Molecular and biochemical characterization of a heat-labile cytotonic enterotoxin fromAeromonas hydrophila

Chopra, Ashok K.; Peterson, Johnny W.; Xu, Xin; Coppenhaver, Dorian H.; Houston, Clifford W.

doi:10.1006/mpat.1996.0068

Cited by 73 publications

(77 citation statements)

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“…To better characterize this 85-to 90-kDa polypeptide, the TCA-precipitated proteins were separated by SDS-12% PAGE, transferred to a polyvinylidene difluoride membrane, and stained by Coomassie blue (11,43). Subsequently, the 85-to 90-kDa band was trypsin digested, and the fragments were subjected to NH 2 -terminal and internal sequencing at UTMB's Protein Chemistry Core Facility.…”

Section: Methodsmentioning

confidence: 99%

Molecular and Functional Characterization of a ToxR-Regulated Lipoprotein from a Clinical Isolate of Aeromonas hydrophila

et al. 2006

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Human diseases caused by species of Aeromonas have been classified into two major groups: septicemia and gastroenteritis. In this study, we reported the molecular and functional characterization of a new virulence factor, ToxR-regulated lipoprotein, or TagA, from a diarrheal isolate, SSU, of Aeromonas hydrophila. The tagA gene of A. hydrophila exhibited 60% identity with that of a recently identified stcE gene from Escherichia coli O157:H7, which encoded a protein (StcE) that provided serum resistance to the bacterium and prevented erythrocyte lysis by controlling classical pathway of complement activation by cleaving the complement C1-esterase inhibitor (C1-INH). We purified A. hydrophila TagA as a histidine-tagged fusion protein (rTagA) from E. coli DE3 strain using a T7 promoter-based pET30 expression vector and nickel affinity column chromatography. rTagA cleaved C1-INH in a time-dependent manner. The tagA isogenic mutant of A. hydrophila, unlike its corresponding wild-type (WT) or the complemented strain, was unable to cleave C1-INH, which is required to potentiate the C1-INH-mediated lysis of host and bacterial cells. We indeed demonstrated colocalization of C1-INH and TagA on the bacterial surface by confocal fluorescence microscopy, which ultimately resulted in increased serum resistance of the WT bacterium. Likewise, we delineated the role of TagA in contributing to the enhanced ability of C1-INH to inhibit the classical complement-mediated lysis of erythrocytes. Importantly, we provided evidence that the tagA mutant was significantly less virulent in a mouse model of infection (60%) than the WT bacterium at two 50% lethal doses, which resulted in 100% mortality within 48 h. Taken together, our data provided new information on the role of TagA as a virulence factor in bacterial pathogenesis. This is the first report of TagA characterization from any species of Aeromonas.Aeromonas hydrophila represents one of the most predominant species within the family Aeromonadaceae that leads to human diseases, such as gastroenteritis, wound infections, and septicemia (21). These pathogens have been isolated from a wide variety of food and water sources and are being recovered with increasing frequency from patients with traveler's diarrhea (9,19,21). Resistance of Aeromonas spp. to water chlorination and to multiple antibiotics has resulted in this organism being placed on the "Contaminant Candidate List" by the Environmental Protection Agency (9). These ubiquitous bacteria produce a large number of virulence factors, many of which have been linked to Aeromonas-associated pathogenesis. Among them are matrix-binding proteins, elastases, proteases, cytotonic and cytotoxic enterotoxins, hemolysins, aldolase, chitinase, lipases/phospholipases, and the ability to form a capsule-like outer layer (10,30,35,45). Aeromonas spp. also possess type IV pili and bundle-forming pili, the latter of which has been shown to be associated with gastroenteritis (23, 29).Our laboratory purified and extensively characterized the cytotoxi...

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Section: Methodsmentioning

confidence: 99%

Molecular and Functional Characterization of a ToxR-Regulated Lipoprotein from a Clinical Isolate of Aeromonas hydrophila

et al. 2006

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“…We demonstrated that these enterotoxins from diarrheal isolate SSU of A. hydrophila were able to cause intestinal fluid loss in a rat/rabbit model (20,58,69). The cytotoxic enterotoxin (Act) is a single-chain, 52-kDa polypeptide that has various biological activities, including hemolysis, cytotoxicity, enterotoxicity, and lethality (18,23).…”

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The Cytotoxic Enterotoxin ofAeromonas hydrophilaInduces Proinflammatory Cytokine Production and Activates Arachidonic Acid Metabolism in Macrophages

