Glucose-6-Phosphate Acts as an Extracellular Signal of SagS To Modulate Pseudomonas aeruginosa c-di-GMP Levels, Attachment, and Biofilm Formation

ABSTRACT In Pseudomonas aeruginosa, the orphan two-component sensor SagS contributes both to transition to biofilm formation and to biofilm cells gaining their heightened tolerance to antimicrobials. However, little is known about the identity of the signals or conditions sensed by SagS to induce the switch to the sessile, drug-tolerant mode of growth. Using a modified Biolog phenotype assay to screen for compounds that modulate attachment in a SagS-dependent manner, we identified glucose-6-phosphate to enhance attachment in a manner dependent on the glucose-6-phosphate concentration and SagS. The stimulatory effect was not limited to the attachment since glucose-6-phosphate likewise enhanced biofilm formation and also enhanced the expression of select biofilm marker genes. Moreover, exposure to glucose-6-phosphate coincided with decreased swarming motility but increased cellular cyclic-di-GMP (c-di-GMP) levels in biofilms. No such response was noted for compounds modulating attachment and biofilm formation in a manner independent of SagS. Modulation of c-di-GMP in response to glucose-6-phosphate was due to the diguanylate cyclase NicD, with NicD also being required for enhanced biofilm formation. The latter was independent of the sensory domain of NicD but dependent on NicD activity, SagS, and the interaction between NicD and SagS. Our findings indicate that glucose-6-phosphate likely mimics a signal or conditions sensed by SagS to activate its motile-sessile switch function. In addition, our findings provide new insight into the interfaces between the ligand-mediated two-component system signaling pathway and c-di-GMP levels. IMPORTANCE Pathogens sense and respond to signals and cues present in their environment, including host-derived small molecules to modulate the expression of their virulence repertoire. Here, we demonstrate that the opportunistic pathogen Pseudomonas aeruginosa responds to glucose-6-phosphate. Since glucose-6-phosphate is primarily made available due to cell lysis, it is likely that glucose-6-phosphate represents a cross-kingdom cell-to-cell signal that enables P. aeruginosa to adapt to the (nutrient-poor) host environment by enhancing biofilm formation, cyclic-di-GMP, and the expression of genes linked to biofilm formation in a concentration- and SagS-dependent manner.

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The Two-Component Sensor KinB Acts as a Phosphatase To Regulate Pseudomonas aeruginosa Virulence

Journal of Bacteriology ◽

10.1128/jb.01168-12 ◽

2012 ◽

Vol 194 (23) ◽

pp. 6537-6547 ◽

Cited By ~ 14

Author(s):

Nikhilesh S. Chand ◽

Anne E. Clatworthy ◽

Deborah T. Hung

Keyword(s):

Pseudomonas Aeruginosa ◽

Response Regulator ◽

Opportunistic Pathogen ◽

Regulatory Control ◽

Primary Role ◽

Chronic Infections ◽

Content Type ◽

Two Component ◽

Sense And Respond ◽

Cognate Response Regulator

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen that is capable of causing both acute and chronic infections.P. aeruginosavirulence is subject to sophisticated regulatory control by two-component systems that enable it to sense and respond to environmental stimuli. We recently reported that the two-component sensor KinB regulates virulence in acuteP. aeruginosainfection. Furthermore, it regulates acute-virulence-associated phenotypes such as pyocyanin production, elastase production, and motility in a manner independent of its kinase activity. Here we show that KinB regulates virulence through the global sigma factor AlgU, which plays a key role in repressingP. aeruginosaacute-virulence factors, and through its cognate response regulator AlgB. However, we show that rather than phosphorylating AlgB, KinB's primary role in the regulation of virulence is to act as a phosphatase to dephosphorylate AlgB and alleviate phosphorylated AlgB's repression of acute virulence.

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The SagS sensory protein modulates biofilm formation and c-di-GMP levels by Pseudomonas aeruginosa in response to glucose-6-phosphate.

