ABSTRACT
SUMMARYA significant increase in the incidence of Candida-mediated infections has been observed in the last decade, mainly due to rising numbers of susceptible individuals. Recently, the World Health Organization published its first fungal pathogen priority list, with Candida species listed in medium, high, and critical priority categories. This review is a synthesis of information and recent advances in our understanding of two of these species-Candida albicans and Candida glabrata. Of these, C. albicans is the most common cause of candidemia around the world and is categorized as a critical priority pathogen. C. glabrata is considered a high-priority pathogen and has become an increasingly important cause of candidemia in recent years. It is now the second most common causative agent of candidemia in many geographical regions. Despite their differences and phylogenetic divergence, they are successful as pathogens and commensals of humans. Both species can cause a broad variety of infections, ranging from superficial to potentially lethal systemic infections. While they share similarities in certain infection strategies, including tissue adhesion and invasion, they differ significantly in key aspects of their biology, interaction with immune cells, host damage strategies, and metabolic adaptations. Here we provide insights on key aspects of their biology, epidemiology, commensal and pathogenic lifestyles, interactions with the immune system, and antifungal resistance.
Subject(s)
Antifungal Agents , Candida albicans , Candida glabrata , Candidiasis , Drug Resistance, Fungal , Candida glabrata/pathogenicity , Humans , Candida albicans/pathogenicity , Candidiasis/microbiology , Antifungal Agents/pharmacology , Antifungal Agents/therapeutic use , Animals , Candidemia/microbiology , Candidemia/epidemiology , Phylogeny , Host-Pathogen InteractionsABSTRACT
Candida glabrata is an important pathogen causing invasive infection associated with a high mortality rate. One mechanism that causes the failure of Candida eradication is an increase in regulatory T cells (Treg), which play a major role in immune suppression and promoting Candida pathogenicity. To date, how C. glabrata induces a Treg response remains unclear. Dendritic cells (DCs) recognition of fungi provides the fundamental signal determining the fate of the T-cell response. This study investigated the interplay between C. glabrata and DCs and its effect on Treg induction. We found that C. glabrata ß-glucan was a major component that interacted with DCs and consequently mediated the Treg response. Blocking the binding of C. glabrata ß-glucan to dectin-1 and complement receptor 3 (CR3) showed that CR3 activation in DCs was crucial for the induction of Treg. Furthermore, a ligand-receptor binding assay showed the preferential binding of C. glabrata ß-glucan to CR3. Our data suggest that C. glabrata ß-glucan potentially mediates the Treg response, probably through CR3-dependent activation in DCs. This study contributes new insights into immune modulation by C. glabrata that may lead to a better design of novel immunotherapeutic strategies for invasive C. glabrata infection.
Subject(s)
Candida glabrata , Dendritic Cells , Macrophage-1 Antigen , T-Lymphocytes, Regulatory , beta-Glucans , Candida glabrata/metabolism , Candida glabrata/pathogenicity , Dendritic Cells/immunology , Dendritic Cells/metabolism , Dendritic Cells/drug effects , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , beta-Glucans/metabolism , beta-Glucans/pharmacology , Animals , Macrophage-1 Antigen/metabolism , Mice , Lectins, C-Type/metabolism , Candidiasis/immunology , Candidiasis/microbiology , Candidiasis/metabolism , Mice, Inbred C57BLABSTRACT
BACKGROUND: The prevalence of Candida glabrata healthcare-associated infections is on the rise worldwide and in Lebanon, Candida glabrata infections are difficult to treat as a result of their resistance to azole antifungals and their ability to form biofilms. OBJECTIVES: The first objective of this study was to quantify biofilm biomass in the most virulent C. glabrata isolates detected in a Lebanese hospital. In addition, other pathogenicity attributes were evaluated. The second objective was to identify the mechanisms of azole resistance in those isolates. METHODS: A mouse model of disseminated systemic infection was developed to evaluate the degree of virulence of 41 azole-resistant C. glabrata collected from a Lebanese hospital. The most virulent isolates were further evaluated alongside an isolate having attenuated virulence and a reference strain for comparative purposes. A DNA-sequencing approach was adopted to detect single nucleotide polymorphisms (SNPs) leading to amino acid changes in proteins involved in azole resistance and biofilm formation. This genomic approach was supported by several phenotypic assays. RESULTS: All chosen virulent isolates exhibited increased adhesion and biofilm biomass compared to the isolate having attenuated virulence. The amino acid substitutions D679E and I739N detected in the subtelomeric silencer Sir3 are potentially involved- in increased adhesion. In all isolates, amino acid substitutions were detected in the ATP-binding cassette transporters Cdr1 and Pdh1 and their transcriptional regulator Pdr1. CONCLUSIONS: In summary, increased adhesion led to stable biofilm formation since mutated Sir3 could de-repress adhesins, while decreased azole susceptibility could result from mutations in Cdr1, Pdh1 and Pdr1.
