Characterization of a novel lysozyme-like protein of Brucella abortus

DEL GIUDICE MG; UGALDE JE; CZIBENER C

Buenos Aires

Congreso; Brucellosis 2011-International Research Conference; 2011

Asociación Argentina de Microbiología

Lysosyme-like enzymes are widely distributed among Gram-negative bacteria. These proteins hydrolyze bonds in the peptidoglycan cell wall and are involved in many pathways that include, among others, lysis during cell division, biogenesis of the flagella and generation of pores in the cell wall for the assembly of macromolecular secretion systems. These lysozyme-like enzymes cleave the β-1,4-glycosidic bond between the N-acetylmuramic acid (MurNAc) and the N-acetylglucosamine (GlcNAc) and have been recently implicated in other microorganisms in the secretion of proteins through the outer membrane. The majority of the known species of the genus Brucella are pathogenic for humans and their virulence is dependent on the capacity to survive and replicate within host cells. With the purpose of identifying novel virulence genes we compared the genome of Brucella abortus with the rest of the sequenced species searching for differences between this and the less pathogenic ones. As a result of this search we identified a region of 15 Kb that is absent in B. ovis, a non-pathogenic species for human. The structural characteristics of this chromosomal region suggest that it could have been horizontally acquired and, thus, is a strong candidate to harbor putative virulence genes. This region contains 20 ORFs, the majority of them with unknown function. One of these ORFs, Bab_1002 (designated sagA), codes for a lysozyme-like protein with homology to peptidoglycan hydrolases. To gain insight into the function of sagA we constructed a deletion mutant and analyzed its intracellular replication capacity in phagocytic as well as non-phagocytic cells. The results showed that the mutant is ten times less invasive than the wild type parental strain in both cell types. In addition, over-expression of the gene in the mutant and wild type strains resulted in structural changes of the smooth lipopolysaccharide suggesting that the expression is probably tightly regulated. Analysis of enzymatic activity using as substrate Micrococcus lysodeikticus and purified recombinant SagA allowed us to confirm that it is a cell wall hydrolase with peptidoglycanase activity. Our results indicate that SagA is a lysozyme-like enzyme probably implicated in the secretion of proteins to the surface of the bacteria that mediate internalization into host cell.