PhoQ Sensor Research Articles

The Salmonellae PhoQ sensor: mechanisms of detection of phagosome signals

Sensor histidine kinases are widely used by prokaryotes to regulate gene expression in response to environmental signals, and such sensing is essential for virulence for plants and animals. PhoQ is one such sensor kinase that is conserved across a variety of Gram-negative pathogens and functions as part of a two-component system with its transcriptional regulator PhoP. Salmonella typhimurium PhoPQ is one of the most studied two-component systems and has been demonstrated to regulate hundreds of genes encoding the majority of virulence properties including intracellular survival, invasion, lipid A structure, resistance to antimicrobial peptides, and phagosome alteration. PhoQ is activated within acidified phagosomes and host tissues. PhoQ binds divalent cations to form bridges with the membrane that maintain the PhoQ repressed state. Antimicrobial peptides and acidic pH activate PhoQ and these signals, present within phagosomes and intestinal tissues, may serve as specific signatures of the host environment for Gram-negative pathogens. This review will focus on recent progress in the molecular details of PhoQ structure and sensing of defined signals. In addition, Pseudomonas aeruginosa and Salmonella typhimurium PhoQ are compared, providing evidence that a subset of PhoQ proteins of environmental organisms evolved more limited sensing capability using a different structural sensing mechanism.

B1500, a small membrane protein, connects the two-component systems EvgS/EvgA and PhoQ/PhoP in Escherichia coli

Two-component signal-transduction systems (TCSs) of bacteria are considered to form an intricate signal network to cope with various environmental stresses. One example of such a network in Escherichia coli is the signal transduction cascade from the EvgS/EvgA system to the PhoQ/PhoP system, where activation of the EvgS/EvgA system promotes expression of PhoP-activated genes. As a factor connecting this signal transduction cascade, we have identified a small inner membrane protein (65 aa), B1500. Expression of the b1500 gene is directly regulated by the EvgS/EvgA system, and b1500 expression from a heterologous promoter simultaneously activated the expression of mgtA and other PhoP regulon genes. This activation was PhoQ/PhoP-dependent and EvgS/EvgA-independent. Furthermore, deletion of b1500 from an EvgS-activated strain suppressed mgtA expression. B1500 is localized in the inner membrane, and bacterial two-hybrid data showed that B1500 formed a complex with the sensor PhoQ. These results indicate that the small membrane protein, B1500, connected the signal transduction between EvgS/EvgA and PhoQ/PhoP systems by directly interacting with PhoQ, thus activating the PhoQ/PhoP system.

Recognition and Integration of Multiple Environmental Signals by the Bacterial Sensor Kinase PhoQ

Two-component regulatory systems are commonly used by bacteria to sense and respond to their environment. In Molecular Cell, Prost et al. describe how low pH activates the Salmonella PhoQ sensor kinase, building on their previous demonstration that the PhoQ periplasmic domain senses magnesium and antimicrobial peptides. These findings are likely to be broadly applicable for understanding how bacteria recognize and transduce the multiple signals characteristic of their complex extracellular environments.

Turned On by a Low pH

Salmonellae , facultative intracellular pathogens that cause gastroenteritis and typhoid fever, respond to the normally inhospitable environment inside macrophage phagosomes--in which pathogens are exposed to antimicrobial peptides, reactive oxygen species, and acidic pH--by activating the transcription of virulence genes. This process, which depends on phagosomal acidification, involves the activation of the membrane-bound sensor kinase PhoQ, which leads to its autophosphorylation and the subsequent phosphorylation of the transcriptional activator PhoP (the PhoPQ system). Using a bacterial strain that expressed a reporter that was transcriptionally activated by PhoP, Prost et al . showed that, in the presence of 1 mM Mg 2+ (the estimated phagosomal concentration), transcription increased when the pH was decreased from 7.5 to 5.5. When PhoQ was reconstituted in vesicles so that the periplasmic sensor domain faced inward and the cytoplasmic kinase domain faced outward, PhoP phosphorylation increased when lumenal pH was decreased from 7.5 to 5.5. Subinhibitory concentrations of antimicrobial peptides have previously been shown to activate PhoQ through a mechanism that may involve displacement of divalent cations, and the effects of antimicrobial peptides and pH on PhoP phosphorylation and PhoPQ-dependent transcription were additive. Nuclear magnetic resonance (NMR) spectroscopy revealed that the conformation of the PhoQ sensor domain was affected by changes in pH and that this conformational change did not depend on loss of divalent cation binding. Thus, the authors propose that PhoQ acts as a pH sensor and that antimicrobial peptides and low pH work together in macrophage phagosomes to activate the PhoPQ system. L. R. Prost, M. E. Daley, V. Le Sage, M. W. Bader, H. Le Moual, R. E. Klevit, S. I. Miller, Activation of the bacterial sensor kinase PhoQ by acidic pH. Mol. Cell 26 , 165-174 (2007). [Online Journal]