Subject(s)
Antifungal Agents , Biofilms , Candida glabrata , Candidiasis , Drug Resistance, Fungal , Mutation , Biofilms/growth & development , Candida glabrata/genetics , Candida glabrata/drug effects , Candida glabrata/isolation & purification , Candida glabrata/pathogenicity , Candida glabrata/physiology , Lebanon , Animals , Mice , Drug Resistance, Fungal/genetics , Antifungal Agents/pharmacology , Humans , Virulence/genetics , Candidiasis/microbiology , Fungal Proteins/genetics , Polymorphism, Single Nucleotide , Disease Models, Animal , Azoles/pharmacology , Microbial Sensitivity Tests , Hospitals , FemaleABSTRACT
Here, we report two paired sets of an index wild-type Candida glabrata bloodstream isolate and subsequent echinocandin-resistant FKS mutant. One paired set exhibited a higher proportion of clumping cells and was more virulent in the invertebrate host Galleria mellonella than the other paired set. No virulence difference between the paired index and FKS strains was observed. These findings imply a potential link of clumping morphology with virulence in C. glabrata that is uncoupled from FKS-mediated echinocandin resistance. IMPORTANCE Candida glabrata is a leading cause of invasive candidiasis. In contrast to other species, it has a high propensity for developing resistance to echinocandins, which are the first-line treatment. Unlike the dimorphic Candida albicans which can grow invasive filamentous hyphae, C. glabrata lacks this ability. Here, we report a link between virulence and clumping cell morphology in two different sets of clinical C. glabrata strains obtained from patients failing echinocandin therapy. One set of paired strains (echinocandin-susceptible and subsequent resistant mutant) had a high proportion of clumping cells in the population and were significantly more virulent than another set which had fewer clumping cells. Additionally, we corroborate that echinocandin resistance does not impart a significant fitness cost. Our findings suggest that clumping morphology may be an important but previously underestimated virulence factor for C. glabrata and also aid our understand for the high prevalence of resistance observed in this species.
Subject(s)
Antifungal Agents/pharmacology , Candida glabrata/growth & development , Candida glabrata/pathogenicity , Candidiasis/microbiology , Drug Resistance, Fungal , Echinocandins/pharmacology , Animals , Candida glabrata/drug effects , Candida glabrata/genetics , Fungal Proteins/genetics , Fungal Proteins/metabolism , Humans , Moths/microbiologyABSTRACT
Abstract Candida glabrata infections are responsible for deaths of people globally. Fluconazole is known to be less effective against C. glabrata, which developed many strategies to evade being destroyed by fluconazole. To achieve enhanced efficacy of fluconazole against C. glabrata, the interaction of fluconazole with sodium bicarbonate was investigated using the CLSI guidelines. The efficacy of fluconazole alone and in combination with sodium bicarbonate was evaluated using the time-kill and phospholipase production assays. Eventually, the expression of PLB was assessed using semi-quantitative RT-PCR to investigate the inhibitory properties of fluconazole alone and in combination with sodium bicarbonate against C. glabrata. The fluconazole/sodium bicarbonate combination displayed synergistic and antagonistic effects (FICI= 0.375-4.25). In C. glabrata ATCC, SN 152, and SN 164, the fluconazole/sodium bicarbonate combination exhibited a significant fungicidal activity (p< 0.05) but antagonistic effect in the case of SN 283. With exception of SN 283, a significant reduction was noted in phospholipase production in clinical isolates of C. glabrata treated with fluconazole/sodium bicarbonate combination. The PLB was down-regulated significantly by 0.168-0.515 fold in C. glabrata treated with fluconazole/sodium bicarbonate. The results suggested fluconazole/sodium bicarbonate to have a potential synergistic interaction in C. glabrata, and the underlying mechanism may be associated with phospholipase gene
Subject(s)
