A Two-Component System FleS/FleR Regulates Multiple Virulence-Related Traits in Pseudomonas aeruginosa

AbstractMicroorganisms commonly use two-component systems (TCSs) to detect specific environmental changes and respond accordingly for their own benefit. However, the regulatory mechanisms and physiological roles of a majority of TCSs are still elusive. In this study, we focused on a previously predicted TCS FleS/FleR in Pseudomonas aeruginosa to systematically investigate its regulation and physiological roles. Loss of fleS or fleR or both genes led to decreased biofilm formation and attenuated motility in PAO1, which could be restored by heterologously complementation of FleR but not FleS, confirming that the sensor kinase FleS and the response regulator FleR constitute a TCS pair. To determine the regulatory spectrum of this TCS, we conducted transcriptome sequencing and comparison between the wild-type strain and the fleR deletion mutant. The result showed that the TCS regulates about 440 genes including most of them are involved in the virulence-related pathways, e.g. siderophore biosynthesis, pyocyanin biosynthesis, type III/VI secretion systems, c-di-GMP metabolism, flagellar assembly etc. In addition to its roles in controlling biofilm formation and motility we have already shown, FleR was demonstrated to regulate the production of virulence factors such as pyocyanin and elastase, mediate stress response to SDS, and autoregulate its own expression. Moreover, EMSA assays revealed that FleR regulates flagellum biosynthesis genes flgBCDE, flgFGHIJKL, filC, which are essential for the bacterial motility, by directly interacting with their promoters. Taken together, these results expanded our understanding on the biological roles of FleS/FleR and provided new insights on its regulatory mechanisms.

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

10.1101/2020.05.13.092544 ◽

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.

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The DNA-binding domain of the Escherichia coli CpxR two-component response regulator is constitutively active and cannot be fully attenuated by fused adjacent heterologous regulatory domains

Microbiology ◽

10.1099/mic.0.28538-0 ◽

2006 ◽

Vol 152 (2) ◽

pp. 431-441 ◽

Cited By ~ 11

Author(s):

Caroline Tapparel ◽

Antoinette Monod ◽

William L. Kelley

Keyword(s):

Escherichia Coli ◽

Dna Binding ◽

Environmental Changes ◽

Response Regulator ◽

Effector Domain ◽

Regulatory Domains ◽

Rapid Responses ◽

Two Component ◽

Two Component Systems ◽

Constitutively Active

Two-component systems (TCS) based on a sensor histidine kinase and a phosphorylated cognate target regulator allow rapid responses to environmental changes. TCS are highly evolutionarily conserved, though in only a few cases are the inducing signals understood. This study focuses on the Escherichia coli CpxR response regulator that responds to periplasmic and outer-membrane stress. N-terminal deletion mutations have been isolated that render the transcription factor constitutively active, indicating that the N terminus functions, in part, to keep the C-terminal winged-helix DNA-binding effector domain in an inactive state. Analysis of truncations spanning the CpxR interdomain region revealed that mutants retaining the α5 helix significantly augment activation. Hybrid proteins obtained by fusing the CpxR effector domain to structurally similar heterologous N-terminal regulatory domains, or even GFP, failed to restore repression to the C-terminal domain. These findings shed light on the mechanism of CpxR effector domain activation and on the investigation of constitutive mutants obtained by truncation in other TCS.

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Novel two-component regulatory systems play a role in biofilm formation of Lactobacillus reuteri rodent isolate 100-23

Microbiology ◽

10.1099/mic.0.071399-0 ◽

2014 ◽

Vol 160 (4) ◽

pp. 795-806 ◽

Cited By ~ 8

Author(s):

Marcia Shu-Wei Su ◽

Michael G. Gänzle

Keyword(s):

