Comparative proteomic analysis of the PhoP regulon in Salmonella enterica serovar Typhi versus Typhimurium

Abstract

Background: S. Typhi, a human-restricted Salmonella enterica serovar, causes a systemic intracellular infection in humans (typhoid fever). In comparison, S. Typhimurium causes gastroenteritis in humans, but causes a systemic typhoidal illness in mice. The PhoP regulon is a well studied two component (PhoP/Q) coordinately regulated network of genes whose expression is required for intracellular survival of S. enterica.

Methodology/principal findings: Using high performance liquid chromatography mass spectrometry (HPLC-MS/MS), we examined the protein expression profiles of three sequenced S. enterica strains: S. Typhimurium LT2, S. Typhi CT18, and S. Typhi Ty2 in PhoP-inducing and non-inducing conditions in vitro and compared these results to profiles of phoP(-)/Q(-) mutants derived from S. Typhimurium LT2 and S. Typhi Ty2. Our analysis identified 53 proteins in S. Typhimurium LT2 and 56 proteins in S. Typhi that were regulated in a PhoP-dependent manner. As expected, many proteins identified in S. Typhi demonstrated concordant differential expression with a homologous protein in S. Typhimurium. However, three proteins (HlyE, STY1499, and CdtB) had no homolog in S. Typhimurium. HlyE is a pore-forming toxin. STY1499 encodes a stably expressed protein of unknown function transcribed in the same operon as HlyE. CdtB is a cytolethal distending toxin associated with DNA damage, cell cycle arrest, and cellular distension. Gene expression studies confirmed up-regulation of mRNA of HlyE, STY1499, and CdtB in S. Typhi in PhoP-inducing conditions.

Conclusions/significance: This study is the first protein expression study of the PhoP virulence associated regulon using strains of Salmonella mutant in PhoP, has identified three Typhi-unique proteins (CdtB, HlyE and STY1499) that are not present in the genome of the wide host-range Typhimurium, and includes the first protein expression profiling of a live attenuated bacterial vaccine studied in humans (Ty800).

Figure 1. Illustration of the PhoQ/PhoP two-component regulatory system in Salmonella enterica serovar Typhimurium.

PhoQ activates PhoP in response to a number of environmental signals including low magnesium. Once activated, PhoP can directly activate its own transcription and the transcription of a number of other genes. A number of directly regulated gene products then regulate additional regulatory cascades, including PmrD, which is able to activate the PmrAB operon independent of iron; SlyA, which regulates genes important to intra-macrophage survival such as pagC and ugtL; IraP which prevents MviA-dependent degradation of RpoS leading to RpoS accumulation and its regulation of genes important for stationary phase survival and resistance to oxidative stress; HilA, which is an inducer of SPI-1 (Salmonella pathogenicity island-1), which contains genes involved in invasion of epithelial cells; and SsrB, which is an inducer of SPI-2 containing genes important in intra-macrophage survival (adapted from Groisman E. and Mouslim C. Nature Reviews Microbiology (2006) 4∶705–709) , . In this figure, underlined genes denote those whose products were detected in our analysis. †: Promoter region contains a typical PhoP box defined as a dyad of (T/G)GTTTA separated by 5 nucleotides. ‡: Presence of an atypical PhoP box defined as a dyad of (T/G)GTTTA separated by 5 nucleotides in the promoter region, allowing four substitutions as long as the following positions were conserved: a thymine in the first dyad half (at position 3) and two conserved thymines and one conserved adenine in the second dyad half at positions 3, 4, and 6, respectively, within 300 nucleotides of the translational start site (see text).