10.5194/biofilms9-122 ◽

2020 ◽

Author(s):

Soyoung Park ◽

Jozef Dingemans ◽

Madison Gowett ◽

Karin Sauer

Keyword(s):

Pseudomonas Aeruginosa ◽

Genetic Analysis ◽

Signaling Pathway ◽

Biofilm Formation ◽

Phosphate Concentration ◽

Dependent Manner ◽

Diguanylate Cyclase ◽

Swarming Motility ◽

Two Component ◽

Insight Into

<p>In <em>Pseudomonas aeruginosa</em>, the orphan two-component sensor SagS contributes to both, the transition to biofilm formation and to biofilm cells gaining their heightened tolerance to antimicrobials. However, little is known about the identity of the signals or conditions sensed by SagS to induce the switch to the sessile, drug tolerant mode of growth. Using a modified Biolog phenotype assay to screen for compounds that modulate attachment in a SagS-dependent manner, we identified glucose-6-phosphate to enhance attachment in a manner dependent on the glucose-6-phosphate concentration and SagS. The stimulatory effect was not limited to the attachment as glucose-6-phosphate likewise enhanced biofilm formation. We show that exposure to glucose-6-phosphate results in decreased swarming motility but increased cellular c-di-GMP levels in biofilms. Genetic analysis indicated that the diguanylate cyclase NicD is an activator of biofilm formation and is not only required for enhanced biofilm formation in response to glucose-6-phosphate but also interacts with SagS. Our findings indicate glucose-6-phosphate to likely mimic a signal or conditions sensed by SagS to activate its motile-sessile switch function. Additionally, our findings provide new insight into the interfaces between the ligand-mediated TCS signaling pathway and c-di-GMP levels.</p>

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HtrA Is Important for Stress Resistance and Virulence in Haemophilus parasuis

Infection and Immunity ◽

10.1128/iai.00147-16 ◽

2016 ◽

Vol 84 (8) ◽

pp. 2209-2219 ◽

Cited By ~ 19

Author(s):

Luhua Zhang ◽

Ying Li ◽

Yiping Wen ◽

Gee W. Lau ◽

Xiaobo Huang ◽

...

Keyword(s):

Biofilm Formation ◽

Protein Quality ◽

Protein A ◽

Opportunistic Pathogen ◽

Dependent Manner ◽

Haemophilus Parasuis ◽

Content Type ◽

Outer Membrane Protein A ◽

Dipeptide Transport ◽

Atp Binding Protein

Haemophilus parasuisis an opportunistic pathogen that causes Glässer's disease in swine, with polyserositis, meningitis, and arthritis. The high-temperature requirement A (HtrA)-like protease, which is involved in protein quality control, has been reported to be a virulence factor in many pathogens. In this study, we showed that HtrA ofH. parasuis(HpHtrA) exhibited both chaperone and protease activities. Finally, nickel import ATP-binding protein (NikE), periplasmic dipeptide transport protein (DppA), and outer membrane protein A (OmpA) were identified as proteolytic substrates for HpHtrA. The protease activity reached its maximum at 40°C in a time-dependent manner. Disruption of thehtrAgene from strain SC1401 affected tolerance to temperature stress and resistance to complement-mediated killing. Furthermore, increased autoagglutination and biofilm formation were detected in thehtrAmutant. In addition, thehtrAmutant was significantly attenuated in virulence in the murine model of infection. Together, these data demonstrate that HpHtrA plays an important role in the virulence ofH. parasuis.

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Thiostrepton Hijacks Pyoverdine Receptors To Inhibit Growth ofPseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00472-19 ◽

2019 ◽

Vol 63 (9) ◽

Cited By ~ 8

Author(s):

Michael R. M. Ranieri ◽

Derek C. K. Chan ◽

Luke N. Yaeger ◽

Madeleine Rudolph ◽

Sawyer Karabelas-Pittman ◽

...

