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We recently identified a novel type III secretion system (T3SS) effector, AexU, from a diarrheal isolate SSU of Aeromonas hydrophila, and demonstrated that mice infected with the DeltaaexU mutant were significantly protected from mortality. Although the NH(2)-terminal domain of this toxin exhibits homology to AexT of A. salmonicida, a fish pathogen, and ExoT/S of Pseudomonas aeruginosa, the COOH-terminal domain of AexU is unique, with no homology to any known proteins in the NCBI database. In this study, we purified the full-length AexU and its NH(2)-terminal (amino acid residues 1-231) and COOH-terminal (amino acid residues 232-512) domains after expression of their corresponding genes in Escherichia coli as histidine-tag fusion proteins using the bacteriophage T7 RNA polymerase/promoter-based pET-30a vector system. The full-length and NH(2)- and COOH-terminal domains of AexU exhibited ADP-ribosyltransferase activity, with the former two exhibiting much higher activity than the latter. These different forms of AexU were also successfully expressed and produced in the HeLa Tet-Off cell system using a pBI-EGFP vector, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and intracellular staining of the toxin using flow cytometric analysis. Production of AexU in HeLa cells resulted in possible actin reorganization and cell rounding, as determined by phalloidin staining and confocal microscopy. Based on electron microscopy, the toxin also caused chromatin condensation, which is indicative of apoptosis. Apoptosis of HeLa cells expressing and producing AexU was confirmed by 7-amino actinomycin D (7-AAD) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] assays, by detection of cytoplasmic histone-associated DNA fragments, and by activation of caspases 3 and 9. These effects were much more pronounced in host cells that expressed and produced the full-length or NH(2)-terminal domain of AexU, compared to those that expressed and produced the COOH-terminal domain or the vector alone. This study represents the first characterization of this novel T3SS effector.

We previously generated a double knockout mutant (act/aopB) of a diarrheal isolate SSU of A. hydrophila, in which the genes encoding Aeromonas outer membrane protein B (AopB), a structural component of the type III secretion system (T3SS), and a type II (T2)-secreted cytotoxic enterotoxin gene (act) were deleted. This mutant exhibited minimal virulence in mice, compared to animals infected with wild-type (WT) A. hydrophila. Based on microarray analyses, WT A. hydrophila altered the expression of 434 and 80 genes in murine macrophages (RAW 264.7) and human colonic epithelial cells (HT-29), respectively. Approximately half of these gene expression alterations were abrogated when host cells were infected instead with the act/aopB mutant. In this study, we used microarrays to examine early host transcriptional responses in spleens of mice infected for 3h with WT A. hydrophila or its act/aopB mutant. Our data indicated that expression of 221 genes was altered (158 up-regulated and 63 down-regulated) in spleens of WT bacteria-infected animals. There were 21 genes that were consistently more highly expressed in WT A. hydrophila-infected mice, compared to mice infected with its act/aopB mutant. Ten of these genes were either induced to a lesser extent (e.g., interleukin-6, macrophage inflammatory protein-2, and cyclooxygenase-2), not altered at all (e.g., killer cell lectin-like receptor subfamily B member A), or down-regulated (e.g., cytochrome P450) in animals infected with A. hydrophila, compared to phosphate-buffered saline-infected control animals, when the mutant was used instead of the WT. We verified the microarray results at the transcript level by performing real-time reverse transcriptase-polymerase chain reaction on selected genes and at the protein level by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. This is the first study demonstrating in vivo gene regulation in mice infected with A. hydrophila and the contribution of virulence factors and host responses to the disease process.

In our previous study, we deleted the gene encoding Aeromonas outer membrane protein B (AopB), a structural component of the type III secretion system (T3SS) from a cytotoxic enterotoxin gene (act)-minus diarrheal isolate SSU of Aeromonas hydrophila. Our laboratory also molecularly characterized the cytotoxic enterotoxin (Act), which is secreted by the bacterium utilizing the type II secretion system (T2SS). The act/aopB mutant exhibited significantly reduced cytotoxicity to cultured cells (e.g. RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells) and was avirulent in mice. In this study, we developed additional A. hydrophila mutants in which T3SS-associated ascV and acrV genes were deleted, either individually or in combination with that of the act gene, to examine host-pathogen interactions. A significant reduction in the induction of inflammatory cytokines and chemokines was noted in the sera of mice infected with these mutants when compared to animals infected with wild-type (WT) A. hydrophila. After infection with the WT and act/aopB mutant, we performed microarray analyses on RNA from the above-mentioned murine macrophages and human colonic epithelial cells to examine global cellular transcriptional responses. Based on three independent experiments, WT A. hydrophila altered the expression of 434 genes in RAW 264.7 cells and 80 genes in HT-29 cells. Alteration in the expression of 209 macrophage and 32 epithelial cell genes was reduced when the act/aopB mutant was used, compared to when cells were infected with the WT bacterium, indicating the involvement of Act and/or AopB in transcriptional regulation of these genes. We verified up-regulation of 15 genes by real-time reverse transcriptase-polymerase chain reaction and confirmed A. hydrophila WT-versus mutant-induced production of cytokines/chemokines in supernatants from RAW 264.7 and HT-29 cells. This is the first description of host cell transcriptional responses to A. hydrophila infection.