Activation of the Bacterial Sensor Kinase PhoQ by Acidic pH

The Salmonellae PhoQ sensor kinase senses the mammalian phagosome environment to activate a transcriptional program essential for virulence. The PhoQ periplasmic domain binds divalent cations, forming bridges with inner membrane phospholipids to maintain PhoQ repression. PhoQ also binds and is activated by cationic antimicrobial peptides. In this work, PhoQ is directly activated by exposure of the sensor domain to pH 5.5. NMR spectroscopy indicates that at acidic pH, the PhoQ periplasmic domain adopts a conformation different from that in the presence of divalent cations or antimicrobial peptides. The conformation is partially simulated by mutation of histidine 157, which is part of an interaction network that distinguishes the repressed conformation. The effects of antimicrobial peptides and pH on PhoQ activity are additive. We propose a model of activation by antimicrobial peptides via disruption of the cation bridges and/or by acidification of the periplasm through destabilization of the interaction network.

A Mg2+-responding RNA That Controls the Expression of a Mg2+ Transporter

Mg2+ is the most abundant divalent cation in biological systems. It is required for ATP-mediated enzymatic reactions and as a stabilizer of ribosomes and membranes. The enteric bacterium Salmonella enterica serovar Typhimurium harbors three Mg2+ transporters and a regulatory system-termed PhoP/PhoQ-whose activity is regulated by the extracytoplasmic levels of Mg2+. We have determined that expression of the PhoP-activated Mg2+ transporter MgtA is also controlled by its 5'-untranslated region (5'UTR). The 5'UTR of the mgtA gene can adopt different stem-loop structures depending on the Mg2+ levels, which determine whether transcription reads through into the mgtA-coding region or stops within the 5'UTR. This makes the mgtA 5'UTR the first example of a cation-responding riboswitch. The initiation of mgtA transcription responds to extracytoplasmic Mg2+, and its elongation into the coding region to cytoplasmic Mg2+, which provides a singular example where the same ligand is sensed in different cellular compartments to regulate disparate steps in gene transcription. The PhoP-activated Mg2+ transporter mgtB is also regulated by Mg2+ in a strain lacking the Mg2+ sensor PhoQ, suggesting the presence of additional Mg2+-responding devices.

Metal Bridges between the PhoQ Sensor Domain and the Membrane Regulate Transmembrane Signaling

Bacterial histidine kinases respond to environmental stimuli by transducing a signal from an extracytosolic sensor domain to a cytosolic catalytic domain. Among them, PhoQ promotes bacterial virulence and is tightly repressed by the divalent cations such as calcium and magnesium. We have determined the crystal structure of the PhoQ sensor domain from Salmonella typhimurium in the Ca2+-bound state, which reveals a highly negatively charged surface that is in close proximity to the inner membrane. This acidic surface binds at least three Ca2+, which mediate the PhoQ-membrane interaction. Mutagenesis analysis indicates that structural integrity at the membrane proximal region of the PhoQ sensor domain promotes metal-mediated repression. We propose that depletion or displacement of divalent cations leads to charge repulsion between PhoQ and the membrane, which initiates transmembrane signaling through a change in orientation between the PhoQ sensor domain and membrane. Therefore, both PhoQ and the membrane are required for extracytosolic sensing and transmembrane signaling.

