How the PhoP/PhoQ System Controls Virulence and Mg2+ Homeostasis: Lessons in Signal Transduction, Pathogenesis, Physiology, and Evolution.
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ABSTRACT:
SUBMITTER: Groisman EA
PROVIDER: S-EPMC8483708 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The rapid emergence of multidrug resistance among bacterial pathogens has become a significant challenge to human health in our century. Therefore, development of next-generation antibacterial compounds is an urgent need. Two-component signal transduction systems (TCS) are stimulus-response coupling devices that allow bacteria to sense and elaborate adaptive responses to changing environmental conditions, including the challenges that pathogenic bacteria face inside the host. The differential presence of TCS, present in bacteria but absent in the animal kingdom, makes them attractive targets in the search for new antibacterial compounds. In Salmonella enterica, the PhoP/PhoQ two-component system controls the expression of crucial phenotypes that define the ability of the pathogen to establish infection in the host. We now report the screening of 686 compounds from a GlaxoSmithKline published kinase inhibitor set in a high-throughput whole-cell assay that targets Salmonella enterica serovar Typhimurium PhoP/PhoQ. We identified a series of quinazoline compounds that showed selective and potent downregulation of PhoP/PhoQ-activated genes and define structural attributes required for their efficacy. We demonstrate that their bioactivity is due to repression of the PhoQ sensor autokinase activity mediated by interaction with its catalytic domain, acting as competitive inhibitors of ATP binding. While noncytotoxic, the hit molecules exhibit antivirulence effect by blockage of S Typhimurium intramacrophage replication. Together, these features make these quinazoline compounds stand out as exciting leads to develop a therapeutic intervention to fight salmonellosis.
| S-EPMC7187569 | biostudies-literature
Project description:The two-component system PhoP/PhoQ controls a large number of genes responsible for a variety of physiological and virulence functions in Salmonella enterica serovar Typhimurium. Here we describe a mechanism whereby the transcriptional activator PhoP elicits expression of dissimilar gene sets when its cognate sensor PhoQ is activated by different signals in the periplasm. We determine that full transcription of over half of the genes directly activated by PhoP requires the Mg(2+) transporter MgtA when the PhoQ inducing signal is low Mg(2+) , but not when PhoQ is activated by mildly acidic pH or the antimicrobial peptide C18G. MgtA promotes the active (i.e. phosphorylated) form of PhoP by removing Mg(2+) from the periplasm, where it functions as a repressing signal for PhoQ. MgtA-dependent expression enhances resistance to the cationic antibiotic polymyxin B. Production of the MgtA protein requires cytoplasmic Mg(2+) levels to drop below a certain threshold, thereby creating a two-tiered temporal response among PhoP-dependent genes.
| S-EPMC3890429 | biostudies-literature
Project description:Transmembrane signaling proteins couple extracytosolic sensors to cytosolic effectors. Here, we examine how binding of Mg2+ to the sensor domain of an E. coli two component histidine kinase (HK), PhoQ, modulates its cytoplasmic kinase domain. We use cysteine-crosslinking and reporter-gene assays to simultaneously and independently probe the signaling state of PhoQ's sensor and autokinase domains in a set of over 30 mutants. Strikingly, conservative single-site mutations distant from the sensor or catalytic site strongly influence PhoQ's ligand-sensitivity as well as the magnitude and direction of the signal. Data from 35 mutants are explained by a semi-empirical three-domain model in which the sensor, intervening HAMP, and catalytic domains can adopt kinase-promoting or inhibiting conformations that are in allosteric communication. The catalytic and sensor domains intrinsically favor a constitutively 'kinase-on' conformation, while the HAMP domain favors the 'off' state; when coupled, they create a bistable system responsive to physiological concentrations of Mg2+. Mutations alter signaling by locally modulating domain intrinsic equilibrium constants and interdomain couplings. Our model suggests signals transmit via interdomain allostery rather than propagation of a single concerted conformational change, explaining the diversity of signaling structural transitions observed in individual HK domains.