Phospholipases/antagonists & inhibitors , Fluconazole/agonists , Sodium Bicarbonate/agonists , Candida glabrata/pathogenicity , Efficacy , InfectionsABSTRACT
Candida glabrata is an opportunistic pathogenic yeast frequently causing infections in humans. Though it lacks typical virulence factors such as hyphal development, C. glabrata contains a remarkably large and diverse set of putative wall adhesins that is crucial for its success as pathogen. Here, we present an analysis of putative adhesins from the homology clusters V and VI. First, sequence similarity network analysis revealed relationships between cluster V and VI adhesins and S. cerevisiae haze protective factors (Hpf). Crystal structures of A-regions from cluster VI adhesins Awp1 and Awp3b reveal a parallel right-handed ß-helix domain that is linked to a C-terminal ß-sandwich. Structure solution of the A-region of Awp3b via single wavelength anomalous diffraction phasing revealed the largest known lanthanide cluster with 21 Gd3+ ions. Awp1-A and Awp3b-A show structural similarity to pectate lyases but binding to neither carbohydrates nor Ca2+ was observed. Phenotypic analysis of awp1Δ, awp3Δ, and awp1,3Δ double mutants did also not confirm their role as adhesins. In contrast, deletion mutants of the cluster V adhesin Awp2 in the hyperadhesive clinical isolate PEU382 demonstrated its importance for adhesion to polystyrene or glass, biofilm formation, cell aggregation and other cell surface-related phenotypes. Together with cluster III and VII adhesins our study shows that C. glabrata CBS138 can rely on a set of 42 Awp1-related adhesins with ß-helix/α-crystallin domain architecture for modifying the surface characteristics of its cell wall.
Subject(s)
Candida glabrata/genetics , Candidiasis/microbiology , Fungal Proteins/chemistry , Biofilms/growth & development , Candida glabrata/metabolism , Candida glabrata/pathogenicity , Cell Adhesion , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Wall/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Humans , Models, Molecular , Virulence FactorsABSTRACT
The opportunistic pathogen Candida glabrata is the second most frequent causative agent of vulvovaginal candidiasis (VVC), a disease that affects 70-75% of women at least once during their life. However, C. glabrata is almost avirulent in mice and normally incapable of inflicting damage to vaginal epithelial cells in vitro. We thus proposed that host factors present in vivo may influence C. glabrata pathogenicity. We, therefore, analyzed the impact of albumin, one of the most abundant proteins of the vaginal fluid. The presence of human, but not murine, albumin dramatically increased the potential of C. glabrata to damage vaginal epithelial cells. This effect depended on macropinocytosis-mediated epithelial uptake of albumin and subsequent proteolytic processing. The enhanced pathogenicity of C. glabrata can be explained by a combination of beneficial effects for the fungus, which includes an increased access to iron, accelerated growth, and increased adhesion. Screening of C. glabrata deletion mutants revealed that Hap5, a key regulator of iron homeostasis, is essential for the albumin-augmented damage potential. The albumin-augmented pathogenicity was reversed by the addition of iron chelators and a similar increase in pathogenicity was shown by increasing the iron availability, confirming a key role of iron. Accelerated growth not only led to higher cell numbers, but also to increased fungal metabolic activity and oxidative stress resistance. Finally, the albumin-driven enhanced damage potential was associated with the expression of distinct C. glabrata virulence genes. Transcriptional responses of the epithelial cells suggested an unfolded protein response (UPR) and ER-stress responses combined with glucose starvation induced by fast growing C. glabrata cells as potential mechanisms by which cytotoxicity is mediated.Collectively, we demonstrate that albumin augments the pathogenic potential of C. glabrata during interaction with vaginal epithelial cells. This suggests a role for albumin as a key player in the pathogenesis of VVC.
