The exopolyphosphatase of Brucella abortus is involved in pathogenesis

IANNINO F; UGALDE JE; IÑON DE IANNINO N

Capital Federal

Congreso; Brucellosis 2011 International Research Conference; 2011

Asociación Argentina de Microbiología

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 polyphosphate kinase (PPK) and hydrolyzed 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. Analysis by electrophoresis in agarose gel indicates that Brucella abortus wild type accumulated polyphosphates (PolyP) of long chain (more than 75 residues of Pi). The ppx mutant showed an increased amount of PolyP compared to the wild type strain suggesting that the mutation is involved in the accumulation of the polymer. 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. The defect observed was not related to major changes in the LPS structure, however 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 infection assays, the B. abortus ppx mutant showed a marked defect in intracellular survival during the initial phase of the infection curve compared to the wild type strain. Double immunofluorescence assays indicated that the ppx mutant attaches to HeLa cells as the wild type strain but is deficient in the internalization process thus indicating that ppx is involved in the penetration of Brucella in non-professional phagocytes. Together, our results suggest that the virulence reduction of the mutant may be due to an altered bacterial membrane that may affect the internalization of Brucella and/or the biogenesis of the intracellular replicative vacuole.