| S-EPMC8719878 | biostudies-literature
Project description:The PhoQ/PhoP two-component system plays an essential role in the response of enterobacteria to the environment of their mammalian hosts. It is known to sense several stimuli that are potentially associated with the host, including extracellular magnesium limitation, low pH, and the presence of cationic antimicrobial peptides. Here, we show that the PhoQ/PhoP two-component systems of Escherichia coli and Salmonella can also perceive an osmotic upshift, another key stimulus to which bacteria become exposed within the host. In contrast to most previously established stimuli of PhoQ, the detection of osmotic upshift does not require its periplasmic sensor domain. Instead, we show that the activity of PhoQ is affected by the length of the transmembrane (TM) helix as well as by membrane lateral pressure. We therefore propose that osmosensing relies on a conformational change within the TM domain of PhoQ induced by a perturbation in cell membrane thickness and lateral pressure under hyperosmotic conditions. Furthermore, the response mediated by the PhoQ/PhoP two-component system was found to improve bacterial growth recovery under hyperosmotic stress, partly through stabilization of the sigma factor RpoS. Our findings directly link the PhoQ/PhoP two-component system to bacterial osmosensing, suggesting that this system can mediate a concerted response to most of the established host-related cues.
| S-EPMC5740661 | biostudies-literature
Project description:UNLABELLED:HAMP domains are α-helical coiled coils that often transduce signals from extracytoplasmic sensing domains to cytoplasmic domains. Limited structural information has resulted in hypotheses that specific HAMP helix movement changes downstream enzymatic activity. These hypotheses were tested by mutagenesis and cysteine cross-linking analysis of the PhoQ histidine kinase, essential for resistance to antimicrobial peptides in a variety of enteric pathogens. These results support a mechanistic model in which periplasmic signals which induce an activation state generate a rotational movement accompanied by a tilt in α-helix 1 which activates kinase activity. Biochemical data and a high-confidence model of the PhoQ cytoplasmic domain indicate a possible physical interaction of the HAMP domain with the catalytic domain as necessary for kinase repression. These results support a model of PhoQ activation in which changes in the periplasmic domain lead to conformational movements in the HAMP domain helices which disrupt interaction between the HAMP and the catalytic domains, thus promoting increased kinase activity. IMPORTANCE:Most studies on the HAMP domain signaling states have been performed with chemoreceptors or the HAMP domain of Af1503. Full-length structures of the HAMP-containing histidine kinases VicK and CpxA or a hybrid between the HAMP domain of Af1503 and the EnvZ histidine kinase agree with the parallel four-helix bundle structure identified in Af1503 and provide snapshots of structural conformations experienced by HAMP domains. We took advantage of the fact that we can easily regulate the activation state of PhoQ histidine kinase to study its HAMP domain in the context of the full-length protein in living cells and provide biochemical evidence for different conformational states experienced by Salmonella enterica serovar Typhimurium PhoQ HAMP domain upon signaling.
| S-EPMC4447245 | biostudies-literature
Project description:In Escherichia coli, Salmonella, and related bacteria, the PhoQ-PhoP system regulates the expression of a large collection of genes in response to conditions of low magnesium or to the presence of certain antimicrobial peptides. We measured transcription of four PhoP-regulated promoters in E. coli that have significantly different PhoP-binding sites. Surprisingly, three promoters show identical responses to magnesium concentrations that range over four orders of magnitude. By analyzing and testing a simple model of transcriptional regulation, we find an explanation for this puzzle and show that these promoters are indeed differentially regulated at sufficiently high levels of stimulus. We then use this analysis to infer an effective level of phosphorylated PhoP as a function of magnesium stimulus. Our results demonstrate that differential regulation generally depends on the strength of the stimulus and highlight how quantitative analysis of stimulus-response curves can be used to infer properties of cell regulatory circuits that cannot be easily obtained from in vitro measurements.