Histidine Kinase ◽

Biofilm Formation ◽

Lactobacillus Reuteri ◽

Response Regulator ◽

Multiple Genes ◽

Regulatory Systems ◽

Two Component ◽

Two Component Systems ◽

Processing Peptidase

This study characterized the two-component regulatory systems encoded by bfrKRT and cemAKR, and assessed their influence on biofilm formation by Lactobacillus reuteri 100-23. A method for deletion of multiple genes was employed to disrupt the genetic loci of two-component systems. The operons bfrKRT and cemAKR showed complementary organization. Genes bfrKRT encode a histidine kinase, a response regulator and an ATP-binding cassette-type transporter with a bacteriocin-processing peptidase domain, respectively. Genes cemAKR code for a signal peptide, a histidine kinase and a response regulator, respectively. Deletion of single or multiple genes in the operons bfrKRT and cemAKR did not affect cell morphology, growth or the sensitivity to various stressors. However, gene disruption affected biofilm formation; this effect was dependent on the carbon source. Deletion of bfrK or cemA increased sucrose-dependent biofilm formation in vitro. Glucose-dependent biofilm formation was particularly increased by deletion of cemK. The expression of cemK and cemR was altered by deletion of bfrK, indicating cross-talk between these two regulatory systems. These results may contribute to our understanding of the genetic factors related to the biofilm formation and competitiveness of L. reuteri in intestinal ecosystems.

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Roles of Two-Component Systems in Pseudomonas aeruginosa Virulence

International Journal of Molecular Sciences ◽

10.3390/ijms222212152 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12152

Author(s):

Maria Sultan ◽

Rekha Arya ◽

Kyeong Kyu Kim

Keyword(s):

Pseudomonas Aeruginosa ◽

Virulence Factors ◽

Opportunistic Pathogen ◽

Secretion Systems ◽

Chronic Infections ◽

Potential Threat ◽

Wide Range ◽

Component Systems ◽

Two Component ◽

Two Component Systems

Pseudomonas aeruginosa is an opportunistic pathogen that synthesizes and secretes a wide range of virulence factors. P. aeruginosa poses a potential threat to human health worldwide due to its omnipresent nature, robust host accumulation, high virulence, and significant resistance to multiple antibiotics. The pathogenicity of P. aeruginosa, which is associated with acute and chronic infections, is linked with multiple virulence factors and associated secretion systems, such as the ability to form and utilize a biofilm, pili, flagella, alginate, pyocyanin, proteases, and toxins. Two-component systems (TCSs) of P. aeruginosa perform an essential role in controlling virulence factors in response to internal and external stimuli. Therefore, understanding the mechanism of TCSs to perceive and respond to signals from the environment and control the production of virulence factors during infection is essential to understanding the diseases caused by P. aeruginosa infection and further develop new antibiotics to treat this pathogen. This review discusses the important virulence factors of P. aeruginosa and the understanding of their regulation through TCSs by focusing on biofilm, motility, pyocyanin, and cytotoxins.

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Genome-Wide Mapping Reveals Complex Regulatory Activities of BfmR in Pseudomonas aeruginosa

Microorganisms ◽

10.3390/microorganisms9030485 ◽

2021 ◽

Vol 9 (3) ◽

pp. 485

Author(s):

Ke Fan ◽

Qiao Cao ◽

Lefu Lan

Keyword(s):

Pseudomonas Aeruginosa ◽

Chromatin Immunoprecipitation ◽

Biofilm Formation ◽

High Throughput Sequencing ◽

Response Regulator ◽

Cellular Functions ◽

Genome Wide ◽

Component Systems ◽

Genes Encoding ◽

Two Component Systems

BfmR is a response regulator that modulates diverse pathogenic phenotypes and induces an acute-to-chronic virulence switch in Pseudomonas aeruginosa, an important human pathogen causing serious nosocomial infections. However, the mechanisms of action of BfmR remain largely unknown. Here, using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), we showed that 174 chromosomal regions of P. aeruginosa MPAO1 genome were highly enriched by coimmunoprecipitation with a C-terminal Flag-tagged BfmR. Integration of these data with global transcriptome analyses revealed that 172 genes in 106 predicted transcription units are potential targets for BfmR. We determined that BfmR binds to and modulates the promoter activity of genes encoding transcriptional regulators CzcR, ExsA, and PhoB. Intriguingly, BfmR bound to the promoters of a number of genes belong to either CzcR or PhoB regulon, or both, indicating that CzcRS and PhoBR two-component systems (TCSs) deeply feed into the BfmR-mediated regulatory network. In addition, we demonstrated that phoB is required for BfmR to promote the biofilm formation by P. aeruginosa. These results delineate the direct BfmR regulon and exemplify the complexity of BfmR-mediated regulation of cellular functions in P. aeruginosa.

