Many gram-negative bacteria use a type III secretion system (TTSS) to deliver effector proteins into host cells. Here we report the characterization of a TTSS chromosomal operon from the diarrheal isolate SSU of Aeromonas hydrophila. We deleted the gene encoding Aeromonas outer membrane protein B (AopB), which is predicted to be involved in the formation of the TTSS translocon, from wild-type (WT) A. hydrophila as well as from a previously characterized cytotoxic enterotoxin gene (act)-minus strain of A. hydrophila, thus generating aopB and act/aopB isogenic mutants. The act gene encodes a type II-secreted cytotoxic enterotoxin (Act) that has hemolytic, cytotoxic, and enterotoxic activities and induces lethality in a mouse model. These isogenic mutants (aopB, act, and act/aopB) were highly attenuated in their ability to induce cytotoxicity in RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells. The act/aopB mutant demonstrated the greatest reduction in cytotoxicity to cultured cells after 4 h of infection, as measured by the release of lactate dehydrogenase enzyme, and was avirulent in mice, with a 90% survival rate compared to that of animals infected with Act and AopB mutants, which caused 50 to 60% of the animals to die at a dose of three 50% lethal doses. In contrast, WT A. hydrophila killed 100% of the mice within 48 h. The effects of these mutations on cytotoxicity could be complemented with the native genes. Our studies further revealed that the production of lactones, which are involved in quorum sensing (QS), was decreased in the act (32%) and aopB (64%) mutants and was minimal (only 8%) in the act/aopB mutant, compared to that of WT A. hydrophila SSU. The effects of act and aopB gene deletions on lactone production could also be complemented with the native genes, indicating specific effects of Act and the TTSS on lactone production. Although recent studies with other bacteria have indicated TTSS regulation by QS, this is the first report describing a correlation between the TTSS and Act of A. hydrophila and the production of lactones.Aeromonas species are emerging human pathogens that cause a wide array of diseases, such as gastroenteritis, wound infections, and septicemia (49, 67). One of the most potent virulence factors of Aeromonas species is the type II-secreted cytotoxic enterotoxin (Act), which possesses hemolytic, cytotoxic, and enterotoxic activities and is lethal in nanogram quantities when injected into mice (12,82).Recently, genes for a type III secretion system (TTSS) were identified in fish isolates of Aeromonas spp. (4,5,6,73,80,84). The TTSS enables many pathogenic gram-negative bacteria to secrete and inject pathogenicity proteins (effectors) into the cytosol of eukaryotic cells via needle-like structures called needle complexes or injectisomes. Overall, type III secretion systems have been grouped into five major families based on phylogenetic analysis of three highly conserved proteins (i.e., homologs of YscN, YscV, and YscC in Yersinia enterocolitica), and, thus f...
Among the various virulence factors produced by Aeromonas hydrophila, a type II secretion system (T2SS)-secreted cytotoxic enterotoxin (Act) and the T3SS are crucial in the pathogenesis of Aeromonas-associated infections. Our laboratory molecularly characterized both Act and the T3SS from a diarrheal isolate, SSU of A. hydrophila, and defined the role of some regulatory genes in modulating the biological effects of Act. In this study, we cloned, sequenced, and expressed the DNA adenine methyltransferase gene of A. hydrophila SSU (dam AhSSU ) in a T7 promoter-based vector system using Escherichia coli ER2566 as a host strain, which could alter the virulence potential of A. hydrophila. Recombinant Dam, designated as M.AhySSUDam, was produced as a histidine-tagged fusion protein and purified from an E. coli cell lysate using nickel affinity chromatography. The purified Dam had methyltransferase activity, based on its ability to transfer a methyl group from S-adenosyl-L-methionine to N 6 -methyladenine-free lambda DNA and to protect methylated lambda DNA from digestion with DpnII but not against the DpnI restriction enzyme. The dam gene was essential for the viability of the bacterium, and overproduction of Dam in A. hydrophila SSU, using an arabinose-inducible, P BAD promoter-based system, reduced the virulence of this pathogen. Specifically, overproduction of M.AhySSUDam decreased the motility of the bacterium by 58%. Likewise, the T3SS-associated cytotoxicity, as measured by the release of lactate dehydrogenase enzyme in murine macrophages infected with the Dam-overproducing strain, was diminished by 55% compared to that of a control A. hydrophila SSU strain harboring the pBAD vector alone. On the contrary, cytotoxic and hemolytic activities associated