Cytotoxic Enterotoxin Research Articles

Putative virulence properties of Aeromonas strains isolated from food, environmental and clinical sources in Italy: A comparative study

The distribution of virulence properties in 142 strains of Aeromonas isolated from diarrhoeic patients, food and surface water in Italy and identified by biochemical and molecular methods was investigated. The virulence properties studied were the presence of genes for the aerolysin (aerA), heat-stable cytotonic enterotoxin (ast), heat-labile cytotonic enterotoxin (alt), cytotoxic enterotoxin (act); and cytotoxicity for Vero cells and adhesion on Hep-2 cells. A. hydrophila and A. caviae were the species most commonly isolated from clinical and environmental samples (9/30; 30.0% and 5/27; 18.5%, respectively) while mesophilic A. salmonicida was most common in food samples (19/80; 23.7%). Out of 142 strains, 86 (60.6%) were positive for at least one of the virulence properties. All the toxin genes were present in 4/18 (22.3%) of clinical strains. Most of the food isolates (54/55; 98.2%) were cytotoxic and most of the environmental strains (12/13; 92.3%) were adhesive. The aerA gene was present in most toxigenic strains (72/86; 83.7%), irrespective of their origin. The growth temperature affected the expression of cytotoxicity and adhesivity. Aeromonas strains from food and surface water frequently had toxin gene patterns similar to those of clinical strains and expressed virulence properties at human body temperature. These findings indicate that aeromonads have the potential to cause human illness and confirm the role of food and water as vehicles for Aeromonas diseases.

Re‐identification of Aeromonas isolates from chironomid egg masses as the potential pathogenic bacteria Aeromonas aquariorum

Egg masses of the non-biting midge Chironomous sp. have recently been found to serve as a reservoir for Vibrio cholerae and Aeromonas species. These insects are widely distributed in freshwater and evidence suggests that they may disseminate pathogenic bacteria species into drinking water systems. In the current study the taxonomy of 26 Aeromonas isolates, previously recovered from chironomid egg masses, was re-evaluated. It was found that 23 isolates, which had previously been identified as Aeromonas caviae, could belong to the recently described species Aeromonas aquariorum by their biochemical traits. To date, A. aquariorum has been found in ornamental fish and also in human extra-intestinal infections. ERIC-PCR genotyping differentiated 11 strains within the 23 A. aquariorum isolates, whose identity was confirmed by their rpoD gene sequences. Strains were found to possess the following virulence-associated genes: alt (90.9%), ahpB (81.8%), pla/lip/lipH3/apl-1/lip (54.5%), fla (27.3%), act/hylA/aerA (27.3%), ascF-ascG (81.8%) and aexT (9%) encoding for the cytotonic heat-labile enterotoxin, elastase, lipase, flagella, cytotoxic enterotoxins, the Type III Secretion System and the AexT toxin delivered by this system respectively. These findings indicate that chironomid egg masses harbour strains of A. aquariorum, which bear an important number of virulence genes, and that this species was misidentified originally as A. caviae.

Electrochemical detection of foodborne pathogen Aeromonas hydrophila by DNA hybridization biosensor

The paper describes an electrochemical DNA biosensor used for the detection of Aeromonas hydrophila. This opportunistic pathogen is recognized as an emerging foodborne hazard and is associated with a variety of virulence factors including production of cytotoxic enterotoxin aerolysin. The genosensor recognition layer was prepared using mixed self-assembled monolayer (SAM) consisting of thiolated single-stranded DNA probe (ssDNA) and diluent molecules – mercaptoalcohol: mercaptoethanol (MCE) or mercaptohexanol (MCH) or mercaptononanol (MCN). The voltammetric examination of double-layer capacitance of biosensor recognition interface supported by chronocoulometric quantitation of DNA present on the electrode surface showed that mixed ssDNA and MCH monolayer revealed the lowest defectiveness. Its double-layer capacitance equaled 4.0 μF cm −2 and ssDNA probe surface coverage reached 8.5 × 10 11 molecules cm −2 of gold electrode surface. Chronocoulometric quantitation of DNA and square wave voltammetry (SWV) measurements of electroactive indicator, methylene blue (MB) were performed to investigate the influence of hybridization reaction time, concentration of target DNA fragments, and presence of non-complementary DNA on the electrochemical response of genosensor recognition interface. The biosensor enabled distinction between the DNA samples isolated from A. hydrophila (present at the concentration of 2.5 μg cm −3) and other microbial DNA.

