scholarly journals A tailored phosphoaspartate probe unravels CprR as a response regulator in Pseudomonas aeruginosa interkingdom signaling

2021 ◽  
Author(s):  
Patrick W. A. Allihn ◽  
Mathias W. Hackl ◽  
Christina Ludwig ◽  
Stephan M. Hacker ◽  
Stephan A. Sieber
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Posttranslational Modification ◽  
Response Regulator ◽  
Peptide Hormone ◽  
Chemical Proteomics ◽  
Bacterial Signaling ◽  
Interkingdom Signaling

Phosphoaspartate (pAsp) is a labile posttranslational modification involved in bacterial signaling. To monitor pAsp we designed a chemical proteomics method and revealed insights into the antimicrobial response triggered by a human peptide hormone.

scholarly journals A Tailored Phosphoaspartate Probe Unravels CprR as a Response Regulator in Pseudomonas Aeruginosa Interkingdom Signaling

2020 ◽  
Author(s):  
Patrick Allihn ◽  
Mathias W. Hackl ◽  
Christina Ludwig ◽  
Stephan M. Hacker ◽  
Stephan A. Sieber
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Response Regulator ◽  
Bacterial Pathogen ◽  
Polymyxin B ◽  
Sequence Motif ◽  
Peptide Antibiotics ◽  
Dynorphin A ◽  
Therapeutic Success ◽  
Adaptive Resistance ◽  
Interkingdom Signaling

Pseudomonas aeruginosa is a difficult-to-treat Gram-negative bacterial pathogen causing life-threatening infections. Adaptive resistance (AR) to cationic peptide antibiotics such as polymyxin B impairs the therapeutic success. This self-protection is mediated by two component systems (TCS) consisting of a membrane-bound histidine kinase and an intracellular response regulator (RR). As phosphorylation of the key RR aspartate residue is transient during signaling and hydrolytically unstable, the study of these systems is challenging. Therefore, we applied a tailored reverse polarity chemical proteomic strategy to capture this transient modification and read-out RR phosphorylation in complex proteomes using a nucleophilic probe. An ideal trapping methodology was developed with a recombinant RR demonstrating the importance of fine-tuned acidic pH values to facilitate the attack on the aspartate carbonyl C-atom and prevent unproductive hydrolysis. Analysis of Bacillus subtilis and P. aeruginosa proteomes revealed the detection of multiple phosphoaspartate sites, which closely resembled the conserved RR sequence motif. With this validated strategy we dissected the signaling of dynorphin A, a human peptide stress hormone, which is sensed by P. aeruginosa to mediate AR. Intriguingly, our methodology identified CprR as an unprecedented RR in dynorphin A interkingdom signaling.

scholarly journals A Tailored Phosphoaspartate Probe Unravels CprR as a Response Regulator in Pseudomonas Aeruginosa Interkingdom Signaling

2020 ◽  
Author(s):  
Patrick Allihn ◽  
Mathias W. Hackl ◽  
Christina Ludwig ◽  
Stephan M. Hacker ◽  
Stephan A. Sieber
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Response Regulator ◽  
Bacterial Pathogen ◽  
Polymyxin B ◽  
Sequence Motif ◽  
Peptide Antibiotics ◽  
Dynorphin A ◽  
Therapeutic Success ◽  
Adaptive Resistance ◽  
Interkingdom Signaling

Pseudomonas aeruginosa is a difficult-to-treat Gram-negative bacterial pathogen causing life-threatening infections. Adaptive resistance (AR) to cationic peptide antibiotics such as polymyxin B impairs the therapeutic success. This self-protection is mediated by two component systems (TCS) consisting of a membrane-bound histidine kinase and an intracellular response regulator (RR). As phosphorylation of the key RR aspartate residue is transient during signaling and hydrolytically unstable, the study of these systems is challenging. Therefore, we applied a tailored reverse polarity chemical proteomic strategy to capture this transient modification and read-out RR phosphorylation in complex proteomes using a nucleophilic probe. An ideal trapping methodology was developed with a recombinant RR demonstrating the importance of fine-tuned acidic pH values to facilitate the attack on the aspartate carbonyl C-atom and prevent unproductive hydrolysis. Analysis of Bacillus subtilis and P. aeruginosa proteomes revealed the detection of multiple phosphoaspartate sites, which closely resembled the conserved RR sequence motif. With this validated strategy we dissected the signaling of dynorphin A, a human peptide stress hormone, which is sensed by P. aeruginosa to mediate AR. Intriguingly, our methodology identified CprR as an unprecedented RR in dynorphin A interkingdom signaling.

