|359-1||Effect of PmrA-dependent wzzst, pbgE2 and pbgE3 genes products in lipid A and O-antigen modifications of Salmonella Typhimurium|
|Autores:||Juan Vicente Farizano (INSIBIO UNT-CONICET - Instituto Superior de Investigaciones Biologicas) ; Maria de Mercedes Pescaretti (INSIBIO UNT-CONICET - Instituto Superior de Investigaciones Biologicas) ; Mariela Analia Torres (INSIBIO UNT-CONICET - Instituto Superior de Investigaciones Biologicas) ; Fabian Enrique Lopez (INSIBIO UNT-CONICET - Instituto Superior de Investigaciones Biologicas) ; Monica Alejandra Delgado (INSIBIO UNT-CONICET - Instituto Superior de Investigaciones Biologicas) |
The Salmonella Typhimurium lipopolysaccharide (LPS) consists of lipid A, non-repeating core oligosaccharide and O-antigen. Previous studies demonstrated that the PmrAB regulatory system promotes the remodeling of lipid A by addition of 4-aminoarabinose (L-Ara4N) and phosphoethanolamine (pEtN), activating ugd, pbgPE operon and pmrC transcription. The O-antigen length determinant wzzfepE and wzzst genes are up-regulated by PmrAB or PmrAB and RcsCDB systems, respectively. These suggest us that Wzzst may participate in many process of bacterial cell. Thus, we demonstrated that under RcsCDB activation, the wzzst gene product is involved in the cell swarming behavior. However, under PmrAB system activation non function for this protein was established. Therefore, we performed MALDI-TOF assay to analyze the lipid A harvested from the wzzst mutant. We found that peaks at m/z 1928, 2166 and 2158 corresponding to lipid A modified with either L-Ara4N or pEtN were absent in the mutant; but it harbor a new peak of m/z 2282, arising from ion of m/z 2158 by addition of a pEtN species. In addition, we observed that in wzzst and wzzfepE mutants an O-antigen portion of lower-molecular-weight (VLMW) was maintained. Therefore, we investigate if the PmrA-dependent PbgE2 and PbgE3 proteins are involved in O-antigen formation, because its function still unknown. To test this, we extract LPS from these mutants and were analyzed in a SDS-PAGE gel. We noticed that these pbgE2 and pbgE3 mutants lack the O-antigen VLMW material, denominated short region (S). To test if PbgE2 and PbgE3 are together required to control the S region formation, we carried out in vivo protein-protein interaction using the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) assay. These result demonstrated that both proteins work together to determine the O-antigen short length. These results allowed us to establish that the S. Typhimurium O-antigen follows a tri-modal length distribution in the outer membrane. Finally, we established that the above mutants display decreases levels of serum-complement and polymyxin B resistance, and phagocytosis and replication within the host. In summary our findings highlight the importance of the PmrAB system in the LPS modification, where lipid A modification and O-antigen chain length distribution are coordinately controlled, manly for bacteria adaptation within the host cell.
Palavras-chave: Salmonella, PmrAB system, Virulence, LPS