CHARACTERIZATION OF THE Ppx GENE OF Brucella abortus

IANNINO F; UGALDE JE; IÑON DE IANNINO N

Puerto Madryn, Chubut, Argentina

Congreso; SAIB 46th Annual Meeting.; 2010

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Inorganic polyphosphate (polyP) is a linear polymer composed of orthophosphate residues (Pi) linked by high energy phosphoanhydride bonds. In bacteria, polyP is synthesized by the polyphospha t e kina s e (PPK) and hydrolyz ed by exopolyphosphatase (PPX). We have cloned and mutagenized the putative ppx gene of the pathogen Brucella abortus. In a previous work we demonstrated that the ppx mutant is affected in J774 intracellular replication and in BALB/c mice infections. Further experiments were carried out in order to characterize the ppx mutant. The B. abortus ppx mutant showed an increased sensitivity to Sarkosyl detergent compared to the wild type. This result indicates that the absence of PPX affects the cell envelope stability of Brucella. SDS-PAGE and Western Blot analysis using outer membrane proteins (OMPs) monoclonal antibodies revealed differences between the ppx mutant and the wild type strain. In HeLa cells and in Bone Marrow-Derived Macrophages infections, the B. abortus ppx mutant showed a marked defect in intracellular survival during the initial phase of the curve compared to the wild type strain. These results suggest that the absence of PPX may affect the internalization of Brucella and/or the biogenesis of the intracellular replicative vacuole. Together, our results suggest that the virulence reduction of the mutant may be due to an altered bacterial membrane.