[Show abstract][Hide abstract] ABSTRACT: We examined the properties of exotoxins produced by Aeromonas trota (A. enteropelogenes), one of the diarrheagenic species of Aeromonadaceae. Nine of 19 A. trota isolates that grew on solid media containing erythrocytes showed hemolytic activity. However, the hemolytic activities of the culture supernatants of these hemolytic strains of A. trota were markedly lower than those of A. sobria when cultured in liquid medium, and the amount of hemolysin detected by immunoblotting using antiserum against the hemolysin produced by A. sobria was also low. A mouse intestine loop assay using living bacterial cells showed that A. trota 701 caused the significant accumulation of fluid, and antiserum against the hemolysin produced suppressed the enterotoxic action of A. trota 701. These results indicated that A. trota 701 was diarrheagenic and the hemolysin produced was the causative agent of the enterotoxic activity of A. trota. The hemolysin in A. sobria was previously shown to be secreted in a preform (inactive form) and be activated when the carboxy-terminal domain was cleaved off by proteases in the culture supernatant. Since mature hemolysin was detected in the culture supernatants of A. trota, we analyzed the extracellular protease produced by A. trota. Fifteen of 19 A. trota isolates that grew on solid media containing skim milk showed proteolytic activity. We subsequently found that most A. trota isolates possessed the serine protease gene, but not the metalloprotease gene. Therefore, we determined the nucleotide sequence of the serine protease gene and its chaperone A. trota gene. The results obtained revealed that the deduced amino acid sequences of serine protease and the chaperone were homologous to those of A. sobria with identities of 83.0% and 75.8%, respectively.
PLoS ONE 01/2014; 9(3):e91149. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previously, we cloned the metalloprotease gene of Aeromonas sobria (amp) and determined its nucleotide sequence (GenBank accession number DQ784565). The protease is composed of 591 amino acid residues. In this study, we purified the mature metalloprotease from the culture supernatant of A. sobria and determined the amino terminal sequence and molecular size of AMP. In addition, we examined the production of AMP diachronically and found that AMP emerges outside of the cell as an intermediate composed of mature and propeptide regions. Subsequently, we determined that the N-terminal amino acid sequence of the intermediate and found that the sequence is identical to that of the mature metalloprotease. This means that the intermediate is composed of a mature AMP region and a C-terminal propeptide. The cross culture experiment of mutants of metalloprotease and serine protease of A. sobria on skim milk agar medium indicates that the intermediate released outside of the cell is inactive and that serine protease produced by A. sobria accelerates the conversion of the intermediate from the inactive to the active form.
Microbiology and Immunology 10/2010; 54(10):596-605. · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To clarify the mechanisms of diarrheal disease induced by Aeromonas sobria, we examined whether prostaglandin E2 (PGE2) was involved in the intestinal secretory action of A. sobria hemolysin by use of a mouse intestinal loop model. The amount of PGE2 in jejunal fluid and the fluid accumulation ratio were directly related to the dose of hemolysin. The increase over time in the level of PGE2 was similar to that of the accumulated fluid. In addition, hemolysin-induced fluid secretion and PGE2 synthesis were inhibited by the selective cyclooxygenase 2 (COX-2) inhibitor NS-398 but not the COX-1 inhibitor SC-560. Western blot analysis revealed that hemolysin increased the COX-2 protein levels but reduced the COX-1 protein levels in mouse intestinal mucosa in vivo. These results suggest that PGE2 functions as an important mediator of diarrhea caused by hemolysin and that PGE2 is produced primarily through a COX-2-dependent mechanism. Subsequently, we examined the relationship between PGE2, cyclic AMP (cAMP), and cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels in mouse intestinal mucosa exposed to hemolysin. Hemolysin increased the levels of cAMP in the intestinal mucosa. NS-398 inhibited the increase in cAMP production, but SC-560 did not. In addition, H-89, a cAMP-dependent protein kinase A (PKA) inhibitor, and glibenclamide, a CFTR inhibitor, inhibited fluid accumulation. Taken together, these results indicate that hemolysin activates PGE2 production via COX-2 and that PGE2 stimulates cAMP production. cAMP then activates PKA, which in turn stimulates CFTR Cl- channels and finally leads to fluid accumulation in the intestines.
Infection and immunity 04/2008; 76(3):1076-82. · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aeromonas sobria hemolysin (ASH) is one of the major virulence factors produced by A. sobria, a causative agent of diarrhea in humans. We investigated the effects of ASH on anion transport in human colonic epithelial cells. ASH increased short circuit currents across the intestinal epithelia, which were suppressed by anion channel antagonists, such as carbonic anhydrase inhibitors, and by the removal of external HCO3-. Iliac fluid accumulation was also inhibited by carbonic anhydrase inhibitors. The results suggest that ASH activates HCO3- secretion, whose level correlates with the severity of diarrhea.
