[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 03/2014; 9(3):e91149. DOI:10.1371/journal.pone.0091149 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We examined the ability of Aeromonas hydrophila to lyse elastin. Eight of 13 strains showed elastolytic activity on agar medium containing elastin and 5 strains did not. In order to examine the involvement of the metalloprotease of A. hydrophila (AMP) in elastolytic activity, we made the amp-deletion mutant strain from an elastolytic strain. The elastolytic activity of the strain decreased with this deletion. The analysis of AMP released into the culture supernatant showed that AMP appeared outside of the cell as the intermediate consisting of a mature domain and carboxy terminal (C-terminal) propeptide domain. Further analysis showed that the intermediate has the ability to lyse elastin and that loss of the C-terminal domain causes loss of the elastolytic activity of the intermediate. We then determined the nucleotide sequence of the amps of all strains used in this study. Phylogenetic analysis revealed that these AMPs were divided into three groups. The AMPs from elastolytic strains belong to group I or group II, and AMPs from non-elastolytic strains belong to group III. The distance between group I and group II is small, but group III is located separately from groups I and II. Comparison of the amino acid residues of the C-terminal domain revealed that there are 13 amino acid residues specific to the C-terminal domain of group III. This indicates that the conformation of the C-terminal propeptide domain formed by these specific amino acid residues is important for AMP to express elastolytic activity.
[Show abstract][Hide abstract] ABSTRACT: Abstract ASP is a serine protease secreted by Aeromonas sobria. ASP cleaves various plasma proteins, which is associated with onset of sepsis complications, such as shock and blood coagulation disorder. To investigate a host defense mechanism against this virulence factor, we examined the plasma for ASP inhibitor(s). Human plasma inhibited ASP activity for azocasein, which was almost completely abolished by treating plasma with methylamine, which inactivates α2-macroglobulin (α2-MG). The ASP-inhibitor complex in ASP-added plasma was not detected by immunoblotting using anti-ASP antibody; however, using gel filtration of the plasma ASP activity for an oligopeptide, the ASP substrate was eluted in the void fraction (Mw>200 000), suggesting ASP trapping by α2-MG. Indeed, human α2-MG inhibited ASP azocaseinolytic activity in a dose-dependent manner, rapidly forming a complex with the ASP. Fibrinogen degradation by ASP was completely inhibited in the presence of α2-MG. α1-Protease inhibitor, antithrombin, and α2-plasmin inhibitor neither inhibited ASP activity nor formed a complex with ASP. Surprisingly, ASP degraded these plasma serine protease inhibitors. Thus, α2-MG is the major ASP inhibitor in the human plasma and can limit ASP virulence activities in A. sobria infection sites. However, as shown by fluorescence correlation spectroscopy, slow ASP inhibition by α2-MG in plasma may indicate insufficient ASP control in vivo.
[Show abstract][Hide abstract] ABSTRACT: ASP is a serine protease secreted by Aeromonas sobria, a sepsis-causing bacterium, and induces sepsis-mimicking disorders through plasma protein cleavage. The pathogen also secretes nASP that has a nick in the carboxy-terminal region. Compared with single-chain ASP (sASP), nASP had near-equivalent activity for small peptide substrates but was less proteolytic. Surprisingly, nASP cleaved proteins more in plasma and was inhibited by human α(2)-macroglobulin more slowly than sASP. Retarded inhibition by α(2)-macroglobulin allows nASP to keep proteolytic activity for longer in the host and exacerbate disorders at Aeromonas sobria infection sites. nASP may be an evolutional form to augment ASP virulence.
[Show abstract][Hide abstract] ABSTRACT: Aeromonas have been isolated from a wide variety of aquatic environments. However the number of Aeromonas in sea water is extremely small compared to that in fresh water. In in vitro culture, Aeromonas can grow in mediums containing NaCl at a concentration of 3.0%, this concentration corresponding to that of sea water. It is unclear why the number of Aeromonas is low in sea water. Exoproteins of bacteria are thought to be important for bacterial growth and survival in the environment. Previously, the present authors have shown that mediums containing 3.0% NaCl suppress production of two proteases, serine protease and metalloprotease. In this experiment, other exoproteins whose production is influenced by the amount of NaCl in the medium were analyzed. A protein whose production is repressed in medium containing 3.0% NaCl was found and purified. Biological assay of the purified protein showed that it degrades tributyrin and hydrolyzes para-nitrophenyl-fatty acylesters. These results show that the protein is a lipase. Subsequently, the nucleotide sequence of the gene encoding the lipase was determined and the amount of mRNA of the lipase gene in the cells measured. It was found that transcription of the gene is not inhibited by NaCl in the medium. This result indicates that the lipase might be synthesized, but the folding process to become an active structure does not progress smoothly in a medium containing 3.0% NaCl.
