Fungal Pathogen Candida Glabrata Research Articles

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.

Evaluation of in-vitro antimicrobial activity of Artemisia  apiacea H. and Scutellaria baicalensis G. extracts.

In traditional Korean medicine, Artemisia apiacea H. (ART) and Scutellaria baicalensis G. (SCU) are combined for the treatment of malaria and other malaria-like diseases. Because SCU is well-known as an antibacterial agent, the antimicrobial effect of a mixture of ART and SCU was investigated. Plant samples were purchased from Kyungdong mart and extracted with 70 % ethanol. The in vitro antimicrobial activity of ART and SCU against pathogenic fungi (Aspergillus niger, Aspergillus oryzae, Candida albicans, Candida tropicalis and Candida glabrata), Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) was evaluated using a broth microdilution assay, modified-disc diffusion and agar dilution methods with further CH2Cl2-fractionated ART, SCU and a mixture of ART/SCU (at a ratio of 3 : 5) (THAN-1). ART and SCU were effective against A. niger, C. albicans, B. subtilis and S. aureus. The range of minimum inhibitory concentration (MIC) values was 0.03125 to 4 mg ml-1 in the ART and SCU treatments. ART exhibited stronger activity than SCU. Interestingly, a 3 : 5 ratio mixture of ART and SCU (THAN-1) showed stronger antimicrobial activity than ART or SCU used individually. A treatment using a mixture of herbs such as THAN-1 would be useful in the suppression of the growth of pathogenic bacterial and fungal strains.

Positive autoregulation and repression of transactivation are key regulatory features of the Candida glabrata Pdr1 transcription factor.

Resistance to azole drugs, the major clinical antifungal compounds, is most commonly due to gain-of-function (GOF) substitution mutations in a gene called PDR1 in the fungal pathogen Candida glabrata. PDR1 encodes a zinc cluster-containing transcription factor. GOF forms of Pdr1 drive high level expression of downstream target gene expression with accompanying azole resistance. PDR1 has two homologous genes in Saccharomyces cerevisiae, called ScPDR1 and ScPDR3. This study provides evidence that the PDR1 gene in C. glabrata represents a blend of the properties found in the two S. cerevisiae genes. We demonstrated that GOF Pdr1 derivatives are overproduced at the protein level and less stable than the wild-type protein. Overproduction of wild-type Pdr1 increased target gene expression but to a lesser extent than GOF derivatives. Site-directed mutagenesis of Pdr1 binding sites in the PDR1 promoter provided clear demonstration that autoregulation of PDR1 is required for its normal function. An internal deletion mutant of Pdr1 lacking its central regulatory domain behaved as a hyperactive transcription factor that was lethal unless conditionally expressed. A full understanding of the regulation of Pdr1 will provide a new avenue of interfering with azole resistance in C.glabrata.

Short-Form Bomanins Mediate Humoral Immunity in Drosophila

The Bomanins (Boms) are a family of a dozen secreted peptides that mediate the innate immune response governed by the Drosophila Toll receptor. We recently showed that deleting a cluster of 10 Bom genes blocks Toll-mediated defenses against a range of fungi and gram-positive bacteria. Here, we characterize the activity of individual Bom family members. We provide evidence that the Boms overlap in function and that a single Bom gene encoding a mature peptide of just 16 amino acids can act largely or entirely independent of other family members to provide phenotypic rescue in vivo. We further demonstrate that the Boms function in Drosophila humoral immunity, mediating the killing of the fungal pathogen Candida glabrata in an in vitro assay of cell-free hemolymph. In addition, we find that the level of antifungal activity both in vivo and in vitro is linked to the level of Bom gene expression. Although Toll dictates expression of the antimicrobial peptides (AMPs) drosomycin and metchnikowin, we find no evidence that Boms act by modifying the expression of the mature forms of these antifungal AMPs.

Benzoxaboroles as Efficient Inhibitors of the β-Carbonic Anhydrases from Pathogenic Fungi: Activity and Modeling Study.