[Show abstract][Hide abstract] ABSTRACT: Subtilisin-like proteases have been grouped into six families based on a sequence of the catalytic domain. One of the six is the kexin family, of which furin is a representative protease. All members of the kexin family, except one, are from eukaryotes. The one prokaryotic protease is a serine protease of Aeromonas sorbria (ASP). Here, we examined the substrate specificity of ASP based on the cleavage of short peptides. The results showed that ASP preferentially cleaves the peptide bond following two basic residues, one of which is Lys, but not the bond following a single basic residue. This indicates that the tertiary structure around the catalytic domain of ASP resembles, but is not identical to that of furin. Prekallikrein was cleaved into four fragments by ASP, indicating that the protein must be cleaved at specific sequences.
[Show abstract][Hide abstract] ABSTRACT: Bacteria produce many types of hemolysin that induce diarrhea by mechanisms that are not completely understood. Aeromonas sobria hemolysin (ASH) is a major virulence factor produced by A. sobria, a human pathogen that causes diarrhea. Since epithelial cells in the intestine are the primary targets of hemolysin, we investigated the effects of ASH on ion transport in human colonic epithelial (Caco-2) cells. ASH increased short-circuit currents (Isc) in a dose-dependent manner, and it also activated a 125I efflux from Caco-2 cells. ASH-induced Isc increases and 125I efflux activations were both suppressed by low Ca2+ levels in the extracellular solution or by pretreatment with the Ca2+ chlelator BAPTA-AM. Intracellular Ca2+ levels were increased by ASH in a biphasic fashion characterized by a rapid sharp increase (peak 1) followed by a sustained low plateau (peak 2). ASH-induced peak 1 was inhibited by pretreatment with pertussis toxin, indicating that Ca2+ was mobilized from intracellular stores, and peak 2 was induced by an influx of extracellular Ca2+. Peak 2 but not peak 1 was related to Cl- secretion. These results indicate that ASH activates Ca2+-dependent Cl- secretion.
[Show abstract][Hide abstract] ABSTRACT: The Aeromonas sobria hemolysin causes diarrhea following infection by this enteropathogenic bacterium. We previously identified the putative receptor for A. sobria hemolysin as a p66 protein on Intestine 407 cells (Microb. Pathog. 27 (1999) 215-221). Here, we have partially purified and obtained a peptide mass fingerprint of p66 which revealed its identity with placental alkaline phosphatase (PLAP). Recombinant PLAP expressed in 293T cells was also found to bind to hemolysin and the binding was found not to be dependent on the N-linked glycosylation of PLAP. By immunohistochemical analysis, PLAP expression was detected in human intestinal mucosa, the target tissue in disease. In addition to PLAP, hemolysin also binds to intestinal alkaline phosphatase (IAP), an enzyme that is also abundantly expressed in intestine. Thus, both PLAP and IAP are very likely involved in the pathogenesis of diarrhea caused by this bacterial toxin.
International Journal of Medical Microbiology 02/2005; 294(7):427-35. · 4.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aeromonas sobria hemolysin (ASH) is one of the major virulence factors produced by A. sobria, a human pathogen that causes diarrhea. We investigated the effects of ASH on Cl(-) transport in human colonic epithelial cells. ASH increased short-circuit currents (Isc) and (125)I efflux from Caco-2 cells, indicating ASH activate Cl(-) secretion. Additions of inhibitors of cyclic AMP dependent Cl(-) channels, glybenclamide and NPPB suppressed the Isc and (125)I efflux increases induced by ASH. And ASH increased the intracellular cyclic AMP concentration. Moreover, ASH stimulated fluid accumulation in the iliac loop test, and glybenclamide and NPPB suppressed this fluid accumulation. Thus, cAMP-dependent Cl(-) secretory pathway could be related with diarrhea induced by A. sobria.
[Show abstract][Hide abstract] ABSTRACT: The hemolysin of Vibrio mimicus(VMH) is a pore-forming toxin with both enterotoxic and hemolytic activity. The hemolysis by VMH is induced by creation of pores in the membrane of erythrocyte; however, the mechanism for the enterotoxic action of VMH has remained unclear. In order to clarify the mechanism, we incubated T84 cells (a human colon carcinoma cell line) with VMH and found that the levels of ATP and cyclic AMP of culture medium increased after exposure of the cells to VMH. Subsequently, we found that the fluid accumulating activity of VMH in a mouse internal loop assay was reduced by administration of glibenclamide, an inhibitor of cyclic AMP-dependent chloride channels, into the intestinal loop. These results suggest that the stimulation of cells to produce nucleotides by VMH is linked to the enterotoxic activity of the toxin.
