Identification and Characterization of VpsR and VpsT Binding Sites in Vibrio cholerae

ABSTRACTThe ability to form biofilms is critical for environmental survival and transmission ofVibrio cholerae, a facultative human pathogen responsible for the disease cholera. Biofilm formation is controlled by several transcriptional regulators and alternative sigma factors. In this study, we report that the two main positive regulators of biofilm formation, VpsR and VpsT, bind to nonoverlapping target sequences in the regulatory region ofvpsL in vitro. VpsR binds to a proximal site (the R1 box) as well as a distal site (the R2 box) with respect to the transcriptional start site identified upstream ofvpsL. The VpsT binding site (the T box) is located between the R1 and R2 boxes. While mutations in the T and R boxes resulted in a decrease invpsLexpression, deletion of the T and R2 boxes resulted in an increase invpsLexpression. Analysis of the role of H-NS invpsLexpression revealed that deletion ofhnsresulted in enhancedvpsLexpression. The level ofvpsLexpression was higher in anhns vpsTdouble mutant than in the parental strain but lower than that in anhnsmutant.In silicoanalysis of the regulatory regions of the VpsR and VpsT targets resulted in the identification of conserved recognition motifs for VpsR and VpsT and revealed that operons involved in biofilm formation andvpsTare coregulated by VpsR and VpsT. Furthermore, a comparative genomics analysis revealed substantial variability in the promoter region of thevpsTandvpsLgenes among extantV. choleraeisolates, suggesting that regulation of biofilm formation is under active selection.IMPORTANCEVibrio choleraecauses cholera and is a natural inhabitant of aquatic environments. One critical factor that is important for environmental survival and transmission ofV. choleraeis the microbe's ability to form biofilms, which are surface-associated communities encased in a matrix composed of the exopolysaccharide VPS (Vibriopolysaccharide), proteins, and nucleic acids. Two proteins, VpsR and VpsT, positively regulate VPS production and biofilm formation. We characterized the structural features of the promoter of thevpsLgene, determined the target sequences recognized by VpsT and VpsR, and analyzed their distribution and conservation patterns in multipleV. choleraeisolates. This work fills a fundamental gap in our understanding of the regulatory mechanisms employed by the master regulators VpsR and VpsT in controlling biofilm matrix production.

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Polymyxin B Resistance and Biofilm Formation in Vibrio cholerae Are Controlled by the Response Regulator CarR

Infection and Immunity ◽

10.1128/iai.02700-14 ◽

2015 ◽

Vol 83 (3) ◽

pp. 1199-1209 ◽

Cited By ~ 38

Author(s):

Kivanc Bilecen ◽

Jiunn C. N. Fong ◽

Andrew Cheng ◽

Christopher J. Jones ◽

David Zamorano-Sánchez ◽

...

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

Lipid A ◽

Response Regulator ◽

Regulatory Region ◽

Regulatory System ◽

Polymyxin B ◽

Content Type ◽

Two Component ◽

Antimicrobial Peptide Resistance

Two-component systems play important roles in the physiology of many bacterial pathogens.Vibrio cholerae's CarRS two-component regulatory system negatively regulates expression ofvps(Vibriopolysaccharide) genes and biofilm formation. In this study, we report that CarR confers polymyxin B resistance by positively regulating expression of thealmEFGgenes, whose products are required for glycine and diglycine modification of lipid A. We determined that CarR directly binds to the regulatory region of thealmEFGoperon. Similarly to acarRmutant, strains lackingalmE,almF, andalmGexhibited enhanced polymyxin B sensitivity. We also observed that strains lackingalmEor thealmEFGoperon have enhanced biofilm formation. Our results reveal that CarR regulates biofilm formation and antimicrobial peptide resistance inV. cholerae.

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Cyclic di-GMP Positively Regulates DNA Repair in Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.00005-18 ◽

2018 ◽

Vol 200 (15) ◽

Cited By ~ 8

Author(s):

Nicolas L. Fernandez ◽

Disha Srivastava ◽

Amanda L. Ngouajio ◽

Christopher M. Waters

Keyword(s):