Subject(s)
Albumins/metabolism , Candida glabrata/pathogenicity , Candidiasis, Vulvovaginal/microbiology , Epithelial Cells/microbiology , Animals , Female , Humans , MiceABSTRACT
BACKGROUND: Vulvovaginal candidiasis (VVC) is a common vaginitis in females. The commonly used diagnostic method, 10% potassium hydroxide (KOH) smear microscopy, makes it not very easy to recognize fungi. METHODS: Vaginal secretions were collected from clinically suspected VVC patients and divided into four groups and examined using KOH, CFW (Calcofluor White), FB 85(fluorescent brightener 85), and culture. The data were statistically analyzed. RESULTS: In total, 110 patients with suspected VVC were recruited. The positive rates of KOH, CFW, FB 85, and the culture method were 68.2%, 64.5%, 61.8%, and 77%, respectively. According to the McNemar test, there was no statistically significant difference between the KOH, CFW, and the FB 85 methods (p > 0.05). However, CFW had a shorter diagnosis time than the KOH method and had a statistically significant difference (p < 0.001). Moreover, CFW has the highest sensitivity, specificity, and accuracy. In morphological recognition, it was easier to recognize fungal structures with CFW and FB 85 than with the KOH. CONCLUSIONS: The fluorescent method is a good method for the diagnosis of VVC. And the fungi can be found more quickly. Similar to CFW, FB 85 is also a potential good fluorescent reagent for the diagnosis of VVC and has potential value for application in clinical fungal infection diseases.
Subject(s)
Benzenesulfonates , Candidiasis, Vulvovaginal/diagnosis , Fluorescent Dyes , Microscopy, Fluorescence/methods , Adult , Candida albicans/pathogenicity , Candida glabrata/pathogenicity , Candida tropicalis/pathogenicity , Candidiasis, Vulvovaginal/microbiology , Female , Humans , Hydroxides , Image Processing, Computer-Assisted , Potassium Compounds , Sensitivity and Specificity , Time FactorsABSTRACT
Loss or inactivation of antivirulence genes is an adaptive strategy in pathogen evolution. Candida glabrata is an important opportunistic pathogen related to baker's yeast, with the ability to both quickly increase its intrinsic high level of azole resistance and persist within phagocytes. During C. glabrata's evolution as a pathogen, the mitochondrial DNA polymerase CgMip1 has been under positive selection. We show that CgMIP1 deletion not only triggers loss of mitochondrial function and a petite phenotype, but increases C. glabrata's azole and endoplasmic reticulum (ER) stress resistance and, importantly, its survival in phagocytes. The same phenotype is induced by fluconazole and by exposure to macrophages, conferring a cross-resistance between antifungals and immune cells, and can be found in clinical isolates despite a slow growth of petite strains. This suggests that petite constitutes a bet-hedging strategy of C. glabrata and, potentially, a relevant cause of azole resistance. Mitochondrial function may therefore be considered a potential antivirulence factor. IMPORTANCE Candida glabrata is an opportunistic pathogen whose incidence has been increasing in the last 40 years. It has risen to become the most prominent non-Candida albicans Candida (NCAC) species to cause candidemia, constituting about one-third of isolates in the United States, and steadily increasing in European countries and in Australia. Despite its clinical importance, C. glabrata's pathogenicity strategies remain poorly understood. Our research shows that loss of mitochondrial function and the resulting petite phenotype is advantageous for C. glabrata to cope with infection-related stressors, such as antifungals and host immune defenses. The (cross-)resistance against both these factors may have major implications in the clinical outcome of infections with this major fungal pathogen.
Subject(s)
Antifungal Agents/pharmacology , Candida glabrata/drug effects , Candida glabrata/genetics , Drug Resistance, Fungal/genetics , Fluconazole/pharmacology , Mitochondria/pathology , Candida glabrata/growth & development , Candida glabrata/pathogenicity , Candidiasis/microbiology , Gene Expression Regulation, Fungal , Humans , Macrophages/microbiology , Microbial Sensitivity Tests , Mitochondria/metabolism , Phagocytes , Phagocytosis , Virulence/geneticsABSTRACT
Aim: Melanin has been linked to pathogenesis in several fungi. They often produce melanin-like pigments in the presence of L-dihydroxyphenylalanine (L-DOPA), but this is poorly studied in Candida glabrata. Methods & materials:C. glabrata was grown in minimal medium with or without L-DOPA supplementation and submitted to a chemical treatment with denaturant and hot acid. Results:C. glabrata turned black when grown in the presence of L-DOPA, whereas cells grown without L-DOPA supplementation remained white. Biophysical properties demonstrated that the pigment was melanin. Melanized C. glabrata cells were effectively protected from azoles and amphotericin B, incubation at 42°C and macrophage killing. Conclusion: In the presence of L-DOPA, C. glabrata produces melanin, increases antifungal resistance and enhances host survival.
