scholarly journals Carbon Starvation Induces the Expression of PprB-Regulated Genes in Pseudomonas aeruginosa

2019 ◽  
Vol 85 (22) ◽  
Author(s):  
Congcong Wang ◽  
Wenhui Chen ◽  
Aiguo Xia ◽  
Rongrong Zhang ◽  
Yajia Huang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Carbon Starvation ◽  
Secretion System ◽  
Surface Adhesion ◽  
Starvation Stress ◽  
Two Component System ◽  
Component System ◽  
Content Type ◽  
Two Component

ABSTRACT Pseudomonas aeruginosa can cause severe infections in humans. This bacterium often adopts a biofilm lifestyle that is hard to treat. In several previous studies, the PprA-PprB two-component system (TCS), which controls the expression of type IVb pili, BapA adhesin, and CupE fimbriae, was shown to be involved in biofilm formation (M. Romero, H. Silistre, L. Lovelock, V. J. Wright, K.-G. Chan, et al., Nucleic Acids Res 46:6823–6840, 2018, https://doi.org/10.1093/nar/gky324; S. de Bentzmann, C. Giraud, C. S. Bernard, V. Calderon, F. Ewald F, et al., PLoS Pathog 8:e1003052, 2012, https://doi.org/10.1371/journal.ppat.1003052). However, signals or environmental conditions that can trigger the PprA-PprB TCS are still unknown, and the molecular mechanisms of PprB-mediated biofilm formation are poorly characterized. Here, we report that carbon starvation stress (CSS) can induce the expression of pprB and genes in the PprB regulon. CSS-induced pprB transcription is mediated by the stress response sigma factor RpoS rather than the two-component sensor PprA. We also observed a strong negative regulation of PprB on the transcription of itself. Further experiments showed that PprB overexpression greatly enhanced cell-cell adhesion (CCA) and cell-surface adhesion (CSA) in P. aeruginosa. Specifically, under the background of PprB overexpression, both the BapA adhesin and CupE fimbriae displayed positive effects on CCA and CSA, while the type IVb pili showed an unexpected negative effect on CCA and no effect on CSA. In addition, expression of the PprB regulon genes were significantly increased in 3-day colony biofilms, indicating a possible carbon limitation state. The CSS-RpoS-PprB-Bap/Flp/CupE pathway identified in this study provides a new perspective on the process of biofilm formation in carbon-limited environments. IMPORTANCE Typically, the determination of the external signals that can trigger a regulatory system is crucial to understand the regulatory logic and inward function of that system. The PprA-PprB two-component system was reported to be involved in biofilm formation in Pseudomonas aeruginosa, but the signals triggering this system are unknown. In this study, we found that carbon starvation stress (CSS) induces transcription of pprB and genes in the PprB regulon through an RpoS-dependent pathway. Increased PprB expression leads to enhanced cell-cell adhesion (CCA) and cell-surface adhesion (CSA) in P. aeruginosa. Both CCA and CSA are largely dependent on the Bap secretion system and are moderately dependent on the CupE fimbriae. Our findings suggest that PprB reinforces the structure of biofilms under carbon-limited conditions, and the Bap secretion system and CupE fimbriae are two potential targets for biofilm treatment.

scholarly journals Carbon starvation induces the expression of PprB-regulated genes inPesudomonas aeruginosa

2019 ◽  
Author(s):  
Congcong Wang ◽  
Wenhui Chen ◽  
Aiguo Xia ◽  
Rongrong Zhang ◽  
Yajia Huang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Surface ◽  
Biofilm Formation ◽  
Carbon Starvation ◽  
Secretion System ◽  
Starvation Stress ◽  
Two Component System ◽  
Component System ◽  
Two Component ◽  
Cell Cell

