The phosphatase activity is the target for Mg2+ regulation of the sensor protein PhoQ in Salmonella

The PhoP/PhoQ two-component system controls the expression of essential virulence traits in the pathogenic bacterium Salmonella enterica serovar Typhimurium. Environmental deprivation of Mg(2+) activates the PhoP/PhoQ signal transduction cascade, which results in an increased expression of genes necessary for survival inside the host. It was previously demonstrated that the interaction of Mg(2+) with the periplasmic domain of PhoQ promotes a conformational change in the sensor protein that leads to the down-regulation of PhoP-activated genes. We have now examined the regulatory effect of Mg(2+) on the putative activities of the membrane-bound PhoQ. We demonstrated that Mg(2+) promotes a phospho-PhoP phosphatase activity in the sensor protein. This activity depends on the intactness of the conserved His-277, suggesting that the phosphatase active site overlaps the H box. The integrity of the N-terminal domain of PhoQ was essential for the induction of the phosphatase activity, because Mg(2+) did not stimulate the release of inorganic phosphate from phospho-PhoP in a fusion protein that lacks this sensing domain. These findings reveal that the sensor PhoQ harbors a phospho-PhoP phosphatase activity, and that this phosphatase activity is the target of the extracellular Mg(2+)-triggered regulation of the PhoP/PhoQ system.     

The PhoP/PhoQ system and its role in Serratia marcescens pathogenesis

Serratia marcescens is able to invade, persist, and multiply inside nonphagocytic cells, residing in nonacidic, nondegradative, autophagosome-like vacuoles. In this work, we have examined the physiological role of the PhoP/PhoQ system and its function in the control of critical virulence phenotypes in S. marcescens. We have demonstrated the involvement of the PhoP/PhoQ system in the adaptation of this bacterium to growth on scarce environmental Mg(2+), at acidic pH, and in the presence of polymyxin B. We have also shown that these environmental conditions constitute signals that activate the PhoP/PhoQ system. We have found that the two S. marcescens mgtE orthologs present a conserved PhoP-binding motif and demonstrated that mgtE1 expression is PhoP dependent, reinforcing the importance of PhoP control in magnesium homeostasis. Finally, we have demonstrated that phoP expression is activated intracellularly and that a phoP mutant strain is defective in survival inside epithelial cells. We have shown that the Serratia PhoP/PhoQ system is involved in prevention of the delivery to degradative/acidic compartments.     

Mutational analysis of the Escherichia coli PhoQ sensor kinase: differences with the Salmonella enterica serovar Typhimurium PhoQ protein and in the mechanism of Mg2+ and Ca2+ sensing

The PhoP-PhoQ two-component system plays a role in Mg2+ homeostasis and/or the virulence properties of a number of bacterial species. A Salmonella enterica serovar Typhimurium PhoQ sensor kinase mutant, in which the threonine at residue 48 in the periplasmic sensor domain is changed to an isoleucine, was shown previously to result in elevated expression of PhoP-activated genes and to affect mouse virulence, epithelial cell invasion, and sensitivity to macrophage killing. We characterized a complete set of proteins having amino acid substitutions at position 48 in the closely related Escherichia coli PhoQ protein. Numerous mutant proteins having amino acid substitutions with side chains of various sizes and characters displayed signaling phenotypes similar to that of the wild-type protein, indicating that interactions mediated by the wild-type threonine side chain are not required for normal protein function. Changes to amino acids with aromatic side chains had little impact on signaling in response to extracellular Mg2+ but resulted in reduced sensitivity to extracellular Ca2+, suggesting that the mechanisms of signal transduction in response to these two divalent cations are different. Surprisingly, the Ile48 protein displayed a defective phenotype rather than the hyperactive phenotype seen with the S. enterica serovar Typhimurium protein. We also describe a mutant PhoQ protein lacking the extracellular sensor domain with a defect in the ability to activate PhoP. The defect does not appear to be due to reduced autokinase activity but rather appears to be due to an effect on the stability of the aspartyl-phosphate bond of phospho-PhoP.     

