Deletion of Vacuolar Proton-translocating ATPase Voa Isoforms Clarifies the Role of Vacuolar pH as a Determinant of Virulence-associated Traits in Candida albicans*

Raines, Summer M.; Rane, Hallie S.; Bernardo, Stella M.; Binder, Jessica; Lee, Samuel A.; Parra, Karlett J.

doi:10.1074/jbc.m112.426197

Cited by 31 publications

(53 citation statements)

References 65 publications

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“…The contribution of several V-ATPase subunits to C. albicans cell biology and virulence has been studied previously (3,4,12,13). Here, we present the study of C. albicans VMA2, which encodes the B subunit of the V-ATPase V 1 domain; this is the first component of the catalytic hexamer of the V 1 complex to be studied in depth in C. albicans.…”

Section: Discussionmentioning

confidence: 99%

“…In order to quantify ATP hydrolysis, 15 g purified vacuolar vesicles was used in an enzymatic assay in which the rate of ATP hydrolysis is coupled to the oxidation of NADH, measured as a loss of A 340 over time (24) in the presence and absence of the V-ATPase inhibitor concanamycin A (12,25). Proton transport of 30 g purified vacuolar vesicles was measured via quenching of 1 M 9-amino-6-chloro-2-methoxyacridine (ACMA) upon the addition of 0.5 mM ATP-1 mM MgSO 4 (MgATP) as described previously (25,26).…”

Section: Methodsmentioning

confidence: 99%

“…Studies of S. cerevisiae have shown that disruption of VMA2 completely inhibits both ATPase activity and proton transport by the V-ATPase (14). We have previously investigated the contribution of several subunits of C. albicans VATPase to cell biology and virulence-related traits (4,12). This is the first study analyzing a subunit of the catalytic hexamer in the V 1 domain of V-ATPase in C. albicans.…”

mentioning

confidence: 99%

“…In addition to its requirement for intraorganellar pH homeostasis, V-ATPase function has been implicated in cytosolic and extracellular pH homeostasis (1,(8)(9)(10)(11), underscoring the far-reaching importance of this pump to overall cellular homeostasis. V-ATPase inhibition also interferes with virulence-related traits in C. albicans, including the secretion of degradative enzymes and yeast-to-hypha transitioning (3,4,12,13). Notably, V-ATPase previously has been shown to be important for in vivo virulence in C. albicans (3, 13); however, the requirement of acidic environmental pH for the growth of VATPase mutants complicates the interpretation of these studies due to the growth-limiting alkaline pH in the bloodstream of the murine host.…”

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confidence: 99%

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The Contribution of Candida albicans Vacuolar ATPase Subunit V ₁ B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

et al. 2014

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Candida albicans vacuoles are central to many critical biological processes, including filamentation and in vivo virulence. The V-ATPase proton pump is a multisubunit complex responsible for organellar acidification and is essential for vacuolar biogenesis and function. To study the function of the V 1 B subunit of C. albicans V-ATPase, we constructed a tetracycline-regulatable VMA2 mutant, tetR-VMA2. Inhibition of VMA2 expression resulted in the inability to grow at alkaline pH and altered resistance to calcium, cold temperature, antifungal drugs, and growth on nonfermentable carbon sources. Furthermore, V-ATPase was unable to fully assemble at the vacuolar membrane and was impaired in proton transport and ATPase-specific activity. VMA2 repression led to vacuolar alkalinization in addition to abnormal vacuolar morphology and biogenesis. Key virulence-related traits, including filamentation and secretion of degradative enzymes, were markedly inhibited. These results are consistent with previous studies of C. albicans V-ATPase; however, differential contributions of the V-ATPase V o and V 1 subunits to filamentation and secretion are observed. We also make the novel observation that inhibition of C. albicans V-ATPase results in increased susceptibility to osmotic stress. Notably, V-ATPase inhibition under conditions of nitrogen starvation results in defects in autophagy. Lastly, we show the first evidence that V-ATPase contributes to virulence in an acidic in vivo system by demonstrating that the tetR-VMA2 mutant is avirulent in a Caenorhabditis elegans infection model. This study illustrates the fundamental requirement of V-ATPase for numerous key virulence-related traits in C. albicans and demonstrates that the contribution of VATPase to virulence is independent of host pH.T he fungal pathogen Candida albicans is the fourth most common cause of hospital-acquired bloodstream infections and is a major cause of catheter-associated infections, sepsis, and devicerelated infections. It is also an extremely common cause of urinary and mucosal infections. Despite its clinical significance, the diagnosis and treatment of disseminated candidiasis remain limited by an incomplete understanding of its molecular pathogenesis. The fungal vacuole, a degradative organelle roughly equivalent to the mammalian lysosome, plays an important role in numerous biological processes in C. albicans, including key aspects of pathogenesis. Previous studies have established that C. albicans mutants compromised in vacuolar function are defective in yeast-to-hypha transitioning, a major virulence-related trait, and exhibit reduced virulence in vivo (1, 2).An essential component of vacuolar biogenesis and function is the vacuolar H ϩ -ATPase (V-ATPase) proton pump, which is a multisubunit complex responsible for the acidification of internal organelles. V-ATPase is located at the vacuolar membrane and throughout the endomembrane system, including prevacuolar compartments and the Golgi complex (1). The acidification of these organelles has sever...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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The Contribution of Candida albicans Vacuolar ATPase Subunit V ₁ B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

