Expression and Secretion of SPI-1 Effector Proteins in DNA Adenine Methylase (dam) Mutants of Salmonella Typhimurium.

GIACOMODONATO MN, UZZAU S, GARCÍA CATTANEO AS, SARNACKI SH, RUBINO S, AND CERQUETTI MC

Victoria, BC, Canada

Conferencia; 2nd ASM Conference on Salmonella: From Pathogenesis to Therapeutics; 2006

American Society for Microbiology

DNA adenine methylase (Dam) protein is a global regulator of bacterial gene expression. Dysregulation of Dam activity is potentially a general strategy for the generation of vaccines against bacterial pathogens. We obtained a Dam mutant of Salmonella serovar Typhimurium, named D220, which bears a defective Dam (ten amino-acid shorter). D220 mutant is highly attenuated and confers a protective immune response. However we observed that D220 mutant has a reduced capacity to invade the murine intestinal epithelium in the ileal loop assay (40 % compared to wild type strain). This partial inability to invade epithelial cells could be related to a reduced secretion of invasion proteins encoded in the Salmonella pathogenicity island 1 (SPI-1). Then, we investigated the involvement of Dam protein on the expression and secretion of SPI-1 proteins essential for invasion, such as SipA, SopA, SopB, SopD and SopE2. Salmonella Typhimurium SSM3213 sopA::3xFLAG sopE2::3xFLAG cat::FLAG, SSM3214 sopD::3xFLAG sipA::3xFLAG cat::FLAG, SSM3215 sopB::3xFLAG cat::FLAG and their derivative dam-231::Tn10dTet were used throughout the experiments. The expression and secretion of these effector proteins were determined in vitro, analyzing different fractions of bacterial culture. Bacterial strains were grown under SPI-1 conditions. Bacterial culture supernatants and pellets were used to investigate secreted-proteins and cell-associated proteins, respectively. Expression and secretion were analysed by SDS-page and immunoblotting with anti-FLAG antibodies. In vitro experiments showed that D220 mutant Salmonella Typhimurium secretes nearly half the amount of SPI-1 effector proteins (with the exception of SopA) that are secreted by the wild type strain. These differences were statistically significant (p < 0.05). We found that the secretion of SipA and SopE2 in D220 dam mutant is reduced by 40 % compared to the virulent strain, whereas the secretion of SopB and SopD is diminished by 38% and 50%, respectively. The amount of SopA detected in the supernatant of D220 culture was only 5% of that seen for the wild type strain (p < 0.01). This fact is likely to be related to the short half-life of SopA rather than a diminished expression of the protein. Indeed, all the effector proteins analyzed (including SopA) were expressed in D220 mutant at comparable levels, around 50% lower than the wild type. Complementation of the dam mutation fully restored the proteins profile observed in the wild type strain. Taken together, our results demonstrate that Dam methylation modulates the SPI-1 encoded type III secretion system.