Microbiology and Immunology 02/2012; 56(5):295-307. DOI:10.1111/j.1348-0421.2012.00445.x · 1.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present authors have previously shown that the serine protease activity of Aeromonas sobria is markedly decreased when A. sobria is cultured in medium containing 3.0% sodium chloride (NaCl, concentration almost equivalent to sea water salinity), and that this occurs because, although the synthesis of ASP is not disturbed by the salt in the medium, the maturation pathway of serine protease of A. sobria (ASP) does not proceed successfully in such a medium. In this study, the effect of salt in the medium on the production of metalloprotease by A. sobria (AMP) was examined. A. sobria produced AMP in the milieu when the bacteria were cultured in medium containing (NaCl) at a concentration of 0.5%. However, AMP was not produced when the bacteria were cultured in salty medium containing 1.5% or more NaCl. To examine how NaCl reduces the production of metalloprotease by A. sobria, the amount of amp mRNA in the cell was measured and it was found that this decreased in proportion to the concentration of NaCl in the medium. The mRNA of amp was not detected in cells cultured in medium containing 1.5% or more NaCl. This means that the transcription of amp is inhibited in salty condition. As described, NaCl in the medium disturbs the maturation pathway of ASP. The mode of action whereby NaCl suppresses AMP activity in A. sobria differs from the mechanism for suppressing ASP activity.
Microbiology and Immunology 01/2011; 55(1):60-5. DOI:10.1111/j.1348-0421.2010.00282.x · 1.31 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. DOI:10.1111/j.1348-0421.2010.00258.x · 1.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ASP is the only bacterial protease in the kexin group of the subtilisin family. Previous studies have revealed that the ORF2 protein encoded at the 3' end of the asp operon is required for ASP to change from a nascent form into an active form in the periplasm. However, the mechanism by which ORF2 makes contact and interacts with ASP in the maturation process remains unknown. The present study examined the effect of mutations in the carboxy-terminal region of ASP on the ASP maturation process. Both deletion-mutation and amino acid-substitution studies have demonstrated that the histidine residue at position 595 (His-595), the sixth residue from the carboxyl terminus of ASP, is highly involved in the generation of active ASP molecules. An analysis by pull-down assay revealed that mutation at His-595 reduces the efficacy of nascent ASP to transition into active ASP by reducing the ability of ASP to make contact and interact with ORF2. Thus, it appears likely that nascent ASP in the periplasm interacts with ORF2 via the carboxy-terminal region, and His-595 of ASP appears to be an indispensable residue in this interaction.
Microbiology and Immunology 12/2009; 53(12):647-57. DOI:10.1111/j.1348-0421.2009.00175.x · 1.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The anaerobic bacterium Aeromonas sobria is known to cause potentially lethal septic shock. We recently proposed that A. sobria serine protease (ASP) is a sepsis-related factor that induces vascular leakage, reductions in blood pressure via kinin release, and clotting via activation of prothrombin. ASP preferentially cleaves peptide bonds that follow dibasic amino acid residues, as do Kex2 (Saccharomyces cerevisiae serine protease) and furin, which are representative kexin family proteases. Here, we revealed the crystal structure of ASP at 1.65 A resolution using the multiple isomorphous replacement method with anomalous scattering. Although the overall structure of ASP resembles that of Kex2, it has a unique extra occluding region close to its active site. Moreover, we found that a nicked ASP variant is cleaved within the occluding region. Nicked ASP shows a greater ability to cleave small peptide substrates than the native enzyme. On the other hand, the cleavage pattern for prekallikrein differs from that of ASP, suggesting the occluding region is important for substrate recognition. The extra occluding region of ASP is unique and could serve as a useful target to facilitate development of novel antisepsis drugs.
[Show abstract][Hide abstract] ABSTRACT: The heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli is an extracellular peptide toxin that evokes watery diarrhea in the host. Two types of STs, STI and STII, have been found. Both STs are synthesized as precursor proteins and are then converted to the active forms with intramolecular disulfide bonds after being released into the periplasm. The active STs are finally translocated across the outer membrane through a tunnel made by TolC. However, it is unclear how the active STs formed in the periplasm are led to the TolC channel. Several transporters in the inner membrane and their periplasmic accessory proteins are known to combine with TolC and form a tripartite transport system. We therefore expect such transporters to also act as a partner with TolC to export STs from the periplasm to the exterior. In this study, we carried out pulse-chase experiments using E. coli BL21(DE3) mutants in which various transporter genes (acrAB, acrEF, emrAB, emrKY, mdtEF, macAB, and yojHI) had been knocked out and analyzed the secretion of STs in those strains. The results revealed that the extracellular secretion of STII was largely decreased in the macAB mutant and the toxin molecules were accumulated in the periplasm, although the secretion of STI was not affected in any mutant used in this study. The periplasmic stagnation of STII in the macAB mutant was restored by the introduction of pACYC184, containing the macAB gene, into the cell. These results indicate that MacAB, an ATP-binding cassette transporter of MacB and its accessory protein, MacA, participates in the translocation of STII from the periplasm to the exterior. Since it has been reported that MacAB cooperates with TolC, we propose that the MacAB-TolC system captures the periplasmic STII molecules and exports the toxin molecules to the exterior.
