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

UNLABELLED: The ability to sense and adapt to temperature fluctuation is critical to the aquatic survival, transmission, and infectivity of Vibrio cholerae, the causative agent of the disease cholera. Little information is available on the physiological changes that occur when V. cholerae experiences temperature shifts. The genome-wide transcriptional profile of V. cholerae upon 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 gene cspV, 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 lacking cspV had significant defects for attachment and type VI-mediated killing on the surface of the aquatic crustacean Daphnia magna Collectively, these studies reveal that cspV is a major regulator of the temperature downshift response and plays an important role in controlling cellular processes crucial to the infectious cycle of V. cholerae IMPORTANCE: Little 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 of V. cholerae and cholera outbreaks. In this study, we showed that V. cholerae reprograms 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

Cyclic di-AMP Acts as an Extracellular Signal That Impacts Bacillus subtilis Biofilm Formation and Plant Attachment

There is a growing appreciation for the impact that bacteria have on higher organisms. Plant roots often harbor beneficial microbes, such as the Gram-positive rhizobacterium Bacillus subtilis, that influence their growth and susceptibility to disease. The ability to form surface-attached microbial communities called biofilms is crucial for the ability of B. subtilis to adhere to and protect plant roots. In this study, strains harboring deletions of the B. subtilis genes known to synthesize and degrade the second messenger cyclic di-adenylate monophosphate (c-di-AMP) were examined for their involvement in biofilm formation and plant attachment. We found that intracellular production of c-di-AMP impacts colony biofilm architecture, biofilm gene expression, and plant attachment in B. subtilis. We also show that B. subtilis secretes c-di-AMP and that putative c-di-AMP transporters impact biofilm formation and plant root colonization. Taken together, our data describe a new role for c-di-AMP as a chemical signal that affects important cellular processes in the environmentally and agriculturally important soil bacterium B. subtilis. These results suggest that the “intracellular” signaling molecule c-di-AMP may also play a previously unappreciated role in interbacterial cell-cell communication within plant microbiomes

A Conserved Regulatory Circuit Controls Large Adhesins in Vibrio cholerae.

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.IMPORTANCEVibrio 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

BamA‐independent CdiA‐mediated adhesion

CdiB/CdiA proteins mediate inter-bacterial competition in a process termed contact-dependent growth inhibition (CDI). Filamentous CdiA exoproteins extend from CDI(+) cells and bind specific receptors to deliver toxins into susceptible target bacteria. CDI has also been implicated in auto-aggregation and biofilm formation in several species, but the contribution of CdiA-receptor interactions to these multi-cellular behaviors has not been examined. Using Escherichia coli isolate EC93 as a model, we show that cdiA and bamA receptor mutants are defective in biofilm formation, suggesting a prominent role for CdiA-BamA mediated cell-cell adhesion. However, CdiA also promotes auto-aggregation in a BamA-independent manner, indicating that the exoprotein possesses an additional adhesin activity. Cells must express CdiA in order to participate in BamA-independent aggregates, suggesting that adhesion could be mediated by homotypic CdiA-CdiA interactions. The BamA-dependent and BamA-independent interaction domains map to distinct regions within the CdiA filament. Thus, CdiA orchestrates a collective behavior that is independent of its growth-inhibition activity. This adhesion should enable 'greenbeard' discrimination, in which genetically unrelated individuals cooperate with one another based on a single shared trait. This kind-selective social behavior could provide immediate fitness benefits to bacteria that acquire the systems through horizontal gene transfer

Biofilm Formation and Detachment in Gram-Negative Pathogens Is Modulated by Select Bile Acids.

Biofilms are a ubiquitous feature of microbial community structure in both natural and host environments; they enhance transmission and infectivity of pathogens and provide protection from human defense mechanisms and antibiotics. However, few natural products are known that impact biofilm formation or persistence for either environmental or pathogenic bacteria. Using the combination of a novel natural products library from the fish microbiome and an image-based screen for biofilm inhibition, we describe the identification of taurine-conjugated bile acids as inhibitors of biofilm formation against both Vibrio cholerae and Pseudomonas aeruginosa. Taurocholic acid (1) was isolated from the fermentation broth of the fish microbiome-derived strain of Rhodococcus erythropolis and identified using standard NMR and MS methods. Screening of the twelve predominant human steroidal bile acid components revealed that a subset of these compounds can inhibit biofilm formation, induce detachment of preformed biofilms under static conditions, and that these compounds display distinct structure-activity relationships against V. cholerae and P. aeruginosa. Our findings highlight the significance of distinct bile acid components in the regulation of biofilm formation and dispersion in two different clinically relevant bacterial pathogens, and suggest that the bile acids, which are endogenous mammalian metabolites used to solubilize dietary fats, may also play a role in maintaining host health against bacterial infection

Bile acid components induce detachment of <i>V</i>. <i>cholerae</i> biofilms.

<p>(A) CSLM images of horizontal (xy) and vertical (xz and yz) projections of biofilm structures formed by the wild-type strain (wt). Biofilms were formed at 30°C for 5 hours. These biofilms were either untreated or exposed to DMSO, 0.4% Ox bile, 150 μM of TCA or TCDCA. CSLM images of biofilms were taken at 5, 7, and 24 hours. (B) Detachment from biofilms was evaluated by quantification of the planktonic population. CFU/mL of cells released from biofilms to the planktonic population was quantified for each condition at 7 h (left) and 24 h (right). Error bars indicate standard deviations of three biological replicates. *<i>P</i> < 0.05, **<i>P</i> < 0.005, n.s., <i>P</i> > 0.05.</p

Bile acid components effect biofilm formation in <i>P</i>. <i>aeruginosa</i>.

<p>(A) CLSM images of horizontal (xy) and vertical (xz and yz) projections of biofilm structures formed by <i>P</i>. <i>aeruginosa</i> Δ<i>wspF</i> mutant in the presence of DMSO control or 50 μM of TLCA. Biofilms were incubated at 37°C and images were taken at 24 hours. (B) CSLM images of preformed biofilms after treatment with either DMSO control or 50 μM of TLCA for 19 hours at 37°C.</p

Structures and screening results for individual bile acids in both <i>V</i>. <i>cholerae</i> and <i>P</i>. <i>aeruginosa</i> biofilm inhibition assays.

<p>BIC₅₀ = Biofilm Inhibitory Concentration required to reduce biofilm coverage by 50%. NA = not active.</p

Bile acid components reduce biofilm formation in <i>V</i>. <i>cholerae</i>.

<p>(A) CLSM images of horizontal (xy) and vertical (xz and yz) projections of biofilm structures formed by the wild-type strain (wt) in the presence of DMSO as a control and 200 μM of TCA or TCDCA. Biofilms were incubated at 30°C and images were taken at 24 hours. (B) Pellicle formed by the rugose strain formed after 2 days of incubation at 30°C with different bile acid components. All assays were repeated with two biological replicates.</p