Crystallization and preliminary X-ray analysis of native and selenomethionyl polymyxin resistance protein D fromE. Coli

Polymyxin B, a cationic antimicrobial peptide has been isolated from Paenibacillus polymyxa. The cationic peptide has polycationic, amphipathic and cyclic unique features. These characteristics of the cationic antimicrobial peptide are thought to contribute to the destruction of gram-negative bacteria by through an initial interaction with the negatively charged surface molecule lipopolysaccharide (LPS), which leads to self promoted uptake across the outer membrane into the negatively charged cytoplasmic membrane of the bacterium. This leads to membrane perturbation and probably translocation of the peptide across the membrane. Some gram-negative bacteria resistant to polymyxin B possess mechanism that modify the LPS through neutralization of its negative charge, which decreases the binding affinity of polymyxin B. Several of the genes regulated by PhoP/PhoQ and PmrA/ PmrB encode enzymes that modify either the acyl chain region or the phosphate group of LPS. In Salmonella, polymyxin B resistance is mainly controlled by the PmrA/PmrB two-component system (Figure 1). PmrA-activated genes encode periplasmic and cause the modification of the LPS. Transcription of PmrA-activated genes is not only promoted by high levels of extra-cytoplasmic Fe, which is sensed by the PmrB protein, also it is activated by the PhoP/PhoQ system in response to low levels of extra-cytoplasmic Mg. For activation of PmrA at the low level Mg condition not only requires the noncognate sensor PhoQ protein but also its cognate regulator PhoP, and the PhoP activated PmrD gene. The PmrD protein appears to control the PmrA/PmrB system at a post-transcriptional level. The PmrD protein activates the PmrA/PmrB system via a protection mechanism through dephosphorylation of PmrA. The protective activity of PmrD protein is suggestive of the behavior displayed by certain members of the 14-3-3 protein family in eukaryotes to prevent the phosphorylated state of CDC25. It has recently been reported that the differential regulation of homologous genes is the mechanism responsible for the divergence of Salmonella enterica and E. coli in their ability to make LPS modifications and mediate resistance to polymyxin B. In contrast to Salmonella, E. coli K-12 induces PmrA-activated gene transcription and polymyxin B resistance in response to Fe, but it makes no response to low levels of Mg. This discrepancy suggests that the structure of E.coli PmrD differs from Salmonella PmrD. In particular, the N-terminal region of PmrD, which differs between the E.coli and Salmonella proteins, is in part responsible for the distinct behaviors of the PmrD proteins (Figure 2). In the present report, we describe the crystallization and preliminary X-ray analysis of E. coli PmrD.

Recognition of Antimicrobial Peptides by a Bacterial Sensor Kinase

PhoQ is a membrane bound sensor kinase important for the pathogenesis of a number of Gram-negative bacterial species. PhoQ and its cognate response regulator PhoP constitute a signal-transduction cascade that controls inducible resistance to host antimicrobial peptides. We show that enzymatic activity of Salmonella typhimurium PhoQ is directly activated by antimicrobial peptides. A highly acidic surface of the PhoQ sensor domain participates in both divalent-cation and antimicrobial-peptide binding as a first step in signal transduction across the bacterial membrane. Identification of PhoQ signaling mutants, binding studies with the PhoQ sensor domain, and structural analysis of this domain can be incorporated into a model in which antimicrobial peptides displace divalent cations from PhoQ metal binding sites to initiate signal transduction. Our findings reveal a molecular mechanism by which bacteria sense small innate immune molecules to initiate a transcriptional program that promotes bacterial virulence.

Salmonella’s Sensor for Host Defense Molecules

The bacterial pathogen Salmonella typhimurium resides within phagosomes in host cells and is able to deflect the host immune response. In this issue of Cell, Bader et al. (2005) decipher an elegant mechanism by which the PhoQ sensor kinase of Salmonella is switched on by host cationic antimicrobial peptides, leading to changes in gene expression that enable Salmonella to combat the host immune response.

