Calcium-dependent intestinal chloride secretion by Vibrio parahaemolyticus thermostable direct hemolysin in a rabbit model

doi:10.1016/0016-5085(95)90324-0

Gastroenterology

Volume 109, Issue 2, August 1995, Pages 381-386

https://doi.org/10.1016/0016-5085(95)90324-0 Get rights and content

Abstract

$Background & Aims :$ Vibrio parahaemolyticus is a major agent of seafood gastroenteritis that induces intestinal secretion in the rabbit through its thermostable direct hemolysin. The aim of this study was to characterize the enterotoxicity of purified hemolysin in vitro. $Methods :$ Rabbit ileum was mounted in Ussing chambers, and changes in potential difference and short-circuit current were monitored after addition of hemolysin. Intracellular calcium concentrations in the nontumoral rat crypt-derived cell line IEC-6 were measured using microspectrofluorometry. $Results :$ In Ussing chamber experiments, mucosal toxin addition up to 50 hemolytic units per milliliter induced a proportional increase of the electrical parameters in normal but not Cl⁻-free Ringer's solution. The response to the toxin was not additive to that of calcium ionophore A23187 and was eliminated by preloading the tissue with 1–2-bis(o-aminophenoxy)ethane N,N,N′,N′-tetraacetic acid (BAPTA), a calcium buffer. In IEC-6 cells, a 10-fold increase in intracellular calcium level was found after addition of hemolysin. Such an increase was totally quenched by BAPTA. Finally, preincubation with trisialoganglioside GT1b, but not monosialoganglioside GM1, eliminated toxin-induced increases in potential difference and short-circuit current. $Conclusions :$ These data support the hypothesis that the thermostable direct hemolysin induces intestinal chloride secretion using GT1b as a putative receptor and Ca²⁺ as a second messenger.