Claudin expression profiles in Epstein-Barr virus-associated nasopharyngeal carcinoma

Claudins are a family of proteins that are structural and functional components of tight junctions and have crucial roles in the maintenance of cellular arrangement, adhesion and paracellular transport. Recent studies have shown that changes and/or loss of claudin expression plays an important role in tumorigenesis and tumor progression, and altered expression of claudins has been reported in various human carcinomas. Non-keratinizing nasopharyngeal carcinoma (NPC) is a common Epstein-Barr virus (EBV)-associated carcinoma with characteristic clinicopathological features. The aim of this study was to investigate claudin expression profiles in EBV-associated non-keratinizing NPC. We analyzed expressions of claudin-1, -2, -3, and -4 in 18 cases of EBV-associated non-keratinizing NPC by immunohistochemical methods. Claudin-1 was expressed in all 18 cases, but claudin-2 was not expressed in any of the 18 cases. Claudin-3 expression was variable, with 8 of the 18 cases (45%) showing no immunoreactivity for claudin-3. Claudin-4 displayed positive immunoreactivity in all cases, even in claudin-3-negative cases. Claudin-3 and -4 are receptors for cytotoxic Clostridium perfringens enterotoxin (CPE) and CPE has emerged as a potential therapeutic target for malignant tumors expressing claudin-3 and/or -4, because CPE specifically and rapidly lyses cells expressing these proteins. Clinically, treatment of distant metastases is a serious problem in EBV-associated non-keratinizing NPC, because frequently there is lymph node involvement and distant metastasis before detection of the primary tumor. Therefore, CPE therapy may be a potential therapeutic target for EBV-associated non-keratinizing NPC, since our results clearly showed claudin-3 and/or -4 expression in all cases studied.

Multiplex PCR Method for Detection of Three Aeromonas Enterotoxin Genes

A novel multiplex PCR method using three sets of specific primers was developed for the detection of the cytotoxic (act), heat-labile (alt), and heat-stable (ast) enterotoxin genes in Aeromonas spp. This assay was used to characterize 35 reference strains as well as 537 food-borne isolates. A total of seven gene pattern combinations were encountered, including act, alt, act/alt, act/alt/ast, act/alt/148-bp amplicon, alt/ast, and alt/148-bp amplicon. The alt gene was detected with 34 reference strains (97%) and occurred singly in 14% of these strains. The frequency of occurrence of the act/alt, act/alt/ast, and alt/ast gene patterns in reference strains was 14 (40%), 2 (6%), and 2 (6%), respectively. An unpredicted amplicon was detected in 11 reference strains (31%). Characterization of this amplicon showed that its size was 148 bp, as generated by the AHLF and AHLR primers, and that it uniquely aligned with the Aeromonas salmonicida A449 genome sequence (GenBank accession number CP000644). This amplicon was named Aeromonas salmonicida subsp. salmonicida hypothetical protein amplicon (AssHPA). In the 537 food-borne isolates, the act and alt genes were most dominant and were detected in 349 (65%) and 452 (84%) isolates, respectively, either alone or in combinations. The act and alt genes occurred singly in 30 (6%) and 128 (24%) of these strains, respectively. The act/alt gene pattern occurred in 315 isolates (59%), whereas the ast gene was always linked to strains exhibiting the act/alt/ast and alt/ast gene combinations in 4 (0.7%) and 5 (0.9%) isolates, respectively. The uniplex amplification of three enterotoxin genes separately confirms the specificity of the unique selected primers. This multiplex PCR is rapid and simple and can detect the presence of three Aeromonas enterotoxin genes in a single assay.