A series of 6-substituted benzoxaboroles were investigated as inhibitors of the β-class carbonic anhydrase from three pathogenic fungi (Cryptococcus neoformans, Candida glabrata, and Malassezia globosa). Independently from the nature of the substituents on the phenyl of the urea/thiourea group, all reported derivatives showed nanomolar inhibitory activities against Can2 and CgNce103 vs micromolar inhibition against MgCA. Selectivity over human CA I and CA II was noticed. The observed structure-activity relationship trends have been rationalized by modeling study of selected compounds into the active site of Can2 and MgCA. The present letter demonstrates that benzoxaborole chemotype may offer interesting opportunities for the inhibition of β-CA from pathogenic fungi and for the development of antifungal agents with a new mechanism of action.

Metal based sulfanilamides: A note on their synthesis, spectral characterization, and antimicrobial activity

Objectives of the current study were synthesis, spectral characterization and biological screening of sulfanilamide derived Schiff bases and their metal based compounds. Sulfanilamide Schiff bases (L 1 –L 3 ) were synthesized by condensation of 4-aminobenzenesulfanilamide with 1-(furan-2-yl)ethanone, 1-(thiophene-2-yl)-ethanone, and 1-acetylindoline-2,3-dione. The ligands were used for preparation of their Co(II), Ni(II), Cu(II), and Zn(II) complexes by using metals chlorides in metal: ligand (1: 2) molar ratio. All metal chelates had octahedral geometry with bidentate ligands. The ligands and their metal complexes were characterized by physical, spectral and analytical data, and screened for in-vitro antibacterial activity against six bacterial pathogens (Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus, and Bacillus subtilis) and for in vitro antifungal activity against six fungal pathogens (Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani, and Candida glabrata). The results of antimicrobial studies revealed that the ligands activity was significantly increased upon chelation.

Unexpected effects of azole transporter inhibitors on antifungal susceptibility in Candida glabrata and other pathogenic Candida species.

The pathogenic fungus Candida glabrata is often resistant to azole antifungal agents. Drug efflux through azole transporters, such as Cdr1 and Cdr2, is a key mechanism of azole resistance and these genes are under the control of the transcription factor Pdr1. Recently, the monoamine oxidase A (MAO-A) inhibitor clorgyline was shown to inhibit the azole efflux pumps, leading to increased azole susceptibility in C. glabrata. In the present study, we have evaluated the effects of clorgyline on susceptibility of C. glabrata to not only azoles, but also to micafungin and amphotericin B, using wild-type and several mutant strains. The addition of clorgyline to the culture media increased fluconazole susceptibility of a C. glabrata wild-type strain, whereas micafungin and amphotericin B susceptibilities were markedly decreased. These phenomena were also observed in other medically important Candida species, including Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida krusei. Expression levels of CDR1, CDR2 and PDR1 mRNAs and an amount of Cdr1 protein in the C. glabrata wild-type strain were highly increased in response to the treatment with clorgyline. However, loss of Cdr1, Cdr2, Pdr1, and a putative clorgyline target (Fms1), which is an ortholog of human MAO-A, or overexpression of CDR1 did not affect the decreased susceptibility to micafungin and amphotericin B in the presence of clorgyline. The presence of other azole efflux pump inhibitors including milbemycin A4 oxime and carbonyl cyanide 3-chlorophenylhydrazone also decreased micafungin susceptibility in C. glabrata wild-type, Δcdr1, Δcdr2, and Δpdr1 strains. These findings suggest that azole efflux pump inhibitors increase azole susceptibility but concurrently induce decreased susceptibility to other classes of antifungals independent of azole transporter functions.

The Fungal Pathogen Candida glabrata Does Not Depend on Surface Ferric Reductases for Iron Acquisition