ABSTRACTPseudomonas aeruginosacan cause severe infections in humans. This bacteria often adopt a biofilm lifestyle that is hard to treat. In several previous studies, the PprA-PprB two-component system (TCS), which controls the expression of type IVb pili, BapA adhesin, and CupE fimbriae, was shown to be involved in biofilm formation. However, signals or environmental conditions that can trigger the PprA-PprB TCS are still unknown, and the molecular mechanisms of PprB-mediated biofilm formation are poorly characterized. Here we report that carbon starvation stress (CCS) can induce the expression ofpprBand genes in the PprB regulon. The stress response sigma factor RpoS, rather than the two-component sensor PprA, was determined to mediate the induction ofpprBtranscription. We also observed a strong negative regulation of PprB to the transcription of itself. Further experiments showed that PprB overexpression greatly enhanced cell-cell adhesion (CCA) and cell-surface adhesion (CSA) inP. aeruginosa. Specially, under the background of PprB overexpression, both of the BapA adhesin and CupE fimbriae displayed positive effect on CCA and CSA, while the type IVb pili showed an unexpected negative effect on CCA and no effect on CSA. In addition, expression of the PprB regulon genes displayed significant increases in 3-day colony biofilms, indicating a possible carbon limitation state in these biofilms. The CSS-RpoS-PprB-Bap/Flp/CupE/Tad pathway identified in this study provides a new perspective on the process of biofilm formation under carbon-limited environments.IMPORTANCETypically, determining the external signals that can trigger a regulatory system is crucial to understand the regulatory logic and inward function of that system. The PprA-PprB two-component system was reported to be involved in biofilm formation inPseudomonas aeruginosa, but the signals that can trigger this system are unknown. In this study, we found that carbon starvation stress (CSS) can induce the transcription ofpprBand genes in PprB regulon, through an RpoS dependent pathway. Increase of PprB expression leads to enhanced cell-cell and cell-surface adhesions inP. aeruginosa,both of which are dependent mainly on the Bap adhesin secretion system and partially on the CupE fimbriae. Our findings suggest that PprB reinforces the structure of biofilms under carbon-limited conditions, and the Bap secretion system and CupE fimbriae are two potential targets for biofilm treatment.

scholarly journals Loss of the Two-Component System TctD-TctE in Pseudomonas aeruginosa Affects Biofilm Formation and Aminoglycoside Susceptibility in Response to Citric Acid

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Patrick K. Taylor ◽  
Li Zhang ◽  
Thien-Fah Mah
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Citric Acid ◽  
Carbon Source ◽  
Biofilm Formation ◽  
Two Component System ◽  
Component System ◽  
Content Type ◽  
Metabolic Versatility ◽  
Two Component ◽  
Biofilm Development

ABSTRACT The two-component system TctD-TctE is important for regulating the uptake of tricarboxylic acids in Pseudomonas aeruginosa. TctD-TctE accomplishes this through derepression of the gene opdH, which encodes a tricarboxylic acid-specific porin. Previous work from our lab revealed that TctD-TctE in P. aeruginosa also has a role in resistance to aminoglycoside antibiotics. The aim of this study was to further characterize the role of TctD-TctE in P. aeruginosa in the presence of citric acid. Here it was found that deletion of P. aeruginosa PA14 TctD-TctE (ΔtctED) resulted in a 4-fold decrease in the biofilm bactericidal concentrations of the aminoglycosides tobramycin and gentamicin when citric acid was present in nutrient media. Tobramycin accumulation assays demonstrated that deletion of TctD-TctE resulted in an increase in the amount of tobramycin retained in biofilm cells. The PA14 wild type responded to increasing concentrations of citric acid by producing less biofilm. In contrast, the amount of ΔtctED mutant biofilm formation remained constant or enhanced. Furthermore, the ΔtctED strain was incapable of growing on citric acid as a sole carbon source and was highly reduced in its ability to grow in the presence of citric acid even when an additional carbon source was available. Use of phenotypic and genetic microarrays found that this growth deficiency of the ΔtctED mutant is unique to citric acid and that multiple metabolic genes are dysregulated. This work demonstrates that TctD-TctE in P. aeruginosa has a role in biofilm development that is dependent on citric acid and that is separate from the previously characterized involvement in resistance to antibiotics. IMPORTANCE Nutrient availability is an important contributor to the ability of bacteria to establish successful infections in a host. Pseudomonas aeruginosa is an opportunistic pathogen in humans causing infections that are difficult to treat. In part, its success is attributable to a high degree of metabolic versatility. P. aeruginosa is able to sense and respond to varied and limited nutrient stress in the host environment. Two-component systems are important sensors-regulators of cellular responses to environmental stresses, such as those encountered in the host. This work demonstrates that the response by the two-component system TctD-TctE to the presence of citric acid has a role in biofilm formation, aminoglycoside susceptibility, and growth in P. aeruginosa.