Detection and characterization of virulence genes and integrons in Aeromonas veronii isolated from catfish

The presence of virulence genes and integrons was determined in 81 strains of Aeromonas veronii isolated from farm-raised catfish. Polymerase chain reaction (PCR) protocols were used to determine the presence of genes for cytotoxic enterotoxin ( act), aerolysin ( aerA), two cytotonic enterotoxins ( ast, alt), lipase ( lip), glycerophospholipid:cholesterol acyltransferase ( gcaT), serine protease ( ser), DNases ( exu), elastase ( ahyB) and the structural gene flagellin ( fla) in the template DNA. Oligonucleotide primers amplified a 231-bp region of the act gene from the template DNA of 97.0% of the isolates. Primers specific for the amplification of the aerA gene amplified a 431-bp region of the aerA gene from the template DNA of 96.0% of the isolates. None of the isolates contained ast or alt genes. Oligonucleotide primers specific for the amplification of lip, gcaT, ser and fla genes, amplified their respective amplicons from 85.0, 78.0, 82.0 and 80.0% of the isolates. None of the isolates contained exu or the elastase genes. Several of the isolates (48.0%) contained class I integrons that confer resistance to multiple antibiotics; various sizes between 0.6 and 3.1 kb were found. None of the isolates contained Class II integrons. Our results indicate that farm-raised catfish may be a source of pathogenic A. veronii and that the potential health risks posed by virulent strains of A. veronii should not be underestimated.

Aeromonas Spp. Human Isolates Induce Apoptosis of Murine Macrophages

Interactions of Aeromonas caviae, Aeromonas veronii biotype sobria, and Aeromonas hydrophila strains isolated from fecal specimens of humans with gastroenteritis on murine macrophages, J774 cells, were investigated. Analyses of cellular morphology and DNA fragmentation in phagocytes infected with these strains exhibited typical characteristic features of cells undergoing apoptosis. We observed the morphological changes, including condensation of nuclear chromatin, formation of apoptotic bodies and blebbing of cell membrane, and fragmentation of nuclear DNA into oligonucleosomal fragments. The lowest apoptotic index did not exceed 25%, whereas the highest reached 78% at 24 h and 96% at 48 h after infection. After incubation of J774 cells with cytotoxic enterotoxin isolated from A. veronii biotype sobria strain, we noted that the toxin was able to trigger cytotoxicity and apoptosis of macrophages. The results indicate that apoptosis could be one of the mechanisms contributing to the development of Aeromonas-associated diarrheal disease.

Multiplex PCR Detection of Enterotoxin Genes in Aeromonas spp. from Suspect Food Samples in Northern Taiwan

Aeromonads possess an array of virulence factors and are causative agents of a number of human infections. Among them, genes of one cytotoxic (Act) and two cytotonic (Alt, Ast) enterotoxins are implicated in a human diarrheal disease. A rapid, specific, simultaneous detection of these enterotoxin genes in suspected food poisoning samples is not yet reported. Hence, a multiplex PCR assay was designed to amplify the cytotoxic (act), heat-labile cytotonic (alt), and heat-stable cytotonic (ast) enterotoxin genes of aeromonads. The PCR assay was tested with 133 Aeromonas spp. isolated from suspect food poisoning samples and retail samples of poultry and fish from wet markets in and around Taipei, Northern Taiwan. The Aeromonas spp. isolates were divided into six genotypes based on absence or presence of one or more enterotoxin genes. Of these 133 isolates, Aeromonas caviae (52.5%) and Aeromonas hydrophila (43.4%) were the most frequently isolated species from food poisoning samples and retail samples, respectively. Among the species, A. hydrophila had a significantly higher proportion for harboring three enterotoxin genes than had the others, whereas Aeromonas encheleia, considered a nonpathogen, was found harboring three enterotoxin genes. The multiplex PCR assays are rapid and specific, and provide a useful tool for the detection and genotyping of enterotoxin genes of aeromonads.

Mutation in the S-ribosylhomocysteinase ( luxS) gene involved in quorum sensing affects biofilm formation and virulence in a clinical isolate of Aeromonas hydrophila