Iron acquisition is a crucial virulence determinant for many bacteria and fungi, including the opportunistic fungal pathogens Candida albicans and C. glabrata. While the diverse strategies used by C. albicans for obtaining iron from the host are well-described, much less is known about the acquisition of this micronutrient from host sources by C. glabrata – a distant relative of C. albicans with closer evolutionary ties to Saccharomyces cerevisiae, which nonetheless causes severe clinical symptoms in humans. Here we show that C. glabrata is much more restricted than C. albicans in using host iron sources, lacking, for example, the ability to grow on transferrin and hemin/hemoglobin. Instead, C. glabrata is able to use ferritin and non-protein-bound iron (FeCl3) as iron sources in a pH-dependent manner. As in other fungal pathogens, iron-dependent growth requires the reductive high affinity (HA) iron uptake system. Typically highly conserved, this uptake mechanism normally relies on initial ferric reduction by cell-surface ferric reductases. The C. glabrata genome contains only three such putative ferric reductases, which were found to be dispensable for iron-dependent growth. In addition and in contrast to C. albicans and S. cerevisiae, we also detected no surface ferric reductase activity in C. glabrata. Instead, extracellular ferric reduction was found in this and the two other fungal species, which was largely dependent on an excreted low-molecular weight, non-protein ferric reductant. We therefore propose an iron acquisition strategy of C. glabrata which differs from other pathogenic fungi, such as C. albicans, in that it depends on a limited set of host iron sources and that it lacks the need for surface ferric reductases. Extracellular ferric reduction by a secreted molecule possibly compensates for the loss of surface ferric reductase activity in the HA iron uptake system.

Widespread Inter- and Intra-Domain Horizontal Gene Transfer of d-Amino Acid Metabolism Enzymes in Eukaryotes

Analysis of the growing number of available fully-sequenced genomes has shown that Horizontal Gene Transfer (HGT) in eukaryotes is more common than previously thought. It has been proposed that genes with certain functions may be more prone to HGT than others, but we still have a very poor understanding of the selective forces driving eukaryotic HGT. Recent work uncovered that d-amino acid racemases have been commonly transferred from bacteria to fungi, but their role in the receiving organisms is currently unknown. Here, we set out to assess whether d-amino acid racemases are commonly transferred to and between eukaryotic groups. For this we performed a global survey that used a novel automated phylogeny-based HGT-detection algorithm (Abaccus). Our results revealed that at least 7.0% of the total eukaryotic racemase repertoire is the result of inter- or intra-domain HGT. These transfers are significantly enriched in plant-associated fungi. For these, we hypothesize a possible role for the acquired racemases allowing to exploit minoritary nitrogen sources in plant biomass, a nitrogen-poor environment. Finally, we performed experiments on a transferred aspartate-glutamate racemase in the fungal human pathogen Candida glabrata, which however revealed no obvious biological role.

Genetic Drivers of Multidrug Resistance in Candida glabrata.

Both the incidence of invasive fungal infections and rates of multidrug resistance associated with fungal pathogen Candida glabrata have increased in recent years. In this perspective, we will discuss the mechanisms underlying the capacity of C. glabrata to rapidly develop resistance to multiple drug classes, including triazoles and echinocandins. We will focus on the extensive genetic diversity among clinical isolates of C. glabrata, which likely enables this yeast to survive multiple stressors, such as immune pressure and antifungal exposure. In particular, over half of C. glabrata clinical strains collected from U.S. and non-U.S. sites have mutations in the DNA mismatch repair gene MSH2, leading to a mutator phenotype and increased frequencies of drug-resistant mutants in vitro. Furthermore, recent studies and data presented here document extensive chromosomal rearrangements among C. glabrata strains, resulting in a large number of distinct karyotypes within a single species. By analyzing clonal, serial isolates derived from individual patients treated with antifungal drugs, we were able to document chromosomal changes occurring in C. glabrata in vivo during the course of antifungal treatment. Interestingly, we also show that both MSH2 genotypes and chromosomal patterns cluster consistently into specific strain types, indicating that C. glabrata has a complex population structure where genomic variants arise, perhaps during the process of adaptation to environmental changes, and persist over time.

Opportunistic fungal pathogen Candida glabrata circulates between humans and yellow-legged gulls.

The opportunistic pathogenic yeast Candida glabrata is a component of the mycobiota of both humans and yellow-legged gulls that is prone to develop fluconazole resistance. Whether gulls are a reservoir of the yeast and facilitate the dissemination of human C. glabrata strains remains an open question. In this study, MLVA genotyping highlighted the lack of genetic structure of 190 C. glabrata strains isolated from either patients in three hospitals or fecal samples collected from gull breeding colonies located in five distinct areas along the French Mediterranean littoral. Fluconazole-resistant isolates were evenly distributed between both gull and human populations. These findings demonstrate that gulls are a reservoir of this species and facilitate the diffusion of C. glabrata and indirect transmission to human or animal hosts via environmental contamination. This eco-epidemiological view, which can be applied to other vertebrate host species, broadens our perspective regarding the reservoirs and dissemination patterns of antifungal-resistant human pathogenic yeast.