AgmR controls transcription of a regulon with several operons essential for ethanol oxidation in Pseudomonas aeruginosa ATCC 17933

Microbiology ◽  
2004 ◽  
Vol 150 (6) ◽  
pp. 1851-1857 ◽  
Author(s):  
Nicole Gliese ◽  
Viola Khodaverdi ◽  
Max Schobert ◽  
Helmut Görisch
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Ethanol Oxidation ◽  
Response Regulator ◽  
Regulatory System ◽  
Pyrroloquinoline Quinone ◽  
Kanamycin Resistance ◽  
Kanamycin Resistance Cassette ◽  
Two Component ◽  
Ethanol Dehydrogenase ◽  
Oxidation System

The response regulator AgmR was identified to be involved in the regulation of the quinoprotein ethanol oxidation system of Pseudomonas aeruginosa ATCC 17933. Interruption of the agmR gene by insertion of a kanamycin-resistance cassette resulted in mutant NG3, unable to grow on ethanol. After complementation with the intact agmR gene, growth on ethanol was restored. Transcriptional lacZ fusions were used to identify four operons which are regulated by the AgmR protein: the exaA operon encodes the pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase, the exaBC operon encodes a soluble cytochrome c 550 and an aldehyde dehydrogenase, the pqqABCDE operon carries the PQQ biosynthetic genes, and operon exaDE encodes a two-component regulatory system which controls transcription of the exaA operon. Transcription of exaA was restored by transformation of NG3 with a pUCP20T derivative carrying the exaDE genes under lac-promoter control. These data indicate that the AgmR response regulator and the exaDE two-component regulatory system are organized in a hierarchical manner. Gene PA1977, which appears to form an operon with the agmR gene, was found to be non-essential for growth on ethanol.

scholarly journals The YebC Family Protein PA0964 Negatively Regulates the Pseudomonas aeruginosa Quinolone Signal System and Pyocyanin Production

2008 ◽  
Vol 190 (18) ◽  
pp. 6217-6227 ◽  
Author(s):  
Haihua Liang ◽  
Lingling Li ◽  
Zhaolin Dong ◽  
Michael G. Surette ◽  
Kangmin Duan
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Response Regulator ◽  
Initial Study ◽  
Pfam Family ◽  
Swarming Motility ◽  
Sensing Systems ◽  
Family Protein ◽  
Pyocyanin Production

ABSTRACT Bacterial pathogenicity is often manifested by the expression of various cell-associated and secreted virulence factors, such as exoenzymes, protease, and toxins. In Pseudomonas aeruginosa, the expression of virulence genes is coordinately controlled by the global regulatory quorum-sensing systems, which includes the las and rhl systems as well as the Pseudomonas quinolone signal (PQS) system. Phenazine compounds are among the virulence factors under the control of both the rhl and PQS systems. In this study, regulation of the phzA1B1C1D1E1 (phzA1) operon, which is involved in phenazine synthesis, was investigated. In an initial study of inducing conditions, we observed that phzA1 was induced by subinhibitory concentrations of tetracycline. Screening of 13,000 mutants revealed 32 genes that altered phzA1 expression in the presence of subinhibitory tetracycline concentrations. Among them, the gene PA0964, designated pmpR ( p qsR-mediated P QS r egulator), has been identified as a novel regulator of the PQS system. It belongs to a large group of widespread conserved hypothetical proteins with unknown function, the YebC protein family (Pfam family DUF28). It negatively regulates the quorum-sensing response regulator pqsR of the PQS system by binding at its promoter region. Alongside phzA1 expression and phenazine and pyocyanin production, a set of virulence factors genes controlled by both rhl and the PQS were shown to be modulated by PmpR. Swarming motility and biofilm formation were also significantly affected. The results added another layer of regulation in the rather complex quorum-sensing systems in P. aeruginosa and demonstrated a clear functional clue for the YebC family proteins.

scholarly journals Target (MexB)- and Efflux-Based Mechanisms Decreasing the Effectiveness of the Efflux Pump Inhibitor D13-9001 in Pseudomonas aeruginosa PAO1: Uncovering a New Role for MexMN-OprM in Efflux of β-Lactams and a Novel Regulatory Circuit (MmnRS) Controlling MexMN Expression

2018 ◽  
Vol 63 (2) ◽  
pp. e01718-18 ◽  
Author(s):  
Srijan Ranjitkar ◽  
Adriana K. Jones ◽  
Mina Mostafavi ◽  
Zachary Zwirko ◽  
Oleg Iartchouk ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Response Regulator ◽  
Heavy Metal Resistance ◽  
Metal Resistance ◽  
Rna Seq ◽  
Efflux Pump Inhibitor ◽  
Content Type ◽  
Regulatory Circuit ◽  
Outer Membrane Channel