Aim: Melanin is a pigment that can help fungi to cause disease. Fungi often produce melanin-like pigments in the presence of L-dihydroxyphenylalanine (L-DOPA), but this is poorly studied in Candida glabrata, a yeast species that can cause human disease. Methods & materials:C. glabrata was grown in minimal medium with or without L-DOPA supplementation and submitted to a chemical treatment to isolate melanin. Results:C. glabrata turned black when grown in the presence of L-DOPA, whereas cells grown without L-DOPA supplementation remained white. Several experiments demonstrated that the black pigment was melanin. Melanized C. glabrata cells were effectively protected from antifungal drugs, incubation at 42°C and killing by cells of the immune system. Conclusion: In the presence of L-DOPA, C. glabrata produces melanin, increases antifungal resistance and has enhanced survival in contact with immunologic defense cells.
Subject(s)
Candida glabrata/pathogenicity , Candidiasis/microbiology , Melanins/metabolism , Amphotericin B/pharmacology , Animals , Antifungal Agents/pharmacology , Azoles/pharmacology , Candida glabrata/drug effects , Candida glabrata/metabolism , Candidiasis/immunology , Cytokines/metabolism , Dihydroxyphenylalanine/metabolism , Drug Resistance, Fungal , Macrophages/immunology , Mice , Microbial Viability , VirulenceABSTRACT
Computer simulations of mathematical models open up the possibility of assessing hypotheses generated by experiments on pathogen immune evasion in human whole-blood infection assays. We apply an interdisciplinary systems biology approach in which virtual infection models implemented for the dissection of specific immune mechanisms are combined with experimental studies to validate or falsify the respective hypotheses. Focusing on the assessment of mechanisms that enable pathogens to evade the immune response in the early time course of a whole-blood infection, the least-square error (LSE) as a measure for the quantitative agreement between the theoretical and experimental kinetics is combined with the Akaike information criterion (AIC) as a measure for the model quality depending on its complexity. In particular, we compare mathematical models with three different types of pathogen immune evasion as well as all their combinations: (i) spontaneous immune evasion, (ii) evasion mediated by immune cells, and (iii) pre-existence of an immune-evasive pathogen subpopulation. For example, by testing theoretical predictions in subsequent imaging experiments, we demonstrate that the simple hypothesis of having a subpopulation of pre-existing immune-evasive pathogens can be ruled out. Furthermore, in this study we extend our previous whole-blood infection assays for the two fungal pathogens Candida albicans and C. glabrata by the bacterial pathogen Staphylococcus aureus and calibrated the model predictions to the time-resolved experimental data for each pathogen. Our quantitative assessment generally reveals that models with a lower number of parameters are not only scored with better AIC values, but also exhibit lower values for the LSE. Furthermore, we describe in detail model-specific and pathogen-specific patterns in the kinetics of cell populations that may be measured in future experiments to distinguish and pinpoint the underlying immune mechanisms.
Subject(s)
Candidiasis/pathology , Immune Evasion/physiology , Models, Theoretical , Staphylococcal Infections/pathology , Candida albicans/pathogenicity , Candida glabrata/pathogenicity , Candidiasis/immunology , Humans , Staphylococcal Infections/immunology , Staphylococcus aureus/pathogenicity , Systems Biology/methodsABSTRACT
Candida glabrata is an emerging fungal pathogen whose success depends on its ability to resist antifungal drugs but also to thrive against host defenses. In this study, the predicted multidrug transporter CgTpo4 (encoded by ORF CAGL0L10912g) is described as a new determinant of virulence in C. glabrata, using the infection model Galleria mellonella. The CgTPO4 gene was found to be required for the C. glabrata ability to kill G. mellonella. The transporter encoded by this gene is also necessary for antimicrobial peptide (AMP) resistance, specifically against histatin-5. Interestingly, G. mellonella's AMP expression was found to be strongly activated in response to C. glabrata infection, suggesting AMPs are a key antifungal defense. CgTpo4 was also found to be a plasma membrane exporter of polyamines, especially spermidine, suggesting that CgTpo4 is able to export polyamines and AMPs, thus conferring resistance to both stress agents. Altogether, this study presents the polyamine exporter CgTpo4 as a determinant of C. glabrata virulence, which acts by protecting the yeast cells from the overexpression of AMPs, deployed as a host defense mechanism.