Dna Repair ◽

Vibrio Cholerae ◽

Biofilm Formation ◽

Binding Assays ◽

Gene Encoding ◽

Repair Gene ◽

Content Type ◽

Dna Repair Gene ◽

High Concentrations

ABSTRACT In Vibrio cholerae, high intracellular cyclic di-GMP (c-di-GMP) concentration are associated with a biofilm lifestyle, while low intracellular c-di-GMP concentrations are associated with a motile lifestyle. c-di-GMP also regulates other behaviors, such as acetoin production and type II secretion; however, the extent of phenotypes regulated by c-di-GMP is not fully understood. We recently determined that the sequence upstream of the DNA repair gene encoding 3-methyladenine glycosylase (tag) was positively induced by c-di-GMP, suggesting that this signaling system might impact DNA repair pathways. We identified a DNA region upstream of tag that is required for transcriptional induction by c-di-GMP. We further showed that c-di-GMP induction of tag expression was dependent on the c-di-GMP-dependent biofilm regulators VpsT and VpsR. In vitro binding assays and heterologous host expression studies show that VpsT acts directly at the tag promoter in response to c-di-GMP to induce tag expression. Last, we determined that strains with high c-di-GMP concentrations are more tolerant of the DNA-damaging agent methyl methanesulfonate. Our results indicate that the regulatory network of c-di-GMP in V. cholerae extends beyond biofilm formation and motility to regulate DNA repair through the VpsR/VpsT c-di-GMP-dependent cascade. IMPORTANCE Vibrio cholerae is a prominent human pathogen that is currently causing a pandemic outbreak in Haiti, Yemen, and Ethiopia. The second messenger molecule cyclic di-GMP (c-di-GMP) mediates the transitions in V. cholerae between a sessile biofilm-forming state and a motile lifestyle, both of which are important during V. cholerae environmental persistence and human infections. Here, we report that in V. cholerae c-di-GMP also controls DNA repair. We elucidate the regulatory pathway by which c-di-GMP increases DNA repair, allowing this bacterium to tolerate high concentrations of mutagens at high intracellular levels of c-di-GMP. Our work suggests that DNA repair and biofilm formation may be linked in V. cholerae.

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VpsR Directly Activates Transcription of Multiple Biofilm Genes in Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.00234-20 ◽

2020 ◽

Vol 202 (18) ◽

Cited By ~ 2

Author(s):

Meng-Lun Hsieh ◽

Christopher M. Waters ◽

Deborah M. Hinton

Keyword(s):

Rna Polymerase ◽

Vibrio Cholerae ◽

Biofilm Formation ◽

Core Promoter ◽

Class Ii ◽

Class I ◽

Dependent Manner ◽

Mobility Shift ◽

Content Type

ABSTRACT Vibrio cholerae biofilm biogenesis, which is important for survival, dissemination, and persistence, requires multiple genes in the Vibrio polysaccharides (vps) operons I and II as well as the cluster of ribomatrix (rbm) genes. Transcriptional control of these genes is a complex process that requires several activators/repressors and the ubiquitous signaling molecule, cyclic di-GMP (c-di-GMP). Previously, we demonstrated that VpsR directly activates RNA polymerase containing σ70 (σ70-RNAP) at the vpsL promoter (PvpsL), which precedes the vps-II operon, in a c-di-GMP-dependent manner by stimulating formation of the transcriptionally active, open complex. Using in vitro transcription, electrophoretic mobility shift assays, and DNase I footprinting, we show here that VpsR also directly activates σ70-RNAP transcription from other promoters within the biofilm formation cluster, including PvpsU, at the beginning of the vps-I operon, PrbmA, at the start of the rbm cluster, and PrbmF, which lies upstream of the divergent rbmF and rbmE genes. In this capacity, we find that VpsR is able to behave both as a class II activator, which functions immediately adjacent/overlapping the core promoter sequence (PvpsL and PvpsU), and as a class I activator, which functions farther upstream (PrbmA and PrbmF). Because these promoters vary in VpsR-DNA binding affinity in the absence and presence of c-di-GMP, we speculate that VpsR’s mechanism of activation is dependent on both the concentration of VpsR and the level of c-di-GMP to increase transcription, resulting in finely tuned regulation. IMPORTANCE Vibrio cholerae, the bacterial pathogen that is responsible for the disease cholera, uses biofilms to aid in survival, dissemination, and persistence. VpsR, which directly senses the second messenger c-di-GMP, is a major regulator of this process. Together with c-di-GMP, VpsR directly activates transcription by RNA polymerase containing σ70 from the vpsL biofilm biogenesis promoter. Using biochemical methods, we demonstrate for the first time that VpsR/c-di-GMP directly activates σ70-RNA polymerase at the first genes of the vps and ribomatrix operons. In this regard, it functions as either a class I or class II activator. Our results broaden the mechanism of c-di-GMP-dependent transcription activation and the specific role of VpsR in biofilm formation.