et al. 2000

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An aerolysin-related cytotoxic enterotoxin (Act) of Aeromonas hydrophila possesses multiple biological activities, which include its ability to lyse red blood cells, destroy tissue culture cell lines, evoke a fluid secretory response in ligated intestinal loop models, and induce lethality in mice. The role of Act in the virulence of the organism has been demonstrated. In this study, we evaluated the potential of Act to induce production of proinflammatory cytokines associated with Act-induced tissue injury and Act's capacity to activate in macrophages arachidonic acid (AA) metabolism that leads to production of eicosanoids (e.g., prostaglandin E 2 [PGE 2 ]). Our data indicated that Act stimulated the production of tumor necrosis factor alpha and upregulated the expression of genes encoding interleukin-1␤ (IL-1␤) and IL-6 in the murine macrophage cell line RAW264.7. Act also activated transcription of the gene encoding inducible nitric oxide synthase. Act evoked the production of PGE 2 coupled to the cyclooxygenase-2 (COX-2) pathway. AA is a substrate for PGE 2 , and Act produced AA from phospholipids by inducing group V secretory phospholipase A 2 . We also demonstrated that Act increased cyclic AMP (cAMP) production in macrophages. cAMP, along with PGE 2 , could potentiate fluid secretion in animal models because of infiltration and activation of macrophages resulting from Act-induced tissue injury. After Act treatment of RAW cells, we detected an increased translocation of NF-B and cAMP-responsive element binding protein (CREB) to the nucleus using gel shift assays. Act also upregulated production of antiapoptotic protein Bcl-2 in macrophages, suggesting a protective role for Bcl-2 against cell death induced by proinflammatory cytokines. The increased expression of genes encoding the proinflammatory cytokines, COX-2, and Bcl-2 appeared correlated with the activation of NF-B and CREB. This is the first report of the detailed mechanisms of action of Act from A. hydrophila.Aeromonas spp. recently have been placed in the family Aeromonadaceae. They cause both intestinal and nonintestinal infections in humans (12), and, unlike gastroenteritis, which generally occurs in young children, these nonintestinal infections are often fatal and involve adults (36). Aeromonas spp. have been cultured from both freshwater and salt water and from many foods. These bacteria have emerged as important human pathogens and are being isolated in an increased incidence from patients with traveler's diarrhea (3,11,28,29,41,44,70). Aeromonas spp. produce an array of virulence factors, and the pathogenesis of Aeromonas infections is therefore complex and multifactorial (2). These virulence factors include hemolysins, cytotoxins, enterotoxins, proteases, lipases/phospholipases, leucocidin, endotoxin, fimbriae or adhesins, and the capacity to form an S-layer (17,45,47). Aeromonas hydrophila has been shown to be invasive for HEp-2 cell monolayers, and the bacterial cells adhere to human erythrocytes (6, 26). Two distinct families of type IV ...

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“…Unlike Act, these cytotonic enterotoxins did not cause degeneration of crypts and villi of the small intestine (15,20,21). The cell lysates from E. coli clones harboring cytotonic enterotoxin genes caused Chinese hamster ovary (CHO) cells to elongate and to produce cyclic AMP, which are typical enterotoxic responses (21).…”

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confidence: 99%

“…The cell lysates from E. coli clones harboring cytotonic enterotoxin genes caused Chinese hamster ovary (CHO) cells to elongate and to produce cyclic AMP, which are typical enterotoxic responses (21). One of these cytotonic enterotoxins was heat labile at 56°C and was referred to as Alt, while the other was heat stable at the same temperature and was designated Ast (20,21). An Ast-related cytotonic enterotoxin gene was first cloned from A. hydrophila (reclassified as A. trota) by Chakraborty et al (13); however, it was not further characterized.…”

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Role of Various Enterotoxins in Aeromonas hydrophila- Induced Gastroenteritis: Generation of Enterotoxin Gene-Deficient Mutants and Evaluation of Their Enterotoxic Activity

2002

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Three enterotoxins from the Aeromonas hydrophila diarrheal isolate SSU have been molecularly characterized in our laboratory. One of these enterotoxins is cytotoxic in nature, whereas the other two are cytotonic enterotoxins, one of them heat labile and the other heat stable. Earlier, by developing an isogenic mutant, we demonstrated the role of a cytotoxic enterotoxin in causing systemic infection in mice. In the present study, we evaluated the role of these three enterotoxins in evoking diarrhea in a murine model by developing various combinations of enterotoxin gene-deficient mutants by marker-exchange mutagenesis. A total of six isogenic mutants were prepared in a cytotoxic enterotoxin gene (act)-positive or -negative background strain of A. hydrophila. We developed two single knockouts with truncation in either the heat-labile (alt) or the heat-stable (ast) cytotonic enterotoxin gene; three double knockouts with truncations of genes encoding (i) alt and ast, (ii) act and alt, and (iii) act and ast genes; and a triple-knockout mutant with truncation in all three genes, act, alt, and ast. The identity of these isogenic mutants developed by double-crossover homologous recombination was confirmed by Southern blot analysis. Northern and Western blot analyses revealed that the expression of different enterotoxin genes in the mutants was correspondingly abrogated. We tested the biological activity of these mutants in a diet-restricted and antibiotic-treated mouse model with a ligated ileal loop assay. Our data indicated that all of these mutants had significantly reduced capacity to evoke fluid secretion compared to that of wild-type A. hydrophila; the triple-knockout mutant failed to induce any detectable level of fluid secretion. The biological activity of selected A. hydrophila mutants was restored after complementation. Taken together, we have established a role for three enterotoxins in A. hydrophila-induced gastroenteritis in a mouse model with the greatest contribution from the cytotoxic enterotoxin Act, followed by the Alt and Ast cytotonic enterotoxins.

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Molecular and biochemical characterization of a heat-labile cytotonic enterotoxin fromAeromonas hydrophila

Cited by 73 publications

References 0 publications

Molecular and Functional Characterization of a ToxR-Regulated Lipoprotein from a Clinical Isolate of Aeromonas hydrophila

Molecular and Functional Characterization of a ToxR-Regulated Lipoprotein from a Clinical Isolate of Aeromonas hydrophila

The Cytotoxic Enterotoxin ofAeromonas hydrophilaInduces Proinflammatory Cytokine Production and Activates Arachidonic Acid Metabolism in Macrophages

Role of Various Enterotoxins in Aeromonas hydrophila- Induced Gastroenteritis: Generation of Enterotoxin Gene-Deficient Mutants and Evaluation of Their Enterotoxic Activity

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