Microbiology and Immunology 02/2005; 49(1):73-8. · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The hemolysin of Vibrio mimicus (VMH) is a pore‐forming toxin with both enterotoxic and hemolytic activity. The hemolysis by VMH is induced by creation of pores in the membrane of erythrocyte; however, the mechanism for the enterotoxic action of VMH has remained unclear. In order to clarify the mechanism, we incubated T84 cells (a human colon carcinoma cell line) with VMH and found that the levels of ATP and cyclic AMP of culture medium increased after exposure of the cells to VMH. Subsequently, we found that the fluid accumulating activity of VMH in a mouse internal loop assay was reduced by administration of glibenclamide, an inhibitor of cyclic AMP‐dependent chloride channels, into the intestinal loop. These results suggest that the stimulation of cells to produce nucleotides by VMH is linked to the enterotoxic activity of the toxin.
Microbiology and Immunology 01/2005; 49(1). · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A previous investigation using the Fur titration assay system showed that Vibrio parahaemolyticus possesses a gene encoding a protein homologous to IutA, the outer-membrane receptor for ferric aerobactin in Escherichia coli. In this study, a 5.6 kb DNA region from the V. parahaemolyticus WP1 genome was cloned and two entire genes, iutA and alcD homologues, were identified which are absent from Vibrio cholerae genomic sequences. The V. parahaemolyticus IutA and AlcD proteins share 43 % identity with the Escherichia coli IutA protein and 24 % identity with the Bordetella bronchiseptica AlcD protein of unknown function, respectively. Primer extension analysis revealed that the iutA gene is transcribed in response to low-iron availability from a putative promoter overlapped with a sequence resembling a consensus E. coli Fur-binding sequence. In agreement with the above finding, V. parahaemolyticus effectively utilized exogenously supplied aerobactin for growth under iron-limiting conditions. Moreover, insertional inactivation of iutA impaired growth in the presence of aerobactin and incapacitated the outer-membrane fraction from iron-deficient cells for binding (55)Fe-labelled aerobactin. These results indicate that the V. parahaemolyticus iutA homologue encodes an outer-membrane protein which functions as the receptor for ferric aerobactin. Southern blot analysis revealed that the iutA homologues are widely distributed in clinical and environmental isolates of V. parahaemolyticus. However, additional genes required for ferric aerobactin transport across the inner membrane remain to be clarified.
[Show abstract][Hide abstract] ABSTRACT: We previously reported that the aerolysin-like hemolysin of Aeromonas sobria stimulates T84 cells to produce cyclic AMP, which then emerges in the culture medium. In order to clarify the mechanism of action of the hemolysin, we examined the involvement of adenosine nucleotide. The results show that the hemolysin stimulates T84 cells to release ATP, which is then converted to adenosine by ectonucleotidase. The adenosine generated might stimulate the P1 adenosine receptors of T84 cells to produce cyclic AMP.
Infection and Immunity 04/2003; 71(3):1557-60. · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: For the successful production of Aeromonas sobria serine protease (ASP), open reading frame 2 (ORF2) protein, encoded at the 3' end of the protease operon, is required. In this study, we examined the action of ORF2 protein. The results showed that the protein associated with ASP in the periplasm and helped ASP to form an active structure.
Journal of Bacteriology 01/2003; 184(24):7058-61. · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intracellular replication of Brucella requires the VirB complex, which is highly similar to conjugative DNA transfer systems. In this study, we show that Brucella internalizes into macrophages by swimming on the cell surface with generalized membrane ruffling for several minutes, after which the bacteria are enclosed by macropinosomes. Lipid raft-associated molecules such as glycosylphosphatidylinositol (GPI)-anchored proteins, GM1 gangliosides and cholesterol were selectively incorporated into macropinosomes containing Brucella. In contrast, lysosomal glycoprotein LAMP-1 and host cell transmembrane protein CD44 were excluded from the macropinosomes. Removing GPI-anchored proteins from the macrophage surface and cholesterol sequestration markedly inhibited the VirB-dependent macropinocytosis and intracellular replication. Our results suggest that the entry route of Brucella into the macrophage determines the intracellular fate of the bacteria that is modulated by lipid raft microdomains.
