Virulence mechanisms of two gram negative bacteria : Studies on Escherichia coli hemolysin HlyA and on the interaction of Brucella abortus with non-phagocytic cells

Abstract: Virulence factors may be classified in two general groups: those that cause damage to the host and those that promote bacterial colonization and invasion. In this study, the tissue damaging Escherichia coli hemolysin HlyA and the mechanism of internalization of the intracellular parasite Brucella abortus were investigated. HIyA is a pore-forming toxin belonging to the family of Gram negative exo-proteins known as RTX (from Repeat in Toxins). HIyA is activated by fatty acylation of two internal lysine residues, by a mechanism requiring acyl carrier protein and HlyC. Site directed mutagenesis studies of hlyC identified amino acid residues essential for HlyC acyl transferase activity as well as other residues that generated simultaneous secretion of bi-, mono- and non-acylated HIyA molecules. HlyC was not detected by Western blot in an E. coli strain encoding hlyC and hIyA, while four different isoforms were found in a strain encoding hlyC alone. As similar hlyC mRNA levels were found in both strains, it was concluded that HlyC was probably degraded by a HlyA dependent mechanism. Degradation of HlyC was performed by multiple protease systems such as Clp, FtsH and Lon. Internalization of B. abortus was studied by fluorescence and transmission electron microscopy. Brucella did not induce major cytoskeletal rearrangements in HeLa cells despite the fact that both, the actin and microtubule networks were needed for its uptake. Treatment of HeLa cells with different bacterial toxins specific for small GTPases together with mutant studies demonstrated that Rho, Rac and Cdc42 were involved in Brucella internalization. Moreover, immunoprecipitation of active Rho, Rac and Cdc42 demonstrated that Brucella is capable of activating Cdc42 during entry to HeLa cells. Analysis by two-dimensional gel electrophoresis of wild type and non virulent B. abortus BvrR-BvrS two component system mutants revealed differences in various protein groups in outer membrane fragments. Two protein groups were chosen for further studies. Western Blot and protein sequencing showed that one group of protein spots, absent in the mutant strains, corresponded to the outer membrane protein Omp3A (Omp25). beta-galactosidase reporter analysis indicated transcriptional regulation of omp3A under the control of BvrR-BvrS. A second family of protein spots had the same sequence and was shown to be an unknown protein, similar to membrane proteins RopA and RopB from Rhizobium melliloti. We also detected variations in the lipid A acylation level in the mutant strains as compared to the wild type strain. Intoxication of HeLa cells with cytotoxic necrotizing factor (CNF) from E. coli increased B. abortus internalization up to 10 fold as compared to non-treated cells, without altering the intracellular replication of wild type and bvrS mutant strains. Entry however was affected, since B. abortus penetrated HeLa cells by membrane ruffles induced by the toxin. It was also established that B. abortus infection does not interfere with the cytokinesis and mitosis processes of its host cell. The use of CNF will help to further elucidate the Brucella internalization process.