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The F1Fo-ATP Synthase β Subunit Is Required for Candida albicans Pathogenicity Due to Its Role in Carbon Flexibility.

ABSTRACT: Previous work has explored link between mitochondrial biology and fungal pathogenicity in F1Fo-ATP synthase in Candida albicans. In this work we have detailed the more specific roles of the F1Fo-ATP synthase β subunit, a key protein subunit of F1Fo-ATP synthase. The ability to assimilate alternative carbons in glucose-limited host niches is known to be a critical factor for infection caused by opportunistic pathogens including C. albicans. The function of the F1Fo-ATP synthase β subunit was characterized through the construction of an ATP2 gene null mutant (atp2Δ/Δ) and the gene-reconstituted strain (atp2Δ/ATP2) in order to understand the link between carbon metabolism and C. albicans pathogenesis. Cell growth, viability, cellular ATP content, mitochondrial membrane potential (ΔΨm), and intracellular ROS were compared between null mutant and control strain. Results showed that growth of the atp2Δ/Δ mutant in synthetic medium was slower than in complex medium. However, the synthetic medium delayed the onset of reduced cell viability and kept cellular ATP content from becoming fully depleted. Consistent with these observations, we identified transcriptional changes in metabolic response that activated other ATP-generating pathways, thereby improving cell viability during the initial phase. Unlike glucose effects, the atp2Δ/Δ mutant exhibited an immediate and sharp reduction in cell viability on non-fermentable carbon sources, consistent with an immediate depletion of cellular ATP content. Along with a reduced viability in non-fermentable carbon sources, the atp2Δ/Δ mutant displayed avirulence in a murine model of disseminated candidiasis as well as lower fungal loads in mouse organs. Regardless of the medium, however, a decrease in mitochondrial membrane potential (ΔΨm) was found in the atp2Δ/Δ mutant but ROS levels remained in the normal range. These results suggest that the F1Fo-ATP synthase β subunit is required for C. albicans pathogenicity and operates by affecting metabolic flexibility in carbon consumption.

SUBMITTER: Li SX

PROVIDER: S-EPMC5974098 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The F(1)F(o)-ATP synthases of alkaliphilic bacteria exhibit latent ATPase activity, and for the thermoalkaliphile Bacillus sp. strain TA2.A1, this activity is intrinsic to the F(1) moiety. To study the mechanism of ATPase inhibition, we developed a heterologous expression system in Escherichia coli to produce TA2F(1) complexes from this thermoalkaliphile. Like the native F(1)F(o)-ATP synthase, the recombinant TA2F(1) was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent lauryldimethylamine oxide. To determine if the C-terminal domain of the epsilon subunit acts as an inhibitor of ATPase activity and if an electrostatic interaction plays a role, a TA2F(1) mutant with either a truncated epsilon subunit [i.e., TA2F(1)(epsilon(DeltaC))] or substitution of basic residues in the second alpha-helix of epsilon with nonpolar alanines [i.e., TA2F(1)(epsilon(6A))] was constructed. Both mutants showed ATP hydrolysis activity at low and high concentrations of ATP. Treatment of the purified F(1)F(o)-ATP synthase and TA2F(1)(epsilon(WT)) complex with proteases revealed that the epsilon subunit was resistant to proteolytic digestion. In contrast, the epsilon subunit of TA2F(1)(epsilon(6A)) was completely degraded by trypsin, indicating that the C-terminal arm was in a conformation where it was no longer protected from proteolytic digestion. In addition, ATPase activity was not further activated by protease treatment when compared to the untreated control, supporting the observation that epsilon was responsible for inhibition of ATPase activity. To study the effect of the alanine substitutions in the epsilon subunit in the entire holoenzyme, we reconstituted recombinant TA2F(1) complexes with F(1)-stripped native membranes of strain TA2.A1. The reconstituted TA2F(o)F(1)(epsilon(WT)) was blocked in ATP hydrolysis and exhibited low levels of ATP-driven proton pumping consistent with the F(1)F(o)-ATP synthase in native membranes. Reconstituted TA2F(o)F(1)(epsilon(6A)) exhibited ATPase activity that correlated with increased ATP-driven proton pumping, confirming that the epsilon subunit also inhibits ATPase activity of TA2F(o)F(1).

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The F1Fo-ATP Synthase β Subunit Is Required for Candida albicans Pathogenicity Due to Its Role in Carbon Flexibility.

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