Keyword(s):

Biofilm Formation ◽

Opportunistic Pathogen ◽

Multidrug Resistant ◽

Clinical Isolates ◽

23S Rrna ◽

Dependent Manner ◽

Peptide Antibiotics ◽

Iron Starvation ◽

Gram Negative ◽

Content Type

ABSTRACTPseudomonas aeruginosais a biofilm-forming opportunistic pathogen and is intrinsically resistant to many antibiotics. In a high-throughput screen for molecules that modulate biofilm formation, we discovered that the thiopeptide antibiotic thiostrepton (TS), which is considered to be inactive against Gram-negative bacteria, stimulatedP. aeruginosabiofilm formation in a dose-dependent manner. This phenotype is characteristic of exposure to antimicrobial compounds at subinhibitory concentrations, suggesting that TS was active againstP. aeruginosa. Supporting this observation, TS inhibited the growth of a panel of 96 multidrug-resistant (MDR)P. aeruginosaclinical isolates at low-micromolar concentrations. TS also had activity againstAcinetobacter baumanniiclinical isolates. The expression of Tsr, a 23S rRNA-modifying methyltransferase from TS producerStreptomyces azureus, intransconferred TS resistance, confirming that the drug acted via its canonical mode of action, inhibition of ribosome function. The deletion of oligopeptide permease systems used by other peptide antibiotics for uptake failed to confer TS resistance. TS susceptibility was inversely proportional to iron availability, suggesting that TS exploits uptake pathways whose expression is increased under iron starvation. Consistent with this finding, TS activity againstP. aeruginosaandA. baumanniiwas potentiated by the FDA-approved iron chelators deferiprone and deferasirox and by heat-inactivated serum. Screening ofP. aeruginosamutants for TS resistance revealed that it exploits pyoverdine receptors FpvA and FpvB to cross the outer membrane. We show that the biofilm stimulation phenotype can reveal cryptic subinhibitory antibiotic activity, and that TS has activity against select multidrug-resistant Gram-negative pathogens under iron-limited growth conditions, similar to those encountered at sites of infection.

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Novel Two-Component System ofStreptococcus sanguinisAffecting Functions Associated with Viability in Saliva and Biofilm Formation

Infection and Immunity ◽

10.1128/iai.00942-17 ◽

2018 ◽

Vol 86 (4) ◽

Cited By ~ 9

Author(s):

Tarsila M. Camargo ◽

Rafael N. Stipp ◽

Lívia A. Alves ◽

Erika N. Harth-Chu ◽

José F. Höfling ◽

...

Keyword(s):

Biofilm Formation ◽

Ex Vivo ◽

Opportunistic Pathogen ◽

Dna Looping ◽

Extracellular Dna ◽

Two Component System ◽

Component System ◽

Content Type ◽

Cell Counts ◽

Two Component

ABSTRACTStreptococcus sanguinisis a pioneer species of teeth and a common opportunistic pathogen of infective endocarditis. In this study, we identified a two-component system,S. sanguinisSptRS (SptRSSs), affectingS. sanguinissurvival in saliva and biofilm formation. Isogenic mutants ofsptRSs(SKsptR) andsptSSs(SKsptS) showed reduced cell counts inex vivoassays of viability in saliva compared to those of parent strain SK36 and complemented mutants. Reduced counts of the mutants in saliva were associated with reduced growth rates in nutrient-poor medium (RPMI) and increased susceptibility to the deposition of C3b and the membrane attach complex (MAC) of the complement system, a defense component of saliva and serum. Conversely,sptRSsandsptSSsmutants showed increased biofilm formation associated with higher levels of production of H2O2and extracellular DNA. Reverse transcription-quantitative PCR (RT-qPCR) comparisons of strains indicated a global role of SptRSSsin repressing genes for H2O2production (2.5- to 15-fold upregulation ofspxB,spxR,vicR,tpk, andackAinsptRSsandsptSSsmutants), biofilm formation, and/or evasion of host immunity (2.1- to 11.4-fold upregulation ofsrtA,pcsB,cwdP,iga, andnt5e). Compatible with the homology of SptRSswith AraC-type regulators, duplicate to multiple conserved repeats were identified in 1,000-bp regulatory regions of downstream genes, suggesting that SptRSsregulates transcription by DNA looping. Significant transcriptional changes in the regulatory genesvicR,spxR,comE,comX, andmecAin thesptRSsandsptSSsmutants further indicated that SptRSSsis part of a regulatory network that coordinates cell wall homeostasis, H2O2production, and competence. This study reveals that SptRSSsis involved in the regulation of crucial functions forS. sanguinispersistence in the oral cavity.

