Water-soluble cranberry extract inhibits Vibrio cholerae biofilm formation possibly through modulating the second messenger 3’, 5’ - Cyclic diguanylate level

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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Cyclic di-GMP Regulates TfoY inVibrio choleraeTo Control Motility by both Transcriptional and Posttranscriptional Mechanisms

Journal of Bacteriology ◽

10.1128/jb.00578-17 ◽

2018 ◽

Vol 200 (7) ◽

Cited By ~ 18

Author(s):

Benjamin R. Pursley ◽

Michael M. Maiden ◽

Meng-Lun Hsieh ◽

Nicolas L. Fernandez ◽

Geoffrey B. Severin ◽

...

Keyword(s):

Transcription Factor ◽

Vibrio Cholerae ◽

Biofilm Formation ◽

Noncoding Rna ◽

Molecular Mechanisms ◽

Second Messenger ◽

Bacterial Motility ◽

Content Type ◽

Intracellular Concentrations ◽

Messenger Molecule

ABSTRACT3′,5′-Cyclic diguanylic acid (c-di-GMP) is a bacterial second messenger molecule that is a key global regulator inVibrio cholerae, but the molecular mechanisms by which this molecule regulates downstream phenotypes have not been fully characterized. One such regulatory factor that may respond to c-di-GMP is the Vc2 c-di-GMP-binding riboswitch that is hypothesized to control the expression of the downstream putative transcription factor TfoY. Although much is known about the physical and structural properties of the Vc2 riboswitch aptamer, the nature of its expression and function inV. choleraehas not been investigated. Here, we show that Vc2 functions as an off switch to inhibit TfoY production at intermediate and high concentrations of c-di-GMP. At low c-di-GMP concentrations, TfoY production is induced to stimulate dispersive motility. We also observed increased transcription oftfoYat high intracellular concentrations of c-di-GMP, but this induction is independent of the Vc2 riboswitch and occurs via transcriptional control of promoters upstream oftfoYby the previously identified c-di-GMP dependent transcription factor VpsR. Our results show that TfoY is induced by c-di-GMP at both low and high intracellular concentrations of c-di-GMP via posttranscriptional and transcriptional mechanisms, respectively. This regulation contributes to the formation of three distinct c-di-GMP signaling states inV. cholerae.IMPORTANCEThe bacterial pathogenVibrio choleraemust transition between life in aquatic environmental reservoirs and life in the gastrointestinal tract. Biofilm formation and bacterial motility, and their control by the second messenger molecule c-di-GMP, play integral roles in this adaptation. Here, we define the third major mechanism by which c-di-GMP controls bacterial motility. This pathway utilizes a noncoding RNA element known as a riboswitch that, when bound to c-di-GMP, inhibits the expression of the transcription factor TfoY. TfoY production switchesV. choleraemotility from a dense to a dispersive state. Our results suggest that the c-di-GMP signaling network ofV. choleraecan exist in at least three distinct states to regulate biofilm formation and motility.

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A Pterin-Dependent Signaling Pathway Regulates a Dual-Function Diguanylate Cyclase-Phosphodiesterase Controlling Surface Attachment in Agrobacterium tumefaciens

mBio ◽

10.1128/mbio.00156-15 ◽

2015 ◽

Vol 6 (4) ◽

Cited By ~ 28

Author(s):

Nathan Feirer ◽

Jing Xu ◽

Kylie D. Allen ◽

Benjamin J. Koestler ◽

Eric L. Bruger ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Signaling Pathway ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Dominant Role ◽