A diarrheal isolate SSU of Aeromonas hydrophila produces a cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. Our laboratory has characterized from the above Aeromonas strain, in addition to Act, the type 3- and T6-secretion systems and their effectors, as well as the genes shown to modulate the production of AI-1-like autoinducers, N-acylhomoserine lactones (AHLs) involved in quorum sensing (QS). In this study, we demonstrated the presence of an S-ribosylhomocysteinase (LuxS)-based autoinducer (AI)-2 QS system in A. hydrophila SSU and its contribution to bacterial virulence. The luxS isogenic mutant of A. hydrophila, which we prepared by marker exchange mutagenesis, showed an alteration in the dynamics and architecture of the biofilm formation, a decrease in the motility of the bacterium, and an enhanced virulence in the septicemic mouse model. Moreover, these effects of the mutation could be complemented. Enhanced production of the biofilm exopolysaccharide and filaments in the mutant strain were presumably the major causes of the observed phenotype. Our earlier studies indicated that the wild-type A. hydrophila with overproduction of DNA adenine methyltransferase (Dam) had significantly reduced motility, greater hemolytic activity associated with Act, and an enhanced ability to produce AI-1 lactones. Furthermore, such a Dam-overproducing strain was not lethal to mice. On the contrary, the luxS mutant with Dam overproduction showed an increased motility and had no effect on lactone production. In addition, the Dam-overproducing luxS mutant strain was not altered in its ability to induce lethality in a mouse model of infection when compared to the parental strain which overproduced Dam. We suggested that an altered gene expression in the luxS mutant of A. hydrophila SSU, as it related to biofilm formation and virulence, might be linked with the interruption of the bacterial metabolic pathway, specifically of methionine synthesis.

Molecular characterization of a functional type VI secretion system from a clinical isolate of Aeromonas hydrophila

Our laboratory recently molecularly characterized the type II secretion system (T2SS)-associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrophila infections was subsequently delineated in in vitro and in vivo models. In this study, we characterized the new type VI secretion system (T6SS) from isolate SSU of A. hydrophila and demonstrated its role in bacterial virulence. Study of the role of T6SS in bacterial virulence is in its infancy, and there are, accordingly, only limited, recent reports directed toward a better understanding its role in bacterial pathogenesis. We have provided evidence that the virulence-associated secretion ( vas) genes vasH (Sigma 54-dependent transcriptional regulator) and vasK (encoding protein of unknown function) are essential for expression of the genes encoding the T6SS and/or they constituted important components of the T6SS. Deletion of the vasH gene prevented expression of the potential translocon hemolysin coregulated protein (Hcp) encoding gene from bacteria, while the vasK gene deletion prevented secretion but not translocation of Hcp into host cells. The secretion of Hcp was independent of the T3SS and the flagellar system. We demonstrated that secreted Hcp could bind to the murine RAW 264.7 macrophages from outside, in addition to its ability to be translocated into host cells. Further, the vasH and vasK mutants were less toxic to murine macrophages and human epithelial HeLa cells, and these mutants were more efficiently phagocytosed by macrophages. We also provided evidence that the expression of the hcp gene in the HeLa cell resulted in apoptosis of the host cells. Finally, the vasH and vasK mutants of A. hydrophila were less virulent in a septicemic mouse model of infection, and animals immunized with recombinant Hcp were protected from subsequent challenge with the wild-type (WT) bacterium. In addition, mice infected with the WT A. hydrophila had circulating antibodies to Hcp, indicating an important role of T6SS in the pathogenesis of A. hydrophila infections. Taken together, we have characterized the T6SS from Aeromonas for the first time and provided new features of this secretion system not yet known for other pathogens.

Identification of a new hemolysin from diarrheal isolate SSU ofAeromonas hydrophila

A clinical strain SSU of Aeromonas hydrophila produces a potent cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. A new gene, which encoded a hemolysin of 439-amino acid residues with a molecular mass of 49 kDa, was identified. This hemolysin (HlyA) was detected based on the observation that the act gene minus mutant of A. hydrophila SSU still had residual hemolytic activity. The new hemolysin gene (hlyA) was cloned, sequenced, and overexpressed in Escherichia coli. The hlyA gene exhibited 96% identity with its homolog found in a recently annotated genome sequence of an environmental isolate, namely the type strain ATCC 7966 of A. hydrophila subspecies hydrophila. The hlyA gene did not exhibit any homology with other known hemolysins and aerolysin genes detected in Aeromonas isolates. However, this hemolysin exhibited significant homology with hemolysin of Vibrio vulnificus as well as with the cystathionine beta synthase domain protein of Shewanella oneidensis. The HlyA protein was activated only after treatment with trypsin and the resulting hemolytic activity was not neutralizable with antibodies to Act. The presence of the hlyA gene in clinical and water Aeromonas isolates was investigated and DNA fingerprint analysis was performed to demonstrate its possible role in Aeromonas virulence.

