Azole Resistance In Candida Glabrata Research Articles

Molecular mechanisms of azole resistance in Candida glabrata isolated from oropharyngeal candidiasis in head and neck cancer patients

ObjectiveThe aim of the current work was to assess the molecular mechanisms of fluconazole-resistant Candida glabrata strains isolated from oropharyngeal candidiasis (OPC) in head and neck patients, as well as evaluation of virulence factors. DesignAntifungal susceptibility pattern of sixty six clinical isolates of C. glabrata were evaluated by broth-microdilution method. The expression of ERG11, CDR1, CDR2, PDR1 genes as well as ERG11 gene capable of possible mutations was also detected in 21 fluconazol-resistant C. glabrata isolates. Phospholipase and proteinase activity of these isolates was estimated, too. The correlation between the virulence factors, antifungal susceptibility patterns and cancer type was also analyzed. ResultsSeven synonymous and four non-synonymous mutations were found in 21 fluconazole-resistant C. glabrata isolates; subsequently, four amino acid substitutions including H257P, Q47H, S487Y and I285N were then reported for the first time. High expression of CDR1 and PDR1 in related to other gene findings were tested in these isolates. Additionally, there was no significant difference between stage of cancer and MIC of all antimicrobial drugs. Significant differences between MIC of fluconazole, voriconazole and cancer types were also, found. The proteinase activity (92.4%) was higher than phospholipase activity in the isolates. Further, no significant difference between proteinase (rs: 0.003), phospholipase (rs: −0.107) activity and fluconazole MICs was observed. ConclusionC. glabrata isolated from OPC in head and neck patients represented high capacities for proteolytic enzymes activity and high mRNA level of CDR1 and PDR1 gene and ERG11 mutations play an important role in azole drug resistance.

In Candida glabrata, ERMES Component GEM1 Controls Mitochondrial Morphology, mtROS, and Drug Efflux Pump Expression, Resulting in Azole Susceptibility

Mitochondrial dysfunction or morphological abnormalities in human pathogenic fungi are known to contribute to azole resistance; however, the underlying molecular mechanisms are unknown. In this study, we investigated the link between mitochondrial morphology and azole resistance in Candida glabrata, which is the second most common cause of human candidiasis worldwide. The ER-mitochondrial encounter structure (ERMES) complex is thought to play an important role in the mitochondrial dynamics necessary for mitochondria to maintain their function. Of the five components of the ERMES complex, deletion of GEM1 increased azole resistance. Gem1 is a GTPase that regulates the ERMES complex activity. Point mutations in GEM1 GTPase domains were sufficient to confer azole resistance. The cells lacking GEM1 displayed abnormalities in mitochondrial morphology, increased mtROS levels, and increased expression of azole drug efflux pumps encoded by CDR1 and CDR2. Interestingly, treatment with N-acetylcysteine (NAC), an antioxidant, reduced ROS production and the expression of CDR1 in Δgem1 cells. Altogether, the absence of Gem1 activity caused an increase in mitochondrial ROS concentration, leading to Pdr1-dependent upregulation of the drug efflux pump Cdr1, resulting in azole resistance.

A gain-of-function mutation in PDR1 of Candida glabrata decreases EPA1 expression and attenuates adherence to epithelial cells through enhancing recruitment of the Mediator subunit Gal11A

Genetic studies have revealed critical roles of transcription factor Pdr1 and the Mediator subunit Gal11A in regulating azole resistance in Candida glabrata. Recently, PDR1 gain-of-function (GOF) mutations have been shown to not only increase azole resistance but also enhance adherence during C. glabrata infection. However, mechanism of how Pdr1 regulates adherence, especially the implication of PDR1 GOF mutations in the regulation of the major adhesin gene EPA1, remains uncharacterized. Initially, we unexpectedly observed that expression of PDR1 harbouring GOF mutation G346D down-regulated EPA1 transcription and attenuated adherence to epithelial cells in different strain backgrounds. Given that PDR1 GOF mutations have been previously regarded as stimulators for adherence of this species, these findings prompted us to explore the regulation of EPA1 by wild-type Pdr1 and Pdr1 harbouring G346D mutation. Epitope tagged version of Pdr1 and Gal11A were utilized to determine the association of Pdr1 and Gal11A with EPA1 promoter. A combination of approaches including deletion, molecular, and biochemical assays showed that EPA1 is a direct target of Pdr1, and demonstrated for the first time that PDR1 G346D mutation decreases EPA1 expression and attenuates adherence to epithelial cells via enhancing recruitment of Gal11A. Taken together, our data propose a critical role of Gal11A in Pdr1-regulated EPA1 expression and adherence to epithelial cells, which could be utilized a novel therapeutic target for the treatment of hyper-adherent C. glabrata infection.

