Clifford W. Houston

Enterotoxins in Aeromonas-associated gastroenteritis

Aeromonas species produce an array of virulence factors, and the pathogenesis of Aeromonas infections is therefore complex and multifactorial. Aeromonas-associated gastroenteritis is especially a problem in young children. The potential involvement of enterotoxins in the pathogenesis of Aeromonas infections is discussed.

The Cytotoxic Enterotoxin of Aeromonas hydrophila Induces Proinflammatory Cytokine Production and Activates Arachidonic Acid Metabolism in Macrophages

ABSTRACT 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 Acts capacity to activate in macrophages arachidonic acid (AA) metabolism that leads to production of eicosanoids (e.g., prostaglandin E2[PGE2]). Our data indicated that Act stimulated the production of tumor necrosis factor alpha and upregulated the expression of genes encoding interleukin-1β (IL-1β) 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 PGE2 coupled to the cyclooxygenase-2 (COX-2) pathway. AA is a substrate for PGE2, and Act produced AA from phospholipids by inducing group V secretory phospholipase A2. We also demonstrated that Act increased cyclic AMP (cAMP) production in macrophages. cAMP, along with PGE2, 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-κB 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-κB and CREB. This is the first report of the detailed mechanisms of action of Act from A. hydrophila.

Molecular Characterization of a Glucose-Inhibited Division Gene, gidA, That Regulates Cytotoxic Enterotoxin of Aeromonas hydrophila

ABSTRACT By using a mini-transposon, we obtained two mutated strains of a diarrheal isolate, SSU, of Aeromonas hydrophila that exhibited a 50 to 53% reduction in the hemolytic activity and 83 to 87% less cytotoxic activity associated with the cytotoxic enterotoxin (Act). Act is a potent virulence factor of A. hydrophila and has been shown to contribute significantly to the development of both diarrhea and septicemia in animal models. Subsequent cloning and DNA sequence analysis revealed that transposon insertion occurred at different locations in these two mutants within the same 1,890-bp open reading frame for the glucose-inhibited division gene (gidA). A similar reduction in hemolytic (46%) and cytotoxic (81%) activity of Act was noted in the gidA isogenic mutant of A. hydrophila that was generated by marker exchange mutagenesis. Northern blot analysis revealed that the transcription of the cytotoxic enterotoxin gene (act) was not altered in the gidA transposon and isogenic mutants. However, by generating a chromosomal act::alkaline phosphatase gene (phoA) reporter construct, we demonstrated significantly reduced phosphatase activity in these mutants, indicating the effect of glucose-inhibited division (GidA) protein in modulating act gene expression at the translational level. The biological effects of Act in the gidA mutants were restored by complementation. The virulence of the gidA mutants in mice was dramatically reduced compared to the those of the wild-type (WT) and complemented strains of A. hydrophila. The histopathological examination of lungs, in particular, indicated severe congestion, alveolar hemorrhage, and acute inflammatory infiltrate in the interstitial compartment and the alveolar spaces when mice were infected with the WT and complemented strains. Minimal-to-mild changes were noted in the lungs with the gidA mutants. Taken together, our data indicate for the first time that GidA regulates the most-potent virulence factor of A. hydrophila, Act.

Differential expression of the enolase gene under in vivo versus in vitro growth conditions of Aeromonas hydrophila

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.

Elevated cAMP in intestinal epithelial cells during experimental cholera and salmonellosis

Cholera and salmonellosis are two diarrheal diseases in which intestinal tissue cyclic adenosine monophosphate (cAMP) concentrations are elevated. Investigations of each experimental disease were initiated to identify the specific intestinal cells containing the elevated cAMP. Epithelial cells were eluted from the mucosa of infected and control intestinal loops of adult rabbits, after which the cAMP content of the epithelial cell fractions and the lamina propria cells was extracted and assayed. The identity of the epithelial cells (in the villus tip-to-crypt cell gradient) was monitored by measuring their intracellular alkaline phosphatase activity, while scanning electron microscopy was used to visualize the effects of infection and cell elution techniques. Clearly, in both experimental cholera and salmonellosis, elevated cAMP levels were associated with crypt epithelial cells. Villus tip epithelial cells from either infection tended to contain less cAMP than those of noninfected control tissue. In Salmonella-infected loops, it was apparent that cAMP was also elevated in lamina propria cell fractions. Lamina propria cells from V. cholerae-infected intestinal loops contained only basal levels of cAMP. In vitro exposure of isolated intestinal cells from normal rabbit intestine to a cell-free lysate of Salmonella resulted in elevation of cAMP in the epithelial cells and lamina propria cells. We conclude that in experimental cholera and salmonellosis, significant elevation of the cAMP levels occurred in intestinal crypt cells, consistent with an enterotoxin-mediated mechanism. In Salmonella-infected loops, it was unclear if the increased concentration of cAMP in lamina propria cells was generated by enterotoxin released from the invasive salmonellae or by prostaglandins formed during the inflammatory response to the bacteria, or by both mechanisms.