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

2018 ◽  
Vol 86 (4) ◽  
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.

scholarly journals ThePseudomonas aeruginosaPilSR Two-Component System Regulates Both Twitching and Swimming Motilities

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Sara L. N. Kilmury ◽  
Lori L. Burrows
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iv Pili ◽  
Geobacter Sulfurreducens ◽  
Type Iv Pilus ◽  
Two Component System ◽  
Component System ◽  
Content Type ◽  
Swimming Motility ◽  
Type Iv ◽  
Two Component

ABSTRACTMotility is an important virulence trait for many bacterial pathogens, allowing them to position themselves in appropriate locations at appropriate times. The motility structures type IV pili and flagella are also involved in sensing surface contact, which modulates pathogenicity. InPseudomonas aeruginosa, the PilS-PilR two-component system (TCS) regulates expression of the type IV pilus (T4P) major subunit PilA, while biosynthesis of the single polar flagellum is regulated by a hierarchical system that includes the FleSR TCS. Previous studies ofGeobacter sulfurreducensandDichelobacter nodosusimplicated PilR in regulation of non-T4P-related genes, including some involved in flagellar biosynthesis. Here we used transcriptome sequencing (RNA-seq) analysis to identify genes in addition topilAwith changes in expression in the absence ofpilR. Among the genes identified were 10 genes whose transcription increased in thepilAmutant but decreased in thepilRmutant, despite both mutants lacking T4P and pilus-related phenotypes. The products of these inversely dysregulated genes, many of which were hypothetical, may be important for virulence and surface-associated behaviors, as mutants had altered swarming motility, biofilm formation, type VI secretion system expression, and pathogenicity in a nematode model. Further, the PilSR TCS positively regulated transcription offleSR, and thus many genes in the FleSR regulon. As a result,pilSRdeletion mutants had defects in swimming motility that were independent of the loss of PilA. Together, these data suggest that in addition to controlling T4P expression, PilSR could have a broader role in the regulation ofP. aeruginosamotility and surface sensing behaviors.IMPORTANCESurface appendages such as type IV pili and flagella are important for establishing surface attachment and infection in a host in response to appropriate cues. The PilSR regulatory system that controls type IV pilus expression inPseudomonas aeruginosahas an established role in expression of the major pilin PilA. Here we provide evidence supporting a new role for PilSR in regulating flagellum-dependent swimming motility in addition to pilus-dependent twitching motility. Further, even though bothpilAandpilRmutants lack PilA and pili, we identified sets of genes downregulated in thepilRmutant and upregulated in apilAmutant as well as genes downregulated only in apilRmutant, independent of pilus expression. This finding suggests that change in the inner membrane levels of PilA is only one of the cues to which PilR responds to modulate gene expression. Identification of PilR as a regulator of multiple motility pathways may make it an interesting therapeutic target for antivirulence compounds.

scholarly journals The Two-Component System CprRS Senses Cationic Peptides and Triggers Adaptive Resistance in Pseudomonas aeruginosa Independently of ParRS