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The Pseudomonas aeruginosa PAO1 Two-Component Regulator CarSR Regulates Calcium Homeostasis and Calcium-Induced Virulence Factor Production through Its Regulatory Targets CarO and CarP

Journal of Bacteriology ◽

10.1128/jb.00963-15 ◽

2016 ◽

Vol 198 (6) ◽

pp. 951-963 ◽

Cited By ~ 19

Author(s):

Manita Guragain ◽

Michelle M. King ◽

Kerry S. Williamson ◽

Ailyn C. Pérez-Osorio ◽

Tatsuya Akiyama ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Virulence Factors ◽

Virulence Factor ◽

Regulatory System ◽

Dependent Manner ◽

Content Type ◽

Factor Production ◽

Two Component ◽

Intracellular Ca ◽

Virulence Factor Production

ABSTRACTPseudomonas aeruginosais an opportunistic human pathogen that causes severe, life-threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or artificial implants. During CF pulmonary infections,P. aeruginosaoften encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed thatP. aeruginosaresponds to externally added Ca2+through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+level, followed by its removal to the basal submicromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+homeostasis are not known. Here, we characterized the genome-wide transcriptional response ofP. aeruginosato elevated [Ca2+] in both planktonic cultures and biofilms. Among the genes induced by CaCl2in strain PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here calledcarSandcarR), while the closely related two-component regulatorsphoPQandpmrABwere repressed by CaCl2addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant ofcarRand performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2are the predicted periplasmic OB-fold protein, PA0320 (here calledcarO), and the inner membrane-anchored five-bladed β-propeller protein, PA0327 (here calledcarP). Mutations in bothcarOandcarPaffected Ca2+homeostasis, reducing the ability ofP. aeruginosato export excess Ca2+. In addition, a mutation incarPhad a pleotropic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+and responding through its regulatory targets that modulate Ca2+homeostasis, surface-associated motility, and the production of the virulence factor pyocyanin.IMPORTANCEDuring infectious disease,Pseudomonas aeruginosaencounters environments with high calcium (Ca2+) concentrations, yet the cells maintain intracellular Ca2+at levels that are orders of magnitude less than that of the external environment. In addition, Ca2+signalsP. aeruginosato induce the production of several virulence factors. Compared to eukaryotes, little is known about how bacteria maintain Ca2+homeostasis or how Ca2+acts as a signal. In this study, we identified a two-component regulatory system inP. aeruginosaPAO1, termed CarRS, that is induced at elevated Ca2+levels. CarRS modulates Ca2+signaling and Ca2+homeostasis through its regulatory targets, CarO and CarP. The results demonstrate thatP. aeruginosauses a two-component regulatory system to sense external Ca2+and relays that information for Ca2+-dependent cellular processes.

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Signal Sensing and Transduction Are Conserved between the Periplasmic Sensory Domains of BifA and SagS

mSphere ◽

10.1128/msphere.00442-19 ◽

2019 ◽

Vol 4 (4) ◽

Author(s):