with Act as well as the protease activity in the culture supernatant of a Dam-overproducing strain were increased by 10-, 3-, and 2.4-fold, respectively, compared to those of the control A. hydrophila SSU strain. The Dam-overproducing strain was not lethal to mice (100% survival) when given by the intraperitoneal route at a dose twice that of the 50% lethal dose, which within 2 to 3 days killed 100% of the animals inoculated with the A. hydrophila control strain. Taken together, our data indicated alteration of A. hydrophila virulence by overproduction of Dam.Aeromonas hydrophila is both a human and an animal pathogen, and the diseases associated with this bacterium in humans include gastroenteritis, wound infections, and septicemia (36, 88). In immuno-compromised individuals, some of the more serious complications associated with A. hydrophila infections are cellulitis, myonecrosis, and ecthyma gangrenosum, which often are fatal (2, 36). A variety of virulence factors/mechanisms, such as the surface layer, capsule, different forms of pili (e.g., type IV [Tap] and bundle-forming pili), toxins, quorum sensing, biofilm formation and, more recently, the type III secretion system (T3SS), were identified in various Aeromonas species that are involved in the pathogenesis...
Aeromonas species are emerging human pathogens, which produce an array of virulence factors and cause diseases ranging from gastroenteritis to systemic infections. These bacteria can be found in food and water and grow well and produce toxins at refrigeration temperatures, which greatly increase the risk of food poisoning. A detailed understanding of host responses to Aeromonas virulence factors is paramount to developing better treatment strategies. One of the important virulence factors of Aeromonas is the cytotoxic enterotoxin, Act, which induces potent inflammatory responses in host cells and is lethal when injected intravenously into mice. Microarray analyses of Act-treated host cells by our laboratory revealed that Act induced host cell signaling and apoptosis of macrophages and colonic epithelial cells. We furthered showed that Act production is regulated by glucose inhibited division gene A (gidA) and an iron-regulated ferric uptake regulatory (fur) gene. In addition to Act, our laboratory recently discovered new virulence factors/mechanisms, including the plasminogen-activating enzyme enolase and a type III secretion system, which contribute to Aeromonas-associated diseases. Current knowledge concerning host responses to these and other Aeromonas virulence factors is discussed.
A cytotoxic enterotoxin (Act) of Aeromonas hydrophila possesses several biological activities, and it induces an inflammatory response in the host. In this study, we used microarrays to gain a global and molecular view of the cellular transcriptional responses to Act and to identify important genes up-regulated by this toxin. Total RNA was isolated at 0, 2, and 12 h from Act-treated macrophages and applied to Affymetrix MGU74 arrays, and the data were processed using a multi-analysis approach to identify genes that might be critical in the inflammatory process evoked by Act. Seventy-six genes were significantly and consistently upregulated. Many of these genes were immune-related, and several were transcription factors, adhesion molecules, and cytokines. Additionally, we identified several apoptosis-associated genes that were significantly upregulated in Act-treated macrophages. Act-induced apoptosis of macrophages was confirmed by annexin V staining and DNA laddering. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay were used to verify increased expression of some inflammatory and apoptosis-associated genes identified by the microarray analysis. To further confirm Act-induced increases in gene expression, real-time RT-PCR was also used for selected genes. Taken together, the array data provided for the first time a global view of Act-mediated signal transduction and clearly demonstrated an inflammatory response and apoptosis mediated by this toxin in host cells at the molecular level.Aeromonas species (spp.) 1 are significant human pathogens that cause both gastrointestinal and non-intestinal diseases in children and adults (1). These bacteria have been isolated from freshwater, salt water, and a variety of foods and are frequently isolated from patients with diarrhea and wound infections. Aeromonas spp. produce an impressive array of virulence factors, including hemolysins, cytotonic and cytotoxic enterotoxins, proteases, lipases, leucocidins, endotoxin, adhesions, and an S-layer (2-4). Our laboratory isolated and molecularly characterized an aerolysin-related cytotoxic enterotoxin (Act)
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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