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

PLoS One. 2019 Feb 14;14(2):e0212202. doi: 10.1371/journal.pone.0212202. eCollection 2019.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Chlorocebus aethiops
  • Coxiella burnetii / physiology*
  • Cricetulus
  • Cyclic AMP / metabolism
  • Guanine Nucleotide Exchange Factors / metabolism*
  • HeLa Cells
  • Host-Pathogen Interactions
  • Humans
  • Membrane Fusion
  • Phagosomes / metabolism
  • Phagosomes / microbiology
  • Q Fever / metabolism*
  • Q Fever / microbiology
  • Vacuoles / metabolism
  • Vacuoles / microbiology*
  • Vero Cells
  • rap GTP-Binding Proteins / metabolism*

Substances

  • Guanine Nucleotide Exchange Factors
  • RAPGEF3 protein, human
  • Cyclic AMP
  • RAP2B protein, human
  • rap GTP-Binding Proteins

Grants and funding

This work was supported by SeCTyP 06/J432 to MIC; Agencia Nacional de Investigación Científica y Tecnológica (PICT2013-0305) to MIC and Agencia Nacional de Investigación Científica y Tecnológica (PICT2016-0443) to MIC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.