Response of Vibrio cholerae to Low-Temperature Shifts: CspV Regulation of Type VI Secretion, Biofilm Formation, and Association with Zooplankton

ABSTRACTThe ability to sense and adapt to temperature fluctuation is critical to the aquatic survival, transmission, and infectivity ofVibrio cholerae, the causative agent of the disease cholera. Little information is available on the physiological changes that occur whenV. choleraeexperiences temperature shifts. The genome-wide transcriptional profile ofV. choleraeupon a shift in human body temperature (37°C) to lower temperatures, 15°C and 25°C, which mimic those found in the aquatic environment, was determined. Differentially expressed genes included those involved in the cold shock response, biofilm formation, type VI secretion, and virulence. Analysis of a mutant lacking the cold shock genecspV, which was upregulated >50-fold upon a low-temperature shift, revealed that it regulates genes involved in biofilm formation and type VI secretion. CspV controls biofilm formation through modulation of the second messenger cyclic diguanylate and regulates type VI-mediated interspecies killing in a temperature-dependent manner. Furthermore, a strain lackingcspVhad significant defects for attachment and type VI-mediated killing on the surface of the aquatic crustaceanDaphnia magna. Collectively, these studies reveal thatcspVis a major regulator of the temperature downshift response and plays an important role in controlling cellular processes crucial to the infectious cycle ofV. cholerae.IMPORTANCELittle is known about how human pathogens respond and adapt to ever-changing parameters of natural habitats outside the human host and how environmental adaptation alters dissemination.Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, experiences fluctuations in temperature in its natural aquatic habitats and during the infection process. Furthermore, temperature is a critical environmental signal governing the occurrence ofV. choleraeand cholera outbreaks. In this study, we showed thatV. choleraereprograms its transcriptome in response to fluctuations in temperature, which results in changes to biofilm formation and type VI secretion system activation. These processes in turn impact environmental survival and the virulence potential of this pathogen.

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The LonA Protease Regulates Biofilm Formation, Motility, Virulence, and the Type VI Secretion System in Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.00741-15 ◽

2016 ◽

Vol 198 (6) ◽

pp. 973-985 ◽

Cited By ~ 21

Author(s):

Andrew Rogers ◽

Loni Townsley ◽

Ana L. Gallego-Hernandez ◽

Sinem Beyhan ◽

Laura Kwuan ◽

...

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

Secretion System ◽

Lon Protease ◽

Human Pathogen ◽

Type Vi Secretion System ◽

Cyclic Diguanylate ◽

Content Type ◽

Infant Mouse ◽

Type Vi Secretion

ABSTRACTThe presence of the Lon protease in all three domains of life hints at its biological importance. The prokaryotic Lon protease is responsible not only for degrading abnormal proteins but also for carrying out the proteolytic regulation of specific protein targets. Posttranslational regulation by Lon is known to affect a variety of physiological traits in many bacteria, including biofilm formation, motility, and virulence. Here, we identify the regulatory roles of LonA in the human pathogenVibrio cholerae. We determined that the absence of LonA adversely affects biofilm formation, increases swimming motility, and influences intracellular levels of cyclic diguanylate. Whole-genome expression analysis revealed that the message abundance of genes involved in biofilm formation was decreased but that the message abundances of those involved in virulence and the type VI secretion system were increased in alonAmutant compared to the wild type. We further demonstrated that alonAmutant displays an increase in type VI secretion system activity and is markedly defective in colonization of the infant mouse. These findings suggest that LonA plays a critical role in the environmental survival and virulence ofV. cholerae.IMPORTANCEBacteria utilize intracellular proteases to degrade damaged proteins and adapt to changing environments. The Lon protease has been shown to be important for environmental adaptation and plays a crucial role in regulating the motility, biofilm formation, and virulence of numerous plant and animal pathogens. We find that LonA of the human pathogenV. choleraeis in line with this trend, as the deletion of LonA leads to hypermotility and defects in both biofilm formation and colonization of the infant mouse. In addition, we show that LonA regulates levels of cyclic diguanylate and the type VI secretion system. Our observations add to the known regulatory repertoire of the Lon protease and the current understanding ofV. choleraephysiology.

