The Rho1 GTPase-activating protein CgBem2 is required for survival of azole stress in Candida glabrata

Exposure to fluconazole leads to activation of the protein kinase C (PKC)-mediated cell wall integrity pathway in C. glabrata.

“We show that exposure to fluconazole leads to activation of the protein kinase C (PKC)mediated cell wall integrity pathway in C. glabrata.”

No growth inhibition was observed on plates supplemented with amphotericin B, sodium chloride, calcium chloride, and terbinafine for the Cgbem2Δ and Cgslt2Δ deletion strains.

“Importantly, no growth inhibition was observed on plates supplemented with amphotericin B, sodium chloride, calcium chloride, and terbinafine for these deletion strains (data not shown).”

C. glabrata cells lacking CgPAN1 were inviable.

“Plasmid shuffling experiments demonstrated that C. glabrata cells lacking CgPAN1 were inviable (data not shown).”

No increase in polyploids was seen for the Cgslt2Δ mutant.

“No increase in polyploids was seen for the Cgslt2⌬ mutant (data not shown).”

Disruption of CgBem2 results in cell wall-related phenotypes.

“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.”

Mutants defective in RNA polymerase II-mediated transcription and multidrug efflux pumps displayed sensitivity to only azoles.

“Mutants defective in RNA polymerase II-mediated transcription and multidrug efflux pumps displayed sensitivity to only azoles (Fig. 2).”

Of the 200 mutants, 91 displayed a distinct dark blue color on plates supplemented with 0.01% methylene blue and 16 μg/ml fluconazole.

“Of these, 91 mutants displayed a distinct dark blue color on plates supplemented with 0.01% methylene blue and 16 g/ml fluconazole (Fig. 1A and data not shown).”

Expression of CgSLT2 and CgRHO1 could not rescue the fluconazole and temperature susceptibility of the Cgbem2Δ mutant.

“Notably, expression of CgSLT2 and CgRHO1 could not rescue the fluconazole and temperature susceptibility of Cgbem2⌬ mutant (supplemental Fig. S2); however, azole sensi- tivity of Cgslt2⌬ was partially restored by plasmid expressing CgRHO1 (supplemental Fig. S2).”

Cgerg4, Cgpan1, Cgbnr1, and Cgcne1 mutants were exquisitely sensitive to the cell membrane-disrupting agent SDS.

“Cgerg4, Cgpan1, Cgbnr1, and Cgcne1 mutants were exquisitely sensitive to cell membranedisrupting agent SDS (Fig. 2).”

A screen of 9134 C. glabrata Tn7 insertion mutants identified three players of Rho GTPase-mediated signaling cascade that are required to sustain viability during fluconazole stress.

“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.”

No altered sensitivity to the polyene antifungal amphotericin B was observed for any of the mutants.

“Importantly, no altered sensitivity to a polyene antifungal amphotericin B was observed for any of the mutants (data not shown).”

DAPI staining of Cgbem2Δ cells revealed that each enlarged cell possessed multiple nuclei, consistent with bud emergence defects.

“DAPI staining of these cells revealed that each enlarged cell possessed multiple nuclei, consistent with bud emergence defects (Fig. 3B and data not shown).”

Tn7 insertion mapping and sequencing analysis of the 20 mutants identified multiple insertions in 7 genes.

“Tn7 insertion mapping and sequencing analysis of these 20 mutants identified multiple insertions in 7 genes (Table 1).”

The Cgbnr1 mutant displayed reduced susceptibility to cell wall stress agents.

“The Cgbnr1 mutant displayed reduced susceptibility to cell wall stress agents (Fig. 2).”

Cgbem2Δ cells display enhanced polyploidy upon fluconazole exposure, with 40% polyploid cells compared with 8% in fluconazole-treated WT cells and 14% in untreated mutant cultures.

“A concomitant increase in cell population with polyploidy (40%) was observed for Cgbem2⌬ compared with the 8 and 14% polyploid cells observed in fluconazole-treated WT cells and untreated mutant cultures, respectively (Fig. 4)”

The Cgswi4 mutant showed resistance to all three azoles tested: fluconazole, clotrimazole, and ketoconazole.

