Poster Presentation 2014 International Biophysics Congress

VCA1008: A new anion-selective trimeric porin of vibrio cholerae (#306)

Carolina L. Goulart 1 , Paulo Mascarello Bisch 1 , Wanda M.A. von Krüger 1 , Fabrice Homblé 2
  1. Institut of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil
  2. Structure et Fonction des Membranes Biologiques, Centre de Biologie Structurale et de Bioinformatique, Faculté des Sciences, Université Libre de Bruxelles, Brussels, Belgium

Vibrio cholerae is the causative agent of cholera, a very severe diarrheal disease. During intestine colonization, the bacterium expresses, among other essential factors to the process, VCA1008, an outer membrane protein induced by phosphate limitation under laboratory conditions1 . VCA1008 is essential to the pathogenesis of Vibrio cholerae O1 biovar El Tor str. N16961, responsible for the great majority of choleracasesworldwide. Furthermore, VCA1008 is required for the bacterium bile salts resistance2.

In this work, the functional properties of VCA1008 were investigated by electrophysiology. For that purpose, a V. cholerae ΔompU strain (derived from N16961) was grown under low phosphate conditions and its outer membrane proteins were extracted. VCA1008 was purified to homogeneity from the outer membrane fraction using DEAE-cellulose, and, then, reconstituted into planar bilayers of E. coli polar lipids.

We show that VCA1008 forms a channel. Its conductance, voltage-dependence and ion selectivity were determined. Experiments on a single unit revealed a trimeric porin with a conductance that varied linearly with KCl concentration, indicating that VCA1008 monomer forms a large pore, which was confirmed by polymer exclusion experiments. Each monomer of VCA1008 shows preference for anions over cations. VCA1008 conductance measurements in the presence of phosphate indicated the protein has a binding site for this anion.

Based on these results we suggest that VCA1008 can promote the influx of anions, especially phosphate, through the outer membrane, which could explain its role in V. cholerae under phosphate deprivation.

  1. von Krüger, W.M., Lery, L.M., Soares, M.R., de Neves-Manta, F.S., Batista e Silva, C.M., Neves-Ferreira, A.G., Perales, J. & Bisch, P.M. (2006) The phosphate-starvation response in Vibrio cholerae O1 and phoB mutant under proteomic analysis: disclosing functions involved in adaptation, survival and virulence. Proteomics 6, 1495-1511.
  2. Goulart C.L., dos Santos G.G., Barbosa L.C., Lery L.M., Bisch P.M., von Krüger W.M. (2010) A ToxR-dependent role for the putative phosphoporin VCA1008 in bile salt resistance in Vibrio cholerae El Tor N16961. Microbiology 156(Pt 10):3011-20.