INSULIN-LIKE GROWTH FACTOR-I PROMOTES MULTIDRUG RESISTANCE IN MCLM COLON CANCER CELLS

Insulin‐like growth factor‐I (IGF‐I) is known as a potent mitogen for a variety of cell types, including colon cancer cell lines. The objective of this study was to determine the effect of IGF‐I on cell death induced by cytotoxic agents actinomycin D (Act‐D), lovastatin (LOV), and doxorubicin (DOX) in the MCLM mouse colon cancer cell line, and the mechanisms involved. Subconfluent monolayer MCLM cells were treated with IGF‐I (25 ng/ml) for 12 h in serum‐free media. Various concentrations of cytotoxic agents then were added to the cells that were incubated continually at 37°C for 24 h. Cell survival was determined with the MTT (3‐[4‐5‐dimenthylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide) assay, which assesses mitochondrial function in living cells. The mRNA expression for multidrug resistance gene‐I (mdr‐I), c‐H‐ras, and manganese superoxide dismutase (MnSOD) in cells treated with IGF‐I was examined by Northern blot or RNase protection assays. The levels of p‐glycoprotein, a drug efflux pump encoded by the mdr‐I gene, were assessed by Western immunoblotting. Results demonstrated that (1) IGF‐I significantly inhibited the cell death and apoptosis of MCLM cells treated with Act‐D, LOV, or DOX; (2) IGF‐I increased mRNA expression for mdr‐I, c‐H‐ras, and MnSOD; (3) the p‐glycoproteins in cells treated with IGF‐I or stably transfected with c‐H‐ras were elevated when compared with control. These results suggest that IGF‐I protects MCLM cells against death induced by cytotoxic agents; this acquired drug resistance may be mediated by multiple mechanisms, including promoting expression of mdr‐I, c‐H‐ras, and MnSOD; whereas, the p‐glycoprotein level stimulated by IGF‐I may result partly from the increase of c‐H‐ras in the cells. J. Cell. Physiol. 175:141–148, 1998.

Aeromonas-associated diarrhea in children

In a 27-month prospective study, Aeromonas spp. were isolated from 7.3% of children with diarrhea and from 2.2% of controls. In 32 patients with diarrhea, ranging in age from 1 to 27 months old, Aeromonas spp. were the only potential bacterial enteropathogens isolated. Principal symptoms of Aeromonas-associated diarrhea were vomiting, fever and bloody stools. Diarrhea was often self-limiting and lasted for 10 days or less in 90% of patients. No secondary spread of diarrhea among close contacts was observed and no clear-cut seasonal patterns of Aeromonas isolation were found. Aeromonas caviae was the most frequently isolated species in fecal samples of patients (24 of 29 isolates) as well as controls (5 of 7 isolates). Cholera toxin cross-reactive cytotoxic enterotoxin was produced by a vast majority of Aeromonas isolates, as compared to a non-cholera toxin cross-reactive cytotonic enterotoxin. In addition no significant correlation was observed between severity of the diarrheal disease and different Aeromonas or the quantity of enterotoxins produced. In our geographic area Aeromonas spp., and A. caviae in particular, seem to be an important and frequent cause of diarrhea in young children.