Antimicrobial metal-based thiophene derived compounds

A novel series of thiophene derived Schiff bases and their transition metal- [Co(II), Cu(II), Zn(II), Ni(II)] based compounds are reported. The Schiff bases act as tridentate ligands toward metal ions via azomethine-N, deprotonated-N of ammine substituents and S-atom of thienyl moiety. The synthesized ligands along with their metal complexes were screened for their in vitro antibacterial activity against six bacterial pathogens (Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus and Bacillus subtilis) and for antifungal activity against six fungal pathogens (Trichophytonlongifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glabrata). The results of antimicrobial studies revealed the free ligands to possess potential activity which significantly increased upon chelation.

Genome engineering in the yeast pathogen Candida glabrata using the CRISPR-Cas9 system.

Among Candida species, the opportunistic fungal pathogen Candida glabrata has become the second most common causative agent of candidiasis in the world and a major public health concern. Yet, few molecular tools and resources are available to explore the biology of C. glabrata and to better understand its virulence during infection. In this study, we describe a robust experimental strategy to generate loss-of-function mutants in C. glabrata. The procedure is based on the development of three main tools: (i) a recombinant strain of C. glabrata constitutively expressing the CRISPR-Cas9 system, (ii) an online program facilitating the selection of the most efficient guide RNAs for a given C. glabrata gene, and (iii) the identification of mutant strains by the Surveyor technique and sequencing. As a proof-of-concept, we have tested the virulence of some mutants in vivo in a Drosophila melanogaster infection model. Our results suggest that yps11 and a previously uncharacterized serine/threonine kinase are involved, directly or indirectly, in the ability of the pathogenic yeast to infect this model host organism.

Candida glabrata encodes a longer variant of the mating type (MAT) alpha2 gene in the mating type-like MTL3 locus, which can form homodimers.

The fungal pathogen Candida glabrata is a haploid asexual yeast. Candida glabrata contains orthologs of the genes that control mating and cell-type identity in other fungi, which encode putative transcription factors localized in the MAT locus in Saccharomyces cerevisiae or MTL in other fungi. Candida glabrata contains three copies of the CgMTL locus but only CgMTL1 correctly expresses the information encoded in it. CgMTL1 can encode the Cg A1: gene ( A: information), or the Cgalpha1 and Cgalpha2 genes (alpha information). CgMTL2 contains an identical copy of the Cg A1: gene. CgMTL3 contains an identical copy of the Cgalpha1 gene but a longer variant of the Cgalpha2 gene that we termed Cgalpha3. In S. cerevisiae diploid cells, that express Sc A: and Scalpha information, Sc A1: and Scalpha2 proteins form a heterodimer, which represses genes expressed only in haploid cells and some genes involved in stress response. We constructed C. glabrata strains that simultaneously express Cg A1: and Cgalpha2 or Cg A1: and Cgalpha3 genes. We did not find any phenotype in these strains when grown under a large variety of stress and nutritional conditions. However, we detected an interaction between Cg A1: and Cgalpha2 but not between Cg A1: and Cgalpha3 by Bimolecular Fluorescence Complementation and co-immunoprecipitation assays.

Roles of vacuolar H+-ATPase in the oxidative stress response of Candida glabrata.