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A Conserved Regulatory Circuit Controls Large Adhesins in Vibrio cholerae

mBio ◽

10.1128/mbio.02822-19 ◽

2019 ◽

Vol 10 (6) ◽

Cited By ~ 6

Author(s):

Giordan Kitts ◽

Krista M. Giglio ◽

David Zamorano-Sánchez ◽

Jin Hwan Park ◽

Loni Townsley ◽

...

Keyword(s):

Cell Surface ◽

Vibrio Cholerae ◽

Biofilm Formation ◽

Diarrheal Disease ◽

Cell Attachment ◽

Surface Display ◽

Second Messenger ◽

Bacterial Biofilm ◽

Content Type ◽

Regulatory Circuit

ABSTRACT The dinucleotide second messenger c-di-GMP has emerged as a central regulator of reversible cell attachment during bacterial biofilm formation. A prominent cell adhesion mechanism first identified in pseudomonads combines two c-di-GMP-mediated processes: transcription of a large adhesin and its cell surface display via posttranslational proteolytic control. Here, we characterize an orthologous c-di-GMP effector system and show that it is operational in Vibrio cholerae, where it regulates two distinct classes of adhesins. Through structural analyses, we reveal a conserved autoinhibition mechanism of the c-di-GMP receptor that controls adhesin proteolysis and present a structure of a c-di-GMP-bound receptor module. We further establish functionality of the periplasmic protease controlled by the receptor against the two adhesins. Finally, transcription and functional assays identify physiological roles of both c-di-GMP-regulated adhesins in surface attachment and biofilm formation. Together, our studies highlight the conservation of a highly efficient signaling effector circuit for the control of cell surface adhesin expression and its versatility by revealing strain-specific variations. IMPORTANCE Vibrio cholerae, the causative agent of the diarrheal disease cholera, benefits from a sessile biofilm lifestyle that enhances survival outside the host but also contributes to host colonization and infectivity. The bacterial second messenger c-di-GMP has been identified as a central regulator of biofilm formation, including in V. cholerae; however, our understanding of the pathways that contribute to this process is incomplete. Here, we define a conserved signaling system that controls the stability of large adhesion proteins at the cell surface of V. cholerae, which are important for cell attachment and biofilm formation. Insight into the regulatory circuit underlying biofilm formation may inform targeted strategies to interfere with a process that renders this bacterium remarkably adaptable to changing environments.

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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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Mannitol and the Mannitol-Specific Enzyme IIB Subunit Activate Vibrio cholerae Biofilm Formation

Applied and Environmental Microbiology ◽

10.1128/aem.01184-13 ◽

2013 ◽

Vol 79 (15) ◽

pp. 4675-4683 ◽

Cited By ~ 29

Author(s):

Patrick Ymele-Leki ◽

Laetitia Houot ◽

Paula I. Watnick

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

Diarrheal Disease ◽

Ectopic Expression ◽

Phosphotransferase System ◽

Content Type ◽

Regulatory Pathways ◽

B Subunit ◽

Multicomponent Signal ◽

Enzyme I

ABSTRACTVibrio choleraeis a halophilic, Gram-negative rod found in marine environments. Strains that produce cholera toxin cause the diarrheal disease cholera.V. choleraeuse a highly conserved, multicomponent signal transduction cascade known as the phosphoenolpyruvate phosphotransferase system (PTS) to regulate carbohydrate uptake and biofilm formation. Regulation of biofilm formation by the PTS is complex, involving many different regulatory pathways that incorporate distinct PTS components. The PTS consists of the general components enzyme I (EI) and histidine protein (HPr) and carbohydrate-specific enzymes II. Mannitol transport byV. choleraerequires the mannitol-specific EII (EIIMtl), which is expressed only in the presence of mannitol. Here we show that mannitol activatesV. choleraebiofilm formation and transcription of thevpsbiofilm matrix exopolysaccharide synthesis genes. This regulation is dependent on mannitol transport. However, we show that, in the absence of mannitol, ectopic expression of the B subunit of EIIMtlis sufficient to activate biofilm accumulation. Mannitol, a common compatible solute and osmoprotectant of marine organisms, is a main photosynthetic product of many algae and is secreted by algal mats. We propose that the ability ofV. choleraeto respond to environmental mannitol by forming a biofilm may play an important role in habitat selection.