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In SilicoDiscovery andIn VitroValidation of Catechol-Containing Sulfonohydrazide Compounds as Potent Inhibitors of the Diguanylate Cyclase PleD

Journal of Bacteriology ◽

10.1128/jb.00742-15 ◽

2015 ◽

Vol 198 (1) ◽

pp. 147-156 ◽

Cited By ~ 14

Author(s):

Silvia Fernicola ◽

Alessandro Paiardini ◽

Giorgio Giardina ◽

Giordano Rampioni ◽

Livia Leoni ◽

...

Keyword(s):

Biofilm Formation ◽

Pathogenic Bacteria ◽

Caulobacter Crescentus ◽

Pharmacophore Model ◽

Second Messenger ◽

Binding Mode ◽

Structural Features ◽

Bacterial Biofilm ◽

Content Type

ABSTRACTBiofilm formation is responsible for increased antibiotic tolerance in pathogenic bacteria. Cyclic di-GMP (c-di-GMP) is a widely used second-messenger signal that plays a key role in bacterial biofilm formation. c-di-GMP is synthesized by diguanylate cyclases (DGCs), a conserved class of enzymes absent in mammals and hence considered attractive molecular targets for the development of antibiofilm agents. Here, the results of a virtual screening approach aimed at identifying small-molecule inhibitors of the DGC PleD fromCaulobacter crescentusare described. A three-dimensional (3D) pharmacophore model, derived from the mode of binding of GTP to the active site of PleD, was exploited to screen the ZINC database of compounds. Seven virtual hits were testedin vitrofor their ability to inhibit the activity of purified PleD by using circular dichroism spectroscopy. Two drug-like molecules with a catechol moiety and a sulfonohydrazide scaffold were shown to competitively inhibit PleD at the low-micromolar range (50% inhibitory concentration [IC50] of ∼11 μM). Their predicted binding mode highlighted key structural features presumably responsible for the efficient inhibition of PleD by both hits. These molecules represent the most potentin vitroinhibitors of PleD identified so far and could therefore result in useful leads for the development of novel classes of antimicrobials able to hamper biofilm formation.IMPORTANCEBiofilm-mediated infections are difficult to eradicate, posing a threatening health issue worldwide. The capability of bacteria to form biofilms is almost universally stimulated by the second messenger c-di-GMP. This evidence has boosted research in the last decade for the development of new antibiofilm strategies interfering with c-di-GMP metabolism. Here, two potent inhibitors of c-di-GMP synthesis have been identifiedin silicoand characterizedin vitroby using the well-characterized DGC enzyme PleD fromC. crescentusas a structural template and molecular target. Given that the protein residues implied as crucial for enzyme inhibition are found to be highly conserved among DGCs, the outcome of this study could pave the way for the future development of broad-spectrum antibiofilm compounds.

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“Quorum Non-Sensing”: Social Cheating and Deception in Vibrio cholerae

Applied and Environmental Microbiology ◽

10.1128/aem.00586-15 ◽

2015 ◽

Vol 81 (11) ◽

pp. 3856-3862 ◽

Cited By ~ 32

Author(s):

David S. Katzianer ◽

Hui Wang ◽

Ryan M. Carey ◽

Jun Zhu

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

Natural Populations ◽

Extracellular Proteases ◽

Content Type ◽

High Frequencies ◽

Environmental Survival ◽

Natural Isolates ◽

External Stimuli ◽

Planktonic Communities

ABSTRACTQuorum sensing (QS) is widely used by bacteria to coordinate behavior in response to external stimuli. InVibrio cholerae, this process is important for environmental survival and pathogenesis, though, intriguingly, a large percentage of natural isolates are QS deficient. Here, we show that QS-deficient mutants can spread as social cheaters by ceasing production of extracellular proteases under conditions requiring their growth. We further show that mutants stimulate biofilm formation and are over-represented in biofilms compared to planktonic communities; on this basis, we suggest that QS-deficient mutants may have the side effect of enhancing environmental tolerance of natural populations due to the inherent resistance properties of biofilms. Interestingly, high frequencies of QS-deficient individuals did not impact production of QS signaling molecules despite mutants being unable to respond to these inducers, indicating that these variants actively cheat by false signaling under conditions requiring QS. Taken together, our results suggest that social cheating may drive QS deficiency emergence withinV. choleraenatural populations.