[Show abstract][Hide abstract] ABSTRACT: Previously, we cloned a protease gene of Aeromonas sobria, determined its nucleotide sequence and established a method of purifying its product. In this study, we examined the properties of the purified protease. The protease was temperature-labile and had an optimal pH of 7.5. Metallo-protease inhibitors and a cysteine protease inhibitor did not block the proteolytic activity of the enzyme. The treatment with reagents to modify sulfhydryl group did not reduce the activity. But, serine protease inhibitors did, showing that it was a serine protease. Subsequently, we examined the ability of the protease to enhance vascular permeability in dorsal skin. The protease showed activity and the reaction was inhibited by a simultaneously injected antihistaminic agent. Histopathological examination showed that mast cells appeared around the site where the protease was injected. These findings show that the vascular permeability-enhancing effect of the protease is due to histamine released at the site. Furthermore, we found that a soybean trypsin inhibitor (Kunitz) did not block the proteolytic action of the protease in vitro, but inhibited its vascular permeability-enhancing activity in skin. This suggests that a trypsin-like protease from skin mediates the activity of the protease to enhance its vascular permeability.
Microbiology and Immunology 02/2002; 46(6):383-90. · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Heat‐stable enterotoxin II of Escherichia coli (STII) is synthesized as a precursor form consisting of pre‐ and mature regions. The pre‐region is cleaved off from the mature region during translocation across the inner membrane, and the mature region emerges in the periplasm. The mature region, composed of 48 amino acid residues, is processed in the periplasm by DsbA to form an intramolecular disulfide bond between Cys‐10 and Cys‐48 and between Cys‐21 and Cys‐36. STII formed with these disulfide bonds is efficiently secreted out of the cell through the secretory system, including TolC. However, it remains unknown which regions of STII are involved in interaction with TolC. In this study, we mutated the STII gene and examined the secretion of these STIIs into the culture supernatant. A deletion of the part covering from amino acid residue 37 to the carboxy terminal end did not markedly reduce the efficiency of secretion of STII into the culture supernatant. On the other hand, the efficiency of secretion of the peptide covering from the amino terminal end to position 18 to the culture supernatant was significantly low. These observations indicated that the central region of STII from amino acid residue 19 to that at position 36 is involved in the secretion of STII into the milieu. The experiment using a dsbA‐deficient strain of E. coli showed that the disulfide bond between Cys‐21 and Cys‐36 by DsbA is necessary for STII to adapt to the structure that can cross the outer membrane.
Microbiology and Immunology 01/2001; 45(5). · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hemolysin of Aeromonas sobria possesses both cytotoxic activity against mammalian cells and enterotoxic activity. Histopathological examination revealed that hemolysin causes diarrhea without damaging the intestinal epithelial cells. And the fluid accumulated in the mouse intestinal loop by the action of the hemolysin is watery. These observations indicated that the enterotoxic activity of hemolysin is not dependent on its cytotoxic activity. To clarify the mechanism of the enterotoxic action of hemolysin, we examined cyclic nucleotide levels in cultured cells exposed to this toxin. These results showed that hemolysin stimulates the production of cyclic AMP in cultured cells and the cyclic AMPs thus produced emerge in the milieu of cells.
[Show abstract][Hide abstract] ABSTRACT: The sequence at the amino terminus region of the hemolysin of Aeromonas sobria is homologous with that of aerolysin of A. hydrophila. However, there is no homology between the two toxins in the sequence at the carboxy terminal region. It has been shown that aerolysin is secreted into culture supernatant as a protoxin. This proaerolysin is activated by the proteolytic removal of a carboxy terminal peptide. However, the role of the carboxy terminal region, which is removed in the activation process, has not been elucidated. In this study, we showed that hemolysin is also secreted as a protoxin into culture supernatant and that prohemolysin is cleaved by the protease of A. sobria between Ser‐446 and Ala‐447, resulting in the removal of a 42 amino acid peptide. The removal of the peptide converts the prohemolysin into active hemolysin. Subsequently, we mutated the hemolysin gene to delete the last several amino acid residues and expressed the genes in Escherichia coli, in order to examine the role of the carboxy terminal region of prohemolysin. The amounts of these mutant hemolysins accumulated in the periplasmic space of E. coli were very low compared with that of the wild‐type. This observation indicated that the carboxy terminal region of prohemolysin contributes to the proteolytic stability of the toxin.
Microbiology and Immunology 01/1999; 43(1). · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract The prostaglandin response of mouse intestinal epithelial cells after exposure to Escherichia coli heat-stable enterotoxin II was examined. The quantity of prostaglandin E2 produced by the intestinal cells was directly related to the dose of heat-stable enterotoxin II. The change in the amount of prostaglandin E2 over time correlated to that of the volume of fluid released into the intestinal lumen. We then demonstrated that administration of heat-stable enterotoxin II into the intestinal loops of mice induced elevation of arachidonic acid and phosphatidic acid levels in intestinal epithelial cells. These results show that heat-stable enterotoxin II stimulates arachidonic acid metabolism in intestinal epithelial cells and that the synthesized prostaglandin E2 functions as a mediator of fluid secretion induced by this enterotoxin.