Overexpression of Clostridium perfringens Enterotoxin Receptors Claudin-3 and Claudin-4 in Uterine Carcinosarcomas

To evaluate the expression levels of claudin-3 and claudin-4, the low- and high-affinity receptors, respectively, for the cytotoxic Clostridium perfringens enterotoxin (CPE) in uterine carcinosarcomas and explore the potential for targeting these receptors in the treatment of this aggressive uterine tumor. We analyzed claudin-3 and claudin-4 receptor expression at mRNA and protein levels in flash frozen and formalin-fixed, paraffin-embedded carcinosarcoma specimens. Recombinant CPE was used as a novel therapy against chemotherapy-resistant carcinosarcoma cell lines in vitro. The therapeutic effect of sublethal doses of CPE was studied in severe combined immunodeficient mouse xenografts harboring large s.c. carcinosarcomas. All flash-frozen carcinosarcoma biopsies (12 of 12) and short-term carcinosarcoma cell lines evaluated overexpressed claudin-3 and claudin-4 by quantitative reverse transcription-PCR. Membranous immunoreactivity for claudin-4 protein expression was documented in 80% (20 of 25) of primary tumors and 100% (6 of 6) of the metastatic carcinosarcomas, whereas negligible staining was found in normal endometrial cells. Regardless of their resistance to chemotherapeutic agents, all short-term carcinosarcoma cell lines tested died within 1 h of exposure to 3.3 microg/mL of CPE in vitro. Intratumoral injections of well-tolerated doses of CPE in large s.c. carcinosarcoma xenografts led to large areas of tumor cell necrosis and tumor disappearance in all treated animals. Claudin-3 and claudin-4 receptors are highly overexpressed in carcinosarcoma. These proteins may offer promising targets for the use of CPE as a novel type-specific therapy against this biologically aggressive variant of endometrial cancer.

Overexpression of claudin‐3 and claudin‐4 receptors in uterine serous papillary carcinoma

Uterine serous papillary carcinoma (USPC) represents a highly aggressive variant of endometrial cancer. Using gene expression profiling, we recently identified high expression of the claudin-3 and claudin-4 receptors in a limited set of USPC. These tight junction proteins represent the low- and high-affinity receptors, respectively, for the cytotoxic Clostridium perfringens enterotoxin (CPE) and are sufficient to mediate CPE binding and trigger subsequent toxin-mediated cytolysis. The potential for targeting this pathway in the treatment of USPC was explored. Claudin-3 and claudin-4 receptor expression was analyzed at the mRNA and protein levels in flash-frozen and formalin-fixed, paraffin-embedded tissue from 20 consecutive USPC patients. The potential of recombinant CPE as a novel therapy against primary, metastatic, and chemotherapy-resistant USPC cell lines was also investigated in vitro. Finally, the in vivo therapeutic effect of sublethal doses of CPE was studied in SCID mouse xenografts harboring subcutaneous and intraperitoneal USPC that expressed claudin-3 and claudin-4. In all, 100% (20 out of 20) of the primary flash-frozen USPC tested overexpressed 1 or both CPE receptors by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). Membranous immunoreactivity for claudin-4 protein expression was documented in the majority of USPC specimens tested by immunohistochemistry, whereas only a low level of membranous staining was found in normal endometrial control tissue samples. When primary and metastatic short-term USPC cell lines were incubated with different concentrations of CPE in vitro, a dose-dependent cytotoxic effect was demonstrated. In vivo, intratumoral injections of well-tolerated doses of CPE in large subcutaneous USPC xenografts led to large areas of tumor cell necrosis and tumor disappearance in all the treated animals, whereas sublethal intraperitoneal injections of CPE had a significant inhibitory effect on tumor progression, with extended survival of animals harboring chemotherapy-resistant intra-abdominal USPC carcinomatosis. Claudin-3 and claudin-4 receptors may offer promising targets for the use of CPE as a novel type-specific therapy against this highly aggressive and chemotherapy-resistant variant of endometrial cancer.

Global transcriptional responses of wild-type Aeromonas hydrophila and its virulence-deficient mutant in a murine model of infection

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 3 h 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.