ABSTRACT Efflux pumps contribute to antibiotic resistance in Gram-negative pathogens. Correspondingly, efflux pump inhibitors (EPIs) may reverse this resistance. D13-9001 specifically inhibits MexAB-OprM in Pseudomonas aeruginosa. Mutants with decreased susceptibility to MexAB-OprM inhibition by D13-9001 were identified, and these fell into two categories: those with alterations in the target MexB (F628L and ΔV177) and those with an alteration in a putative sensor kinase of unknown function, PA1438 (L172P). The alterations in MexB were consistent with reported structural studies of the D13-9001 interaction with MexB. The PA1438L172P alteration mediated a >150-fold upregulation of MexMN pump gene expression and a >50-fold upregulation of PA1438 and the neighboring response regulator gene, PA1437. We propose that these be renamed mmnR and mmnS for MexMN regulator and MexMN sensor, respectively. MexMN was shown to partner with the outer membrane channel protein OprM and to pump several β-lactams, monobactams, and tazobactam. Upregulated MexMN functionally replaced MexAB-OprM to efflux these compounds but was insusceptible to inhibition by D13-9001. MmnSL172P also mediated a decrease in susceptibility to imipenem and biapenem that was independent of MexMN-OprM. Expression of oprD, encoding the uptake channel for these compounds, was downregulated, suggesting that this channel is also part of the MmnSR regulon. Transcriptome sequencing (RNA-seq) of cells encoding MmnSL172P revealed, among other things, an interrelationship between the regulation of mexMN and genes involved in heavy metal resistance.

scholarly journals The Pseudomonas aeruginosa Sensor Kinase KinB Negatively Controls Alginate Production through AlgW-Dependent MucA Proteolysis

2009 ◽  
Vol 191 (7) ◽  
pp. 2285-2295 ◽  
Author(s):  
F. Heath Damron ◽  
Dongru Qiu ◽  
Hongwei D. Yu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Response Regulator ◽  
Negative Regulator ◽  
Loss Of Function ◽  
Cystic Fibrosis Mutation ◽  
Strain Pao1 ◽  
Alginate Production ◽  
Mucoid Conversion ◽  
Σ Factor ◽  
Lung Infections

ABSTRACT Mucoidy, or overproduction of the exopolysaccharide known as alginate, in Pseudomonas aeruginosa is a poor prognosticator for lung infections in cystic fibrosis. Mutation of the anti-σ factor MucA is a well-accepted mechanism for mucoid conversion. However, certain clinical mucoid strains of P. aeruginosa have a wild-type (wt) mucA. Here, we describe a loss-of-function mutation in kinB that causes overproduction of alginate in the wt mucA strain PAO1. KinB is the cognate histidine kinase for the transcriptional activator AlgB. Increased alginate production due to inactivation of kinB was correlated with high expression at the alginate-related promoters P algU and P algD . Deletion of alternative σ factor RpoN (σ54) or the response regulator AlgB in kinB mutants decreased alginate production to wt nonmucoid levels. Mucoidy was restored in the kinB algB double mutant by expression of wt AlgB or phosphorylation-defective AlgB.D59N, indicating that phosphorylation of AlgB was not required for alginate overproduction when kinB was inactivated. The inactivation of the DegS-like protease AlgW in the kinB mutant caused loss of alginate production and an accumulation of the hemagglutinin (HA)-tagged MucA. Furthermore, we observed that the kinB mutation increased the rate of HA-MucA degradation. Our results also indicate that AlgW-mediated MucA degradation required algB and rpoN in the kinB mutant. Collectively, these studies indicate that KinB is a negative regulator of alginate production in wt mucA strain PAO1.

scholarly journals The two-component system CopRS maintains femtomolar levels of free copper in the periplasm of Pseudomonas aeruginosa using a phosphatase-based mechanism

2020 ◽  
Author(s):  
Lorena Novoa-Aponte ◽  
Fernando C. Soncini ◽  
José M. Argüello
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Phosphatase Activity ◽  
Response Regulator ◽  
Redox Enzymes ◽  
Two Component System ◽  
Component System ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
Systems Control