We investigated the potential of the cytotoxic enterotoxin (Act) of Aeromonas hydrophila to bind to 1869 human and 4319 yeast proteins, using protein microarray technology. Act was capable of binding nine different human proteins, including the SNARE complex scaffolding protein synaptosomal-associated protein 23 (SNAP23), galectin-3, and guanylate kinase 1 (GUK-1). Act was also able to bind to four of the yeast proteins examined, which included the vesicle tethering protein Vsp52. We verified interaction of Act with murine and human SNAP23, galectin-3, and GUK-1 by sandwich Western blot analysis. In order to determine the physiological relevance of Act binding to these three proteins, we performed small interfering RNA (siRNA) gene knockdown experiments in RAW 264.7 cells, a murine macrophage cell line in which Act-induced signaling and cell death is well characterized. Based on real-time reverse transcriptase-polymerase chain reaction, siRNA transfection of RAW 264.7 cells with specific oligonucleotides reduced the expression of genes encoding SNAP23, galectin-3, and GUK-1 by 62, 63, and 99%, respectively. Knockdown of galectin-3 and SNAP23, but not GUK-1, significantly reduced Act-induced apoptosis of host cells, as determined by TUNEL (TdT-mediated dUTP nick end labeling) assay, lactate dehydrogenase release, Giemsa staining, and reduction in activation of caspase 3, compared to toxin-treated macrophages that were transfected with a random sequence control siRNA. We also performed these assays using a human intestinal epithelial cell line (HT-29) and observed a similar trend of galectin-3 and SNAP23 association with Act-induced apoptosis. This is the first report of putative protein binding partners for this toxin and potential mediators/regulators of Act-induced apoptosis.

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.

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.

A cytotoxic enterotoxin (Act) of Aeromonas hydrophila possesses several biological activities, induces an inflammatory response in the host, and causes apoptosis of murine macrophages. In this study, we utilized five target cell types (a murine macrophage cell line (RAW 264.7), bone marrow-derived transformed macrophages, murine peritoneal macrophages, and two human intestinal epithelial cell lines (T84 and HT-29)) to investigate the effect of Act on mitogen-activated protein kinase (MAPK) pathways and mechanisms leading to apoptosis. As demonstrated by immunoprecipitation/kinase assays or Western blot analysis, Act activated stress-associated p38, c-Jun NH(2)-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2) in these cells. Act also induced phosphorylation of upstream MAPK factors (MAPK kinase 3/6 (MKK3/6), MKK4, and MAP/ERK kinase 1 (MEK1)) and downstream effectors (MAPK-activated protein kinase-2, activating transcription factor-2, and c-Jun). Act evoked cell membrane blebbing, caspase 3-cleavage, and activation of caspases 8 and 9 in these cells. In macrophages that do not express functional tumor necrosis factor receptors, apoptosis and caspase activities were significantly decreased. Immunoblotting of host whole cell lysates revealed Act-induced up-regulation of apoptosis-related proteins, including the mitochondrial proteins cytochrome c and apoptosis-inducing factor. However, mitochondrial membrane depolarization was not detected in response to Act. Taken together, the data demonstrated for the first time Act-induced activation of MAPK signaling and classical caspase-associated apoptosis in macrophages and intestinal epithelial cells. Given the importance of MAPK pathways and apoptosis in inflammation-associated diseases, this study provided new insights into the mechanism of action of Act on host cells.

We performed microarray analyses of murine peritoneal macrophages to examine cellular transcriptional responses to a cytotoxic enterotoxin of Aeromonas hydrophila. While 66% of altered genes were common to both primary macrophages and the murine macrophage cell line RAW 264.7, Act caused expression changes of 28 genes specifically in murine peritoneal macrophages.

Aeromonas hydrophila is an emerging human pathogen that leads to gastroenteritis and other invasive diseases. By using a murine peritoneal culture (MPC) model, we identified via restriction fragment differential display PCR (RFDDPCR) five genes of A. hydrophila that were differentially expressed under in vivo versus in vitro growth conditions. The gene encoding enolase was among those five genes that were differentially up regulated. Enolase is a glycolytic enzyme and its surface expression was recently shown to be important in the pathogenesis of a gram-positive bacterium Streptococcus pyogenes. By Western blot analysis and Immunogold staining, we demonstrated secretion and surface expression of enolase in A. hydrophila. We also showed that the whole cells of A. hydrophila had strong enolase activity. Using an enzyme-linked immunosorbant assay and sandwich Western blot analysis, we demonstrated binding of enolase to human plasminogen, which is involved in the fibrinolytic system of the host. We cloned the A. hydrophila enolase gene, which exhibited 62% homology at the DNA level and 57% homology at the amino acid level when compared to S. pyogenes enolase. This is a first report describing the increased expression of enolase gene in vivo that could potentially contribute to the pathogenesis of A. hydrophila infections., (Copyright 2003 Elsevier Science Ltd.)