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The Two-Component System CopRS Maintains Subfemtomolar Levels of Free Copper in the Periplasm of Pseudomonas aeruginosa Using a Phosphatase-Based Mechanism

mSphere ◽

10.1128/msphere.01193-20 ◽

2020 ◽

Vol 5 (6) ◽

pp. e01193-20

Author(s):

Lorena Novoa-Aponte ◽

Cheng Xu ◽

Fernando C. Soncini ◽

José M. Argüello

Keyword(s):

Pseudomonas Aeruginosa ◽

Phosphatase Activity ◽

Response Regulator ◽

Redox Enzymes ◽

Two Component System ◽

Component System ◽

Content Type ◽

Component Systems ◽

Two Component ◽

Two Component Systems

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 region of CusS, a cytoplasmic phosphotransfer domain of the sensor 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 CopR in a nonphosphorylated state when the periplasmic Cu levels are below the activation threshold of CopS. 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 exhibits maximal expression of the CopRS regulon, lower intracellular Cu+ levels, and increased Cu tolerance compared to wild-type cells. The invariant phosphoacceptor residue His235 of CopS was not required for the phosphatase activity itself but was 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 supports the different binding stoichiometries in the two systems. Interestingly, CopS binds Cu+/2+ with 3 × 10−14 M affinity, pointing to the absence of free (hydrated) Cu+/2+ in the periplasm.IMPORTANCE Copper 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 paper shows that the sensor kinase of the copper-sensing two-component system present in Pseudomonadales exhibits a signal-dependent phosphatase activity controlling the activation of its cognate response regulator, distinct from previously described periplasmic Cu sensors. Importantly, the data show that the system is activated by copper levels compatible with the absence of free copper in the cell periplasm. These observations emphasize the diversity of molecular mechanisms that have evolved in bacteria to manage the copper cellular distribution.

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Mutation-induced remodeling of the BfmRS two-component system in Pseudomonas aeruginosa clinical isolates

Science Signaling ◽

10.1126/scisignal.aaz1529 ◽

2020 ◽

Vol 13 (656) ◽

pp. eaaz1529

Author(s):

Qiao Cao ◽

Nana Yang ◽

Yanhui Wang ◽

Chenchen Xu ◽

Xue Zhang ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Response Regulator ◽

Clinical Isolates ◽

Missense Mutations ◽

Chronic Infections ◽

Two Component System ◽

Component System ◽

Transcriptional Regulatory ◽

Two Component

Genetic mutations are a primary driving force behind the adaptive evolution of bacterial pathogens. Multiple clinical isolates of Pseudomonas aeruginosa, an important human pathogen, have naturally evolved one or more missense mutations in bfmS, which encodes the sensor histidine kinase of the BfmRS two-component system (TCS). A mutant BfmS protein containing both the L181P and E376Q substitutions increased the phosphorylation and thus the transcriptional regulatory activity of its cognate downstream response regulator, BfmR. This reduced acute virulence and enhanced biofilm formation, both of which are phenotypic changes associated with a chronic infection state. The increased phosphorylation of BfmR was due, at least in part, to the cross-phosphorylation of BfmR by GtrS, a noncognate sensor kinase. Other spontaneous missense mutations in bfmS, such as A42E/G347D, T242R, and R393H, also caused a similar remodeling of the BfmRS TCS in P. aeruginosa. This study highlights the plasticity of TCSs mediated by spontaneous mutations and suggests that mutation-induced activation of BfmRS may contribute to host adaptation by P. aeruginosa during chronic infections.

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Pseudomonas aeruginosa biofilm formation on endotracheal tubes requires multiple two-component systems

Journal of Medical Microbiology ◽

10.1099/jmm.0.001199 ◽

2020 ◽

Vol 69 (6) ◽

pp. 906-919 ◽

Cited By ~ 2

Author(s):

