The cAMP effectors, Rap2b and EPAC, are involved in the regulation of the development of the Coxiella burnetii containing vacuole by altering the fusogenic capacity of the vacuole.

María Eugenia Mansilla Pareja,Milton Osmar Aguilera,Esteban Robledo,María Isabel Colombo,Maria Celeste Gaurón,Zhao-Qing Luo

doi:10.1371/journal.pone.0212202

Abstract

Cyclic Adenosine 3′,5′-monophosphate (cAMP) is a key second messenger known to directly regulate not only the protein kinase A (PKA) activity but also other important molecules such as the exchange protein activated by cAMP (EPAC), which is as a guanine nucleotide exchange factor (GEF) of the low molecular weight GTPase, Rap2. Coxiella burnetii is a Gram negative bacterium that survives and grows in a large Coxiella replicative vacuole (CRV), which displays lysosomal and autophagic features. In this report, we present evidence that both, EPAC and its downstream effector Rap2b, were recruited to the CRV. The transient over-expression of the Rap2b wt protein, but not its inactive mutant Rap2b ΔAAX, markedly inhibited the development of the large CRV. Additionally, Rap2b wtinhibited the fusion of early Coxiella phagosomes with the fully developed CRV, indicating that homotypic fusion events are altered in the presence of high levels of Rap2b wt. Likewise, the fusion of endosome/lysosomal compartments (heterotypic fusions) with the large CRV was also affected by the over-expression of this GTPase. Interestingly, cell overexpression of Rap2b wt markedly decreased the levels of the v-SNARE, Vamp7, suggesting that this down-regulation impairs the homotypic and heterotypic fusions events of the Coxiella vacuole.

Highlights

Coxiella burnetii, the etiological agent of the Q-fever, is an obligate intracellular bacterium
We have demonstrated that the direct activation of exchange protein activated by cAMP (EPAC) and/ or Rap2b inhibits the autophagic response induced by the toxin
We determined if this was a common mechanism involved in the regulation of other intracellular pathogens, we wondered whether these molecules play a role in development of the Coxiella replicative vacuole (CRV) in C. burnetii infected cells[26]

Summary

Introduction

The etiological agent of the Q-fever, is an obligate intracellular bacterium. This microorganism has a biphasic life cycle in which the small cell variant (SCV) and the large cell variant (LCV) alternate. The LCV form is generated in the lysosomal-like large replicative compartment (C. burnetii-replicative vacuoles, CRVs) of infected cells, where the acidic microenvironment favors its replication [1]. We have demonstrated that C. burnetii invades and travels within the host cell through classical endocytic/phagocytic. EPAC, Rap2b and Coxiella burnetii analysis, decision to publish, or preparation of the manuscript

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Results

Conclusion