SopB effector protein of Salmonella Typhimurium is translocated in mesenteric lymph nodes during murine salmonellosis

Abstract

Salmonella Typhimurium harbors two Salmonella pathogenicity islands (SPIs), each encoding a type three secretion system for virulence proteins. Although there is increasing evidence of postinvasion roles for SPI-1, it has been generally accepted that SPI-1 genes are downregulated following the invasion process. Here, we analyzed the expression and translocation of SopB in vitro, in cell culture and in vivo. To this end, a sopB-FLAG-tagged strain of Salmonella Typhimurium was obtained by epitope tagging. Tagged proteins were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting with anti-FLAG antibodies. SopB expression was observed in vitro under cultured conditions that mimic the intestinal niche and different intracellular environments. In agreement, bacteria isolated from infected monolayers expressed and translocated SopB for at least 24 h postinoculation. For in vivo experiments, BALB/c mice were inoculated intraperitoneally with the tagged strain of Salmonella Typhimurium. Infecting bacteria and infected cells were recovered from mesenteric lymph nodes. Our results showed that SopB continues to be synthesized in vivo during 5 days after inoculation. Interestingly, translocation of SopB was detected in the cytosol of cells isolated from lymph nodes 1 day after infection. Altogether, these findings indicate that the expression and translocation of SopB during Salmonella infection is not constrained to the initial host-bacteria encounter in the intestinal environment as defined previously.

A second wave of Salmonella T3SS1 activity prolongs the lifespan of infected epithelial cells

“…Previously, the continued presence of T3SS1 effectors post-invasion has been explained by effector persistence in the intracellular environment [48] or delivery by the T3SS2 [25,26,49]. The main reason that the role of T3SS1 post-invasion is not well understood is because mutants lacking T3SS1 activity are unable to infect host cells.…”

“…SopB delivery by cytosolic bacteria requires continued transcription and translation of the molecule by the bacterium post-invasion [50]. Furthermore, T3SS2 has no role in delivery of SopB or SipA to infected host cells, although that has previously been suggested as a mechanism for sustained delivery both in vitro and in vivo [25,26,49]. Using a murine systemic infection model, we confirmed that SPI1 effectors are present in cells containing large numbers of cytosolic bacteria in vivo .…”

Dam methylation regulates the expression of SPI-5-encoded sopB gene in Salmonella enterica serovar Typhimurium

“…Animals received a high dose of Salmonella that, in turn, yielded a sufficient number of infecting bacteria (to be recovered to investigate SopB expression), and also provided an adequate amount of infected cells (to be isolated to determine SopB translocation). In a previous report, we demonstrated that 24 h after inoculation, SopB continues to be expressed by wild type infecting bacteria recovered from MLN [25]. Once again, intracellular Salmonella dam mutant expressed lower levels of SopB respect to the wild type strain ( p < 0.05) ( Fig.…”

“…We have shown earlier that Salmonella effectors involved in invasion processes continue to be expressed in vivo during systemic infection [25,34]. For instance, SopB protein was found in both phases of Salmonella infection [6,25]. However, the amount of SopB detected at day 5 was lower than that observed in bacteria recovered at 12 h postintraperitoneally inoculation [34,35].…”

“…In vitro and in vivo experiments showed that Dam methylation also modulates SopB expression under intracellular conditions. We have shown earlier that Salmonella effectors involved in invasion processes continue to be expressed in vivo during systemic infection [25,34]. For instance, SopB protein was found in both phases of Salmonella infection [6,25].…”