Divakar Badal ◽

Abhijith Vimal Jayarani ◽

Mohammed Ameen Kollaran ◽

Aloke Kumar ◽

Varsha Singh

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Type Species ◽

Content Type ◽

Endotracheal Tubes ◽

Link Type ◽

Component Systems ◽

Two Component ◽

Two Component Systems

Introduction. Indwelling medical devices such as endotracheal tubes (ETTs), urinary catheters, vascular access devices, tracheostomies and feeding tubes are often associated with hospital-acquired infections. Bacterial biofilm formed on the ETTs in intubated patients is a significant risk factor associated with ventilator-associated pneumonia. Pseudomonas aeruginosa is one of the four frequently encountered bacteria responsible for causing pneumonia, and the biofilm formation on ETTs. However, understanding of biofilm formation on ETT and interventions to prevent biofilm remains lagging. The ability to sense and adapt to external cues contributes to their success. Thus, the biofilm formation is likely to be influenced by the two-component systems (TCSs) that are composed of a membrane-associated sensor kinase and an intracellular response regulator. Aim. This study aims to establish an in vitro method to analyse the P. aeruginosa biofilm formation on ETTs, and identify the TCSs that contribute to this process. Methodology. In total, 112 P. aeruginosa PA14 TCS mutants were tested for their ability to form biofilm on ETTs, their effect on quorum sensing (QS) and motility. Results. Out of 112 TCS mutants studied, 56 had altered biofilm biomass on ETTs. Although the biofilm formation on ETTs is QS-dependent, none of the 56 loci controlled quorum signal. Of these, 18 novel TCSs specific to ETT biofilm were identified, namely, AauS, AgtS, ColR, CopS, CprR, NasT, KdpD, ParS, PmrB, PprA, PvrS, RcsC, PA14_11120, PA14_32580, PA14_45880, PA14_49420, PA14_52240, PA14_70790. The set of 56 included the GacS network, TCS proteins involved in fimbriae synthesis, TCS proteins involved in antimicrobial peptide resistance, and surface-sensing. Additionally, several of the TCS-encoding genes involved in biofilm formation on ETTs were found to be linked to flagellum-dependent swimming motility. Conclusions. Our study established an in vitro method for studying P. aeruginosa biofilm formation on the ETT surfaces. We also identified novel ETT-specific TCSs that could serve as targets to prevent biofilm formation on indwelling devices frequently used in clinical settings.

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

Microbiology ◽

10.1099/mic.0.26882-0 ◽

2004 ◽

Vol 150 (6) ◽

pp. 1851-1857 ◽

Cited By ~ 21

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.

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Multiple Mechanisms of Action for Inhibitors of Histidine Protein Kinases from Bacterial Two-Component Systems

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.7.1693 ◽

1999 ◽

Vol 43 (7) ◽

pp. 1693-1699 ◽

Cited By ~ 130

Author(s):

Jamese J. Hilliard ◽

Raul M. Goldschmidt ◽

Lisa Licata ◽

Ellen Z. Baum ◽

Karen Bush

Keyword(s):

Pathogenic Bacteria ◽

Response Regulator ◽

Membrane Integrity ◽

Bacterial Killing ◽

Gram Positive Bacteria ◽

Component Systems ◽

Two Component ◽

Two Component Systems ◽

Bacterial Cell Membrane ◽

Cellular Incorporation

ABSTRACT Many pathogenic bacteria utilize two-component systems consisting of a histidine protein kinase (HPK) and a response regulator (RR) for signal transduction. During the search for novel inhibitors, several chemical series, including benzoxazines, benzimidazoles, bis-phenols, cyclohexenes, trityls, and salicylanilides, were identified that inhibited the purified HPK-RR pairs KinA-Spo0F and NRII-NRI, with 50% inhibitory concentrations (IC50s) ranging from 1.9 to >500 μM and MICs ranging from 0.5 to >16 μg/ml for gram-positive bacteria. However, additional observations suggested that mechanisms other than HPK inhibition might contribute to antibacterial activity. In the present work, representative compounds from the six different series of inhibitors were analyzed for their effects on membrane integrity and macromolecular synthesis. At 4× MIC, 17 of 24 compounds compromised the integrity of the bacterial cell membrane within 10 min, as measured by uptake of propidium iodide. In this set, compounds with lower IC50s tended to cause greater membrane disruption. Eleven of 12 compounds inhibited cellular incorporation of radiolabeled thymidine and uridine >97% in 5 min and amino acids >80% in 15 min. The HPK inhibitor that allowed >25% precursor incorporation had no measurable MIC (>16 μg/ml). Fifteen of 24 compounds also caused hemolysis of equine erythrocytes. Thus, the antibacterial HPK inhibitors caused a rapid decrease in cellular incorporation of RNA, DNA, and protein precursors, possibly as a result of the concomitant disruption of the cytoplasmic membrane. Bacterial killing by these HPK inhibitors may therefore be due to multiple mechanisms, independent of HPK inhibition.

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