| S-EPMC2042202 | biostudies-literature
Project description:The phoP-phoQ operon of Salmonella typhimurium is a member of the family of two-component regulatory systems and controls expression of the phoN gene that codes for nonspecific acid phosphatase and the genes involved in the pathogenicity of the bacterium. The phoP-phoQ operon of Escherichia coli was cloned on a plasmid vector by complementation of a phoP mutant, and the 4.1-kb nucleotide sequence, which includes the phoP-phoQ operon and its flanking regions, was determined. The phoP-phoQ operon was mapped at 25 min on the standard E. coli linkage map by hybridization with the Kohara mini set library of the E. coli chromosome (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). The predicted phoP and phoQ gene products consist of 223 and 486 amino acids with estimated molecular masses of 25,534 and 55,297 Da, respectively, which correspond well with the sizes of the PhoP and PhoQ proteins identified by the maxicell method. The amino acid sequences of PhoP and PhoQ of E. coli were 93 and 86% identical, respectively, to those of S. typhimurium.
| S-EPMC205742 | biostudies-other
Project description:The PhoQ/PhoP signaling system responds to low magnesium and the presence of certain cationic antimicrobial peptides. It regulates genes important for growth under these conditions, as well as additional genes important for virulence in many gram-negative pathogens. PhoQ is a sensor kinase that phosphorylates and activates the transcription factor PhoP. Since feedback inhibition is a common theme in stress-response circuits, we hypothesized that some members of the PhoP regulon may play such a role in the PhoQ/PhoP pathway. We therefore screened for PhoP-regulated genes that mediate feedback in this system. We found that deletion of mgrB (yobG), which encodes a 47 amino acid peptide, results in a potent increase in PhoP-regulated transcription. In addition, over-expression of mgrB decreased transcription at both high and low concentrations of magnesium. Localization and bacterial two-hybrid studies suggest that MgrB resides in the inner-membrane and interacts directly with PhoQ. We further show that MgrB homologs from Salmonella typhimurium and Yersinia pestis also repress PhoP-regulated transcription in these organisms. In cell regulatory circuits, feedback has been associated with modulating the induction kinetics and/or the cell-to-cell variability in response to stimulus. Interestingly, we found that elimination of MgrB-mediated feedback did not have a significant effect on the kinetics of reporter protein production and did not decrease the variability in expression among cells. Our results indicate MgrB is a broadly conserved membrane peptide that is a critical mediator of negative feedback in the PhoQ/PhoP circuit. This new regulator may function as a point of control that integrates additional input signals to modulate the activity of this important signaling system.
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Project description:Homo-oligomerisation of proteins is a ubiquitous phenomenon whose exact role remains unclear in many cases. To identify novel functions, this paper provides an exploration of general dynamical mathematical models of homo-oligomerisation. Simulation and analysis of these models show that homo-oligomerisation on its own allows for a remarkable variety of complex dynamic and steady state regulatory behaviour such as transient overshoots or homeostatic control of monomer concentration. If post-translational modifications are considered, however, conventional mass action kinetics lead to thermodynamic inconsistencies due to asymmetric combinatorial expansion of reaction routes. Introducing a conservation principle to balance rate equations re-establishes thermodynamic consistency. Using such balanced models it is shown that oligomerisation can lead to bistability by enabling pseudo-multisite modification and kinetic pseudo-cooperativity via multi-enzyme regulation, thereby constituting a novel motif for bistable modification reactions. Due to these potential signal processing capabilities, homo-oligomerisation could play far more versatile roles in signal transduction than previously appreciated.
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Project description:The PhoQP two-component system is a signaling complex essential for bacterial virulence and cationic antimicrobial peptide resistance. PhoQ is the histidine kinase chemoreceptor of this tandem machine and assembles in a homodimer conformation spanning the bacterial inner membrane. Currently, a full understanding of the PhoQ signal transduction is hindered by the lack of a complete atomistic structure. In this study, an atomistic model of the key transmembrane (TM) domain is assembled by using molecular simulations, guided by experimental cross-linking data. The formation of a polar pocket involving Asn202 in the lumen of the tetrameric TM bundle is crucial for the assembly and solvation of the domain. Moreover, a concerted displacement of the TM helices at the periplasmic side is found to modulate a rotation at the cytoplasmic end, supporting the transduction of the chemical signal through a combination of scissoring and rotational movement of the TM helices.
| S-EPMC3554529 | biostudies-literature