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The mucosal disruptions are the structural correlate to the functional barrier defect, indicated by a drop in TER. In general, hemolytic bacteria can induce intestinal malabsorption (Songserm et al., 2002), active ion secretion (Raimondi et al., 1995) and epithelial lesions by cell death phenomena (Hutto and Wannemuehler, 1999). In experimental treatment of mouse colon with HlyA-containing supernatants, epithelial restitution was delayed after induction of single-cell lesions, pointing to an inhibition of epithelial repair mechanisms (Günzel et al., 2006).
Zinc treatment is beneficial for infectious diarrhea or colitis. This study aims to characterize the pathomechanisms of the epithelial barrier dysfunction caused by alpha-hemolysin (HlyA)-expressing Escherichia coli in the colon mucosa and the mitigating effects of zinc ions.
We performed Ussing chamber experiments on porcine colon epithelium and infected the tissues with HlyA-producing E. coli. Colon mucosa from piglets was obtained from a feeding trial with defined normal or high dose zinc feeding (pre-conditioning). Additional to the zinc feeding, zinc was added to the luminal compartment of the Ussing chamber. Transepithelial electrical resistance (TER) was measured during the infection of the living tissue and subsequently the tissues were immuno-stained for confocal microscopy.
Zinc applied to the luminal compartment was effective in preventing from E. coli-induced epithelial barrier dysfunction in Ussing chamber experiments. In contrast, zinc pre-conditioning of colon mucosae when zinc ions were missing subsequently in the luminal compartment was not sufficient to prevent epithelial barrier impairment during E. coli infection. The pathological changes caused by E. coli HlyA were alterations of tight junction proteins claudin-4 and claudin-5, focal leak formation, and cell exfoliation which reflected the paracellular barrier defect measured by a reduced TER. In microscopic analysis of luminal zinc-treated mucosae these changes were absent.
In conclusion, continuous presence of unbound zinc ions in the luminal compartment is essential for the protective action of zinc against E. coli HlyA. This suggests the usage of zinc as therapeutic regimen, while prophylactic intervention by high dietary zinc loads may be less useful.
First detection of the plasmid-mediated colistin resistance gene mcr-1 in virulent Vibrio parahaemolyticus
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V. parahaemolyticus is the leading cause of foodborne bacterial poisoning in China (Li et al., 2014). This organism bearing the tdh gene, which encodes thermostable direct haemolysin (TDH), is generally pathogenic to humans and responsible for the vast majority of clinical cases (Raimondi et al., 1995). Although most cases of V. parahaemolyticus infections are self-limiting, antimicrobial therapy is required for severe or prolonged infections (Wong et al., 2012).
The plasmid-mediated colistin resistance gene mcr-1 has been identified in various Enterobacteriaceae species, which poses a great challenge to the public health. The present study aimed to investigate the prevalence of mcr-1 in Vibrio parahaemolyticus isolated from food samples in China, and to conduct a comprehensive analysis on the molecular characterization of V. parahaemolyticus isolate carrying mcr-1-harboring plasmid. A total of 646 V. parahaemolyticus strains isolated from 2531 food samples collected in retail markets in 34 different cities in China were screened for colistin resistance. Of the 646 V. parahaemolyticus isolates tested, 25 (2.5%) exhibited colistin resistance. The mcr-1 gene was detected in one colistin-resistant V. parahaemolyticus isolate, VP181, obtained from a shrimp sample collected in Hong Kong. The mcr-1 gene was located on a transferable IncX4 plasmid with size of ~40 kb. A Class A β-lactamase gene, bla_CARB-17 and the plasmid-mediated quinolone resistance (PMQR) gene qnrVC5 were detected in the mcr-1-positive V. parahaemolyticus isolate VP181. Virulence gene assays indicated that tdh was detected in VP181 by PCR. This is the first report of the occurrence of plasmid-encoded mcr-1 in virulent V. parahaemolyticus strain. Our findings indicate horizontal transfer of this gene to non-Enterobacteriaceae gram-negative bacteria, which warrants further investigation because of the public health threat it poses.
Molecular mechanisms of Vibrio parahaemolyticus pathogenesis
2019, Microbiological Research
Citation Excerpt :
The mechanism of cytotoxicity of TDH is different from its hemolytic activity (Naim et al., 2001). High concentration of TDH increases the concentration of calcium ions in intestinal epithelial cells, leading to the opening of the chloride channel, and increased secretion of chloride ions in the intestinal cells (Raimondi et al., 1995; Takahashi et al., 2000a, b; Takahashi et al., 2001). trh shares 54.8–68.8% homology with tdh, and is present in a small number of clinical isolates (Kishishita et al., 1992).
Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that is mainly distributed in the seafood such as fish, shrimps and shellfish throughout the world. V. parahaemolyticus can cause diseases in marine aquaculture, leading to huge economic losses to the aquaculture industry. More importantly, it is also the leading cause of seafood-borne diarrheal disease in humans worldwide. With the development of animal model, next-generation sequencing as well as biochemical and cell biological technologies, deeper understanding of the virulence factors and pathogenic mechanisms of V. parahaemolyticus has been gained. As a globally transmitted pathogen, the pathogenicity of V. parahaemolyticus is closely related to a variety of virulence factors. This article comprehensively reviewed the molecular mechanisms of eight types of virulence factors: hemolysin, type III secretion system, type VI secretion system, adhesion factor, iron uptake system, lipopolysaccharide, protease and outer membrane proteins. This review comprehensively summarized our current understanding of the virulence factors in V. parahaemolyticus, which are potentially new targets for the development of therapeutic and preventive strategies.
Structure, function and regulation of the thermostable direct hemolysin (TDH) in pandemic Vibrio parahaemolyticus
2018, Microbial Pathogenesis
Citation Excerpt :
TDH has been also shown to be involved in the enterotoxigenic effect on small intestine and the watery diarrhea induced by V. parahaemolyticus infection [49–53]. Interaction of TDH with the colonic epithelial cells elevates the concentration of Ca2+ inside the cells, resulting in the secretion of Cl− into the mucosal side of the epithelial layer from the serosal [50–52]. Although Ca2+ is a second messenger of the protein kinase C (PKC) phosphorylation pathway, Ca2+-dependent Cl− secretion is not associated with this pathway [53].
Vibrio parahaemolyticus is a leading cause of seafood-associated bacterial gastroenteritis. The pathogen produces the thermostable direct hemolysin (TDH), which is the sole cause of the Kanagawa phenomenon (KP), a special β-type haemolysis in the Wagatsuma agar. TDH also exerts several other biological activities, the major includes lethal toxicity, cytotoxicity, and enterotoxicity. The structure and roles of TDH and the transcriptional regulation of tdh genes, are summarized in this review, which will give a better understanding of the pathogenesis of V. parahaemolyticus.
Foodborne Pathogen-Produced Toxins and Their Signal Transduction
2018, Foodborne Diseases
Foodborne diseases impose a high public health burden globally. The majority of bacterial infections lead to pandemics. Dominant pathogens produce one or more toxins targeting signal transduction of the host, which finally bring about pathophysiological changes and lead to disease(s). Some inhibit protein synthesis, some are neurotoxic, and some target different cellular functions. Evolution of all these toxin moieties enables pathogen(s) to emerge and target the host. Targets and modes of action are mostly known to develop prophylaxis; however, the corresponding defensive actions in a host are not yet well understood.
Glycolipids and Lectins in Endocytic Uptake Processes
2016, Journal of Molecular Biology
A host of endocytic processes has been described at the plasma membrane of eukaryotic cells. Their categorization has most commonly referenced cytosolic machinery, of which the clathrin coat has occupied a preponderant position. In what concerns the intramembrane constituents, the focus of interest has been on phosphatidylinositol lipids and their capacity to orchestrate endocytic events on the cytosolic leaflet of the membrane. The contribution of extracellular determinants to the construction of endocytic pits has received much less attention, despite the fact that (glyco)sphingolipids are exoplasmic leaflet fabric of membrane domains, termed rafts, whose contributions to predominantly clathrin-independent internalization processes are well recognized. Furthermore, sugar-binding proteins, termed lectins, and sugar modifications on extracellular domains of proteins have also been linked, have also been linked to the uptake of endocytic cargos at the plasma membrane. In this review, we summarize these contributions by extracellular determinants to the endocytic process. We thus propose a molecular hypothesis—termed the GL-Lect hypothesis —on how GlycoLipids and Lectins drive the formation of compositional nanoenvironments from which the endocytic uptake of glycosylated cargo proteins is operated via clathrin-independent carriers. Finally, we position this hypothesis within the global context of endocytic pathway proposals that have emerged in the recent years.