“Surprisingly, Cgswi4 mutant showed resistance to all the three azoles tested, fluconazole, clotrimazole, and ketoconazole (Fig. 2 and data not shown); the molecular basis of this phenotype is not clear and warrants further investigation.”

A Cgbem2Δ-reconstituted strain restored wild-type-like cell cycle profiles upon fluconazole exposure.

“Notably, a Cgbem2⌬-reconstituted strain restored wild-typelike cell cycle profiles upon fluconazole exposure (Fig. 4).”

Disruption of the RhoGAP domain-containing protein CgBem2 results in bud emergence defects.

“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 Hsp90 inhibitor geldanamycin renders fluconazole a fungicidal drug in C. glabrata.

“Last, we report that Hsp90 inhibitor geldanamycin renders fluconazole a fungicidal drug in C. glabrata.”

YPD-cultured WT cells showed a faint band corresponding to 23-kDa CgRho1 in affinity pulldown assays.

“Beads coated with RhoA binding domain of the RhoA effector protein, Rhotekin, which binds to only the active, GTP-bound RhoA form, were used, and a faint band corresponding to 23-kDa CgRho1 was observed in YPDcultured WT cells.”

Disruption of CgBem2 results in azole susceptibility.

“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.”

A screen of 9134 C. glabrata Tn7 insertion mutants identified two proteins implicated in actin cytoskeleton biogenesis and ergosterol biosynthesis that are required to sustain viability during fluconazole stress.

“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.”

A screen of 9134 C. glabrata Tn7 insertion mutants identified two components of the RNA polymerase II mediator complex that are required to sustain viability during fluconazole stress.

“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.”

Disruption of CgBem2 results in constitutive activation of the CgRho1-regulated CgPkc1 signaling cascade.

“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 the Cgbem2Δ mutant upon fluconazole treatment could be partially rescued by the PKC inhibitor staurosporine.

“The viability loss of Cgbem2⌬ mutant upon fluconazole treatment could be partially rescued by the PKC inhibitor staurosporine.”

The Cgbem2Δ strain has a growth defect in YPD medium (doubling time = 95 min) compared with the WT strain (doubling time = 72 min).

“Next, phenotypic characterization of Cgbem2⌬ and Cgslt2⌬ mutants revealed a growth defect for Cgbem2⌬ strains in YPD medium (doubling time ϭ 95 min) compared with the WT strain (doubling time ϭ 72 min) (Fig. 3A).”

CgBEM2 is required for the transcriptional activation of genes encoding multidrug efflux pumps in response to fluconazole exposure.

“Additionally, we present evidence that CgBEM2 is required for the transcriptional activation of genes encoding multidrug efflux pumps in response to fluconazole exposure.”

A typical logarithmic-phase culture of Cgbem2Δ accumulated a high percentage (20-25%) of oversized, large, un/small-budded cells.

“A typical logarithmic-phase culture of Cgbem2⌬ accumulated a high percentage (20 -25%) of the cells as oversized, large, un/small-budded cells (Fig. 3, B and C, and data not shown).”

Expression of CgSLT2 restored the growth defect and viability loss of Cgslt2Δ in the presence of fluconazole and clotrimazole.

“Expression of CgSLT2 restored the growth defect and viability loss of Cgslt2⌬ in the presence of fluconazole and clotrimazole (supplemental Figs.S1 and S2).”

Azole sensitivity of Cgslt2Δ was partially restored by a plasmid expressing CgRHO1.

“Notably, expression of CgSLT2 and CgRHO1 could not rescue the fluconazole and temperature susceptibility of Cgbem2⌬ mutant (supplemental Fig. S2); however, azole sensi- tivity of Cgslt2⌬ was partially restored by plasmid expressing CgRHO1 (supplemental Fig. S2).”

We were unable to observe a consistent activation of Rho1 GTPase by RhoA binding domain pulldown assays.

“Similarly, fluconazole treatment led to an ϳ2-fold increase in the CgRho1 levels in the membrane fractions of WT cells (supplemental Fig. S3); however, we were unable to observe a consistent activation of Rho1 GTPase by RhoA binding domain pulldown assays (data not shown).”

A synergistic cidal effect of fluconazole was observed with geldanamycin in WT and Cgbem2Δ cells with both survival rates being ~15%.

