Effects of growth conditions on the expression of virulence factors by Salmonella enterica subspecies 1 serovar typhimurium
Salmonella enterica subspecies 1 serovar Typhimurium is a frequent cause of gastroenteritis and diarrhoea in humans. As diarrhoea is one of the major health problems in the world, it is of great interest to understand Salmonella pathogenesis and the factors controlling it. Upon infection of its host, S. Typhimurium enters epithelial cells. At the beginning of this process several effector proteins are translocated into the host cell through a type III secretion system encoded on the Salmonella pathogenicity island 1 (SPI-1). These virulence factors modulate signal transduction pathways inside the host cell and lead to the internalisation of the bacterial cell. Most of the virulence factors involved in this process have been identified, e.g. the SPI-1 type III secretion apparatus protein PrgH and the effector proteins SopE and SptP. However, little is known about the environmental growth factors controlling their expression and, in the case of effector proteins, their translocation.
In this study the influence of different conditions on the expression of PrgH, SopE and SptP as well as on the secretion of the latter two was examined. Salmonella enterica subspecies 1 serovar Typhimurium was grown in batch cultures and in continuous culture at different dilution rates. Growth in Luria-Bertani (LB) broth containing different NaCl concentrations as well as in glucose- and ammonia limited mineral medium was examined. Furthermore, the influence of aerobic and anaerobic conditions during growth in LB was tested. In addition, carbon utilisation patterns were examined using Biolog microplates. The expression and secretion of proteins were analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting.
In present study the main factor found controlling virulence factor expression was specific growth rate. At specific growth rates equal to or smaller than 0.2 h-1 very little or no PrgH, SptP and SopE were produced in continuous cultures containing LB medium. They were strongly expressed and the effector proteins also secreted at fast specific growth rates (0.7, 1, 1.25 h-1). The expression of PrgH and SopE but not of SptP was reduced again at a specific growth rate of 1.5 h-1. These results indicate a maximal expression at intermediate to high specific growth rates and a repression at low or very high specific growth rates.
In addition, these proteins were found earlier in the growth cycle and in higher amounts if LB batch cultures had been inoculated 1:20 with stationary cells than 1:100 with exponentially growing cells. Contradictory to some other studies, only a minor effect of high osmolarity conditions (0.3 M NaCl) in LB on the expression of virulence factors was seen and none of the examined proteins were expressed under obligate anaerobic conditions in LB batch cultures. However, a weak expression was found in anaerobic LB continuous cultures.
Furthermore, it could be shown that some unidentified substances, which are vital for the expression of PrgH and SopE but not of SptP, are not available in glucose- and ammonialimited mineral medium.
This study contributes to the knowledge about the regulation of virulence factors in S. Typhimurium by environmental factors. Starting from the observed crucial role of the specific growth rate, there is a chance that more detailed investigations might unravel the trigger which transforms S. Typhimurium in a (hyper)virulent state.