Subject(s)
Candida glabrata/genetics , Candidiasis/microbiology , Fungal Proteins/genetics , Multidrug Resistance-Associated Proteins/genetics , Polyamines/pharmacology , Pore Forming Cytotoxic Proteins/pharmacology , Antifungal Agents/metabolism , Antifungal Agents/pharmacology , Candida glabrata/drug effects , Candida glabrata/metabolism , Candida glabrata/pathogenicity , Candidiasis/drug therapy , Candidiasis/metabolism , Drug Resistance, Fungal , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Genes, Fungal , Histatins/metabolism , Histatins/pharmacology , Humans , Multidrug Resistance-Associated Proteins/metabolism , Polyamines/metabolism , Pore Forming Cytotoxic Proteins/metabolism , VirulenceABSTRACT
Candida glabrata is a clinically relevant human pathogen with the ability to form high recalcitrant biofilms that contribute to the establishment and persistence of infection. A defining trait of biofilms is the auto-produced matrix, which is suggested to have structural, virulent and protective roles. Thus, elucidation of matrix components, their function and modulation by the host environment is crucial to disclose their role in C. glabrata pathogenesis. As a major step toward this end, this study aimed to reveal, for the first time, the matrix proteome of C. glabrata biofilms, to characterize it with bioinformatic tools and to study its modulation by the environmental pH (acidic and neutral). The results showed the presence of several pH-specific matrix proteins (51 acidic- and 206 neutral-specific) and also proteins commonly found at both pH conditions (236). Of note, several proteins related to mannan and ß-glucan metabolism, which have a potential role in the delivery/organization of carbohydrates in the matrix, were found in both pH conditions but in much higher quantity under the neutral environment. Additionally, several virulence-related proteins, including epithelial adhesins, yapsins and moonlighting enzymes, were found among matrix proteins. Importantly, several proteins seem to have a non-canonical secretion pathway and Pdr1 was found to be a potential regulator of matrix proteome. Overall, this study indicates a relevant impact of environmental cues in the matrix proteome and provides a unique resource for further functional investigation of matrix proteins, contributing to the identification of potential targets for the development of new therapies against C. glabrata biofilms.
Subject(s)
Biofilms , Candida glabrata/metabolism , Fungal Proteins/isolation & purification , Hydrogen-Ion Concentration , Proteome , Candida glabrata/drug effects , Candida glabrata/genetics , Candida glabrata/pathogenicity , Carbohydrate Metabolism , Cell Adhesion , Computational Biology , Culture Media/pharmacology , Gene Expression Regulation, Fungal , Protein Interaction Mapping , Transcription Factors/metabolism , VirulenceABSTRACT
How cells respond to DNA damage is key to maintaining genome integrity or facilitating genetic change. In fungi, DNA damage responses have been extensively characterized in the model budding yeast Saccharomyces cerevisiae, which is generally not pathogenic. However, it is not clear how closely these responses resemble those in fungal pathogens, in which genetic change plays an important role in the evolutionary arms race between pathogen and host and the evolution of antifungal drug resistance. A close relative of S. cerevisiae, Candida glabrata, is an opportunistic pathogen that displays high variability in chromosome structure among clinical isolates and rapidly evolves antifungal drug resistance. The mechanisms facilitating such genomic flexibility and evolvability in this organism are unknown. Recently we characterized the DNA damage response of C. glabrata and identified several features that distinguish it from the well characterized DNA damage response of S. cerevisiae. First, we discovered that, in contrast to the established paradigm, C. glabrata effector kinase Rad53 is not hyperphosphorylated upon DNA damage. We also uncovered evidence of an attenuated DNA damage checkpoint response, wherein in the presence of DNA damage C. glabrata cells did not accumulate in S-phase and proceeded with cell division, leading to aberrant mitoses and cell death. Finally, we identified evidence of transcriptional rewiring of the DNA damage response of C. glabrata relative to S. cerevisiae, including an upregulation of genes involved in mating and meiosis-processes that have not been reported in C. glabrata. Together, these results open new possibilities and raise tantalizing questions of how this major fungal pathogen facilitates genetic change.
