous growth. A major impediment for the clinical therapy of C. glabrata infections is its high intrinsic resistance to several antifungal drugs, especially azoles. Further, the development of antifungal resistance, particularly during prolonged and prophylactic therapies is diminishing efficacies of therapeutic interventions. In addition, C. glabrata harbors a large repertoire of adhesins involved in the adherence to host epithelia. Interestingly, genome plasticity, phenotypic switching or the remarkable ability to persist and survive inside host immune cells further contribute to the pathogenicity of C. glabrata. In this comprehensive review, we want to emphasize and discuss the mechanisms underlying virulence and drug resistance of C. glabrata, and discuss its ability to escape from the host immune surveillance or persist inside host cells.

Regulation of Candida glabrata oxidative stress resistance is adapted to host environment

The human fungal pathogen Candida glabrata is related to Saccharomyces cerevisiae but has developed high resistance against reactive oxygen species. We find that induction of conserved genes encoding antioxidant functions is dependent on the transcription factors CgYap1 and CgSkn7 which cooperate for promoter recognition. Superoxide stress resistance of C. glabrata is provided by superoxide dismutase CgSod1, which is not dependent on CgYap1/Skn7. Only double mutants lacking both CgSod1 and CgYap1 were efficiently killed by primary mouse macrophages. Our results suggest that in C. glabrata the regulation of key genes providing stress protection is adopted to meet a host–pathogen situation.

From Saccharomyces cerevisiae to Candida glabrata in a few easy steps: important adaptations for an opportunistic pathogen

The opportunistic human fungal pathogen Candida glabrata is closely related to Saccharomyces cerevisiae, yet it has evolved to survive within mammalian hosts. Which traits help C. glabrata to adapt to this different environment? Which specific responses are crucial for its survival in the host? The main differences seem to include an extended repertoire of adhesin genes, high drug resistance, an enhanced ability to sustain prolonged starvation and adaptations of the transcriptional wiring of key stress response genes. Here, we discuss the properties of C. glabrata with a focus on the differences to related fungi.

Autophagy supportsCandida glabratasurvival during phagocytosis

The opportunistic human fungal pathogen Candida glabrata is confronted with phagocytic cells of the host defence system. Survival of internalized cells is thought to contribute to successful dissemination. We investigated the reaction of engulfed C. glabrata cells using fluorescent protein fusions of the transcription factors CgYap1 and CgMig1 and catalase CgCta1. The expression level and peroxisomal localization of catalase was used to monitor the metabolic and stress status of internalized C. glabrata cells. These reporters revealed that the phagocytosed C. glabrata cells were exposed to transient oxidative stress and starved for carbon source. Cells trapped within macrophages increased their peroxisome numbers indicating a metabolic switch. Prolonged phagocytosis caused a pexophagy-mediated decline in peroxisome numbers. Autophagy, and in particular pexophagy, contributed to survival of C. glabrata during engulfment. Mutants lacking CgATG11 or CgATG17, genes required for pexophagy and non-selective autophagy, respectively, displayed reduced survival rates. Furthermore, both CgAtg11 and CgAtg17 contribute to survival, since the double mutant was highly sensitive to engulfment. Inhibition of peroxisome formation by deletion of CgPEX3 partially restored viability of CgATG11 deletion mutants during engulfment. This suggests that peroxisome formation and maintenance might sequester resources required for optimal survival. Mobilization of intracellular resources via autophagy is an important virulence factor that supports the viability of C. glabrata in the phagosomal compartment of infected innate immune cells.

The high-osmolarity glycerol response pathway in the human fungal pathogen Candida glabrata strain ATCC 2001 lacks a signaling branch that operates in baker's yeast.

The high-osmolarity glycerol (HOG) mitogen-activated protein (MAP) kinase pathway mediates adaptation to high-osmolarity stress in the yeast Saccharomyces cerevisiae. Here we investigate the function of HOG in the human opportunistic fungal pathogen Candida glabrata. C. glabrata sho1Delta (Cgsho1Delta) deletion strains from the sequenced ATCC 2001 strain display severe growth defects under hyperosmotic conditions, a phenotype not observed for yeast sho1Delta mutants. However, deletion of CgSHO1 in other genetic backgrounds fails to cause osmostress hypersensitivity, whereas cells lacking the downstream MAP kinase Pbs2 remain osmosensitive. Notably, ATCC 2001 Cgsho1Delta cells also display methylglyoxal hypersensitivity, implying the inactivity of the Sln1 branch in ATCC 2001. Genomic sequencing of CgSSK2 in different C. glabrata backgrounds demonstrates that ATCC 2001 harbors a truncated and mutated Cgssk2-1 allele, the only orthologue of yeast SSK2/SSK22 genes. Thus, the osmophenotype of ATCC 2001 is caused by a point mutation in Cgssk2-1, which debilitates the second HOG pathway branch. Functional complementation experiments unequivocally demonstrate that HOG signaling in yeast and C. glabrata share similar functions in osmostress adaptation. In contrast to yeast, however, Cgsho1Delta mutants display hypersensitivity to weak organic acids such as sorbate and benzoate. Hence, CgSho1 is also implicated in modulating weak acid tolerance, suggesting that HOG signaling in C. glabrata mediates the response to multiple stress conditions.

Superinfection and the evolution of parasite virulence

Earlier ideas that parasites evolve toward becoming harmless to their hosts have, in recent years, given way to more analytic studies, focused on the 'basic reproductive rate', R0, of individual parasites. In general, the biology of the parasite life cycle will lead to constraining relations between virulence (parasite-associated host death or reduction in fertility) and transmissibility: the maximum R0 may then be attained by virulence being high, or low, or at some intermediate level, depending on the details of the constraining relations. Such studies have not generally included superinfection (where an already-infected host is infected by another parasite). Here we propose a general, but simple, model of superinfection, which is amenable to analytical treatment. In such models selection does not simply act to maximize R0; superinfection leads to selection for higher levels of virulence, highly polymorphic parasite populations and very complicated dynamics. We calculate the equilibrium distribution of parasite strains and the maximum level of virulence that can be maintained by superinfection. We also note the equivalence between our 'superinfection model' and recent approaches to the study of the meta-population dynamics of multi-species interactions.