The Rho1 GTPase-activating Protein CgBem2 Is Required for Survival of Azole Stress in Candida glabrata

Abstract

Invasive fungal infections are common clinical complications of neonates, critically ill, and immunocompromised patients worldwide. Candida species are the leading cause of disseminated fungal infections, with Candida albicans being the most prevalent species. Candida glabrata, the second/third most common cause of candidemia, shows reduced susceptibility to a widely used antifungal drug fluconazole. Here, we present findings from a screen of 9134 C. glabrata Tn7 insertion mutants for altered survival profiles in the presence of fluconazole. We have identified two components of RNA polymerase II mediator complex, three players of Rho GTPase-mediated signaling cascade, and two proteins implicated in actin cytoskeleton biogenesis and ergosterol biosynthesis that are required to sustain viability during fluconazole stress. We show that exposure to fluconazole leads to activation of the protein kinase C (PKC)-mediated cell wall integrity pathway in C. glabrata. Our data demonstrate that disruption of a RhoGAP (GTPase activating protein) domain-containing protein, CgBem2, results in budemergence defects, azole susceptibility, and constitutive activation of CgRho1-regulated CgPkc1 signaling cascade and cell wall-related phenotypes. The viability loss of Cgbem2⌬ mutant upon fluconazole treatment could be partially rescued by the PKC inhibitor staurosporine. Additionally, we present evidence that CgBEM2 is required for the transcriptional activation of genes encoding multidrug efflux pumps in response to fluconazole exposure. Last, we report that Hsp90 inhibitor geldanamycin renders fluconazole a fungicidal drug in C. glabrata.

Identification of Components of the SUMOylation Machinery in Candida glabrata

“…Maps of proteins along with their domains were generated using DOG (Domain Graph) (54). Zymolyase digestion, CgSlt2 phosphorylation, and adherence analysis were performed as described previously (38,51).…”

“…Cell wall integrity in C. glabrata is maintained by protein kinase C (PKC)-mediated signal transduction pathway, and mutants with cell wall defects have previously been reported to exhibit constitutively activated PKC signaling cascade (38). To examine the status of the PKC-dependent signaling pathway in the Cgulp2⌬ mutant, we checked phosphorylation levels of CgSlt2, which is the terminal mitogen-activated protein kinase (MAPK) of the PKC pathway, in the mutant.…”

Pivotal Role for a Tail Subunit of the RNA Polymerase II Mediator Complex CgMed2 in Azole Tolerance and Adherence in Candida glabrata

“…We have recently reported two putative C. glabrata tail module subunits, CgMed2 and CgPgd1, to be essential for retaining viability during azole stress (25). Here, using a combined approach of deletion, molecular, and biochemical analyses, we show that Cg-MED2 is required for both basal and acquired resistance to azole antifungals in C. glabrata.…”

“…Primers for quantitative real-time PCR (qPCR) analysis were designed using Primer3 Plus software and are listed in Table S2 in the supplemental material. Total RNA was isolated using the acid phenol extraction method, and quantitative reverse transcription-PCR was performed as described previously (25). The C. glabrata glyceraldehyde-3-phosphate dehydrogenase (CgGAPDH) gene transcript level was used as a normalization control, and the threshold cycle (C T ) values either between the wild-type and mutant cultures or between cultures grown in the presence and absence of fluconazole were compared.…”

“…The C. glabrata Cgmed2⌬ deletion strain was generated using the homologous recombinationbased strategy, as described previously (25), by replacing the CgMED2 open reading frame (ORF) (CAGL0C04477g, 1.1 kb) with a cassette-containing the nat1 gene, which codes for nourseothricin acetyltransferase and confers resistance to nourseothricin. Nourseothricin-resistant yeast transformants were checked for gene replacement by PCR after colony purification.…”