Relative Contribution of the ABC Transporters Cdr1, Pdh1, and Snq2 to Azole Resistance in Candida glabrata.

The utility of the azole antifungals for the treatment of invasive candidiasis is severely hampered by azole resistance in Candida glabrata This resistance is mediated almost exclusively by activating mutations in the zinc cluster transcription factor Pdr1, which controls the genes encoding the multidrug resistance transporters Cdr1, Pdh1, and Snq2. However, the specific relative contributions of these transporters to resistance are not known. To address this question, the SAT1 flipper method was used to delete CDR1, PDH1, and SNQ2 in a strain of C. glabrata engineered to carry a clinically relevant activating mutation in PDR1 Susceptibility testing was performed according to the CLSI guidelines, with minor modifications, and confirmed with Etest strips. Of the single-transporter-deletion strains, only the CDR1 deletion resulted in a decreased azole MIC. The deletion of PDH1 in combination with CDR1 resulted in a moderate decrease in MIC compared to that observed with the deletion of CDR1 alone. SNQ2 deletion only decreased the MIC in the triple-deletion strain in the absence of both CDR1 and PDH1 The deletion of all three transporters in combination decreased the MIC to the level observed in the PDR1 deletion strains for some, but not all, azoles tested, which indicates that additional Pdr1 targets likely play a minor role in this process. These results indicate that while Cdr1 is the most important Pdr1-mediated multidrug resistance transporter for azole resistance in this clinical isolate, all three of these transporters contribute to its high-level resistance to the azole antifungals.

Sequence modification of the master regulator Pdr1 interferes with its transcriptional autoregulation and confers altered azole resistance in Candida glabrata.

The transcriptional regulator Pdr1 plays a positive role in regulating azole drug resistance in Candida glabrata. Previous studies have shown the importance of the carboxyl (C)-terminal sequence of Pdr1 in fulfilling its function, as this region mediates interactions between Pdr1 and the co-activator Gal11A and is crucial for activation of Pdr1 targets. However, mechanisms of how Pdr1 is regulated, especially implication of its C-terminus in the regulatory activity, remain uncharacterized. In this study, we unexpectedly observed that the C-terminal modification of Pdr1 in an azole-resistant clinical isolate harboring a single GOF mutation, resulted in adverse effects such as decreased expression levels of Pdr1, downregulation of Pdr1 targets and azole hypersensitivity. Importantly, the C-terminal 3×FLAG tagging significantly decreased the binding of Pdr1 to the pleiotropic drug response elements in its own promoter, promoted an irregular cellular mislocalization and thereby disrupted the transcriptional autoregulation of this master regulator. Unexpectedly, the aberrant cytoplasmic localization caused a non-functional interaction with Gal11A, a co-activator involved in drug resistance. Based on these findings, we proposed that C-terminal sequence of Pdr1 is vital for its stability and functionality, and targeting regulation of this region may represent a promising future strategy for combating C. glabrata infection and drug resistance.

Milbemycin A4 oxime as a probe of azole transport in Candida glabrata.

Azole resistance in Candida glabrata, a pathogenic yeast, has prompted studies of compounds that have therapeutic potential by reversing azole resistance. Milbemycin A4 oxime blocked azole efflux and enhanced azole susceptibility fourfold in 28 clinical isolates of C.glabrata. Specificity of the milbemycin A4 oxime effect depended on the drug transporter and the substrate being effluxed. The major effect of milbemycin A4 oxime was inhibition of azole and rhodamine 6G efflux by the ATP-binding cassette (ABC) transporters CgCDR1 and PDH1. Milbemycin A4 oxime effect did not extend to oligomycin, transported by the ABC transporter YOR1 or to benomyl, transported by the major facilitator superfamily transporter, CgFLR1. Milbemycin A4 oxime did not suppress transcription of CgCDR1 but increased CgCDR1 expression 126-fold. Selectivity of the effect is compatible with the concept that milbemycin A4 oxime may interact directly with one or more drug-binding sites of the major azole transporters.

