scholarly journals Exploring Environmental Control of Cyclic di-GMP Signaling in Vibrio cholerae by Using theEx VivoLysate Cyclic di-GMP Assay (TELCA)

2013 ◽  
Vol 79 (17) ◽  
pp. 5233-5241 ◽  
Author(s):  
Benjamin J. Koestler ◽  
Christopher M. Waters
Keyword(s):  
Vibrio Cholerae ◽  
Environmental Control ◽  
Ex Vivo ◽  
Signaling Networks ◽  
Diguanylate Cyclase ◽  
Systematic Analysis ◽  
Content Type ◽  
Extracellular Signals ◽  
Novel Method ◽  
Low Cell Density

ABSTRACTVibrio choleraesenses its environment, including the surrounding bacterial community, using both the second messenger cyclic di-GMP (c-di-GMP) and quorum sensing (QS) to regulate biofilm formation and other bacterial behaviors. Cyclic di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes.V. choleraeencodes a complex network of 61 enzymes predicted to mediate changes to the levels of c-di-GMP in response to extracellular signals, and the transcription of many of these enzymes is influenced by QS. Because of the complexity of the c-di-GMP signaling system inV. cholerae, it is difficult to determine if modulation of intracellular c-di-GMP in response to different stimuli is driven primarily by changes in c-di-GMP synthesis or hydrolysis. Here, we describe a novel method, named theex vivolysate c-di-GMP assay (TELCA), that systematically measures total DGC and PDE cellular activity. We show thatV. choleraegrown in different environments exhibits significantly different intracellular levels of c-di-GMP, and we used TELCA to determine that these differences correspond to changes in both c-di-GMP synthesis and hydrolysis. Furthermore, we show that the increased concentration of c-di-GMP at low cell density is primarily due to increased DGC activity due to the DGC CdgA. Our findings highlight the idea that modulation of both total DGC and PDE activity alters the intracellular concentration of c-di-GMP, and we present a new method that is widely applicable to the systematic analysis of complex c-di-GMP signaling networks.

scholarly journals The Two-Component Signal Transduction System VxrAB Positively Regulates Vibrio cholerae Biofilm Formation

2017 ◽  
Vol 199 (18) ◽  
Author(s):  
Jennifer K. Teschler ◽  
Andrew T. Cheng ◽  
Fitnat H. Yildiz
Keyword(s):  
Signal Transduction ◽  
Vibrio Cholerae ◽  
Histidine Kinase ◽  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Response Regulator ◽  
Systematic Analysis ◽  
Content Type ◽  
Two Component ◽  
Two Component Signal Transduction

ABSTRACT Two-component signal transduction systems (TCSs), typically composed of a sensor histidine kinase (HK) and a response regulator (RR), are the primary mechanism by which pathogenic bacteria sense and respond to extracellular signals. The pathogenic bacterium Vibrio cholerae is no exception and harbors 52 RR genes. Using in-frame deletion mutants of each RR gene, we performed a systematic analysis of their role in V. cholerae biofilm formation. We determined that 7 RRs impacted the expression of an essential biofilm gene and found that the recently characterized RR, VxrB, regulates the expression of key structural and regulatory biofilm genes in V. cholerae. vxrB is part of a 5-gene operon, which contains the cognate HK vxrA and three genes of unknown function. Strains carrying ΔvxrA and ΔvxrB mutations are deficient in biofilm formation, while the ΔvxrC mutation enhances biofilm formation. The overexpression of VxrB led to a decrease in motility. We also observed a small but reproducible effect of the absence of VxrB on the levels of cyclic di-GMP (c-di-GMP). Our work reveals a new function for the Vxr TCS as a regulator of biofilm formation and suggests that this regulation may act through key biofilm regulators and the modulation of cellular c-di-GMP levels. IMPORTANCE Biofilms play an important role in the Vibrio cholerae life cycle, providing protection from environmental stresses and contributing to the transmission of V. cholerae to the human host. V. cholerae can utilize two-component systems (TCS), composed of a histidine kinase (HK) and a response regulator (RR), to regulate biofilm formation in response to external cues. We performed a systematic analysis of V. cholerae RRs and identified a new regulator of biofilm formation, VxrB. We demonstrated that the VxrAB TCS is essential for robust biofilm formation and that this system may regulate biofilm formation via its regulation of key biofilm regulators and cyclic di-GMP levels. This research furthers our understanding of the role that TCSs play in the regulation of V. cholerae biofilm formation.