Jozef Dingemans ◽

Rebecca E. Al-Feghali ◽

Holger Sondermann ◽

Karin Sauer

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Structural Similarity ◽

Opportunistic Pathogen ◽

Sequence Conservation ◽

Sensor Kinase ◽

Antibiotic Tolerance ◽

Bacterial Cells ◽

Chronic Infections ◽

Content Type

ABSTRACT The hybrid sensor kinase SagS of Pseudomonas aeruginosa plays a key role in the transition from the planktonic to the biofilm mode of growth. Recently, we have shown that distinct sets of residues in its periplasmic HmsP sensory domain are involved in the regulation of biofilm formation or antibiotic tolerance. Interestingly, the HmsP domain of the phosphodiesterase BifA shows great predicted structural similarity to that of SagS, despite moderate sequence conservation and only a number of residues involved in SagS signaling being conserved between both proteins. Based on this observation, we hypothesized that BifA and SagS may use similar mechanisms to sense and transduce signals perceived at their periplasmic HmsP domains and, therefore, may be interchangeable. To test this hypothesis, we constructed SagS hybrids in which the HmsP domain of SagS was replaced by that of BifA (and vice versa) or by the DISMED2 sensory domain of NicD. The SagS-BifA hybrid restored attachment and biofilm formation by the ΔbifA mutant. Likewise, while the NicD-SagS hybrid was nonfunctional, the BifA-SagS hybrid partially restored pathways leading to biofilm formation and antibiotic tolerance in a ΔsagS mutant background. Furthermore, alanine substitution of key residues previously associated with the biofilm formation and antibiotic tolerance pathways of SagS impaired signal transduction by the BifA-SagS hybrid in a similar way to SagS. In conclusion, our data indicate that the nature of the sensory domain is important for proper functionality of the cytoplasmic effector domains and that signal sensing and transduction are likely conserved in SagS and BifA. IMPORTANCE Biofilms have been associated with more than 60% of all recalcitrant and chronic infections and can render bacterial cells up to a thousand times more resistant to antibiotics than planktonic cells. Although it is known that the transition from the planktonic to the biofilm mode of growth involves two-component regulatory systems, increased c-di-GMP levels, and quorum sensing systems among others, the exact signaling events that lead to biofilm formation remain unknown. In the opportunistic pathogen Pseudomonas aeruginosa, the hybrid sensor kinase SagS regulates biofilm formation and antibiotic tolerance through two independent pathways via distinct residues in its periplasmic sensory domain. Interestingly, the sensory domains of SagS and BifA show great predicted structural similarity despite moderate sequence conservation. Here we show that the sensory domains of BifA and SagS are functionally interchangeable and that they use a similar mechanism of signal sensing and transduction, which broadens our understanding of how bacteria perceive and transduce signals when transitioning to the biofilm mode of growth.

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PilZ Domain Protein FlgZ Mediates Cyclic Di-GMP-Dependent Swarming Motility Control in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00196-16 ◽

2016 ◽

Vol 198 (13) ◽

pp. 1837-1846 ◽

Cited By ~ 55

Author(s):

Amy E. Baker ◽

Andreas Diepold ◽

Sherry L. Kuchma ◽

Jessie E. Scott ◽

Dae Gon Ha ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Fluorescent Protein ◽

Bacterial Species ◽

Dependent Manner ◽

Swarming Motility ◽

Content Type ◽

Bacterial Surface ◽

Important Regulator ◽

Green Fluorescent

ABSTRACTThe second messenger cyclic diguanylate (c-di-GMP) is an important regulator of motility in many bacterial species. InPseudomonas aeruginosa, elevated levels of c-di-GMP promote biofilm formation and repress flagellum-driven swarming motility. The rotation ofP. aeruginosa's polar flagellum is controlled by two distinct stator complexes, MotAB, which cannot support swarming motility, and MotCD, which promotes swarming motility. Here we show that when c-di-GMP levels are elevated, swarming motility is repressed by the PilZ domain-containing protein FlgZ and by Pel polysaccharide production. We demonstrate that FlgZ interacts specifically with the motility-promoting stator protein MotC in a c-di-GMP-dependent manner and that a functional green fluorescent protein (GFP)-FlgZ fusion protein shows significantly reduced polar localization in a strain lacking the MotCD stator. Our results establish FlgZ as a c-di-GMP receptor affecting swarming motility byP. aeruginosaand support a model wherein c-di-GMP-bound FlgZ impedes motility via its interaction with the MotCD stator.IMPORTANCEThe regulation of surface-associated motility plays an important role in bacterial surface colonization and biofilm formation. c-di-GMP signaling is a widespread means of controlling bacterial motility, and yet the mechanism whereby this signal controls surface-associated motility inP. aeruginosaremains poorly understood. Here we identify a PilZ domain-containing c-di-GMP effector protein that contributes to c-di-GMP-mediated repression of swarming motility byP. aeruginosa. We provide evidence that this effector, FlgZ, impacts swarming motility via its interactions with flagellar stator protein MotC. Thus, we propose a new mechanism for c-di-GMP-mediated regulation of motility for a bacterium with two flagellar stator sets, increasing our understanding of surface-associated behaviors, a key prerequisite to identifying ways to control the formation of biofilm communities.