“A synergistic cidal effect of fluconazole was observed with geldanamycin in WT and Cgbem2⌬ cells with both survival rates being ϳ15% (Fig. 7B).”

• AssumptionRests on an assumption: Geldanamycin specifically inhibits CgHsp90 in C. glabrata.
• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

CgBem2R1957A expression could not rescue the growth defects of Cgbem2Δ cells.

“CgBem2R1957A expression could neither rescue the growth defects of Cgbem2⌬ cells nor did its expression in WT cells lead to elevated susceptibility to azoles and high temperature (Fig. 6D and data not shown).”

• AssumptionRests on an assumption: The CgBem2R1957A mutation abolishes GAP activity.

A 20% increase in β-glucan amount was seen in drug-treated WT cells.

“A 20% increase in ␤-glucan amount was seen in drug-treated WT cells (Fig. 7A).”

Increasing the dosage of neither CgSlt2 nor CgRho1 had any effect on the thermal and azole stress sensitivity of the Cgbem2Δ mutant.

“Intriguingly, increasing the dosage of neither CgSlt2 nor CgRho1 had any effect on the thermal and azole stress sensitivity of Cgbem2⌬ mutant (supplemental Fig. S2); the basis for this observation is not clear.”

Three mutants carrying a Tn7 insertion in CgBEM2 were identified in the screen for altered fluconazole survival profiles.

“In the screen for altered fluconazole survival profiles, three mutants carrying Tn7 insertion in CgBEM2 were identified”

Growth in the presence of caffeine resulted in higher levels of GTP-bound CgRho1 in WT cells.

“Growth in the presence of cell wall stress agent caffeine resulted in higher levels of GTP-bound form of CgRho1 in WT cells (Fig. 5A and data not shown).”

Addition of staurosporine increased viability of fluconazole-treated Cgbem2Δ cells (43% vs 29%).

“Furthermore, the addition of staurosporine strikingly increased viability of fluconazole-treated Cgbem2⌬ cells (43% viability for fluconazole-and staurosporinetreated compared with 29% survival for fluconazole alone) (Fig. 7B).”

• AssumptionRests on an assumption: Staurosporine specifically inhibits CgPkc1 signaling in C. glabrata.
• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

Fluconazole induced transcriptional activation of CgERG11 (3-4-fold) and CgSLT2 (2.5-fold) in both WT and Cgbem2Δ cells.

“Notably, fluconazole induced the transcriptional activation of CgERG11 (3-4-fold) and CgSLT2 (2.5-fold) in both WT and Cgbem2⌬ cells (Fig. 8A)”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

Expression of CgBEM2 in Cgbem2Δ cells led to wild-type β-glucan levels.

“Expression of CgBEM2 in Cgbem2⌬ cells led to wild-type ␤-glucan levels (Fig. 7A)”

Fluconazole treatment led to an approximately 2-fold increase in CgRho1 levels in the membrane fractions of WT cells.

“Similarly, fluconazole treatment led to an ϳ2-fold increase in the CgRho1 levels in the membrane fractions of WT cells (supplemental Fig. S3); however, we were unable to observe a consistent activation of Rho1 GTPase by RhoA binding domain pulldown assays (data not shown).”

Approximately a 3-fold increase in total CgSlt2 levels was observed upon drug exposure.

“Additionally, we observed approximately a 3-fold increase in total CgSlt2 levels upon drug exposure (Fig. 5B).”

Cgbem2Δ cells displayed 50% higher β-glucan content in their cell walls even under standard growth conditions.

“In contrast, Cgbem2⌬ cells displayed 50% higher ␤-glucan content in their cell walls even under standard growth conditions”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

All three CgBEM2 Tn7 mutants had constitutively active CgSlt2.

“and a constitutively active CgSlt2 (Fig. 6B)”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

Wild-type cells responded to fluconazole exposure by elevating expression of genes coding for drug transporters (Cdr1 and Cdr2) by 4-6-fold.

“Consistent with earlier reports, wildtype cells responded to fluconazole exposure by elevating the expression of genes coding for drug transporters (Cdr1 and Cdr2) by 4 -6-fold (Fig. 8A).”

Levels of phosphorylated CgSlt2 remained unchanged in WT and Cgbem2Δ cells expressing mutated CgBem2 protein compared with untransformed cultures.