2012 ◽  
Vol 56 (12) ◽  
pp. 6212-6222 ◽  
Author(s):  
Lucía Fernández ◽  
Håvard Jenssen ◽  
Manjeet Bains ◽  
Irith Wiegand ◽  
W. James Gooderham ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Peptides ◽  
Outer Membrane ◽  
Dependent Manner ◽  
Two Component System ◽  
Concentration Dependent Manner ◽  
Component System ◽  
Content Type ◽  
Wide Range ◽  
Two Component

ABSTRACTCationic antimicrobial peptides pass across the outer membrane by interacting with negatively charged lipopolysaccharide (LPS), leading to outer membrane permeabilization in a process termed self-promoted uptake. Resistance can be mediated by the addition of positively charged arabinosamine through the action of thearnBCADTEFoperon. We recently described a series of two-component regulators that lead to the activation of thearnoperon after recognizing environmental signals, including low-Mg2+(PhoPQ, PmrAB) or cationic (ParRS) peptides. However, some peptides did not activate thearnoperon through ParRS. Here, we report the identification of a new two-component system, CprRS, which, upon exposure to a wide range of antimicrobial peptides, triggered the expression of the LPS modification operon. Thus, mutations in thecprRSoperon blocked the induction of thearnoperon in response to several antimicrobial peptides independently of ParRS but did not affect the response to low Mg2+. Distinct patterns ofarninduction were identified. Thus, the responses to polymyxins were abrogated by eitherparRorcprRmutations, while responses to other peptides, including indolicidin, showed differential dependency on the CprRS and ParRS systems in a concentration-dependent manner. It was further demonstrated that, following exposure to inducing antimicrobial peptides,cprRSmutants did not become adaptively resistant to polymyxins as was observed for wild-type cells. Our microarray studies demonstrated that the CprRS system controlled a quite modest regulon, indicating that it was quite specific to adaptive peptide resistance. These findings provide greater insight into the complex regulation of LPS modification inPseudomonas aeruginosa, which involves the participation of at least 4 two-component systems.

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

mSphere ◽  
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.

scholarly journals Two-Component System VicRK Regulates Functions Associated with Establishment of Streptococcus sanguinis in Biofilms

2014 ◽  
Vol 82 (12) ◽  
pp. 4941-4951 ◽  
Author(s):  
Julianna J. Moraes ◽  
Rafael N. Stipp ◽  
Erika N. Harth-Chu ◽  
Tarsila M. Camargo ◽  
José F. Höfling ◽  
...  
Keyword(s):  
Cell Wall ◽  
Biofilm Formation ◽  
Extracellular Dna ◽  
Aerobic Growth ◽  
Two Component System ◽  
Component System ◽  
Content Type ◽  
Streptococcus Sanguinis ◽  
Hydrolytic Activities ◽  
Two Component

ABSTRACTStreptococcus sanguinisis a commensal pioneer colonizer of teeth and an opportunistic pathogen of infectious endocarditis. The establishment ofS. sanguinisin host sites likely requires dynamic fitting of the cell wall in response to local stimuli. In this study, we investigated the two-component system (TCS) VicRK inS. sanguinis(VicRKSs), which regulates genes of cell wall biogenesis, biofilm formation, and virulence in opportunistic pathogens. AvicKknockout mutant obtained from strain SK36 (SKvic) showed slight reductions in aerobic growth and resistance to oxidative stress but an impaired ability to form biofilms, a phenotype restored in the complemented mutant. The biofilm-defective phenotype was associated with reduced amounts of extracellular DNA during aerobic growth, with reduced production of H2O2, a metabolic product associated with DNA release, and with inhibitory capacity ofS. sanguiniscompetitor species. No changes in autolysis or cell surface hydrophobicity were detected in SKvic. Reverse transcription-quantitative PCR (RT-qPCR), electrophoretic mobility shift assays (EMSA), and promoter sequence analyses revealed that VicR directly regulates genes encoding murein hydrolases (SSA_0094,cwdP, andgbpB) andspxB, which encodes pyruvate oxidase for H2O2production. Genes previously associated withspxBexpression (spxR,ccpA,ackA, andtpK) were not transcriptionally affected in SKvic. RT-qPCR analyses ofS. sanguinisbiofilm cells further showed upregulation of VicRK targets (spxB,gbpB, andSSA_0094) and other genes for biofilm formation (gtfPandcomE) compared to expression in planktonic cells. This study provides evidence that VicRKSsregulates functions crucial forS. sanguinisestablishment in biofilms and identifies novel VicRK targets potentially involved in hydrolytic activities of the cell wall required for these functions.