ABSTRACTTwo component systems control periplasmic Cu+ homeostasis in Gram-negative bacteria. In characterized systems such as Escherichia coli CusRS, upon Cu+ binding to the periplasmic sensing domain of CusS, a cytoplasmic phosphotransfer domain phosphorylates the response regulator CusR. This drives the expression of efflux transporters, chaperones, and redox enzymes to ameliorate metal toxic effects. Here, we show that the Pseudomonas aeruginosa two component sensor histidine kinase CopS exhibits a Cu-dependent phosphatase activity that maintains a non-phosphorylated CopR when the periplasmic Cu levels are below its activation threshold. Upon Cu+ binding to the sensor, the phosphatase activity is blocked and the phosphorylated CopR activates transcription of the CopRS regulon. Supporting the model, mutagenesis experiments revealed that the ΔcopS strain showed constitutive high expression of the CopRS regulon, lower intracellular Cu+ levels, and larger Cu tolerance when compared to wild type cells. The invariant phospho-acceptor residue His235 of CopS was not required for the phosphatase activity itself, but necessary for its Cu-dependency. To sense the metal, the periplasmic domain of CopS binds two Cu+ ions at its dimeric interface. Homology modeling of CopS based on CusS structure (four Ag+ binding sites) clearly explains the different binding stoichiometries in both systems. Interestingly, CopS binds Cu+/2+ with 30 × 10−15 M affinities, pointing to the absence of free (hydrated) Cu+/2+ in the periplasm.IMPORTANCECopper is a micronutrient required as cofactor in redox enzymes. When free, copper is toxic, mismetallating proteins, and generating damaging free radicals. Consequently, copper overload is a strategy that eukaryotic cells use to combat pathogens. Bacteria have developed copper sensing transcription factors to control copper homeostasis. The cell envelope is the first compartment that has to cope with copper stress. Dedicated two component systems control the periplasmic response to metal overload. This manuscript shows that the copper sensing two component system present in Pseudomonadales exhibits a signal-dependent phosphatase activity controlling the activation of the response regulator, distinct from previously described periplasmic Cu sensors. Importantly, the data show that the sensor is activated by copper levels compatible with the absence of free copper in the cell periplasm. This emphasizes the diversity of molecular mechanisms that have evolved in various bacteria to manage the copper cellular distribution.

scholarly journals What a Dinner Party! Mechanisms and Functions of Interkingdom Signaling in Host-Pathogen Associations

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Melissa M. Kendall ◽  
Vanessa Sperandio
Keyword(s):  
Host Defense ◽  
Single Species ◽  
Signaling Networks ◽  
Chemical Signaling ◽  
Bacterial Signaling ◽  
Bidirectional Signaling ◽  
Interkingdom Signaling ◽  
Important Regulator ◽  
Host Signals

ABSTRACTChemical signaling between cells is an effective way to coordinate behavior within a community. Although cell-to-cell signaling has mostly been studied in single species, it is now appreciated that the sensing of chemical signals across kingdoms can be an important regulator of nutrient acquisition, virulence, and host defense. In this review, we focus on the role of interkingdom signaling in the interactions that occur between bacterial pathogens and their mammalian hosts. We discuss the quorum-sensing (QS) systems and other mechanisms used by these bacteria to sense, respond to, and modulate host signals that include hormones, immune factors, and nutrients. We also describe cross talk between these signaling pathways and strategies used by the host to interfere with bacterial signaling, highlighting the complex bidirectional signaling networks that are established across kingdoms.

scholarly journals The Atypical Response Regulator AtvR Is a New Player in Pseudomonas aeruginosa Response to Hypoxia and Virulence

2017 ◽  
Vol 85 (8) ◽  
Author(s):  
Gilberto Hideo Kaihami ◽  
Leandro Carvalho Dantas Breda ◽  
José Roberto Fogaça de Almeida ◽  
Thays de Oliveira Pereira ◽  
Gianlucca Gonçalves Nicastro ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Strain ◽  
Wild Type Strain ◽  
Environmental Changes ◽  
Response Regulator ◽  
Classical Pathway ◽  
Wild Type ◽  
Macrophage Infection ◽  
Content Type ◽  
Successful Infection

ABSTRACT Two-component systems are widespread in bacteria, allowing adaptation to environmental changes. The classical pathway is composed of a histidine kinase that phosphorylates an aspartate residue in the cognate response regulator (RR). RRs lacking the phosphorylatable aspartate also occur, but their function and contribution during host-pathogen interactions are poorly characterized. AtvR (PA14_26570) is the only atypical response regulator with a DNA-binding domain in the opportunistic pathogen Pseudomonas aeruginosa. Macrophage infection with the atvR mutant strain resulted in higher levels of tumor necrosis factor alpha secretion as well as increased bacterial clearance compared to those for macrophages infected with the wild-type strain. In an acute pneumonia model, mice infected with the atvR mutant presented increased amounts of proinflammatory cytokines, increased neutrophil recruitment to the lungs, reductions in bacterial burdens, and higher survival rates in comparison with the findings for mice infected with the wild-type strain. Further, several genes involved in hypoxia/anoxia adaptation were upregulated upon atvR overexpression, as seen by high-throughput transcriptome sequencing (RNA-Seq) analysis. In addition, atvR was more expressed in hypoxia in the presence of nitrate and required for full expression of nitrate reductase genes, promoting bacterial growth under this condition. Thus, AtvR would be crucial for successful infection, aiding P. aeruginosa survival under conditions of low oxygen tension in the host. Taken together, our data demonstrate that the atypical response regulator AtvR is part of the repertoire of transcriptional regulators involved in the lifestyle switch from aerobic to anaerobic conditions. This finding increases the complexity of regulation of one of the central metabolic pathways that contributes to Pseudomonas ubiquity and versatility.

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