Shigella flexneri infection: pathogenesis and vaccine development

Shigella flexneri is a gram-negative bacterium which causes the most communicable of bacterial dysenteries, shigellosis. Shigellosis causes 1.1 million deaths and over 164 million cases each year, with the majority of cases occurring in the children of developing nations. The pathogenesis of S. flexneri is based on the bacteria's ability to invade and replicate within the colonic epithelium, which results in severe inflammation and epithelial destruction. The molecular mechanisms used by S. flexneri to cross the epithelial barrier, evade the host's immune response and enter epithelial cells have been studied extensively in both in vitro and in vivo models. Consequently, numerous virulence factors essential to bacterial invasion, intercellular spread and the induction of inflammation have been identified in S. flexneri. The inflammation produced by the host has been implicated in both the destruction of the colonic epithelium and in controlling and containing the Shigella infection. The host's humoral response to S. flexneri also appears to be important in protecting the host, whilst the role of the cellular immune response remains unclear. The host's immune response to shigellosis is serotype-specific and protective against reinfection by the same serotype, making vaccination a possibility. Since the 1940s vaccines for S. flexneri have been developed with little success, however, the growing understanding of S. flexneri's pathogenesis and the host's immune response is assisting in the generation of more refined vaccine strategies. Current research encompasses a variety of vaccine types, which despite disparity in their efficacy and safety in humans represent promising progress in S. flexneri vaccine development.     

Characterization of the Salmonella typhimurium pagC/pagD chromosomal region.

The PhoP/PhoQ two-component system regulates Salmonella typhimurium genes that are essential to bacterial virulence and survival within macrophages. The best characterized of these PhoP-activated genes (pag) is pagC, which encodes a 188-amino-acid envelope protein (W. S. Pulkkinen and S. I. Miller, J. Bacteriol. 173:86-93, 1991). We here report the identification of four genes (pagD, envE, msgA, and envF) located 5' to pagC. Each gene is transcribed from its own promoter, two of which (msgA and pagD) were defined by primer extension analysis. Three of these genes (pagD, envE, and envF) are predicted to encode envelope proteins. The pagD gene is transcribed in a direction opposite from that of and adjacent to pagC and is positively regulated by PhoP/PhoQ. Transposon insertions within pagD and msgA attenuate bacterial virulence and survival within macrophages; however, deletion of pagD has no effect on virulence. The product of the envF gene is predicted to be a lipoprotein on the basis of the presence of a consensus lipid attachment site. The low G + C content of these genes and the homology of msgA to Shigella plasmid DNA suggest that this region may have been acquired by horizontal transmission.     

Influence of PhoP and Intra-Species Variations on Virulence of Yersinia pseudotuberculosis during the Natural Oral Infection Route

The two-component regulatory system PhoP/PhoQ has been shown to (i) control expression of virulence-associated traits, (ii) confer survival and growth within macrophages and (iii) play a role in Yersinia infections. However, the influence of PhoP on virulence varied greatly between different murine models of infection and its role in natural oral infections with frequently used representative isolates of Y. pseudotuberculosis was unknown. To address this issue, we constructed an isogenic set of phoP ⁺ and phoP ⁻ variants of strain IP32953 and YPIII and analyzed the impact of PhoP using in vitro functionality experiments and a murine oral infection model, whereby we tested for bacterial dissemination and influence on the host immune response. Our results revealed that PhoP has a low impact on virulence, lymphatic and systemic organ colonization, and on immune response modulation by IP32953 and YPIII, indicating that PhoP is not absolutely essential for oral infections but may be involved in fine-tuning the outcome. Our work further revealed certain strain-specific differences in virulence properties, which do not strongly rely on the function of PhoP, but affect tissue colonization, dissemination and/or persistence of the bacteria. Highlighted intra-species variations may provide a potential means to rapidly adjust to environmental changes inside and outside of the host.