scholarly journals MetR-Regulated Vibrio cholerae Metabolism Is Required for Virulence

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Ryan W. Bogard ◽  
Bryan W. Davies ◽  
John J. Mekalanos
Keyword(s):  
Amino Acid ◽  
Vibrio Cholerae ◽  
Virulence Factor ◽  
Current Knowledge ◽  
Intestinal Colonization ◽  
Wild Type ◽  
Pathogenic Potential ◽  
Content Type ◽  
Mouse Intestine

ABSTRACTLysR-type transcriptional regulators (LTTRs) are the largest, most diverse family of prokaryotic transcription factors, with regulatory roles spanning metabolism, cell growth and division, and pathogenesis. Using a sequence-defined transposon mutant library, we screened a panel ofV. choleraeEl Tor mutants to identify LTTRs required for host intestinal colonization. Surprisingly, out of 38 LTTRs, only one severely affected intestinal colonization in the suckling mouse model of cholera: the methionine metabolism regulator, MetR. Genetic analysis of genes influenced by MetR revealed thatglyA1andmetJwere also required for intestinal colonization. Chromatin immunoprecipitation of MetR and quantitative reverse transcription-PCR (qRT-PCR) confirmed interaction with and regulation ofglyA1, indicating that misregulation ofglyA1is likely responsible for the colonization defect observed in themetRmutant. TheglyA1mutant was auxotrophic for glycine but exhibited wild-type trimethoprim sensitivity, making folate deficiency an unlikely cause of its colonization defect. MetJ regulatory mutants are not auxotrophic but are likely altered in the regulation of amino acid-biosynthetic pathways, including those for methionine, glycine, and serine, and this misregulation likely explains its colonization defect. However, mutants defective in methionine, serine, and cysteine biosynthesis exhibited wild-type virulence, suggesting that these amino acids can be scavenged in vivo. Taken together, our results suggest that glycine biosynthesis may be required to alleviate an in vivo nutritional restriction in the mouse intestine; however, additional roles for glycine may exist. Irrespective of the precise nature of this requirement, this study illustrates the importance of pathogen metabolism, and the regulation thereof, as a virulence factor.IMPORTANCEVibrio choleraecontinues to be a severe cause of morbidity and mortality in developing countries. Identification ofV. choleraefactors critical to disease progression offers the potential to develop or improve upon therapeutics and prevention strategies. To increase the efficiency of virulence factor discovery, we employed a regulator-centric approach to multiplex our in vivo screening capabilities and allow whole regulons inV. choleraeto be interrogated for pathogenic potential. We identified MetR as a new virulence regulator and serine hydroxymethyltransferase GlyA1 as a new MetR-regulated virulence factor, both required byV. choleraeto colonize the infant mouse intestine. Bacterial metabolism is a prerequisite to virulence, and current knowledge of in vivo metabolism of pathogens is limited. Here, we expand the known role of amino acid metabolism and regulation in virulence and offer new insights into the in vivo metabolic requirements ofV. choleraewithin the mouse intestine.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

scholarly journals A Putative Cro-Like Repressor Contributes to Arylomycin Resistance in Staphylococcus aureus

2015 ◽  
Vol 59 (6) ◽  
pp. 3066-3074 ◽  
Author(s):  
Arryn Craney ◽  
Floyd E. Romesberg
Keyword(s):  
Staphylococcus Aureus ◽  
Ex Vivo ◽  
Transcriptional Regulator ◽  
Signal Peptidase ◽  
Health Concern ◽  
Resistant Bacteria ◽  
Type I ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Wide Range

ABSTRACTAntibiotic-resistant bacteria are a significant public health concern and motivate efforts to develop new classes of antibiotics. One such class of antibiotics is the arylomycins, which target type I signal peptidase (SPase), the enzyme responsible for the release of secreted proteins from their N-terminal leader sequences. Despite the essentiality, conservation, and relative accessibility of SPase, the activity of the arylomycins is limited against some bacteria, including the important human pathogenStaphylococcus aureus. To understand the origins of the limited activity againstS. aureus, we characterized the susceptibility of a panel of strains to two arylomycin derivatives, arylomycin A-C16and its more potent analog arylomycin M131. We observed a wide range of susceptibilities to the two arylomycins and found that resistant strains were sensitized by cotreatment with tunicamycin, which inhibits the first step of wall teichoic acid synthesis. To further understand howS. aureusresponds to the arylomycins, we profiled the transcriptional response ofS. aureusNCTC 8325 to growth-inhibitory concentrations of arylomycin M131 and found that it upregulates the cell wall stress stimulon (CWSS) and an operon consisting of a putative transcriptional regulator and three hypothetical proteins. Interestingly, we found that mutations in the putative transcriptional regulator are correlated with resistance, and selection for resistanceex vivodemonstrated that mutations in this gene are sufficient for resistance. The results begin to elucidate howS. aureuscopes with secretion stress and how it evolves resistance to the inhibition of SPase.

A Novel Method for Ex-Vivo Latex Angiography of the Mouse Brain

2021 ◽  
pp. 109342
Author(s):  
Keyan Peterson ◽  
Stephanie Coffman ◽  
Stacey Wolfe ◽  
Zhidan Xiang
Keyword(s):  
Mouse Brain ◽  
Ex Vivo ◽  
Novel Method

scholarly journals Specific Control of Pseudomonas aeruginosa Surface-Associated Behaviors by Two c-di-GMP Diguanylate Cyclases

mBio ◽  
2010 ◽  
Vol 1 (4) ◽  
Author(s):  
Judith H. Merritt ◽  
Dae-Gon Ha ◽  
Kimberly N. Cowles ◽  
Wenyun Lu ◽  
Diana K. Morales ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Extracellular Polysaccharide ◽  
Diguanylate Cyclase ◽  
Flagellar Motility ◽  
Critical Question ◽  
Cyclic Diguanylate ◽  
Critical Signal ◽  
Content Type ◽  
Wide Range

ABSTRACT The signaling nucleotide cyclic diguanylate (c-di-GMP) regulates the transition between motile and sessile growth in a wide range of bacteria. Understanding how microbes control c-di-GMP metabolism to activate specific pathways is complicated by the apparent multifold redundancy of enzymes that synthesize and degrade this dinucleotide, and several models have been proposed to explain how bacteria coordinate the actions of these many enzymes. Here we report the identification of a diguanylate cyclase (DGC), RoeA, of Pseudomonas aeruginosa that promotes the production of extracellular polysaccharide (EPS) and contributes to biofilm formation, that is, the transition from planktonic to surface-dwelling cells. Our studies reveal that RoeA and the previously described DGC SadC make distinct contributions to biofilm formation, controlling polysaccharide production and flagellar motility, respectively. Measurement of total cellular levels of c-di-GMP in ∆roeA and ∆sadC mutants in two different genetic backgrounds revealed no correlation between levels of c-di-GMP and the observed phenotypic output with regard to swarming motility and EPS production. Our data strongly argue against a model wherein changes in total levels of c-di-GMP can account for the specific surface-related phenotypes of P. aeruginosa. IMPORTANCE A critical question in the study of cyclic diguanylate (c-di-GMP) signaling is how the bacterial cell integrates contributions of multiple c-di-GMP-metabolizing enzymes to mediate its cognate functional outputs. One leading model suggests that the effects of c-di-GMP must, in part, be localized subcellularly. The data presented here show that the phenotypes controlled by two different diguanylate cyclase (DGC) enzymes have discrete outputs despite the same total level of c-di-GMP. These data support and extend the model in which localized c-di-GMP signaling likely contributes to coordination of the action of the multiple proteins involved in the synthesis, degradation, and/or binding of this critical signal.

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

Microbiology ◽  
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.

JMM Profile: Vibrio cholerae: an opportunist of human crises

2021 ◽  
Vol 70 (9) ◽  
Author(s):  
Trudy-Ann Grant ◽  
Deepak Balasubramanian ◽  
Salvador Almagro-Moreno
Keyword(s):  
Vibrio Cholerae ◽  
Disease Transmission ◽  
Type Species ◽  
Diarrhoeal Disease ◽  
Health Surveillance ◽  
Clean Water ◽  
Aetiological Agent ◽  
Public Infrastructure ◽  
Content Type ◽  
Primary Mode

Vibrio cholerae O1 is the aetiological agent of the severe diarrhoeal disease cholera. Annually, there are an estimated 1–4 million cholera cases worldwide and over 140 000 deaths. The primary mode of disease transmission is through the consumption of water or food contaminated with the bacterium. Although cholera patients can be treated effectively using rehydration therapy, the disease remains a major scourge in areas with limited access to clean water and proper sanitation. Its continued prevalence highlights the failure of socioeconomic policies leading to wealth disparities, fragile and dated public infrastructure, and lack of appropriate health surveillance.

