Tn<i>7</i>-Based Genome-Wide Random Insertional Mutagenesis of <i>Candida glabrata</i>

Castaño, Irene; Kaur, Rupinder; Pan, Shih Jung; Cregg, Robert; Peñas, Alejandro De Las; Guo, Nini; Biery, Matthew C.; Craig, Nancy L.; Cormack, Brendan P.

doi:10.1101/gr.848203

Cited by 73 publications

(77 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Plates were incubated at 30°C, growth profiles were scored after 24 -48 h, and mutants were selected. Integration of Tn7 insertion in the mutant genome via homologous recombination was confirmed by PCR as described previously (29).…”

Section: Methodsmentioning

confidence: 99%

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

Borah¹,

Shivarathri

Kaur

2011

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

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

Borah¹,

Shivarathri

Kaur

2011

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…The URA3 marker (from pHOBST-URA3 [35]) was ligated between the SmaI and EcoRI sites of the vector, and a 1-kb sequence immediately downstream of the EPA1 ORF was amplified from genomic DNA and inserted at the PacI site, creating pMS15. pMS15 contained an extended region of genomic sequence around EPA1(-3HA) which, together with the internal URA3 marker positioned immediately downstream of the EPA1 ORF, was excised as a ϳ6.8-kb fragment by digestion with XbaI and PacI and transformed into C. glabrata using a modified lithium acetate method (8). (The URA3 marker added 1.2 kb distance between EPA1 and the telomere, but this was too small to affect silencing significantly [see Discussion].…”

Section: Methodsmentioning

confidence: 99%

Heterogeneous Expression of the Virulence-Related Adhesin Epa1 between Individual Cells and Strains of the Pathogen Candida glabrata

et al. 2012

View full text Add to dashboard Cite

We investigated the relevance of gene expression heterogeneity to virulence properties of a major fungal pathogen, Candida glabrata. The organism's key virulence-associated factors include glycosylphosphatidylinositol-anchored adhesins, encoded subtelomerically by the EPA gene family. Individual-cell analyses of expression revealed very striking heterogeneity for Epa1, an adhesin that mediates ϳ95% of adherence to epithelial cells in vitro. The heterogeneity in Epa1 was markedly greater than that known for other yeast genes. Sorted cells expressing high or low levels of Epa1 exhibited high and low adherence to epithelial cells, indicating a link between gene expression noise and potential virulence. The phenotypes of sorted subpopulations reverted to mixed phenotypes within a few generations. Variation in single-cell Epa1 protein and mRNA levels was correlated, consistent with transcriptional regulation of heterogeneity. Sir-dependent transcriptional silencing was the primary mechanism driving heterogeneous Epa1 expression in C. glabrata BG2, but not in CBS138 (ATCC 2001). Inefficient silencing in the latter strain was not due to a difference in EPA1 sequence or (sub)telomere length and was overcome by ectopic SIR3 expression. Moreover, differences between strains in the silencing dependence of EPA1 expression were evident across a range of clinical isolates, with heterogeneity being the greatest in strains where EPA1 was subject to silencing. The study shows how heterogeneity can impact the virulence-related properties of C. glabrata cell populations, with potential implications for microbial pathogenesis more broadly.

show abstract

“…To assess the degree of silencing of the URA3 gene inserted at different positions through-VOL. 9,2010 SILENCING BY Ku AND Rif1 AT THE C. glabrata MTL3 LOCUS 1603…”

Section: Methodsmentioning

confidence: 99%

“…To assess whether the MTL loci are transcriptionally silenced, we inserted a URA3 reporter gene at different positions throughout the MTL1, MTL2, and MTL3 loci using a modified Tn7 transposon carrying the URA3 gene (9). For this, we mutagenized in vitro each of the plasmids containing each MTL locus (pSD10, pAP699, and pAP695) with Tn7 UKR as described previously (9).…”

Section: Methodsmentioning

confidence: 99%

Subtelomeric Silencing of the MTL3 Locus of Candida glabrata Requires yKu70, yKu80, and Rif1 Proteins

et al. 2010

Self Cite

View full text Add to dashboard Cite

Candida glabrata is a haploid opportunistic fungal pathogen that is phylogenetically related to Saccharomyces cerevisiae. Even though C. glabrata has no known sexual cycle, it contains, like S. cerevisiae, three mating type-like loci (MTL) called MTL1, MTL2, and MTL3, as well as most of the genes required for mating, meiosis, and sporulation. MTL1 is localized at an internal position on chromosome B and is thought to be the locus corresponding to the MAT locus in S. cerevisiae. MTL2 and MTL3 are localized close to two telomeres on different chromosomes (29.4 kb from Chr E-L and 10.5 kb from Chr B-L, respectively). By using URA3 reporter gene insertions at the three MTL loci, we found that in contrast to the case for S. cerevisiae, only MTL3 is subject to transcriptional silencing while MTL2 is transcriptionally active, and this is in agreement with previously reported data. We found that the silencing of MTL3 is nucleated primarily at the left telomere of chromosome B and spreads over 12 kb to MTL3, rather than nucleating at flanking, closely positioned cis-acting silencers, like those flanking HMR and HML of S. cerevisiae. Interestingly, the silencing of MTL3 absolutely requires the yKu70, yKu80, and Rif1 proteins, in sharp contrast to the silencing of the HM loci of S. cerevisiae. In addition, we found that several cell type-specific genes are expressed in C. glabrata regardless of the presence, or even absence, of mating type information at any of the MTL loci.Candida glabrata is a haploid yeast found as a normal part of the mammalian microflora, but in recent years it also has emerged as a common opportunistic pathogen of humans, and now it accounts for about 12% of all Candida infections worldwide, second only to Candida albicans (29,36,37,39,50). C. glabrata and Saccharomyces cerevisiae are closely related phylogenetically, and both genomes conserve a high degree of synteny, whereas C. albicans is more distantly related (6,14).Sexual reproduction has been documented in many fungal species; however, several human fungal pathogens either do not reproduce sexually or very rarely do so (33). As the genomes of more fungal species are sequenced it has become clear, however, that the vast majority of the species have highly conserved genes involved in sexual reproduction, and yet some species have not been observed to mate (5, 6, 16). In some cases, a cryptic sexual cycle and even same-sex mating have been discovered recently, as is the case for C. albicans and Aspergillus fumigatus (1, 34). C. glabrata has no known sexual cycle even though it contains the vast majority of the genes required for mating (6,14), and some mating type identity is maintained (32).In (21,26). In addition to the MAT locus, the S. cerevisiae chromosome III contains two silent loci, HMR and HML, located near each telomere (ϳ22.7 and ϳ13.0 kb, respectively). The HMR and HML loci usually contain identical copies of MATa and MAT␣, respectively, that are normally maintained efficiently repressed through a chromatin-based mechanism called sil...

show abstract

Tn7-Based Genome-Wide Random Insertional Mutagenesis of Candida glabrata

Cited by 73 publications

References 30 publications

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

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

Heterogeneous Expression of the Virulence-Related Adhesin Epa1 between Individual Cells and Strains of the Pathogen Candida glabrata

Subtelomeric Silencing of the MTL3 Locus of Candida glabrata Requires yKu70, yKu80, and Rif1 Proteins

Contact Info

Product

Resources

About