Mutations within the Catalytic Motif of DNA Adenine Methyltransferase (Dam) of Aeromonas hydrophila Cause the Virulence of the Dam-Overproducing Strain To Revert to That of the Wild-Type Phenotype

In this study, we demonstrated that the methyltransferase activity associated with Dam was essential for attenuation of Aeromonas hydrophila virulence. We mutated aspartic acid and tyrosine residues to alanine within the conserved DPPY catalytic motif of Dam and transformed the pBAD/damD/A, pBAD/damY/A, and pBAD/damAhSSU (with the native dam gene) recombinant plasmids into the Escherichia coli GM33 (dam-deficient) strain. Genomic DNA (gDNA) isolated from either of the E. coli GM33 strains harboring the pBAD vector with the mutated dam gene was resistant to DpnI digestion and sensitive to DpnII restriction endonuclease cutting. These findings were contrary to those with the gDNA of E. coli GM33 strain containing the pBAD/damAhSSU plasmid, indicating nonmethylation of E. coli gDNA with mutated Dam. Overproduction of mutated Dam in A. hydrophila resulted in bacterial motility, hemolytic and cytotoxic activities associated with the cytotoxic enterotoxin (Act), and protease activity similar to that of the wild-type (WT) bacterium, which harbored the pBAD vector and served as a control strain. On the contrary, overproduction of native Dam resulted in decreased bacterial motility, increased Act-associated biological effects, and increased protease activity. Lactone production, an indicator of quorum sensing, was increased when the native dam gene was overexpressed, with its levels returning to that of the control strain when the dam gene was mutated. These effects of Dam appeared to be mediated through a regulatory glucose-inhibited division A protein. Infection of mice with the mutated Dam-overproducing strains resulted in mortality rates similar to those for the control strain, with 100% of the animals dying within 2 to 3 days with two 50% lethal doses (LD50s) of the WT bacterium. Importantly, immunization of mice with a native-Dam-overproducing strain at the same LD50 did not result in any lethality and provided protection to animals after subsequent challenge with a lethal dose of the control strain.

Deletion of the genes encoding the type III secretion system and cytotoxic enterotoxin alters host responses to Aeromonas hydrophila infection

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.

Potential involvement of galectin-3 and SNAP23 in Aeromonas hydrophila cytotoxic enterotoxin-induced host cell apoptosis

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.

DNA Adenine Methyltransferase Influences the Virulence of Aeromonas hydrophila

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.

The Type III Secretion System and Cytotoxic Enterotoxin Alter the Virulence of Aeromonas hydrophila

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.

Treatment of Chemotherapy-Resistant Human Ovarian Cancer Xenografts in C.B-17/SCID Mice by Intraperitoneal Administration of Clostridium perfringens Enterotoxin

Ovarian cancer remains the most lethal gynecologic malignancy in the United States. Although many patients with advanced-stage disease initially respond to standard combinations of surgical and cytotoxic therapy, nearly 90% develop recurrence and inevitably die from the development of chemotherapy-resistant disease. The discovery of novel and effective therapy against chemotherapy-resistant/recurrent ovarian cancer remains a high priority. Using expression profiling, we and others have recently found claudin-3 and claudin-4 genes to be highly expressed in ovarian cancer. Because these tight junction proteins have been described as the low- and high-affinity receptors, respectively, for the cytotoxic Clostridium perfringens enterotoxin (CPE), in this study we investigated the level of expression of claudin-3 and/or claudin-4 in chemotherapy-naive and chemotherapy-resistant primary human ovarian cancers as well as their sensitivity to CPE treatment in vitro. We report that 100% (17 of 17) of the primary ovarian tumors tested overexpress one or both CPE receptors by quantitative reverse transcription-PCR. All ovarian tumors showed a dose-dependent cytotoxic effect to CPE in vitro. Importantly, chemotherapy-resistant/recurrent ovarian tumors were found to express claudin-3 and claudin-4 genes at significantly higher levels when compared with chemotherapy-naive ovarian cancers. All primary ovarian tumors tested, regardless of their resistance to chemotherapeutic agents, died within 24 hours to the exposure to 3.3 microg/mL CPE in vitro. In addition, we have studied the in vivo efficacy of i.p. CPE therapy in SCID mouse xenografts in a highly relevant clinical model of chemotherapy-resistant freshly explanted human ovarian cancer (i.e., OVA-1). Multiple i.p. administration of sublethal doses of CPE every 3 days significantly inhibited tumor growth in 100% of mice harboring 1 week established OVA-1. Repeated i.p. doses of CPE also had a significant inhibitory effect on tumor progression with extended survival of animals harboring large ovarian tumor burdens (i.e., 4-week established OVA-1). Our findings suggest that CPE may have potential as a novel treatment for chemotherapy-resistant/recurrent ovarian cancer.