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Siderophore piracy enhances Vibrio cholerae environmental survival and pathogenesis

Microbiology ◽

10.1099/mic.0.000975 ◽

2020 ◽

Vol 166 (11) ◽

pp. 1038-1046 ◽

Cited By ~ 1

Author(s):

Hyuntae Byun ◽

I-Ji Jung ◽

Jiandong Chen ◽

Jessie Larios Valencia ◽

Jay Zhu

Keyword(s):

Vibrio Cholerae ◽

Type Species ◽

Iron Acquisition ◽

Content Type ◽

Iron Restriction ◽

Environmental Survival ◽

Link Type ◽

Adult Mice ◽

Polymicrobial Communities

Vibrio cholerae, the aetiological agent of cholera, possesses multiple iron acquisition systems, including those for the transport of siderophores. How these systems benefit V. cholerae in low-iron, polymicrobial communities in environmental settings or during infection remains poorly understood. Here, we demonstrate that in iron-limiting conditions, co-culture of V. cholerae with a number of individual siderophore-producing microbes significantly promoted V. cholerae growth in vitro. We further show that in the host environment with low iron, V. cholerae colonizes better in adult mice in the presence of the siderophore-producing commensal Escherichia coli . Taken together, our results suggest that in aquatic reservoirs or during infection, V. cholerae may overcome environmental and host iron restriction by hijacking siderophores from other microbes.

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In VitroAnalyses of the Effects of Heparin and Parabens on Candida albicans Biofilms and Planktonic Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05061-11 ◽

2011 ◽

Vol 56 (1) ◽

pp. 148-153 ◽

Cited By ~ 15

Author(s):

Marisa H. Miceli ◽

Stella M. Bernardo ◽

T. S. Neil Ku ◽

Carla Walraven ◽

Samuel A. Lee

Keyword(s):

Candida Albicans ◽

Metabolic Activity ◽

Biofilm Formation ◽

High Dose ◽

Dependent Manner ◽

Planktonic Cells ◽

Content Type ◽

Heparin Sodium ◽

The Individual

ABSTRACTInfections and thromboses are the most common complications associated with central venous catheters. Suggested strategies for prevention and management of these complications include the use of heparin-coated catheters, heparin locks, and antimicrobial lock therapy. However, the effects of heparin onCandida albicansbiofilms and planktonic cells have not been previously studied. Therefore, we sought to determine thein vitroeffect of a heparin sodium preparation (HP) on biofilms and planktonic cells ofC. albicans. Because HP contains two preservatives, methyl paraben (MP) and propyl paraben (PP), these compounds and heparin sodium without preservatives (Pure-H) were also tested individually. The metabolic activity of the mature biofilm after treatment was assessed using XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction and microscopy. Pure-H, MP, and PP caused up to 75, 85, and 60% reductions of metabolic activity of the mature preformedC. albicansbiofilms, respectively. Maximal efficacy against the mature biofilm was observed with HP (up to 90%) compared to the individual compounds (P< 0.0001). Pure-H, MP, and PP each inhibitedC. albicansbiofilm formation up to 90%. A complete inhibition of biofilm formation was observed with HP at 5,000 U/ml and higher. When tested against planktonic cells, each compound inhibited growth in a dose-dependent manner. These data indicated that HP, MP, PP, and Pure-H havein vitroantifungal activity againstC. albicansmature biofilms, formation of biofilms, and planktonic cells. Investigation of high-dose heparin-based strategies (e.g., heparin locks) in combination with traditional antifungal agents for the treatment and/or prevention ofC. albicansbiofilms is warranted.

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A Single B-Repeat of Staphylococcus epidermidis Accumulation-Associated Protein Induces Protective Immune Responses in an Experimental Biomaterial-Associated Infection Mouse Model

Clinical and Vaccine Immunology ◽

10.1128/cvi.00306-14 ◽

2014 ◽

Vol 21 (9) ◽

pp. 1206-1214 ◽

Cited By ~ 12

Author(s):

Lin Yan ◽

Lei Zhang ◽

Hongyan Ma ◽

David Chiu ◽

James D. Bryers

Keyword(s):