Subject(s)
Candida glabrata/genetics , Candidiasis/genetics , DNA Damage/genetics , Genetic Variation/genetics , Candida glabrata/pathogenicity , Candidiasis/microbiology , Drug Resistance, Fungal/genetics , Genes, Mating Type, Fungal/genetics , Humans , Meiosis/geneticsABSTRACT
Experimental evolution is an experiment class of its own; instead of requiring an a priori hypothesis, the genetic adaptation of microbes to defined environments tells us about the underlying pathways and mechanisms. Such experiments are often deceptively simple in their design, based on a single abiotic stressor and what is in essence a long-term continuous culture. However, they generally provide a starting point to thorough follow-up analyses (which are specific for the organism at hand and not part of this method chapter). In this chapter, we describe a method to use a biotic stressor which is frequently encountered by pathogenic fungi-macrophage-like cells-in a serial passaging regime. Experimental evolution under such conditions can reveal new virulence attributes and mechanisms by selecting for adaptive mutations against the host cell-induced stress.It is important to note that every evolution experiment is different, and these techniques should be taken as a general guideline to be adapted to different organisms and questions. Then, it is a powerful tool with many potential applications in pathobiology research.
Subject(s)
Candida albicans/pathogenicity , Candida glabrata/pathogenicity , Cell Proliferation , Evolution, Molecular , Macrophages/microbiology , Animals , Candida albicans/genetics , Candida albicans/growth & development , Candida glabrata/genetics , Candida glabrata/growth & development , Gene Expression Regulation, Fungal , Host-Pathogen Interactions , Mice , Mutation , Phagocytosis , RAW 264.7 Cells , Time Factors , VirulenceABSTRACT
The mechanisms by which Candida glabrata resists host defense peptides and caspofungin are incompletely understood. To identify transcriptional regulators that enable C. glabrata to withstand these classes of stressors, a library of 215 C. glabrata transcriptional regulatory deletion mutants was screened for susceptibility to both protamine and caspofungin. We identified eight mutants that had increased susceptibility to both host defense peptides and caspofungin. Of these mutants, six were deleted for genes that were predicted to specify proteins involved in histone modification. These genes were ADA2, GCN5, SPT8, HOS2, RPD3, and SPP1 Deletion of ADA2, GCN5, and RPD3 also increased susceptibility to mammalian host defense peptides. The Δada2 and Δgcn5 mutants had increased susceptibility to other stressors, such as H2O2 and SDS. In the Galleria mellonella model of disseminated infection, the Δada2 and Δgcn5 mutants had attenuated virulence, whereas in neutropenic mice, the virulence of the Δada2 and Δrpd3 mutants was decreased. Thus, histone modification plays a central role in enabling C. glabrata to survive host defense peptides and caspofungin, and Ada2 and Rpd3 are essential for the maximal virulence of this organism during disseminated infection.
Subject(s)
Candida glabrata/genetics , Candida glabrata/pathogenicity , Fungal Proteins/genetics , Host-Pathogen Interactions/genetics , Transcription Factors/genetics , Virulence/genetics , Gene Deletion , Genetic Variation , Humans , MutationABSTRACT
Candida glabrata is an opportunistic human fungal pathogen and is frequently present in the human microbiome. It has a high relative resistance to environmental stresses and several antifungal drugs. An important component involved in microbial stress tolerance is trehalose. In this work, we characterized the three C. glabrata trehalase enzymes Ath1, Nth1 and Nth2. Single, double and triple deletion strains were constructed and characterized both in vitro and in vivo to determine the role of these enzymes in virulence. Ath1 was found to be located in the periplasm and was essential for growth on trehalose as sole carbon source, while Nth1 on the other hand was important for oxidative stress resistance, an observation which was consistent by the lower survival rate of the NTH1 deletion strain in human macrophages. No significant phenotype was observed for Nth2. The triple deletion strain was unable to establish a stable colonization of the gastrointestinal (GI) tract in mice indicating the importance of having trehalase activity for colonization in the gut.