STB5 Is a Negative Regulator of Azole Resistance in Candida glabrata

The opportunistic yeast pathogen Candida glabrata is recognized for its ability to acquire resistance during prolonged treatment with azole antifungals (J. E. Bennett, K. Izumikawa, and K. A. Marr. Antimicrob. Agents Chemother. 48:1773-1777, 2004). Resistance to azoles is largely mediated by the transcription factor PDR1, resulting in the upregulation of ATP-binding cassette (ABC) transporter proteins and drug efflux. Studies in the related yeast Saccharomyces cerevisiae have shown that Pdr1p forms a heterodimer with another transcription factor, Stb5p. In C. glabrata, the open reading frame (ORF) designated CAGL0I02552g has 38.8% amino acid identity with STB5 (YHR178w) and shares an N-terminal Zn(2)Cys(6) binuclear cluster domain and a fungus-specific transcriptional factor domain, prompting us to test for homologous function and a possible role in azole resistance. Complementation of a Δyhr178w (Δstb5) mutant with CAGL0I02552g resolved the increased sensitivity to cold, hydrogen peroxide, and caffeine of the mutant, for which reason we designated CAGl0I02552g CgSTB5. Overexpression of CgSTB5 in C. glabrata repressed azole resistance, whereas deletion of CgSTB5 caused a modest increase in resistance. Expression analysis found that CgSTB5 shares many transcriptional targets with CgPDR1 but, unlike the latter, is a negative regulator of pleiotropic drug resistance, including the ABC transporter genes CDR1, PDH1, and YOR1.

Characterization of the Multi-drug Efflux Systems of Pathogenic Fungi Using Functional Hyperexpression in Saccharomyces cerevisiae

Systemic fungal infections, caused by a wide variety of fungi, contribute to high mortality in humans with immunocompromised conditions. However, there are few classes of antifungal drugs available, limiting therapeutic options. Azoles are the most commonly used class of antifungals to treat many fungal infections, but resistance to azoles can be induced or, for some fungi, is an inherent property. One of major mechanisms of azole resistance is overexpression of drug efflux pumps in fungal cell membranes, such as the energy-dependent ATP-binding cassette (ABC) transporters. We have developed a protein hyperexpression system to facilitate functional analysis of efflux pumps using a Saccharomyces cerevisiae strain as the host for heterologous expression. The system is well suited for the hyperexpression of individual fungal ABC transporters for structural and functional studies. Furthermore, the recombinant yeast strains expressing heterologous membrane proteins can be used to screen for compounds that overcome fungal drug resistance. Also in this review, the mechanisms of azole resistance in Candida glabrata and C. krusei will be considered in relation to the recent increase in the incidence of Candida infections caused by non-albicans Candida. The development of possible novel antifungal agents will also be discussed.

Antifungal Resistance of Candida glabrata Vaginal Isolates and Development of a Quantitative Reverse Transcription-PCR-Based Azole Susceptibility Assay

A multiplex quantitative reverse transcription-PCR assay was developed to detect azole resistance in Candida glabrata, an important opportunistic pathogen that develops resistance rapidly. Resistance was defined as a >or=3-fold increase in CDR1 expression by this assay, which proved to be 100% sensitive and 95% specific in comparison to the gold standard broth microdilution assay.

P1963 Effects of MG3290, a selective histone deacetylase inhibitor, on azole resistance in Candida glabrata

P1963 Effects of MG3290, a selective histone deacetylase inhibitor, on azole resistance in Candida glabrata

Proteomic analysis of experimentally induced azole resistance in Candida glabrata

The aim of the present study was to identify changes in the proteome of a laboratory-derived azole-resistant strain of Candida glabrata compared with its susceptible parent strain in an effort to identify proteins that are differentially expressed in association with azole resistance. Soluble and membrane protein fractions were isolated from mutant strain F15 (fluconazole MIC>128 mg/L) and parent strain 66032 (fluconazole MIC=16 mg/L) grown to mid-log phase. Soluble proteins were resolved by both two-dimensional (2D) and one-dimensional (1D) polyacrylamide gel electrophoresis (GE) whereas membrane proteins were resolved by 1D GE. Spots or bands representing differentially expressed proteins were identified by matrix-assisted desorption ionization-time of flight mass spectroscopy (MALDI-TOF MS) and peptide mass fingerprinting. A total of 22 proteins were found to be more abundantly represented, and 3 proteins were found to be less abundantly represented, in strain F15 compared with strain 66032. These included up-regulation of the ATP-binding cassette transporter Cdr1p, the ergosterol biosynthesis enzyme Erg11p, proteins involved in glycolysis and glycerol metabolism, and proteins involved in the response to oxidative stress and cadmium exposure. In addition to transcriptional regulation of Cdr1p, this study identified the differential expression of several proteins that may contribute to azole resistance and suggests the possibility for a post-transcriptional mechanism for increased expression of Erg11p.