Staphylococcus Epidermidis ◽

Biofilm Formation ◽

Porous Scaffold ◽

The United States ◽

Protein Vaccine ◽

Weight Changes ◽

Biofilm Inhibition ◽

Content Type ◽

Accumulation Associated Protein

ABSTRACTNosocomial infections are the fourth leading cause of morbidity and mortality in the United States, resulting in 2 million infections and ∼100,000 deaths each year. More than 60% of these infections are associated with some type of biomedical device.Staphylococcus epidermidisis a commensal bacterium of the human skin and is the most common nosocomial pathogen infecting implanted medical devices, especially those in the cardiovasculature.S. epidermidisantibiotic resistance and biofilm formation on inert surfaces make these infections hard to treat. Accumulation-associated protein (Aap), a cell wall-anchored protein ofS. epidermidis, is considered one of the most important proteins involved in the formation ofS. epidermidisbiofilm. A small recombinant protein vaccine comprising a single B-repeat domain (Brpt1.0) ofS. epidermidisRP62A Aap was developed, and the vaccine's efficacy was evaluatedin vitrowith a biofilm inhibition assay andin vivoin a murine model of biomaterial-associated infection. A high IgG antibody response againstS. epidermidisRP62A was detected in the sera of the mice after two subcutaneous immunizations with Brpt1.0 coadministered with Freund's adjuvant. Sera from Brpt1.0-immunized mice inhibitedin vitroS. epidermidisRP62A biofilm formation in a dose-dependent pattern. After receiving two immunizations, each mouse was surgically implanted with a porous scaffold disk containing 5 × 106CFU ofS. epidermidisRP62A. Weight changes, inflammatory markers, and histological assay results after challenge withS. epidermidisindicated that the mice immunized with Brpt1.0 exhibited significantly higher resistance toS. epidermidisRP62A implant infection than the control mice. Day 8 postchallenge, there was a significantly lower number of bacteria in scaffold sections and surrounding tissues and a lower residual inflammatory response to the infected scaffold disks for the Brpt1.0-immunized mice than for of the ovalbumin (Ova)-immunized mice.

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Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis

Infection and Immunity ◽

10.1128/iai.05773-11 ◽

2011 ◽

Vol 80 (1) ◽

pp. 3-13 ◽

Cited By ~ 27

Author(s):

Chen Li ◽

Kurniyati ◽

Bo Hu ◽

Jiang Bian ◽

Jianlan Sun ◽

...

Keyword(s):

Porphyromonas Gingivalis ◽

Biofilm Formation ◽

Adult Population ◽

Transcriptional Analysis ◽

Wild Type ◽

Content Type ◽

Multiple Organs ◽

Biochemical Analyses

ABSTRACTThe oral bacteriumPorphyromonas gingivalisis a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide.P. gingivalisexhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence ofP. gingivalisremain elusive. In this report, we found thatP. gingivalisencodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed thatPG0352is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPgis an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that thePG0352deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type,in vitrostudies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement.In vivostudies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPgis an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity ofP. gingivalis, and it can potentially serve as a new target for developing therapeutic agents againstP. gingivalisinfection.

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Two regulatory factors of Vibrio cholerae activating the mannose-sensitive haemagglutinin pilus expression is important for biofilm formation and colonization in mice

Microbiology ◽

10.1099/mic.0.001098 ◽

2021 ◽

Vol 167 (10) ◽

Author(s):

Mengting Shi ◽

Yue Zheng ◽

Xianghong Wang ◽

Zhengjia Wang ◽

Menghua Yang

Keyword(s):

Mouse Model ◽

Vibrio Cholerae ◽

Biofilm Formation ◽

Type Species ◽

Wild Type ◽

Expression Library ◽

Content Type ◽

Genomic Expression ◽

Infant Mouse ◽

Link Type

Vibrio cholerae the causative agent of cholera, uses a large number of coordinated transcriptional regulatory events to transition from its environmental reservoir to the host intestine, which is its preferred colonization site. Transcription of the mannose-sensitive haemagglutinin pilus (MSHA), which aids the persistence of V. cholerae in aquatic environments, but causes its clearance by host immune defenses, was found to be regulated by a yet unknown mechanism during the infection cycle of V. cholerae . In this study, genomic expression library screening revealed that two regulators, VC1371 and VcRfaH, are able to positively activate the transcription of MSHA operon. VC1371 is localized and active in the cell membrane. Deletion of vc1371 or VcrfaH genes in V. cholerae resulted in less MshA protein production and less efficiency of biofilm formation compared to that in the wild-type strain. An adult mouse model showed that the mutants with vc1371 or VcrfaH deletion colonized less efficiently than the wild-type; the VcrfaH deletion mutant showed less colonization efficiency in the infant mouse model. The findings strongly suggested that the two regulators, namely VC1371 and VcRfaH, which are involved in the regulation of MSHA expression, play an important role in V. cholerae biofilm formation and colonization in mice.

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