Attenuation of the sensing capabilities of PhoQ in transition to obligate insect-bacterial association

Sodalis glossinidius, a maternally inherited endosymbiont of the tsetse fly, maintains genes encoding homologues of the PhoP-PhoQ two-component regulatory system. This two-component system has been extensively studied in facultative bacterial pathogens and is known to serve as an environmental magnesium sensor and a regulator of key virulence determinants. In the current study, we show that the inactivation of the response regulator, phoP, renders S. glossinidius sensitive to insect derived cationic antimicrobial peptides (AMPs). The resulting mutant strain displays reduced expression of genes involved in the structural modification of lipid A that facilitates resistance to AMPs. In addition, the inactivation of phoP alters the expression of type-III secretion system (TTSS) genes encoded within three distinct chromosomal regions, indicating that PhoP-PhoQ also serves as a master regulator of TTSS gene expression. In the absence of phoP, S. glossinidius is unable to superinfect either its natural tsetse fly host or a closely related hippoboscid louse fly. Furthermore, we show that the S. glossinidius PhoQ sensor kinase has undergone functional adaptations that result in a substantially diminished ability to sense ancestral signals. The loss of PhoQ's sensory capability is predicted to represent a novel adaptation to the static symbiotic lifestyle, allowing S. glossinidius to constitutively express genes that facilitate resistance to host derived AMPs.     

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.     

Is PhoR–PhoP partner fidelity strict? PhoR is required for the activation of the pho regulon in Streptomyces coelicolor

Two-component regulatory systems play a key role in the cell metabolism adaptation to changing nutritional and environmental conditions. The fidelity between the two cognate proteins of a two-component system is important since it determines whether a specific response regulator integrates the signals transmitted by different sensor kinases. Phosphate regulation in Streptomyces coelicolor is mostly mediated by the PhoR-PhoP two-component system. Previous studies elucidated the mechanisms that control phosphate regulation as well as the genes directly regulated by the response regulator PhoP (pho regulon) in this organism. However, the role of the histidine kinase PhoR in Streptomyces coelicolor had not been unveiled so far. In this work, we report the characterization of a non-polar ΔphoR deletion mutant in S. coelicolor that keeps its native promoter. Induction of the phoRP operon was dependent upon phosphorylation of PhoP, but the ΔphoR mutant expressed phoP at a basal level. RT-PCR and reporter luciferase assays demonstrated that PhoR plays a key role in the activation of the pho regulon in this organism. Our results point towards a strict cognate partner specificity in terms of the phosphorylation of PhoP by PhoR thus corroborating the tight interaction between the two-components of this system.     

Constitutive expression of the phoP regulon attenuates Salmonella virulence and survival within macrophages.

The phoP genetic locus is a two-component regulatory system (phoP-phoQ) that controls the expression of genes essential for Salmonella typhimurium virulence and survival within macrophages. Strains with a phoP constitutive mutation (phenotype PhoPC) showed up to 10-fold greater expression of phoP-activated genes (pag loci) than did strains with a wild-type phoP locus (phenotype PhoP+). While the phoP constitutive mutation resulted in increased expression of pag loci, it also dramatically reduced the expression of other protein species. Comparison of the protein content of PhoP+ and PhoPC strains by two-dimensional protein gel electrophoresis demonstrated that at least 40 separate protein species were changed in expression as a result of this mutation. The PhoPC S. typhimurium were found to be attenuated for virulence and survival within macrophages. This finding suggests that a balanced PhoP-PhoQ regulatory response, which allows expression of phoP-repressed as well as -activated genes, is required for full virulence of S. typhimurium. We have further shown that small numbers of PhoPC bacteria can be used as a live attenuated vaccine to protect against mouse typhoid. As few as 15 PhoPC bacteria protected mice against challenge with 10(5) 50% lethal doses of wild-type organisms, suggesting that important protective antigens are regulated by the PhoP-PhoQ virulence regulon.     

Role of two novel two-component regulatory systems in development and phosphatase expression in Myxococcus xanthus

We have cloned a two-component regulatory system (phoR2-phoP2) of Myxococcus xanthus while searching for genes that encode proteins with phosphatase activity, where phoR2 encodes the histidine kinase and phoP2 encodes the response regulator. A second system, phoR3-phoP3, was identified and isolated by using phoP2 as a probe. These two systems are quite similar, sharing identities along the full-length proteins of 52% on the histidine kinases and 64% on the response regulators. The predicted structures of both kinases suggest that they are anchored to the membrane, with the sensor domains being located in the periplasmic space and the kinase domains in the cytoplasm. The response regulators (PhoP2 and PhoP3) exhibit a helix-loop-helix motif typical of DNA-binding proteins in the effector domains located in the C-terminal region. Studies on two single-deletion mutants and one double-deletion mutant have revealed that these systems are involved in development. Mutant fruiting bodies are not well packed, originating loose and flat aggregates where some myxospores do not reshape properly, and they remain as elongated cells. These systems are also involved in the expression of Mg-independent acid and neutral phosphatases, which are expressed during development. The neutral phosphatase gene is especially dependent on PhoP3. Neither PhoP2 nor PhoP3 regulates the expression of alkaline phosphatases and the pph1 gene.     

Characterization of SfPgdA, a Shigella flexneri peptidoglycan deacetylase required for bacterial persistence within polymorphonuclear neutrophils

Peptidoglycan deacetylases protect the Gram-positive bacteria cell wall from host lysozymes by deacetylating peptidoglycan. Sequence analysis of the genome of Shigella flexneri predicts a putative polysaccharide deacetylase encoded by the plasmidic gene orf185, renamed here SfpgdA. We demonstrated a peptidoglycan deacetylase (PGD) activity with the purified SfPgdA in vitro. To investigate the role SfPgdA in virulence, we constructed a SfpgdA mutant and studied its phenotype in vitro. The mutant showed an increased sensitivity to lysozyme compared to the parental strain. Moreover, the mutant was rapidly killed by polymorphonuclear neutrophils (PMNs). Specific substitution of histidines residues 120 and 125, located within the PGD catalytic domain, by phenylalanine abolished SfPgdA function. SfPgdA expression is controlled by PhoP. Mutation of phoP increases sensitivity to lysozyme compared to the SfpgdA mutant. Here, we confirmed that SfPgdA expression is enhanced under low magnesium concentration and not produced by the phoP mutant. Ectopic expression of SfPgdA in the phoP mutant restored lysozyme resistance and parental bacterial persistence within PMNs. Together our results indicate that PG deacetylation mechanism likely contributes to Shigella persistence by subverting detection by the host immune system.