Signal-dependent Binding of the Response Regulators PhoP and PmrA to Their Target Promoters in Vivo

Low Mg2+ promotes phosphorylation of the response regulators PhoP and PmrA and transcription of their activated genes in Salmonella enterica. Using chromatin immunoprecipitation, we have now determined that the PhoP and PmrA proteins are recruited to the regulatory region of their target genes when bacteria experience low Mg2+ but not when they are grown in high Mg2+. Even when the PhoP protein was artificially produced at 4-fold higher levels than the wild-type strain, promoter occupancy required the low Mg2+ signal. Substitution of the predicted phosphorylation site Asp-52 with a valine residue abolished phosphorylation of the PhoP protein, resulting in loss of PhoP binding to target promoters and transcription of PhoP-activated genes. Our results indicate that the promoter binding ability of the PhoP and PmrA proteins occurring in low Mg2+ is correlated with phosphorylation of these proteins in vivo.

The H Box-harboring Domain Is Key to the Function of the Salmonella enterica PhoQ Mg2+-sensor in the Recognition of Its Partner PhoP

In two-component signaling systems, the transduction strategy relies on a conserved His-Asp phosphoryl exchange between the sensor histidine kinase and its cognate response-regulator, and structural and functional consensus motifs are found when comparing either the diverse histidine kinases or response regulators present in a single cell. Therefore, the mechanism that guarantees the specific recognition between partners of an individual pair is essential to unequivocally generate the appropriate adaptive response. Based on sequence alignments with other histidine kinases, we dissected the Salmonella enterica Mg2+-sensor PhoQ in different subdomains and examined by in vivo and in vitro assays its interaction with the associated response regulator PhoP. This signal transduction system allows Salmonella to withstand environmental Mg2+ limitation by triggering gene expression that is vital throughout the infective cycle in the host. Using resonant mirror biosensor technology, we calculated the kinetic and equilibrium binding constants and determined that the His-phosphotransfer domain is essential for the PhoQ specific recognition and interaction with PhoP. Additionally, we show the role of this domain in the bimolecular transphosphorylation and provide evidence that this region undergoes dimerization.

Mg2+ Sensing by the Mg2+ Sensor PhoQ of Salmonella enterica

The PhoP/PhoQ two-component regulatory system governs the adaptation to low Mg2+ environments and virulence in several Gram-negative species. During growth in low Mg2+, the sensor PhoQ modifies the activity of the response regulator PhoP promoting gene transcription, whereas growth in high Mg2+ represses transcription of PhoP-activated genes. The PhoQ protein harbors a periplasmic domain of 146 amino acid residues that binds Mg2+in vitro and is required for Mg2+-mediated repression in vivo. Here, we identify periplasmic mutants of the Salmonella PhoQ protein that allow transcription of PhoP-activated genes even under high Mg2+ concentrations. When expressed in a strain harboring a PhoP variant that is phosphorylated from acetyl phosphate, some of the mutants failed to repress PhoP-promoted transcription in high Mg2+, whereas others displayed a wild-type ability to do so. Mutant PhoQ proteins that allowed expression of PhoP-activated genes in high Mg2+ displayed a pattern of iron-mediated cleavage in vitro that was different from that displayed by wild-type PhoQ, indicative of altered Mg2+ binding. A PhoQ protein with the conserved histidine residue (H277) substituted by alanine could not promote transcription of PhoP-activated genes in low Mg2+ but could turn off expression in response to high Mg2+. Our studies demonstrate that residues G93, W97, H120 and T156 are required for a wild-type response to Mg2+, and suggest that Mg2+ binding to the periplasmic domain regulates several activities in the PhoQ protein.

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.

Comparison of the Pseudomonas aeruginosa andEscherichia coli PhoQ Sensor Domains: EVIDENCE FOR DISTINCT MECHANISMS OF SIGNAL DETECTION

