Expression and Translocation of SPI-1 Effector Proteins of Salmonella Typhimurium during Systemic Infection in Mice

64. GIACOMODONATO MN, UZZAU S, BACCIU D, CACCURI R, SARNACKI SH, RUBINO S, AND CERQUETTI MC

Orlando, Florida, EEUU

Congreso; 106th General Meeting of the American Society for Microbiology; 2006

American Society for Microbiology

Salmonella enterica serovar Typhimurium SPI-1 is essential for invasion of non-phagocytic cells, whereas SPI-2 is required for intracellular survival and proliferation in phagocytes. However, some SPI-1 effectors are  induced upon invasion into both phagocytic and non-phagocytic cells, suggesting that they may also be required following translocation across the intestinal epithelium. To analyze whether SPI-1 effector proteins participate in the late stages of murine salmonellosis, we investigated in vivo the expression and secretion of SipA, SopA, SopB, SopD and SopE2 during systemic infection of mice using tagged (3xFLAG) strains of serovar Typhimurium. Initially, tagged strains were grown in vitro under SPI-1 or SPI-2 conditions and different fractions of bacterial cultures, 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. SipA, SopA, SopB, SopD and SopE2 expression was observed in vitro under SPI-1 conditions. Lowering pH from 6.0 to 4.5, levels compatible with those of the late endosome, drastically reduced the expression of SipA and SopA (p<0.05). In addition, effector secretion into culture medium only occurred under SPI-1 conditions. In a mice model of systemic infection, internalized bacteria were recovered from MLN and spleen, 18 hours after intraperitoneal infection. Our results showed that serovar Typhimurium colonizing MLNs and spleen expressed all the SPI-1 effector proteins under study. SopA was detected in the lowest amount. Interestingly, SopB, SopD and