Second Messenger ◽

Dual Function ◽

Diguanylate Cyclase ◽

Cyclic Diguanylate ◽

Content Type

ABSTRACTThe motile-to-sessile transition is an important lifestyle switch in diverse bacteria and is often regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). In general, high c-di-GMP concentrations promote attachment to surfaces, whereas cells with low levels of signal remain motile. In the plant pathogenAgrobacterium tumefaciens, c-di-GMP controls attachment and biofilm formation via regulation of a unipolar polysaccharide (UPP) adhesin. The levels of c-di-GMP inA. tumefaciensare controlled in part by the dual-function diguanylate cyclase-phosphodiesterase (DGC-PDE) protein DcpA. In this study, we report that DcpA possesses both c-di-GMP synthesizing and degrading activities in heterologous and native genetic backgrounds, a binary capability that is unusual among GGDEF-EAL domain-containing proteins. DcpA activity is modulated by a pteridine reductase called PruA, with DcpA acting as a PDE in the presence of PruA and a DGC in its absence. PruA enzymatic activity is required for the control of DcpA and through this control, attachment and biofilm formation. Intracellular pterin analysis demonstrates that PruA is responsible for the production of a novel pterin species. In addition, the control of DcpA activity also requires PruR, a protein encoded directly upstream of DcpA with a predicted molybdopterin-binding domain. PruR is hypothesized to be a potential signaling intermediate between PruA and DcpA through an as-yet-unidentified mechanism. This study provides the first prokaryotic example of a pterin-mediated signaling pathway and a new model for the regulation of dual-function DGC-PDE proteins.IMPORTANCEPathogenic bacteria often attach to surfaces and form multicellular communities called biofilms. Biofilms are inherently resilient and can be difficult to treat, resisting common antimicrobials. Understanding how bacterial cells transition to the biofilm lifestyle is essential in developing new therapeutic strategies. We have characterized a novel signaling pathway that plays a dominant role in the regulation of biofilm formation in the model pathogenAgrobacterium tumefaciens. This control pathway involves small metabolites called pterins, well studied in eukaryotes, but this is the first example of pterin-dependent signaling in bacteria. The described pathway controls levels of an important intracellular second messenger (cyclic diguanylate monophosphate) that regulates key bacterial processes such as biofilm formation, motility, and virulence. Pterins control the balance of activity for an enzyme that both synthesizes and degrades the second messenger. These findings reveal a complex, multistep pathway that modulates this enzyme, possibly identifying new targets for antibacterial intervention.

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Cyclic Diguanylate Regulates Vibrio cholerae Virulence Gene Expression

Infection and Immunity ◽

10.1128/iai.73.9.5873-5882.2005 ◽

2005 ◽

Vol 73 (9) ◽

pp. 5873-5882 ◽

Cited By ~ 201

Author(s):

Anna D. Tischler ◽

Andrew Camilli

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

Bacterial Species ◽

Single Amino Acid ◽

Transcriptional Level ◽

Phosphodiesterase Activity ◽

High Concentration ◽

Cyclic Diguanylate ◽

Virulence Gene Expression ◽

Infant Mouse

ABSTRACT The cyclic dinucleotide second messenger cyclic diguanylate (c-diGMP) has been implicated in regulation of cell surface properties in several bacterial species, including Vibrio cholerae. Expression of genes required for V. cholerae biofilm formation is activated by an increased intracellular c-diGMP concentration. The response regulator VieA, which contains a domain responsible for degradation of c-diGMP, is required to maintain a low concentration of c-diGMP and repress biofilm formation. The VieSAB three-component signal transduction system was, however, originally identified as a regulator of ctxAB, the genes encoding cholera toxin (CT). Here we show that the c-diGMP phosphodiesterase activity of VieA is required to enhance CT production. This regulation occurred at the transcriptional level, and ectopically altering the c-diGMP concentration by expression of diguanylate cyclase or phosphodiesterase enzymes also affected ctxAB transcription. The c-diGMP phosphodiesterase activity of VieA was also required for maximal transcription toxT but did not influence the activity of ToxR or expression of TcpP. Finally, a single amino acid substitution in VieA that increases the intracellular c-diGMP concentration led to attenuation in the infant mouse model of cholera. Since virulence genes including toxT and ctxA are repressed by a high concentration of c-diGMP, while biofilm genes are activated, we suggest that c-diGMP signaling is important for the transition of V. cholerae from the environment to the host.

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Faculty Opinions recommendation of Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm formation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1005403.240509 ◽

2004 ◽

Author(s):

Drusilla L Burns

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

Cyclic Diguanylate

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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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Functional Specialization in Vibrio cholerae Diguanylate Cyclases: Distinct Modes of Motility Suppression and c-di-GMP Production

mBio ◽

10.1128/mbio.00670-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 15

Author(s):

David Zamorano-Sánchez ◽

Wujing Xian ◽

Calvin K. Lee ◽

Mauro Salinas ◽

Wiriya Thongsomboon ◽

...