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Cyclic di-GMP Increases Catalase Production and Hydrogen Peroxide Tolerance in Vibrio cholerae

Applied and Environmental Microbiology ◽

10.1128/aem.01043-19 ◽

2019 ◽

Vol 85 (18) ◽

Cited By ~ 3

Author(s):

Nicolas L. Fernandez ◽

Christopher M. Waters

Keyword(s):

Vibrio Cholerae ◽

Catalase Activity ◽

Biofilm Formation ◽

Salt Water ◽

Bacterial Pathogen ◽

Second Messenger ◽

Signaling Network ◽

Dependent Manner ◽

Environmental Persistence ◽

Content Type

ABSTRACT Vibrio cholerae is a Gram-negative bacterial pathogen that causes the disease cholera, which affects nearly 1 million people each year. In between outbreaks, V. cholerae resides in fresh and salt water environments, where it is able to persist through changes in temperature, oxygen, and salinity. One key characteristic that promotes environmental persistence of V. cholerae is the ability to form multicellular communities, called biofilms, that often adhere to biotic and abiotic sources. Biofilm formation in V. cholerae is positively regulated by the dinucleotide second messenger cyclic dimeric GMP (c-di-GMP). While most research on the c-di-GMP regulon has focused on biofilm formation or motility, we hypothesized that the c-di-GMP signaling network encompassed a larger set of effector functions than reported. We found that high intracellular c-di-GMP increased catalase activity ∼4-fold relative to strains with unaltered c-di-GMP. Genetic studies demonstrated that c-di-GMP mediated catalase activity was due to increased expression of the catalase-encoding gene katB. Moreover, c-di-GMP mediated regulation of catalase activity and katB expression required the c-di-GMP dependent transcription factors VpsT and VpsR. Lastly, we found that high c-di-GMP increased survival after H2O2 challenge in a katB-, vpsR-, and vpsT-dependent manner. Our results indicate that antioxidant production is regulated by c-di-GMP uncovering a new node in the growing VpsT and VpsR c-di-GMP signaling network of V. cholerae. IMPORTANCE As a result of infection with V. cholerae, patients become dehydrated, leading to death if not properly treated. The aquatic environment is the natural reservoir for V. cholerae, where it can survive alterations in temperature, salinity, and oxygen. The second messenger molecule c-di-GMP is an important signal regulating host and aquatic environmental persistence because it controls whether V. cholerae will form a biofilm or disperse through flagellar motility. In this work, we demonstrate another function of c-di-GMP in V. cholerae biology: promoting tolerance to the reactive oxygen species H2O2 through the differential regulation of catalase expression. Our results suggest a mechanism where c-di-GMP simultaneously controls biofilm formation and antioxidant production, which could promote persistence in human and marine environments.

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A Metalloprotease Secreted by the Type II Secretion System Links Vibrio cholerae with Collagen

Journal of Bacteriology ◽

10.1128/jb.02329-14 ◽

2015 ◽

Vol 197 (6) ◽

pp. 1051-1064 ◽

Cited By ~ 17

Author(s):

Bo R. Park ◽

Ryszard A. Zielke ◽

Igor H. Wierzbicki ◽

Kristie C. Mitchell ◽

Jeffrey H. Withey ◽

...

Keyword(s):