Bombesin: an activator of specific Aeromonas antibody secretion in rat intestine

The effect of bombesin (BBS) in modulating the secretion of specific Aeromonasantibodies in rat intestine was determined. Rats were immunized with the culture supernatant of Aeromonas hydrophila,isolate SSU. This culture supernatant contained a number of toxins that may be considered virulence factors. After 24 days of immunization, rats were anesthetized and a 10-cm intestinal segment was perfused with phosphate-buffered saline. The effluents were collected for measurement of IgA and IgG by the enzyme-linked immunosorbent assay. When compared with the effect of intravenous administration of normal saline in the control group, intravenous injection of BBS (20 μg/kg) in the experimental group caused a significant increase in rat intestinal IgA and IgG in perfusates. The stimulatory effects of BBS on the presence of IgA and IgG were depressed partially by proglumide, a receptor antagonist of cholecystokinin (CCK) and gastrin. Treatment with pentagastrin (250 μg/kg) accelerated intestinal secretion of IgA, but failed to stimulate intestinal IgG secretion. In addition, intravenous injection of CCK-8 (120 ng/kg) evoked the intestinal secretion of either IgA or IgG. These findings demonstrated that BBS, gastrin, and CCK can stimulate antibody secretion in rat intestine and the stimulatory effect of BBS may be mediated partially via release of CCK and gastrin. These results suggest that neuropeptides such as BBS and gastrointestinal hormones, eg, CCK and gastrin, may participate in the regulation of intestinal secretion of IgA and IgG antibodies, respectively, in rats.

Cloning and expression of putative cytotonic enterotoxin-encoding genes from Aeromonas hydrophila

A genomic library from a diarrheal isolate, SSU, of Aeromonas hydrophila was constructed in a cosmid vector, pHC79, and in bacteriophage lambda EMBL3. Cell lysates from various Escherichia coli clones containing the recombinant cosmid were examined for their ability to elongate Chinese hamster ovary (CHO) cells, which is a typical enterotoxic response. Based on restriction analysis, a 4.0-kb SalI DNA fragment from one of the clones that exhibited enterotoxic activity was subcloned into a bacteriophage T7 RNA polymerase/promoter hyperexpression system. The cell lysate from this E. coli [pSL24] clone caused CHO cells to elongate and revealed the presence of a major 35-kDa polypeptide by [35S]methionine labeling and sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis (PAGE). The toxin was biologically heat labile, losing all activity within 20 min at 56 degrees C. In addition, another enterotoxin-producing clone, E. coli[pSBS32], was isolated from cosmid and lambda bacteriophage libraries. We localized this heat-stable (56 degrees C/20 min) enterotoxin to a 4.8-kb SalI-BamHI fragment. Both enterotoxins caused elevation of cyclic adenosine monophosphate (cAMP) in CHO cells. The DNA fragments encoding these enterotoxins did not hybridize with each other. However, a 4.8-kb SalI-BamHI DNA fragment encoding a heat-stable enterotoxin hybridized to a 3.5-kb BamHI DNA fragment of a plasmid, pHPC100, that contained a cytotonic enterotoxin-encoding gene isolated from A. trota. Our data suggest Aeromonas species produce different structural types of cytotonic enterotoxins that are functionally similar.

Effect of cultural conditions on the presence of a cholera-toxin cross-reactive factor in culture filtrates of Aeromonas hydrophila

Culture filtrates ofAeromonas hydrophila isolate SSU were shown to contain an extracellular product that antigenically cross-reacts with cholera enterotoxin as determined by the enzyme-linked immunosorbent assay (ELISA). We have termed this extracellular product “cholera-toxin cross-reactive (CTC) factor” until further characterization is accomplished. Various cultural conditions were examined to determine maximum CTC factor production. The results indicate that optimal conditions for the highest levels of extracellular CTC factor produced byA. hydrophila grown in CYE broth occurred when cultures were incubated at 37°C with shaking at 100 rpm. CYE culture filtrates caused fluid accumulation in rabbit ligated intestinal loops. Heating the culture filtrates at 56°C for 10 min resulted in the loss of biological activity and antigenic integrity of the CTC factor as determined by the rabbit ligated intestinal loop assay and theELISA, respectively. The data presented in this study suggest that culture filtrate containing this molecule, or aggregate of molecules, may play a role in the pathogenesis ofA. hydrophila-mediated disease. Further studies including the purification of this factor(s) presently are being conducted in our laboratory.