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

Cell Tracking ◽

Regulation Mechanism ◽

Cyclic Diguanylate ◽

Content Type ◽

Bacterial Signaling ◽

Diguanylate Cyclases ◽

Motility Regulation ◽

Gmp Production

ABSTRACT Vibrio cholerae biofilm formation and associated motility suppression are correlated with increased concentrations of cyclic diguanylate monophosphate (c-di-GMP), which are in turn driven by increased levels and/or activity of diguanylate cyclases (DGCs). To further our understanding of how c-di-GMP modulators in V. cholerae individually and collectively influence motility with cellular resolution, we determined how DGCs CdgD and CdgH impact intracellular c-di-GMP levels, motility, and biofilm formation. Our results indicated that CdgH strongly influences swim speed distributions; cells in which cdgH was deleted had higher average swim speeds than wild-type cells. Furthermore, our results suggest that CdgD, rather than CdgH, is the dominant DGC responsible for postattachment c-di-GMP production in biofilms. Lipopolysaccharide (LPS) biosynthesis genes were found to be extragenic bypass suppressors of the motility phenotypes of strains ΔcdgD and ΔcdgH. We compared the motility regulation mechanism of the DGCs with that of Gmd, an LPS O-antigen biosynthesis protein, and discovered that comodulation of c-di-GMP levels by these motility effectors can be positively or negatively cooperative rather than simply additive. Taken together, these results suggest that different environmental and metabolic inputs orchestrate DGC responses of V. cholerae via c-di-GMP production and motility modulation. IMPORTANCE Cyclic diguanylate monophosphate (c-di-GMP) is a broadly conserved bacterial signaling molecule that affects motility, biofilm formation, and virulence. Although it has been known that high intracellular concentrations of c-di-GMP correlate with motility suppression and biofilm formation, how the 53 predicted c-di-GMP modulators in Vibrio cholerae collectively influence motility is not understood in detail. Here we used a combination of plate assays and single-cell tracking methods to correlate motility and biofilm formation outcomes with specific enzymes involved in c-di-GMP synthesis in Vibrio cholerae, the causative agent of the disease cholera.

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Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm formation

Molecular Microbiology ◽

10.1111/j.1365-2958.2004.04155.x ◽

2004 ◽

Vol 53 (3) ◽

pp. 857-869 ◽

Cited By ~ 358

Author(s):

Anna D. Tischler ◽

Andrew Camilli

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

Cyclic Diguanylate

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The bioactive lipid (S)-sebastenoic acid impacts motility and dispersion in Vibrio cholerae

Canadian Journal of Chemistry ◽

10.1139/cjc-2017-0334 ◽

2018 ◽

Vol 96 (2) ◽

pp. 196-203

Author(s):

Christopher J.A. Warner ◽

Mauro Salinas ◽

David Zamorano-Sánchez ◽

Walter M. Bray ◽

R. Scott Lokey ◽

...

Keyword(s):

Vibrio Cholerae ◽

High Throughput ◽

Biofilm Formation ◽

Healthcare Systems ◽

Bacterial Motility ◽

Cyclic Diguanylate ◽

Gram Negative ◽

Chemical Genetic ◽

Genetic Probes ◽

Screening Platform

Although Gram-negative bacterial pathogens continue to impart a substantial burden on global healthcare systems, much remains to be understood about aspects of basic physiology in these organisms. In recent years, cyclic-diguanylate (c-di-GMP) has emerged as a key regulator of a number of important processes related to pathogenicity, including biofilm formation, motility, and virulence. In an effort to discover chemical genetic probes for studying Vibrio cholerae we have developed a new motility-based high-throughput screen to identify compounds that modulate c-di-GMP levels. Using this new screening platform, we tested a library of microbially derived marine natural products extracts, leading to the discovery of the bioactive lipid (S)-sebastenoic acid. The evaluation of the effect of this new compound on bacterial motility, vpsL expression, and biofilm formation implied that (S)-sebastenoic acid may alter phenotypes associated to c-di-GMP signaling in V. cholerae.

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