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^☆: Supported in part by a grant “Dottorato di Ricerca in Scienze Pediatriche” from the Italian Ministry for Science Advancement (MURST) and also by National Institutes of Health grant AI 19716 (to J.B.K.)

^☆☆: Presented in part at the annual meeting of the American Gastroenterological Association in New Orleans, May 15–18, 1994.

^∗∗: Dr. Raimondi's present address is: Dipartimento di Pediatria, Universitá Federico II, via S. Pansini 5, 80131 Napoli, Italy.

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Gastroenterology

Abstract

References (19)

The purification of the Kanagawa haemolysin from Vibrio parahaemolyticus

FEMS Microbiol Lett

Diarrhea in ciguatera fish poisoning: preliminary evaluation of pathophysiological mechanisms

Gastroenterology

Practical aspects of measuring ²⁺ with fluorescent indicators

Methods Cell Biol

Enterotoxic effect of stool supernatant of Cryptosporidium infected calves on human jejunum

Gastroenterology

Vibrio parahaemolyticus' epidemiology, ecology and biology

Purification and characterization of an hemolysin produced by Vibrio parahaemolyticus

J Infect Dis

Enteropathogenic activity of Vibrio parahaemolyticus

Demonstration of the cardiotoxicity of the thermostable direct hemolysin (lethal toxin) produced by Vibrio Parahaemolyticus

Infect Immun

Stopping the spontaneous beating of cultured mouse and rat myocardial cells by a toxin (thermostable direct hemolysin) from Vibrio parahaemolyticus

Recent Adv Stud Cardiac Struct Metab

Cited by (42)

Zinc prevents intestinal epithelial barrier dysfunction induced by alpha-hemolysin-producing Escherichia coli 536 infection in porcine colon

First detection of the plasmid-mediated colistin resistance gene mcr-1 in virulent Vibrio parahaemolyticus

Molecular mechanisms of Vibrio parahaemolyticus pathogenesis

Structure, function and regulation of the thermostable direct hemolysin (TDH) in pandemic Vibrio parahaemolyticus

Foodborne Pathogen-Produced Toxins and Their Signal Transduction

Glycolipids and Lectins in Endocytic Uptake Processes

Alimentary tractCalcium-dependent intestinal chloride secretion by Vibrio parahaemolyticus thermostable direct hemolysin in a rabbit model☆,☆☆

Abstract

FEMS Microbiol Lett

Gastroenterology

Methods Cell Biol

Gastroenterology

Vibrio parahaemolyticus' epidemiology, ecology and biology

Purification and characterization of an hemolysin produced by Vibrio parahaemolyticus

J Infect Dis

Enteropathogenic activity of Vibrio parahaemolyticus

Demonstration of the cardiotoxicity of the thermostable direct hemolysin (lethal toxin) produced by Vibrio Parahaemolyticus

Infect Immun

Stopping the spontaneous beating of cultured mouse and rat myocardial cells by a toxin (thermostable direct hemolysin) from Vibrio parahaemolyticus

Recent Adv Stud Cardiac Struct Metab

Alimentary tract
Calcium-dependent intestinal chloride secretion by Vibrio parahaemolyticus thermostable direct hemolysin in a rabbit model☆,☆☆