Vacuolar H(+)-ATPase (V-ATPase) is responsible for the acidification of eukaryotic intracellular compartments and plays an important role in oxidative stress response (OSR), but its molecular bases are largely unknown. Here, we investigated how V-ATPase is involved in the OSR by using a strain lacking VPH2, which encodes an assembly factor of V-ATPase, in the pathogenic fungus Candida glabrata The loss of Vph2 resulted in increased H2O2 sensitivity and intracellular reactive oxygen species (ROS) level independently of mitochondrial functions. The Δvph2 mutant also displayed growth defects under alkaline conditions accompanied by the accumulation of intracellular ROS and these phenotypes were recovered in the presence of the ROS scavenger N-acetyl-l-cysteine. Both expression and activity levels of mitochondrial manganese superoxide dismutase (Sod2) and catalase (Cta1) were decreased in the Δvph2 mutant. Phenotypic analyses of strains lacking and overexpressing these genes revealed that Sod2 and Cta1 play a predominant role in endogenous and exogenous OSR, respectively. Furthermore, supplementation of copper and iron restored the expression of SOD2 specifically in the Δvph2 mutant, suggesting that the homeostasis of intracellular cupper and iron levels maintained by V-ATPase was important for the Sod2-mediated OSR. This report demonstrates novel roles of V-ATPase in the OSR in C. glabrata.

Prevalent mutator genotype identified in fungal pathogen Candida glabrata promotes multi-drug resistance.

The fungal pathogen Candida glabrata has emerged as a major health threat since it readily acquires resistance to multiple drug classes, including triazoles and/or echinocandins. Thus far, cellular mechanisms promoting the emergence of resistance to multiple drug classes have not been described in this organism. Here we demonstrate that a mutator phenotype caused by a mismatch repair defect is prevalent in C. glabrata clinical isolates. Strains carrying alterations in mismatch repair gene MSH2 exhibit a higher propensity to breakthrough antifungal treatment in vitro and in mouse models of colonization, and are recovered at a high rate (55% of all C. glabrata recovered) from patients. This genetic mechanism promotes the acquisition of resistance to multiple antifungals, at least partially explaining the elevated rates of triazole and multi-drug resistance associated with C. glabrata. We anticipate that identifying MSH2 defects in infecting strains may influence the management of patients on antifungal drug therapy.

Utilising established SDL-screening methods as a tool for the functional genomic characterisation of model and non-model organisms.

The trend for large-scale genetic and phenotypic screens has revealed a wealth of information on biological systems. A major challenge is understanding how genes function and putative roles in networks. The majority of current gene knowledge is garnered from studies utilising the model yeast Saccharomyces cerevisiae. We demonstrate that synthetic dosage lethal genetic array methodologies can be used to study genetic networks in other yeasts, namely the fungal pathogen Candida glabrata, which has limited forward genetic tools, due to the lack of 'natural' mating. We performed two SDL screens in S. cerevisiae, overexpressing the transcriptional regulator UME6 as bait in the first screen and its C. glabrata ortholog CAGL0F05357g in the second. Analysis revealed that SDL maps share 204 common interactors, with 10 genetic interactions unique to C. glabrata indicating a level of genetic rewiring, indicative of linking genotype to phenotype in fungal pathogens. This was further validated by incorporating our results into the global genetic landscape map of the cell from Costanzo et al. to identify common and novel gene attributes. This data demonstrated the utility large data sets and more robust analysis made possible by interrogating exogenous genes in the context of the eukaryotic global genetic landscape.

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.

Characterization of Cell Wall Proteins in Saccharomyces cerevisiae Clinical Isolates Elucidates Hsp150p in Virulence.

The budding yeast Saccharomyces cerevisiae has recently been described as an emerging opportunistic fungal pathogen. Fungal cell wall mannoproteins have been demonstrated to be involved in adhesion to inert surfaces and might be engaged in virulence. In this study, we observed four clinical isolates of S. cerevisiae with relatively hydrophobic cell surfaces. Yeast cell wall subproteome was evaluated quantitatively by liquid chromatography/tandem mass spectrometry. We identified totally 25 cell wall proteins (CWPs) from log-phase cells, within which 15 CWPs were quantified. The abundance of Scw10p, Pst1p, and Hsp150p/Pir2p were at least 2 folds higher in the clinical isolates than in S288c lab strain. Hsp150p is one of the members in Pir family conserved in pathogenic fungi Candida glabrata and Candida albicans. Overexpression of Hsp150p in lab strain increased cell wall integrity and potentially enhanced the virulence of yeast. Altogether, these results demonstrated that quantitative cell wall subproteome was analyzed in clinical isolates of S. cerevisiae, and several CWPs, especially Hsp150p, were found to be expressed at higher levels which presumably contribute to strain virulence and fungal pathogenicity.

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.