Subject(s)
Candida glabrata/enzymology , Candida glabrata/genetics , Fungal Proteins/genetics , Gastrointestinal Tract/microbiology , Stress, Physiological/genetics , Trehalase/genetics , Animals , Candida glabrata/drug effects , Candida glabrata/pathogenicity , Female , Fungal Proteins/metabolism , Humans , Hydrogen Peroxide/pharmacology , Macrophages/microbiology , Mice , Mice, Inbred BALB C , Oxidative Stress/genetics , RAW 264.7 Cells , Trehalase/classification , Trehalase/metabolism , VirulenceABSTRACT
BACKGROUND: Candida auris infections have recently emerged worldwide, and this species is highly capable of colonization and is associated with high levels of mortality. However, strain-dependent differences in colonization capabilities and virulence have not yet been reported. OBJECTIVES: In the present study, we aimed to clarify the differences between clinically isolated invasive and non-invasive strains of C. auris. METHODS: We evaluated colonization, dissemination, and survival rates in wild C57BL/6J mice inoculated with invasive or non-invasive strains of C. auris under cortisone acetate immunosuppression, comparing with those of Candida albicans and Candida glabrata infections. We also evaluated the potency of biofilm formation. RESULTS: Stool fungal burdens were significantly higher in mice inoculated with the invasive strains than in those infected with the non-invasive strain. Along with intestinal colonization, liver and kidney fungal burdens were also significantly higher in mice inoculated with the invasive strains. In addition, histopathological findings revealed greater dissemination and colonization of the invasive strains. Regarding biofilm-forming capability, the invasive strain of C. auris exhibited a significantly higher capacity of producing biofilms. Moreover, inoculation with the invasive strains resulted in significantly greater loss of body weight than that noted following infection with the non-invasive strain. CONCLUSIONS: Invasive strains showed higher colonization capability and rates of dissemination from gastrointestinal tracts under cortisone acetate immunosuppression than non-invasive strains, although the mortality rates caused by C. auris were lower than those caused by C. albicans.
Subject(s)
Candida/physiology , Candidiasis, Invasive/pathology , Candidiasis/pathology , Gastrointestinal Tract/microbiology , Animals , Biofilms/growth & development , Candida/pathogenicity , Candida albicans/pathogenicity , Candida albicans/physiology , Candida glabrata/pathogenicity , Candida glabrata/physiology , Candidiasis/microbiology , Candidiasis, Invasive/microbiology , Gastrointestinal Tract/pathology , Male , Mice , Mice, Inbred C57BL , VirulenceABSTRACT
La fístula traqueoesofágica consiste en una comunicación anómala entre el sistema respiratorio y el digestivo, que puede ser a causa de una enfermedad congénita, o bien secundaria a una patología tal como la infecciosa, la tumoral o postraumática. Entre las etiologías infecciosas, Candida o Aspergillus son dos hongos que provocan esofagitis fúngica, la cual podría complicarse con esta entidad, aunque son casos extremadamente raros. El diagnóstico de las fístulas se basa en la clínica, las pruebas de imagen como la TC y la endoscopia, donde se podrá apreciar la solución de continuidad. Este defecto debe ser tratado mediante cirugía o endoscópicamente
The tracheoesophageal fistula consists of an abnormal communication between the respiratory and digestive systems, which may be due to a congenital disease or secondary to a pathology such as infectious, tumorous or post-traumatic. Among the infectious etiologies, Candida or Aspergillus are two fungi that cause fungal esophagitis, which could be complicated by this entity, although they are extremely rare cases. The diagnosis of fistulas is based on the clinic, imaging tests such as CT and endoscopy, where the solution of continuity can be appreciated. This defect must be treated surgically or endoscopically, a technique that is currently on the rise due to its fewer complications
Subject(s)
Humans , Female , Middle Aged , Tracheoesophageal Fistula/diagnostic imaging , Tracheoesophageal Fistula/microbiology , Candida glabrata/pathogenicity , Candidiasis/complications , Esophagitis/complications , Rare Diseases , Tomography, X-Ray Computed , Endoscopy, Gastrointestinal , Esophagitis/microbiologyABSTRACT
BACKGROUND: Candida glabrata yeast is the second cause of candidiasis worldwide. Differs from other yeasts since assimilates only glucose and trehalose (a characteristic used in rapid identification tests for this pathogen) by secreting into the medium a highly active acid trehalase encoded by the CgATH1 gene. OBJECTIVE: This study aimed to characterise the function of the acid trehalase in the physiopathology of C. glabrata. METHODS: Gene deletion was performed to obtain a mutant ath1Δ strain, and the ability of the ath1Δ strain to grow in trehalase, or the presence of trehalase activity in the ath1Δ yeast cells, was verified. We also tested the virulence of the ath1Δ strain in a murine model of infection. FINDINGS: The ath1Δ mutant strain grows normally in the presence of glucose, but loses its ability to grow in trehalose. Due to the high acid trehalase activity present in wild-type cells, the cytoplasmic neutral trehalase activity is only detected in the ath1Δ strain. We also observed a significantly lower virulence of the ath1Δ strain in a murine model of infection with either normal or immunocompromised mice. MAIN CONCLUSIONS: The acid trehalase is involved in the hydrolysis of external trehalose by C. glabrata, and the enzyme also plays a major virulence role during infectivity.