Abstract
During infection of mammalian hosts, facultative intracellular pathogens have to adjust rapidly to different environmental conditions encountered during passage through the gastrointestinal tract and following uptake into epithelial cells and macrophages. Successful establishment within the host therefore requires the coordinated expression of a large number of virulence genes necessary for the adaptation between the extracellular and intracellular phases of infection. In this study we show that the bacterial signal molecule, ppGpp, plays a major role in mediating the environmental signals involved in the regulation of both the extracellular and intracellular virulence gene programs. Under oxygen limiting conditions, we observed a strong ppGpp dependence for invasion gene expression, the result of severe reductions in expression of the Salmonella pathogenicity island (SPI) 1 transcriptional regulator genes hilA, C, and D and invF. Overexpression of the non-SPI1-encoded regulator RtsA restored hilA expression in the absence of ppGpp. SPI2-encoded genes, required for intracellular proliferation in macrophages, were activated in the wild type strain under aerobic, late log phase growth conditions. The expression of SPI2 genes was also shown to be ppGpp-dependent under these conditions. The results from this study suggest a mechanism for the alternate regulation of the opposing extracellular and intracellular virulence gene programs and indicate a remarkable specificity for ppGpp in the regulation of genes involved in virulence compared with the rest of the genome. This is the first demonstration that this highly conserved regulatory system is involved in bacterial virulence gene expression on a global scale.
Highlights
Salmonella enterica serovar Typhimurium (Salmonella typhimurium) is a major pathogen of animals and man, causing gastroenteritis characterized by inflammatory diarrhea in humans
⌬relA⌬spoT strain of S. typhimurium showed severe reductions in the expression of genes involved in invasion of host cells and an avirulent phenotype in susceptible mice [23]
S. typhimurium virulence genes as well as global gene expression patterns. Because it was not known which of the environmental signaling pathways involved in virulence gene expression were affected by the absence of ppGpp, we compared the expression profiles obtained from wild type and ⌬relA⌬spoT strains grown under four different growth conditions: low oxygen cultures grown in L-broth containing 0.3 M NaCl, previously reported to activate SPI1 invasion genes [25, 26], and the separated growth conditions of either L-broth with low oxygen, or aerated
Summary
A recent study found that an S. typhimurium ⌬relA⌬spoT strain was severely attenuated in susceptible BALB/c mice, was effectively noninvasive for epithelial cells in vitro, and played a crucial role in the regulation of SPI1 and the spv virulence plasmid genes in S. typhimurium [23, 24] These results suggested that ppGpp may play a larger role in virulence gene expression in Salmonella. Because ppGpp is part of a highly conserved regulatory system for mediating the growth response to environmental conditions, this mechanism may represent a common strategy whereby facultative intracellular pathogens regulate the virulence gene programs required for invasion and later survival and persistence within host cells to match the capacity for growth
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