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The Vancomycin Resistance-Associated Regulatory System VraSR Modulates Biofilm Formation of Staphylococcus epidermidis in an ica -Dependent Manner

mSphere ◽

10.1128/msphere.00641-21 ◽

2021 ◽

Author(s):

Youcong Wu ◽

Yuanyuan Meng ◽

Lian Qian ◽

Baixing Ding ◽

Haiyan Han ◽

...

Keyword(s):

Staphylococcus Epidermidis ◽

Biofilm Formation ◽

Regulatory System ◽

Vancomycin Resistance ◽

Dependent Manner ◽

Content Type ◽

Two Component ◽

Complex Procedure ◽

Hospital Acquired

S. epidermidis is a leading cause of hospital-acquired catheter-related infections, and its pathogenicity depends mostly on its ability to form biofilms on implants. The biofilm formation is a complex procedure that involves multiple regulating factors. Here, we show that a vancomycin resistance-associated two-component regulatory system, VraSR, plays an important role in modulating S. epidermidis biofilm formation and tolerance to stress.

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TosR-Mediated Regulation of Adhesins and Biofilm Formation in UropathogenicEscherichia coli

mSphere ◽

10.1128/msphere.00222-18 ◽

2018 ◽

Vol 3 (3) ◽

Cited By ~ 7

Author(s):

Courtney L. Luterbach ◽

Valerie S. Forsyth ◽

Michael D. Engstrom ◽

Harry L. T. Mobley

Keyword(s):

Urinary Tract ◽

Cross Talk ◽

Biofilm Formation ◽

Dependent Manner ◽

Rna Seq ◽

Concentration Dependent Manner ◽

Content Type ◽

E Coli ◽

Expression Of Genes ◽

Invasive Infections

ABSTRACTUropathogenicEscherichia colistrains utilize a variety of adherence factors that assist in colonization of the host urinary tract. TosA (typeonesecretionA) is a nonfimbrial adhesin that is predominately expressed during murine urinary tract infection (UTI), binds to kidney epithelial cells, and promotes survival during invasive infections. ThetosRCBDAEFoperon encodes the secretory machinery necessary for TosA localization to theE. colicell surface, as well as the transcriptional regulator TosR. TosR binds upstream of thetosoperon and in a concentration-dependent manner either induces or repressestosAexpression. TosR is a member of the PapB family of fimbrial regulators that can participate in cross talk between fimbrial operons. TosR also binds upstream of thepapoperon and suppresses PapA production. However, the scope of TosR-mediated cross talk is understudied and may be underestimated. To quantify the global effects of TosR-mediated regulation on theE. coliCFT073 genome, we induced expression oftosR, collected mRNA, and performed high-throughput RNA sequencing (RNA-Seq). These findings show that production of TosR affected the expression of genes involved with adhesins, including P, F1C, and Auf fimbriae, nitrate-nitrite transport, microcin secretion, and biofilm formation.IMPORTANCEUropathogenicE. colistrains cause the majority of UTIs, which are the second most common bacterial infection in humans. During a UTI, bacteria adhere to cells within the urinary tract, using a number of different fimbrial and nonfimbrial adhesins. Biofilms can also develop on the surfaces of catheters, resulting in complications such as blockage. In this work, we further characterized the regulator TosR, which links both adhesin production and biofilm formation and likely plays a crucial function during UTI and disseminated infection.

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