“Additionally, the levels of phosphorylated CgSlt2 remained unchanged in WT and Cgbem2⌬ cells expressing mutated CgBem2 protein compared with the untransformed cultures (supplemental Fig. S4).”

• AssumptionRests on an assumption: The CgBem2R1957A mutation abolishes GAP activity.

Levels of total Rho1 were nearly identical across all samples.

“Notably, levels of total Rho1 were nearly identical across all samples (Fig. 5A).”

In silico structural analysis predicted that CgBem2 possesses a Ras-GEF, a RasGEF-N, a PH, and a RhoGAP domain.

“In silico structural analysis predicted CgBem2 to possess a Ras-GEF, a RasGEF-N, a PH (pleckstrin homology), and a RhoGAP domain (Fig. 6A).”

Steady-state RNA levels of CgCDR1, CgCDR2, CgSNQ2, and CgPDR1 in the absence of drug were approximately equivalent in all strains.

“Steady-state RNA levels of CgCDR1, CgCDR2, CgSNQ2, and CgPDR1 in the absence of drug were approximately equivalent in all strains (data not shown)”

For WT cells in medium containing fluconazole, addition of the PKC inhibitor staurosporine reduced recoverable viable cells from 90 to 15%.

“For WT cells in medium containing fluconazole, the addition of the PKC inhibitor staurosporine reduced the recoverable viable cells from 90 to 15% (Fig. 7B).”

• AssumptionRests on an assumption: Staurosporine specifically inhibits CgPkc1 signaling in C. glabrata.

YPD-cultured Cgbem2Δ cells displayed an approximately 3-fold higher amount of membrane-associated CgRho1.

“YPD- cultured Cgbem2⌬ cells displayed an ϳ3-fold higher amount of membrane-associated CgRho1 (supplemental Fig. S3), reinforcing the result that CgBem2 negatively regulates CgRho1, and mutants deleted for CgBEM2 contain higher levels of constitutively active CgRho1.”

Fluconazole-treated cells of Cgbem2Δ showed neither induction of genes coding for ABC transporters nor for the transcriptional regulator.

“In contrast, fluconazole-treated cells of Cgbem2⌬ showed neither induction of the genes coding for ABC transporters nor for the transcriptional regulator (Fig. 8A).”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

Ectopic expression of CgBEM2 in Cgbem2Δ mutant resulted in phosphorylated CgSlt2 levels comparable with those of WT cells.

“Furthermore, ectopic expression of CgBEM2 in Cgbem2⌬ mutant resulted in phosphorylated CgSlt2 levels comparable with those of the WT cells (Fig. 5B).”

A modest reduction in β-glucan was seen in fluconazole-cultured Cgbem2Δ cells.

“and a modest reduction was seen in fluconazolecultured cells (Fig. 7A)”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

9% of Cgbem2Δ cells could exclude trypan blue in the presence of FK506 and fluconazole compared with 29% in the presence of fluconazole alone.

“Consistent with those results, 9% of Cgbem2⌬ cells could exclude trypan blue in the presence of FK506 and fluconazole compared with 29% in the presence of fluconazole alone (Fig. 7B).”

• AssumptionRests on an assumption: FK506 specifically inhibits calcineurin signaling in C. glabrata.
• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

About 2.5-fold induction was seen in expression levels of CgSnq2 and CgPdr1.

“About 2.5-fold induction was seen in the expression levels of the third drug transporter, CgSnq2, and of the transcriptional factor CgPdr1 (Fig. 8A).”

Cgbem2Δ cells grew better in medium containing fluconazole and staurosporine than in medium containing fluconazole alone.

“In contrast, the Cgbem2⌬ cells grew better in medium containing fluconazole and staurosporine than in medium containing fluconazole alone.”

• AssumptionRests on an assumption: Staurosporine specifically inhibits CgPkc1 signaling in C. glabrata.
• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

Cgbem2Δ cells exhibited a modest survival defect in medium containing geldanamycin alone.

“Interestingly, Cgbem2⌬ cells exhibited a modest survival defect in medium containing geldanamycin alone (Fig. 7B)”

• AssumptionRests on an assumption: Geldanamycin specifically inhibits CgHsp90 in C. glabrata.
• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

Energy-dependent extrusion of R6G was not significantly elevated after fluconazole treatment in Cgbem2Δ mutant.