et al. 2014

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“…S2 in the supplemental material). To determine if the azole tolerance of the vps21⌬/⌬ mutant depends upon V-ATPase activity, we deleted both alleles of VPH1, a gene encoding a vacuole-specific subunit of the V-ATPase (27). However, the loss of VPH1 did not affect the susceptibility of the wild-type control strain to fluconazole or the trailing growth of the vps21⌬/⌬ mutant (Fig.…”

Section: Azole Tolerance Of the C Albicans Vps21⌬/⌬ Mutant Does Not mentioning

confidence: 99%

Endosomal Trafficking Defects Can Induce Calcium-Dependent Azole Tolerance in Candida albicans

Luna-Tapia

Tournu

Peters

et al. 2016

Antimicrob Agents Chemother

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The azole antifungals arrest fungal growth through inhibition of ergosterol biosynthesis. We recently reported that a Candida albicans vps21⌬/⌬ mutant, deficient in membrane trafficking through the late endosome/prevacuolar compartment (PVC), continues to grow in the presence of the azoles despite the depletion of cellular ergosterol. Here, we report that the vps21⌬/⌬ mutant exhibits less plasma membrane damage upon azole treatment than the wild type, as measured by the release of a cytoplasmic luciferase reporter into the culture supernatant. Our results also reveal that the vps21⌬/⌬ mutant has abnormal levels of intracellular Ca 2؉ and, in the presence of fluconazole, enhanced expression of a calcineurin-responsive RTA2-GFP reporter. Furthermore, the azole tolerance phenotype of the vps21⌬/⌬ mutant is dependent upon both extracellular calcium levels and calcineurin activity. These findings underscore the importance of endosomal trafficking in determining the cellular consequences of azole treatment and indicate that this may occur through modulation of calcium-and calcineurin-dependent responses.

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Proton Transport and pH Control in Fungi

Kane

2016

Advances in Experimental Medicine and Biology

101

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Despite diverse and changing extracellular environments, fungi maintain a relatively constant cytosolic pH and numerous organelles of distinct lumenal pH. Key players in fungal pH control are V-ATPases and the P-type proton pump Pma1. These two proton pumps act in concert with a large array of other transporters and are highly regulated. The activities of Pma1 and the V-ATPaseare coordinated under some conditions, suggesting that pH in the cytosol and organelles is not controlled independently. Genomic studies, particularly in the highly tractable S. cerevisiae, are beginning to provide a systems-level view of pH control, including transcriptional responses to acid or alkaline ambient pH and definition of the full set of regulators required to maintain pH homeostasis. Genetically encoded pH sensors have provided new insights into localized mechanisms of pH control, as well as highlighting the dynamic nature of pH responses to the extracellular environment. Recent studies indicate that cellular pH plays a genuine signaling role that connects nutrient availability and growth rate through a number of mechanisms. Many of the pH control mechanisms found in S. cerevisiae are shared with other fungi, with adaptations for their individual physiological contexts. Fungi deploy certain proton transport and pH control mechanisms not shared with other eukaryotes; these regulators of cellular pH are potential antifungal targets. This re view describes current and emerging knowledge proton transport and pH control mechanisms in S. cerevisiae and briefly discusses how these mechanisms vary among fungi.

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Deletion of Vacuolar Proton-translocating ATPase Voa Isoforms Clarifies the Role of Vacuolar pH as a Determinant of Virulence-associated Traits in Candida albicans*

Cited by 31 publications

References 65 publications

The Contribution of Candida albicans Vacuolar ATPase Subunit V ₁ B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

The Contribution of Candida albicans Vacuolar ATPase Subunit V ₁ B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

Endosomal Trafficking Defects Can Induce Calcium-Dependent Azole Tolerance in Candida albicans

Proton Transport and pH Control in Fungi

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Deletion of Vacuolar Proton-translocating ATPase Voa Isoforms Clarifies the Role of Vacuolar pH as a Determinant of Virulence-associated Traits in Candida albicans*

Cited by 31 publications

References 65 publications

The Contribution of Candida albicans Vacuolar ATPase Subunit V 1 B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

The Contribution of Candida albicans Vacuolar ATPase Subunit V 1 B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

Endosomal Trafficking Defects Can Induce Calcium-Dependent Azole Tolerance in Candida albicans

Proton Transport and pH Control in Fungi

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Resources

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The Contribution of Candida albicans Vacuolar ATPase Subunit V ₁ B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

The Contribution of Candida albicans Vacuolar ATPase Subunit V ₁ B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH