scholarly journals Sublingual Adjuvant Delivery by a Live AttenuatedVibrio cholerae-Based Antigen Presentation Platform

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Julie Liao ◽  
Jacob A. Gibson ◽  
Bradley S. Pickering ◽  
Paula I. Watnick
Keyword(s):  
Immune Response ◽  
Antigen Presentation ◽  
Vibrio Cholerae ◽  
Matrix Protein ◽  
Diarrheal Disease ◽  
Mucosal Immune Response ◽  
Mucosal Adjuvant ◽  
Content Type ◽  
Heterologous Antigens ◽  
Biofilm Matrix

ABSTRACTA sublingually delivered heterologous antigen presentation platform that does not depend on antigen or adjuvant purification would be of great benefit in protection against diarrheal disease. In proof-of-concept studies, we previously showed that when a fusion protein comprised of theVibrio choleraebiofilm matrix protein RbmA and the B subunit of cholera toxin (R-CTB) is expressed from a plasmid withinV. cholerae, R-CTB is sequestered in the biofilm matrix, leading to decoration of the cell surface. Sublingual delivery of live attenuated R-CTB-decorated cells results in a mucosal immune response to CTB. To improve the immune response to diarrheal antigens presented by this platform, we have engineered our live attenuated vaccine to express the mucosal adjuvant mmCT (i.e., multiply mutated CT). Here we report that delivery of this adjuvant via sublingual administration of our vaccine enhances the mucosal immune response toV. choleraeLPS and elicits a systemic and mucosal immune response to CTB. However, provision of R-CTB with mmCT selectively blunts the mucosal immune response to CTB. We propose that mmCT delivered by this live attenuatedVibrio choleraevaccine platform may serve as a mucosal adjuvant for heterologous antigens, provided they are not too similar to mmCT.IMPORTANCEDiarrheal disease is the most common infectious disease of children in the developing world. Our goal is to develop a diarrheal antigen presentation platform based on wholeVibrio choleraecells that does not depend on protein purification. We have previously shown the feasibility of genetically fusing antigens to theV. choleraebiofilm matrix protein RbmA for presentation on the cell surface. A mucosal adjuvant could improve immunogenicity of such a vaccine at the mucosal surface. Here we engineer a live attenuatedV. choleraevaccine to constitutively synthesize mmCT, a nontoxic form of cholera toxin. When this vaccine is delivered sublingually,in vivo-synthesized mmCT acts as both an adjuvant and antigen. This could greatly increase the magnitude and duration of the immune response elicited by codelivered heterologous antigens.

scholarly journals Neutrophils Are Essential for Containment of Vibrio cholerae to the Intestine during the Proinflammatory Phase of Infection

2012 ◽  
Vol 80 (8) ◽  
pp. 2905-2913 ◽  
Author(s):  
Jessica Queen ◽  
Karla J. Fullner Satchell
Keyword(s):  
Immune Response ◽  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Innate Immune ◽  
Infection Model ◽  
Proinflammatory Response ◽  
Necrosis Factor Alpha ◽  
Live Attenuated Vaccine ◽  
Content Type

ABSTRACTCholera is classically considered a noninflammatory diarrheal disease, in comparison to invasive enteric organisms, although there is a low-level proinflammatory response during early infection withVibrio choleraeand a strong proinflammatory reaction to live attenuated vaccine strains. Using an adult mouse intestinal infection model, this study examines the contribution of neutrophils to host defense to infection. Nontoxigenic El Tor O1V. choleraeinfection is characterized by the upregulation of interleukin-6 (IL-6), IL-10, and macrophage inflammatory protein 2 alpha in the intestine, indicating an acute innate immune response. Depletion of neutrophils from mice with anti-Ly6G IA8 monoclonal antibody led to decreased survival of mice. The role of neutrophils in protection of the host is to limit the infection to the intestine and control bacterial spread to extraintestinal organs. In the absence of neutrophils, the infection spread to the spleen and led to increased systemic levels of IL-1β and tumor necrosis factor alpha, suggesting the decreased survival in neutropenic mice is due to systemic shock. Neutrophils were found not to contribute to either clearance of colonizing bacteria or to alter the local immune response. However, when genes for secreted accessory toxins were deleted, the colonizing bacteria were cleared from the intestine, and this clearance is dependent upon neutrophils. Thus, the requirement for accessory toxins in virulence is negated in neutropenic mice, which is consistent with a role of accessory toxins in the evasion of innate immune cells in the intestine. Overall, these data support that neutrophils impact disease progression and suggest that neutrophil effectiveness can be manipulated through the deletion of accessory toxins.

scholarly journals A Biofilm Matrix-Associated Protease Inhibitor ProtectsPseudomonas aeruginosafrom Proteolytic Attack

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Boo Shan Tseng ◽  
Courtney Reichhardt ◽  
Gennifer E. Merrihew ◽  
Sophia A. Araujo-Hernandez ◽  
Joe J. Harrison ◽  
...  
Keyword(s):  
Immune Response ◽  
Immune System ◽  
Protease Inhibitor ◽  
Neutrophil Elastase ◽  
Matrix Protein ◽  
Lipid Vesicles ◽  
Matrix Proteins ◽  
Host Immune System ◽  
Content Type ◽  
Biofilm Matrix

ABSTRACTPseudomonas aeruginosaproduces an extracellular biofilm matrix that consists of nucleic acids, exopolysaccharides, lipid vesicles, and proteins. In general, the protein component of the biofilm matrix is poorly defined and understudied relative to the other major matrix constituents. While matrix proteins have been suggested to provide many functions to the biofilm, only proteins that play a structural role have been characterized thus far. Here we identify proteins enriched in the matrix ofP. aeruginosabiofilms. We then focused on a candidate matrix protein, the serine protease inhibitor ecotin (PA2755). This protein is able to inhibit neutrophil elastase, a bactericidal enzyme produced by the host immune system duringP. aeruginosabiofilm infections. We show that ecotin binds to the key biofilm matrix exopolysaccharide Psl and that it can inhibit neutrophil elastase when associated with Psl. Finally, we show that ecotin protects both planktonic and biofilmP. aeruginosacells from neutrophil elastase-mediated killing. This may represent a novel mechanism of protection for biofilms to increase their tolerance against the innate immune response.IMPORTANCEProteins associated with the extracellular matrix of bacterial aggregates called biofilms have long been suggested to provide many important functions to the community. To date, however, only proteins that provide structural roles have been described, and few matrix-associated proteins have been identified. We developed a method to identify matrix proteins and characterized one. We show that this protein, when associated with the biofilm matrix, can inhibit a bactericidal enzyme produced by the immune system during infection and protect biofilm cells from death induced by the enzyme. This may represent a novel mechanism of protection for biofilms, further increasing their tolerance against the immune response. Together, our results are the first to show a nonstructural function for a confirmed matrix-interacting protein.

scholarly journals Host Intestinal Signal-Promoted Biofilm Dispersal Induces Vibrio cholerae Colonization

2014 ◽  
Vol 83 (1) ◽  
pp. 317-323 ◽  
Author(s):  
Amanda J. Hay ◽  
Jun Zhu
Keyword(s):  
Bile Salt ◽  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Virulence Gene ◽  
Human Infection ◽  
Content Type ◽  
Abiotic Degradation ◽  
Contaminated Food ◽  
Free Media ◽  
Biofilm Matrix

Vibrio choleraecauses human infection through ingestion of contaminated food and water, leading to the devastating diarrheal disease cholera.V. choleraeforms matrix-encased aggregates, known as biofilms, in the native aquatic environment. While the formation ofV. choleraebiofilms has been well studied, little is known about the dispersal from biofilms, particularly upon entry into the host. In this study, we found that the exposure of mature biofilms to physiologic levels of the bile salt taurocholate, a host signal for the virulence gene induction ofV. cholerae, induces an increase in the number of detached cells with a concomitant decrease in biofilm mass. Scanning electron microscopy micrographs of biofilms exposed to taurocholate revealed an altered, perhaps degraded, appearance of the biofilm matrix. The inhibition of protein synthesis did not alter rates of detachment, suggesting thatV. choleraeundergoes a passive dispersal. Cell-free media from taurocholate-exposed biofilms contains a larger amount of free polysaccharide, suggesting an abiotic degradation of biofilm matrix by taurocholate. Furthermore, we found thatV. choleraeis only able to induce virulence in response to taurocholate after exit from the biofilm. Thus, we propose a model in whichV. choleraeingested as a biofilm has coopted the host-derived bile salt signal to detach from the biofilm and go on to activate virulence.

scholarly journals Vibrio cholerae Utilizes Direct sRNA Regulation in Expression of a Biofilm Matrix Protein

PLoS ONE ◽  
2014 ◽  
Vol 9 (7) ◽  
pp. e101280 ◽  
Author(s):  
Tianyan Song ◽  
Dharmesh Sabharwal ◽  
Jyoti Mohan Gurung ◽  
Andrew T. Cheng ◽  
Annika E. Sjöström ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Matrix Protein ◽  
Biofilm Matrix

scholarly journals Pushing beyond the Envelope: the Potential Roles of OprF inPseudomonas aeruginosaBiofilm Formation and Pathogenicity

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Erin K. Cassin ◽  
Boo Shan Tseng
Keyword(s):  
Extracellular Matrix ◽  
Outer Membrane ◽  
Biofilm Formation ◽  
Matrix Protein ◽  
Cell Envelope ◽  
Outer Membrane Vesicles ◽  
Extracellular Dna ◽  
Future Research ◽  
Content Type ◽  
Biofilm Matrix

ABSTRACTThe ability ofPseudomonas aeruginosato form biofilms, which are communities of cells encased in a self-produced extracellular matrix, protects the cells from antibiotics and the host immune response. While some biofilm matrix components, such as exopolysaccharides and extracellular DNA, are relatively well characterized, the extracellular matrix proteins remain understudied. Multiple proteomic analyses of theP. aeruginosasoluble biofilm matrix and outer membrane vesicles, which are a component of the matrix, have identified OprF as an abundant matrix protein. To date, the few reports on the effects ofoprFmutations on biofilm formation are conflicting, and little is known about the potential role of OprF in the biofilm matrix. The majority of OprF studies focus on the protein as a cell-associated porin. As a component of the outer membrane, OprF assumes dual conformations and is involved in solute transport, as well as cell envelope integrity. Here, we review the current literature on OprF inP. aeruginosa, discussing how the structure and function of the cell-associated and matrix-associated protein may affect biofilm formation and pathogenesis in order to inform future research on this understudied matrix protein.

scholarly journals Type III Secretion Is Essential for the Rapidly Fatal Diarrheal Disease Caused by Non-O1, Non-O139 Vibrio cholerae

mBio ◽  
2011 ◽  
Vol 2 (3) ◽  
Author(s):  
Ok S. Shin ◽  
Vincent C. Tam ◽  
Masato Suzuki ◽  
Jennifer M. Ritchie ◽  
Roderick T. Bronson ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Intestinal Epithelium ◽  
Type Iii Secretion ◽  
Diarrheal Disease ◽  
Secretion Systems ◽  
Content Type ◽  
Type Iii ◽  
Severe Diarrhea ◽  
Toxin Coregulated Pilus

ABSTRACTCholera is a severe diarrheal disease typically caused by O1 serogroup strains ofVibrio cholerae. The pathogenicity of all pandemicV. choleraeO1 strains relies on two critical virulence factors: cholera toxin, a potent enterotoxin, and toxin coregulated pilus (TCP), an intestinal colonization factor. However, certain non-O1, non-O139V. choleraestrains, such as AM-19226, do not produce cholera toxin or TCP, yet they still cause severe diarrhea. The molecular basis for the pathogenicity of non-O1, non-O139V. choleraehas not been extensively characterized, but many of these strains encode related type III secretion systems (TTSSs). Here, we used infant rabbits to assess the contribution of the TTSS to non-O1, non-O139V. choleraepathogenicity. We found that all animals infected with wild-type AM-19226 developed severe diarrhea even more rapidly than rabbits infected withV. choleraeO1. UnlikeV. choleraeO1 strains, which do not damage the intestinal epithelium in rabbits or humans, AM-19226 caused marked disruptions of the epithelial surface in the rabbit small intestine. TTSS proved to be essential for AM-19226 virulence in infant rabbits; an AM-19226 derivative deficient for TTSS did not elicit diarrhea, colonize the intestine, or induce pathological changes in the intestine. Deletion of either one of the two previously identified or two newly identified AM-19226 TTSS effectors reduced but did not eliminate AM-19226 pathogenicity, suggesting that at least four effectors contribute to this strain’s virulence. In aggregate, our results suggest that the TTSS-dependent virulence in non-O1, non-O139V. choleraerepresents a new type of diarrheagenic mechanism.IMPORTANCECholera, which is caused byVibrio cholerae, is an important cause of diarrheal disease in many developing countries. The mechanisms of virulence of nonpandemic strains that can cause a diarrheal illness are poorly understood. AM-19226, like several other pathogenic, nonpandemicV. choleraestrains, carries genes that encode a type III secretion system (TTSS), but not cholera toxin (CT) or toxin coregulated pilus (TCP). In this study, we used infant rabbits to study AM-19226 virulence. Infant rabbits orally inoculated with this strain rapidly developed a fatal diarrheal disease, which was accompanied by marked disruptions of the intestinal epithelium. This strain’s TTSS proved essential for its pathogenicity, and there was no diarrhea, intestinal pathology, or colonization in rabbits infected with a TTSS mutant. The effector proteins translocated by the TTSS all appear to contribute to AM-19226 virulence. Thus, our study provides insight intoin vivomechanisms by which a novel TTSS contributes to diarrheal disease caused by nonpandemic strains ofV. cholerae.

scholarly journals A Small Unstructured Region in Vibrio cholerae ToxT Mediates the Response to Positive and Negative Effectors and ToxT Proteolysis

2014 ◽  
Vol 197 (3) ◽  
pp. 654-668 ◽  
Author(s):  
Joshua J. Thomson ◽  
Sarah C. Plecha ◽  
Jeffrey H. Withey
Keyword(s):  
Vibrio Cholerae ◽  
Virulence Factors ◽  
Unsaturated Fatty Acids ◽  
Diarrheal Disease ◽  
Virulence Gene ◽  
Site Directed Mutagenesis ◽  
Content Type ◽  
Terminal Domain ◽  
Virulence Gene Regulation ◽  
Host Signals

Vibrio choleraeis the causative agent of the severe diarrheal disease cholera. The production of the virulence factors that are required for human disease is controlled by a complex network of transcriptional and posttranscriptional regulators. ToxT is the transcription regulator that directly controls the production of the two major virulence factors, toxin-coregulated pilus (TCP) and cholera toxin (CT). The solved crystal structure of ToxT revealed an unstructured region in the N-terminal domain between residues 100 and 110. This region and the surrounding amino acids have been previously implicated in ToxT proteolysis, resistance to inhibition by negative effectors, and ToxT dimerization. To better characterize this region, site-directed mutagenesis was performed to assess the effects on ToxT proteolysis and bile sensitivity. This analysis identified specific mutations within this unstructured region that prevent ToxT proteolysis and other mutations that reduce inhibition by bile and unsaturated fatty acids. In addition, we found that mutations that affect the sensitivity of ToxT to bile also affect the sensitivity of ToxT to its positive effector, bicarbonate. These results suggest that a small unstructured region in the ToxT N-terminal domain is involved in multiple aspects of virulence gene regulation and response to human host signals.

scholarly journals Characterization of the Immune Response to Vibrio cholerae Infection in a Natural Host Model

2021 ◽  
Vol 11 ◽  
Author(s):  
Dustin A. Farr ◽  
Dhrubajyoti Nag ◽  
Jeffrey H. Withey
Keyword(s):  
Immune Response ◽  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Natural Host ◽  
Zebrafish Model ◽  
Mucin Production ◽  
Immunological Markers ◽  
Contaminated Food ◽  
Life Threatening ◽  
El Tor

The gram-negative bacterium Vibrio cholerae causes the life-threatening diarrheal disease cholera, which is spread through the ingestion of contaminated food or water. Cholera epidemics occur largely in developing countries that lack proper infrastructure to treat sewage and provide clean water. Numerous vertebrate fish species have been found to be natural V. cholerae hosts. Based on these findings, zebrafish (Danio rerio) have been developed as a natural host model for V. cholerae. Diarrheal symptoms similar to those seen in humans are seen in zebrafish as early as 6 hours after exposure. Our understanding of basic zebrafish immunology is currently rudimentary, and no research has been done to date exploring the immune response of zebrafish to V. cholerae infection. In the present study, zebrafish were infected with either pandemic El Tor or non-pandemic, environmental V. cholerae strains and select immunological markers were assessed to determine cellular immunity and humoral immunity. Significant increases in the gene expression of two transcription factors, T-bet and GATA3, were observed in response to infection with both V. cholerae strains, as were levels of mucosal related antibodies. Additionally, the cytokine IL-13 was shown to be significantly elevated and paralleled the mucin output in zebrafish excretions, strengthening our knowledge of IL-13 induced mucin production in cholera. The data presented here further solidify the relevancy of the zebrafish model in studying V. cholerae, as well as expanding its utility in the field of cholera immunology.

scholarly journals A Conserved Regulatory Circuit Controls Large Adhesins in Vibrio cholerae

mBio ◽  
2019 ◽  
Vol 10 (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.

scholarly journals Vibrio cholerae at the Intersection of Immunity and the Microbiome

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Ana A. Weil ◽  
Rachel L. Becker ◽  
Jason B. Harris
Keyword(s):  
Immune Response ◽  
Small Intestine ◽  
Immune Responses ◽  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Intestinal Microbiome ◽  
Secretory Diarrhea ◽  
Content Type ◽  
New Approaches

ABSTRACT Vibrio cholerae is a noninvasive pathogen that colonizes the small intestine and produces cholera toxin, causing severe secretory diarrhea. Cholera results in long lasting immunity, and recent studies have improved our understanding of the antigenic repertoire of V. cholerae. Interactions between the host, V. cholerae, and the intestinal microbiome are now recognized as factors which impact susceptibility to cholera and the ability to mount a successful immune response to vaccination. Here, we review recent data and corresponding models to describe immune responses to V. cholerae infection and explain how the host microbiome may impact the pathogenesis of V. cholerae. In the ongoing battle against cholera, the intestinal microbiome represents a frontier for new approaches to intervention and prevention.

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