“however, energy-dependent extrusion of R6G was not significantly elevated after fluconazole treatment in Cgbem2⌬ mutant (Fig. 8B).”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

These 7 genes belong to one of four biological processes: RNA polymerase II-mediated transcription, ergosterol biosynthesis, actin cytoskeleton organization, and Rho1-mediated signaling.

“These genes belong to one of the four biological processes: RNA polymerase II-mediated transcription, ergosterol biosynthesis, actin cytoskeleton organization, and Rho1-mediated signaling”

Disruption of putative RNA polymerase II mediator complex subunits encoded by CgMED2 and CgPGD1 results in loss of viability during azole stress.

“we report for the first time that disruption of putative RNA polymerase II mediator complex subunits encoded by CgMED2 and CgPGD1 results in loss of viability during azole stress.”

Blast analysis revealed the presence of seven small Rho GTPases and seven proteins containing RhoGAP domain in the C. glabrata genome.

“Blast analysis revealed the presence of seven small Rho GTPases and seven proteins containing RhoGAP domain in the C. glabrata genome (supplemental Table S3).”

Of seven mutants selected from the genome-wide screen, three had Tn7 insertions in genes implicated in Rho GTPase-mediated signal transduction cascade.

“Of seven mutants selected from the genome-wide screen, three had Tn7 insertions in the genes implicated in Rho GTPase-mediated signal transduction cascade (Table 1).”

Fluconazole-and staurosporine-treated cells failed to display a significant increase in efflux of R6G in response to glucose.

“Similarly, fluconazole-and staurosporinetreated cells failed to display a significant increase in the efflux of R6G in response to glucose (Fig. 8D)”

• AssumptionRests on an assumption: Staurosporine specifically inhibits CgPkc1 signaling in C. glabrata.

Drug-treated cultures of reconstituted Cgbem2Δ/CgBEM2 strain displayed up-regulation of CDR genes similar to WT cells.

“Importantly, drug-treated cultures of reconstituted Cgbem2⌬/CgBEM2 strain displayed up-regulation of CDR genes similar to that of the WT cells (data not shown).”

WT cells showed a 4-fold energy-dependent enhanced efflux of R6G upon fluconazole treatment.

“As shown in Fig. 8B, WT cells showed a 4-fold energy-dependent enhanced efflux of R6G upon fluconazole treatment.”

The screen of 9134 random Tn7 insertional mutants identified a total of 7 genes required specifically for survival of C. glabrata during fluconazole stress.

“This screen of 9134 random Tn7 insertional mutants identified a total of 7 genes required specifically for survival of C. glabrata during fluconazole stress (Table 1).”

Cgbem2 mutants take up R6G at a higher rate than WT cells.

“Quantitative uptake and accumulation assays revealed that Cgbem2 mutants take up R6G at a higher rate than the WT cells (sup- plemental Fig. S6 and data not shown)”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

Staurosporine-and geldanamycin-mediated inhibition of CgPkc1 and CgHsp90, respectively, led to survival defects for wild-type cells in the presence of fluconazole.

“In the present study, we show that despite the lack of intrinsic antifungal activity, staurosporine-and geldanamycin-mediated inhibition of CgPkc1 and CgHsp90, respectively, led to survival defects for wild-type cells in the presence of fluconazole”

• AssumptionRests on an assumption: Staurosporine specifically inhibits CgPkc1 signaling in C. glabrata.
• AssumptionRests on an assumption: Geldanamycin specifically inhibits CgHsp90 in C. glabrata.

The incapacity of Cgbem2Δ to survive fluconazole stress is in part due to altered progression through the cell cycle.

“suggesting that the incapacity of Cgbem2⌬ to survive fluconazole stress is in part due to altered progression through the cell cycle.”

Phenotypic profiling data suggest that components of the CWI pathway, actin cytoskeleton organization machinery, sterol biosynthesis, and mediator complex are required for tolerance/resistance to the azole class of antifungals in C. glabrata.

“Altogether, the phenotypic profiling data suggest that components of CWI pathway, actin cytoskeleton organization machinery, sterol biosynthesis, and mediator complex, are required for tolerance/resistance to the azole class of antifungals in C. glabrata.”

