OUTER MEMBRANE VESICLES IN Serratia marcescens

ROBERTO E. BRUNA; JAVIER FERNANDO MARISCOTTI; ELEONORA GARCÍA VÉSCOVI

Rosario

Congreso; IX Congreso de Microbiología General SAMIGE 2013; 2013

Sociedad Argentina de Microbiología General

Nearly all Gram negative bacteria, both pathogenic and non pathogenic, release outer membrane vesicles (OMVs) into their environment as a natural process. These OMVs are composed of lipopolysaccharide (LPS), phospholipids, outer membrane proteins and periplasmic components. In the last years, several studies have suggested a number of potential roles for vesiculization, incluiding bacterial envelope stress response, protection against antimicrobial and toxic components, binding and delivery of DNA, and in pathogenic species, transport of virulence factors, and evasion and modulation of the host immune response. Serratia marcescens is a Gram negative enteric bacillus. It is a pathogen with a remarkably wide host range, acting in humans as an opportunistic pathogen. Despite its clinical prevalence, mechanisms of Serratia pathogenesis remain unclear. In our previous work, we have shown that Serratia produces OMVs in a thermoregulated fashion, with a significant increase at 30°C with respect to 37°C. In addition, comparative proteomic analysis of OMVs and outer membrane fraction revealed that a number of components were selectively enriched in the first one, while others appeared to be excluded. These facts can be taken as strong evidence to asses that production of vesicles is a regulated phenomenon. In this work, we have set up an optimized protocol for preparing S. marcescens OMVs from stationary and exponential growth phases. This procedure includes a final step of ultracentrifugation in a sucrose density gradient, which allowed us to leave behind non vesicular contaminating components that usually cosediment with the OMVs -such as flagella- that can interfere with future studies of vesicle proteome and host cell interaction/reactivity to OMVs. We also demonstrate that our OMVs preparation provokes citotoxicity against a monolayer of epithelial CHO cells, by destabilizing the integrity of the eukaryotic plasmatic membrane. On the other hand, we designed and tested a small-scale based vesicle quantification method, to be used in a random mutagenesis screening for detecting overproducing and underproducing vesicle mutants in order to identify genes involved in the modulation of OMVs production.