Vibrio Cholerae ◽

Type I Collagen ◽

Diarrheal Disease ◽

Type Ii Secretion ◽

Type I ◽

Type Ii ◽

Marine Animals ◽

Natural Habitats ◽

Content Type ◽

Functional Components

Vibrio choleraeis autochthonous to various aquatic niches and is the etiological agent of the life-threatening diarrheal disease cholera. The persistence ofV. choleraein natural habitats is a crucial factor in the epidemiology of cholera. In contrast to the well-studiedV. cholerae-chitin connection, scarce information is available about the factors employed by the bacteria for the interaction with collagens. Collagens might serve as biologically relevant substrates, because they are the most abundant protein constituents of metazoan tissues andV. choleraehas been identified in association with invertebrate and vertebrate marine animals, as well as in a benthic zone of the ocean where organic matter, including collagens, accumulates. Here, we describe the characterization of theV. choleraeputative collagenase, VchC, encoded by open reading frame VC1650 and belonging to the subfamily M9A peptidases. Our studies demonstrate that VchC is an extracellular collagenase degrading native type I collagen of fish and mammalian origin. Alteration of the predicted catalytic residues coordinating zinc ions completely abolished the protein enzymatic activity but did not affect the translocation of the protease by the type II secretion pathway into the extracellular milieu. We also show that the protease undergoes a maturation process with the aid of a secreted factor(s). Finally, we propose thatV. choleraeis a collagenovorous bacterium, as it is able to utilize collagen as a sole nutrient source. This study initiates new lines of investigations aiming to uncover the structural and functional components of theV. choleraecollagen utilization program.

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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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Identification and Characterization of OscR, a Transcriptional Regulator Involved in Osmolarity Adaptation in Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.01540-08 ◽

2009 ◽

Vol 191 (13) ◽

pp. 4082-4096 ◽

Cited By ~ 44

Author(s):

Nicholas J. Shikuma ◽

Fitnat H. Yildiz

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

Transcriptional Regulator ◽

Dependent Manner ◽

Genome Wide ◽

Low Salt ◽

Adaptation Response ◽

Matrix Production ◽

Identification And Characterization

ABSTRACT Vibrio cholerae is a facultative human pathogen. In its aquatic habitat and as it passes through the digestive tract, V. cholerae must cope with fluctuations in salinity. We analyzed the genome-wide transcriptional profile of V. cholerae grown at different NaCl concentrations and determined that the expression of compatible solute biosynthesis and transporter genes, virulence genes, and genes involved in adhesion and biofilm formation is differentially regulated. We determined that salinity modulates biofilm formation, and this response was mediated through the transcriptional regulators VpsR and VpsT. Additionally, a transcriptional regulator controlling an osmolarity adaptation response was identified. This regulator, OscR (osmolarity controlled regulator), was found to modulate the transcription of genes involved in biofilm matrix production and motility in a salinity-dependent manner. oscR mutants were less motile and exhibited enhanced biofilm formation only under low-salt conditions.

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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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Indole Inhibits ToxR Regulon Expression inVibrio cholerae

Infection and Immunity ◽

10.1128/iai.00776-18 ◽

2019 ◽

Vol 87 (3) ◽

Cited By ~ 7

Author(s):

Mondraya F. Howard ◽

X. Renee Bina ◽

James E. Bina

Keyword(s):

Virulence Factor ◽

Virulence Gene ◽

Dependent Manner ◽

Content Type ◽

Virulence Gene Expression ◽

Type Vi Secretion ◽

Biofilm Production ◽

Factor Production ◽

Periplasmic Domain ◽

Virulence Factor Production

ABSTRACTIndole is a degradation product of tryptophan that functions as a signaling molecule in many bacteria. This includesVibrio cholerae, where indole was shown to regulate biofilm and type VI secretion in nontoxigenic environmental isolates. Indole is also produced by toxigenicV. choleraestrains in the human intestine, but its significance in the host is unknown. We investigated the effects of indole on toxigenicV. choleraeO1 El Tor during growth under virulence inducing conditions. The indole transcriptome was defined by RNA sequencing and showed widespread changes in the expression of genes involved in metabolism, biofilm production, and virulence factor production. In contrast, genes involved in type VI secretion were not affected by indole. We subsequently found that indole repressed genes involved inV. choleraepathogenesis, including the ToxR virulence regulon. Consistent with this, indole inhibited cholera toxin and toxin-coregulated pilus production in a dose-dependent manner. The effects of indole on virulence factor production and biofilm were linked to ToxR and the ToxR-dependent regulator LeuO. The expression ofleuOwas increased by exogenous indole and linked to repression of the ToxR virulence regulon. This process was dependent on the ToxR periplasmic domain, suggesting that indole was a ToxR agonist. This conclusion was further supported by results showing that the ToxR periplasmic domain contributed to indole-mediated increased biofilm production. Collectively, our results suggest that indole may be a niche-specific cue that can function as a ToxR agonist to modulate virulence gene expression and biofilm production inV. cholerae.

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