scholarly journals An Osmoregulatory Mechanism Operating through OmpR and LrhA Controls the Motile-Sessile Switch in the Plant Growth-Promoting BacteriumPantoea alhagi

2019 ◽  
Vol 85 (10) ◽  
Author(s):  
Shuyu Li ◽  
Hong Liang ◽  
Zhiyan Wei ◽  
Haonan Bai ◽  
Mengyun Li ◽  
...  
Keyword(s):  
Plant Growth ◽  
Osmotic Stress ◽  
Biofilm Formation ◽  
Two Component System ◽  
Component System ◽  
Content Type ◽  
Two Component ◽  
Plant Growth Promoting ◽  
Growth Promoting ◽  
Osmoregulatory Mechanism

ABSTRACTAdaptation to osmotic stress is crucial for bacterial growth and survival in changing environments. Although a large number of osmotic stress response genes have been identified in various bacterial species, how osmotic changes affect bacterial motility, biofilm formation, and colonization of host niches remains largely unknown. In this study, we report that the LrhA regulator is an osmoregulated transcription factor that directly binds to the promoters of theflhDC,eps, andopgGHoperons and differentially regulates their expression, thus inhibiting motility and promoting exopolysaccharide (EPS) production, synthesis of osmoregulated periplasmic glucans (OPGs), biofilm formation, and root colonization of the plant growth-promoting bacteriumPantoea alhagiLTYR-11Z. Further, we observed that the LrhA-regulated OPGs control RcsCD-RcsB activation in a concentration-dependent manner, and a high concentration of OPGs induced by increased medium osmolarity is maintained to achieve the high level of activation of the Rcs phosphorelay, which results in enhanced EPS synthesis and decreased motility inP. alhagi. Moreover, we showed that the osmosensing regulator OmpR directly binds to the promoter oflrhAand promotes its expression, whilelrhAexpression is feedback inhibited by the activated Rcs phosphorelay system. Overall, our data support a model wherebyP. alhagisenses environmental osmolarity changes through the EnvZ-OmpR two-component system and LrhA to regulate the synthesis of OPGs, EPS production, and flagellum-dependent motility, thereby employing a hierarchical signaling cascade to control the transition between a motile lifestyle and a biofilm lifestyle.IMPORTANCEMany motile bacterial populations form surface-attached biofilms in response to specific environmental cues, including osmotic stress in a range of natural and host-related systems. However, cross talk between bacterial osmosensing, swimming, and biofilm formation regulatory networks is not fully understood. Here, we report that the pleiotropic regulator LrhA inPantoea alhagiis involved in the regulation of flagellar motility, biofilm formation, and host colonization and responds to osmotic upshift. We further show that this sensing relies on the EnvZ-OmpR two-component system that was known to detect changes in external osmotic stress. The EnvZ-OmpR-LrhA osmosensing signal transduction cascade is proposed to increase bacterial fitness under hyperosmotic conditions inside the host. Our work proposes a novel regulatory mechanism that links osmosensing and motile-sessile lifestyle transitions, which may provide new approaches to prevent or promote the formation of biofilms and host colonization inP. alhagiand other bacteria possessing a similar osmoregulatory mechanism.

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