Global transcriptional defects in Cgbem2Δ mutant are ruled out.

“thus ruling out global transcriptional defects in Cgbem2⌬ mutant.”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

A constitutively activated CgPkc1-mediated CWI pathway leads to increased β-glucan levels in the cell wall of Cgbem2Δ mutant.

“indicating that a constitutively activated CgPkc1-mediated CWI pathway leads to increased ␤-glucan levels in the cell wall of Cgbem2⌬ mutant.”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

CgBem2 protein is required for response to azole antifungals.

“indicating that CgBem2 protein is required for response to azole antifungals and for controlled activation of CgPkc1-mediated signaling.”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

There is cross-talk between PKC-mediated CWI and Hsp90-mediated cell survival in C. glabrata.

“indicating a cross-talk between PKC-mediated CWI and Hsp90-mediated cell survival in C. glabrata.”

• AssumptionRests on an assumption: Geldanamycin specifically inhibits CgHsp90 in C. glabrata.

CgBem2 protein is required for controlled activation of CgPkc1-mediated signaling.

“indicating that CgBem2 protein is required for response to azole antifungals and for controlled activation of CgPkc1-mediated signaling.”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

Cgbem2Δ cells have an inability to further activate CgFks2 in response to fluconazole treatment.

“implying an inability to further activate CgFks2 in response to the drug treatment and an imbalance in the polysaccharide composition of cell wall.”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

The mutant CgBem2R1957A allele was expressed at levels comparable to wild-type CgBEM2 in both WT and Cgbem2Δ transformants.

“however, qRT-PCR revealed that the mutant allele was expressed at levels comparable to those of the wild-type CgBEM2 in both WT and Cgbem2⌬ transformants (data not shown).”

A functional GAP domain is required for the role of CgBem2 in conferring tolerance to thermal, cell wall, and azole stress in C. glabrata.

“Altogether, these results indicate a requirement for a functional GAP domain for the role of CgBem2 in conferring tolerance to thermal, cell wall, and azole stress in C. glabrata.”

• AssumptionRests on an assumption: The CgBem2R1957A mutation abolishes GAP activity.

Rho1-dependent CgPkc1-mediated CWI signaling cascade is constitutively active in C. glabrata mutants lacking CgBem2.

“Collectively, these data indicate that Rho1-dependent CgPkc1-mediated CWI signaling cascade is constitutively active in C. glabrata mutants lacking CgBem2.”

Cgbem2Δ cells have an imbalance in the polysaccharide composition of the cell wall.

“implying an inability to further activate CgFks2 in response to the drug treatment and an imbalance in the polysaccharide composition of cell wall.”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

Enhanced susceptibility of Cgbem2Δ cells toward fluconazole, despite their polyploidy nature, probably indicates impaired ability to maintain genomic stability due to defective morphogenesis.

“However, the enhanced susceptibility of Cgbem2⌬ cells toward fluconazole, despite their polyploidy nature, probably indicates impaired ability to maintain genomic stability due to defective morphogenesis.”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

Staurosporine-mediated inhibition of CgPkc1 signaling impedes activation of multidrug efflux pumps upon fluconazole exposure.

“indicating that staurosporine-mediated inhibition of CgPkc1 signaling impedes the activation of multidrug efflux pumps upon fluconazole exposure.”

• AssumptionRests on an assumption: Staurosporine specifically inhibits CgPkc1 signaling in C. glabrata.

Downstream components of the CgPkc1-mediated CWI pathway are not involved in actin organization and cell morphogenesis in C. glabrata.

“the unique budding, thermal, and morphogenetic defects of Cgbem2 mutants suggest that downstream components of CgPkc1-mediated CWI pathway are not involved in actin organization and cell morphogenesis in C. glabrata.”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

Staurosporine-mediated partial rescue of fluconazole-induced viability loss in Cgbem2Δ cells implicates CgPkc1 signaling.

“but the staurosporinemediated partial rescue of fluconazole-induced viability loss in Cgbem2⌬ cells implicates CgPkc1 signaling.”

• AssumptionRests on an assumption: Staurosporine specifically inhibits CgPkc1 signaling in C. glabrata.