The PhoP-PhoQ two-component system is present in a number of Gram-negative bacteria where it has roles in Mg(2+) homeostasis and virulence. PhoQ is a transmembrane histidine kinase that activates PhoP-mediated regulation of a set of genes when the extracellular concentration of divalent cations is low. Divalent cations are thought to interact directly with the periplasmic PhoQ sensor domain. The PhoP-PhoQ systems of Escherichia coli and Pseudomonas aeruginosa are similar in their biological response to extracellular divalent cations; however, their sensor domains display little sequence identity. Here we have begun to explore the consequences of this sequence divergence by comparing the biophysical properties of the P. aeruginosa PhoQ sensor domain with the corresponding E. coli sensor domain. Unlike the E. coli protein, the P. aeruginosa PhoQ sensor domain undergoes changes in the circular dichroism and fluorescence spectra as well as destabilization of its dimeric form in response to divalent cations. These results suggest that distinct mechanisms of signal detection are utilized by these proteins. A hybrid protein in which the E. coli sensor domain has been substituted with the corresponding P. aeruginosa sensor domain responds normally to the presence of extracellular divalent cations in vivo in E. coli. Thus, despite apparent differences in the structural response to its stimulus, the P. aeruginosa sensor domain transduces signals to the E. coli PhoQ cytoplasmic kinase domain in a manner that mimics normal E. coli PhoQ function.

The pleiotropic two-component regulatory system PhoP-PhoQ.

PhoP-PhoQ is a two-component system that governs virulence, mediates the adaptation to Mg2+-limiting environments, and regulates numerous cellular activities in several gram-negative species. It consists of the inner membrane sensor PhoQ and the cytoplasmic regulator PhoP. The PhoP-PhoQ system is

Acetyl phosphate-dependent activation of a mutant PhoP response regulator that functions independently of its cognate sensor kinase

The two-component system is a signal communication network generally consisting of a sensor kinase that receives inputs from the environment and modifies the phosphorylated state of a response regulator that executes an adaptive behavior. PhoP is a response regulator that controls virulence gene expression in Salmonella enterica. Transcription of PhoP-regulated genes is modulated by the Mg2+ levels detected by the sensor PhoQ. Here, we describe a PhoP mutant protein, PhoP∗, that functions in the absence of its cognate sensor, thereby allowing transcription of PhoP-activated genes independently of the Mg2+ concentration in the environment. The PhoP∗ protein harbors a S93N substitution in the response regulator receiver domain. PhoP∗-mediated transcription is abolished by either mutation of the aspartate residue that is conserved among response regulators as the site of phosphorylation or inactivation of the pta-encoded phosphotransacetylase. This enzyme mediates the production of acetyl phosphate, which has been shown to serve as a low molecular mass phosphate donor for certain response regulators. The purified PhoP∗ protein autophosphorylated from acetyl phosphate more efficiently than the wild-type PhoP protein in vitro. The PhoP∗ protein retained the capacity to interact with the PhoQ protein, which promoted phosphorylation of the PhoP∗ protein in vitro and abolished PhoP∗-mediated transcription under high Mg2+ concentrations in vivo. Cumulatively, our results uncover a role of PhoQ in transcriptional repression during growth in millimolar Mg2+ and define a mutant response regulator form with an increased capacity to be phosphorylated by acetyl phosphate.

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.

Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes.

Survival of Salmonella typhimurium within macrophage phagosomes requires the coordinate expression of bacterial gene products. This report examines the contribution of phagosomal pH as a signal for expression of genes positively regulated by the S. typhimurium virulence regulators PhoP and PhoQ. Several hours after bacterial phagocytosis by murine bone marrow-derived macrophages, PhoP-activated gene transcription increased 50- to 77-fold. In contrast, no difference in PhoP-activated gene expression was observed after infection of cultured epithelial cells, suggesting that the membrane sensor PhoQ recognized signals unique to macrophage phagosomes. The increase in PhoP-regulated gene expression was abolished when macrophage culture medium contained NH4Cl or chloroquine, weak bases that raise the pH of acidic compartments. Measurements of pH documented that S. typhimurium delayed and attenuated acidification of its intracellular compartment. Phagosomes containing S. typhimurium required 4-5 hr to reach pH < 5.0. In contrast, within 1 hr vacuoles containing heat-killed bacteria were measured at pH < 4.5. The eventual acidification of phagosomes to pH < 5.0 correlated with the period of maximal PhoP-dependent gene expression. These observations implicate phagosome acidification as an intracellular inducer of PhoP-regulated gene expression and suggest that Salmonella survival is dependent on its ability to attenuate phagosome acidification.