scholarly journals Overlapping Roles for Interleukin-36 Cytokines in Protective Host Defense against MurineLegionella pneumophilaPneumonia

2018 ◽  
Vol 87 (1) ◽  
Author(s):  
Yuta Nanjo ◽  
Michael W. Newstead ◽  
Tetsuji Aoyagi ◽  
Xianying Zeng ◽  
Kazuhisa Takahashi ◽  
...  
Keyword(s):  
Host Defense ◽  
Legionella Pneumophila ◽  
Ex Vivo ◽  
Inflammatory Cells ◽  
Bacterial Clearance ◽  
Content Type ◽  
Cytokines And Chemokines ◽  
M1 Polarization ◽  
Life Threatening ◽  
Deficient Mice

ABSTRACTLegionella pneumophilacauses life-threatening pneumonia culminating in acute lung injury. Innate and adaptive cytokines play an important role in host defense againstL. pneumophilainfection. Interleukin-36 (IL-36) cytokines are recently described members of the larger IL-1 cytokine family known to exert potent inflammatory effects. In this study, we elucidated the role for IL-36 cytokines in experimental pneumonia caused byL. pneumophila. Intratracheal (i.t.) administration ofL. pneumophilainduced the upregulation of both IL-36α and IL-36γ mRNA and protein production in the lung. Compared to the findings forL. pneumophila-infected wild-type (WT) mice, the i.t. administration ofL. pneumophilato IL-36 receptor-deficient (IL-36R−/−) mice resulted in increased mortality, a delay in lung bacterial clearance, increasedL. pneumophiladissemination to extrapulmonary organs, and impaired glucose homeostasis. Impaired lung bacterial clearance in IL-36R−/−mice was associated with a significantly reduced accumulation of inflammatory cells and the decreased production of proinflammatory cytokines and chemokines.Ex vivo, reduced expression of costimulatory molecules and impaired M1 polarization were observed in alveolar macrophages isolated from infected IL-36R−/−mice compared to macrophages from WT mice. WhileL. pneumophila-induced mortality in IL-36α- or IL-36γ-deficient mice was not different from that in WT animals, antibody-mediated neutralization of IL-36γ in IL-36α−/−mice resulted in mortality similar to that observed in IL-36R−/−mice, indicating redundant and overlapping roles for these cytokines in experimental murineL. pneumophilapneumonia.

scholarly journals High Frequency of a Novel Filamentous Phage, VCYϕ, within an Environmental Vibrio cholerae Population

2011 ◽  
Vol 78 (1) ◽  
pp. 28-33 ◽  
Author(s):  
Hong Xue ◽  
Yan Xu ◽  
Yan Boucher ◽  
Martin F. Polz
Keyword(s):  
Vibrio Cholerae ◽  
Sequence Similarity ◽  
Host Population ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Filamentous Phage ◽  
Nucleotide Polymorphisms ◽  
Environmental Biology ◽  
Content Type ◽  
Filamentous Phages

ABSTRACTEnvironmentalVibrio choleraestrains isolated from a coastal brackish pond (Oyster Pond, Woods Hole, MA) carried a novel filamentous phage, VCYϕ, which can exist as a host genome integrative form (IF) and a plasmid-like replicative form (RF). Outside the cell, the phage displays a morphology typical ofInovirus, with filamentous particles ∼1.8 μm in length and 7 nm in width. Four independent RF isolates had identical genomes, except for 8 single nucleotide polymorphisms clustered in two regions. The overall genome size is 7,103 bp with 11 putative open reading frames organized into three functional modules (replication, structure and assembly, and regulation). VCYϕ shares sequence similarity with other filamentous phages (including cholera disease-associated CTX) in a highly mosaic manner, indicating evolution by horizontal gene transfer and recombination. VCYϕ integrates in the vicinity of the putative translation initiation factor Sui1 in chromosome II ofV. cholerae. A screen of 531 closely related host isolates showed that ∼40% harbored phages, with 27% and 13% carrying the IF and RF, respectively. The relative frequencies of the RF and IF differed among strains isolated from the pond or lagoon of Oyster Pond, suggesting that the host habitat influences intracellular phage biology. The overall high prevalence within the host population shows that filamentous phages can be an important component of the environmental biology ofV. cholerae.

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