CgBem2 plays a crucial role in regulation of activity of drug efflux pumps.

“Collectively, the qRT-PCR and R6G efflux analyses demonstrate that CgBem2 plays a crucial role in the regulation of activity of drug efflux pumps”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

Multiple signaling cascades operate to ensure cell survival during azole stress in C. glabrata.

“implying that multiple signaling cascades operate to ensure cell survival during azole stress in C. glabrata.”

CgBem2 functions as a GAP for CgRho1.

“The morphology defects and presence of higher amounts of active (GTP-bound) Rho GTPase and of phosphorylated CgSlt2 in mutants lacking CgBEM2 suggest that CgBem2 functions as a GAP for CgRho1, consistent with its known role in S. cerevisiae (53).”

• cites [53] Peterson, 1994, J. Cell Biol. sourceNot checked yet

  Not checked in the free run.

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

CgBem2, a negative regulator of CgRho1 GTPase, is essential for survival of azole stress.

“In conclusion, we report for the first time that CgBem2, a negative regulator of CgRho1 GTPase, is essential for survival of azole stress and for the up-regulation of genes coding for multidrug transporters upon fluconazole exposure.”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

Expression and activity of multidrug transporters in response to fluconazole is modulated by disruption of CgBem2, a negative regulator of CgRho1.

“Our data demonstrate that expression and activity of multidrug transporters in response to fluconazole is modulated by the disruption of CgBem2, a negative regulator of CgRho1.”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

Defective regulation of these transporters may partially explain increased fluconazole susceptibility associated with CgBEM2 disruption.

“and defective regulation of these transporters may partially explain the increased fluconazole susceptibility associated with CgBEM2 disruption.”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

These four processes play a crucial role in survival of azole stress in C. glabrata.

“suggesting that these processes play a crucial role in survival of azole stress in C. glabrata.”

There is broad conservation of cellular mechanisms to survive antifungal drug stress in fungal species.

“and suggest a broad conservation of cellular mechanisms to survive antifungal drug stress in fungal species.”

• cites [18] Bonilla, 2002, EMBO J. sourceNot checked yet

  Not checked in the free run.

• cites [22] LaFayette, 2010, PLoS Pathog. sourceNot checked yet

  Not checked in the free run.

• cites [50] Bonilla, 2003, Mol. Biol. Cell sourceNot checked yet

  Not checked in the free run.

A C. glabrata mutant carrying a Tn7 insertion in the CDR1 gene showed no significant increase in R6G efflux upon drug exposure in response to glucose pulse.

“A C. glabrata mutant carrying a Tn7 insertion in the CDR1 gene was used as a negative control, and no significant increase in R6G efflux was seen upon drug exposure in response to glucose pulse (data not shown).”

CgBem2 is essential for up-regulation of genes coding for multidrug transporters upon fluconazole exposure.

“In conclusion, we report for the first time that CgBem2, a negative regulator of CgRho1 GTPase, is essential for survival of azole stress and for the up-regulation of genes coding for multidrug transporters upon fluconazole exposure.”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

Staurosporine-mediated inhibition of CgPkc1 signaling in wild-type cells phenocopies the effects of CgBem2 deletion.

“Intriguingly, staurosporine-mediated inhibition of CgPkc1 signaling in wild-type cells phenocopies the effects of CgBem2 deletion, as staurosporine-treated wild-type cells were found to be compromised for their response to azole stress (Figs.7B and 8, C and D).”

• AssumptionRests on an assumption: Staurosporine specifically inhibits CgPkc1 signaling in C. glabrata.

CgBem2 is required for transcriptional activation of genes coding for ABC transporters and their transcriptional regulator upon fluconazole exposure.

“These data indicate that CgBem2 is required for the transcriptional activation of the genes coding for ABC transporters and their transcriptional regulator upon fluconazole exposure.”

• AssumptionRests on an assumption: Tn7 insertions in CgBEM2 result in loss of CgBem2 function.

These functions of Bem2 seem to be conserved between S. cerevisiae and C. glabrata.

“These functions of Bem2 seem to be conserved between S. cerevisiae and C. glabrata, as the mutants lacking Bem2 in C. glabrata displayed many biochemical and cell biological aspects that match the phenotypes in S. cerevisiae.”