Journal of bacteriology 10/2008; 190(23):7693-8. DOI:10.1128/JB.00853-08 · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aeromonas sobria causes pus and edema at sites of infection. However, the mechanisms underlying these effects have not been elucidated. C5a, the amino-terminal fragment of the complement 5th component (C5), mimics these events. To investigate the involvement of C5a in the pathophysiology of A. sobria infection, we examined release of C5a from human C5 by a serine protease (ASP), a putative virulence factor secreted by this bacterium. C5 incubated with enzymatically active ASP induced neutrophil migration in a dose-dependent manner from an ASP concentration of 3 nM and in an incubation time-dependent manner in as little as 7 min, with neutrophil accumulation in guinea pigs at intradermal injection sites and neutrophil superoxide release. These effects on neutrophils were inhibited by a C5a-receptor antagonist. The ASP incubation mixture with C5 but not C3 elicited vascular leakage in a dose- and incubation time-dependent manner, which was inhibited by a histamine H(1)-receptor antagonist. Together with these C5a-like activities, ASP cleaved C5 to release only one C5a Ag, the m.w. of which was similar to that of C5a. Immunoblotting using an anti-C5a Ab revealed generation of a C5a-like fragment from human plasma incubated with ASP. These results suggest that ASP-elicited neutrophil migration and vascular leakage via C5a production from C5 could occur in vivo, which was supported by that ASP did not affect functions of C5a and neutrophil C5a receptor. Through C5a generation, ASP could be associated with the induction of pus and edema caused by infection with this bacterium.
The Journal of Immunology 10/2008; 181(5):3602-8. DOI:10.4049/jimmunol.181.5.3602 · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aeromonas sobria infection often advances to sepsis, in which interaction of bacterial components with plasma proteins possibly causes various disorders. This bacterium releases a serine protease (ASP), a putative virulence factor, and binds to fibrinogen. To study the ASP effect on fibrinogen, we incubated fibrinogen or plasma with ASP and investigated their clotting elicited by thrombin, which converts fibrinogen to a fibrin clot. Enzymatically active ASP retarded plasma clotting in a dose-dependent manner starting at an ASP concentration of 10 nM. ASP also retarded fibrinogen clotting at 3 nM and above, which appeared to correspond to ASP cleavage of fibrinogen at the A alpha-chain. Consistent with containing serine protease activity for an ASP-specific substrate, the culture supernatant of an ASP gene-introduced strain retarded plasma and fibrinogen clotting more than that of the wild-type strain. The culture supernatant of an ASP gene-disrupted strain that releases negligible serine protease activity for the ASP-specific substrate did not affect plasma clotting. These results indicate that ASP is the main fibrinogenolytic protease released from A. sobria. Impaired plasma clottability induction through fibrinogen degradation is a new virulence activity of ASP and may contribute to hemorrhagic tendencies in sepsis caused by infection with this bacterium.
[Show abstract][Hide abstract] ABSTRACT: Vancomycin is mainly used as an antibacterial agent of last resort, but recently vancomycin-resistant bacterial strains have been emerging. Although new antimicrobials have been developed in order to overcome drug-resistant bacteria, many are structurally complex beta-lactams or quinolones. In this study, we aimed to create new anti-drug-resistance antibacterials which can be synthesized in a few steps from inexpensive starting materials. Since sulfa drugs function as p-aminobenzoic acid mimics and inhibit dihydropteroate synthase (DHPS) in the folate pathway, we hypothesized that sulfa derivatives would act as folate metabolite-mimics and inhibit bacterial folate metabolism. Screening of our sulfonanilide libraries, including benzenesulfonanilide-type cyclooxygenase-1-selective inhibitors, led us to discover benzenesulfonanilides with potent anti-methicillin-resistant Staphylococcus aureus (MRSA)/vancomycin-resistant Enterococcus (VRE) activity, that is, N-3,5-bis(trifluoromethyl)phenyl-3,5-dichlorobenzenesulfonanilide (16b) [MIC=0.5microg/mL (MRSA), 1.0microg/mL (VRE)], and 3,5-bis(trifluoromethyl)-N-(3,5-dichlorophenyl)benzenesulfonanilide (16c) [MIC=0.5microg/mL (MRSA), 1.0microg/mL (VRE)]. These compounds are more active than vancomycin [MIC=2.0microg/mL (MRSA), 125microg/mL (VRE)], but do not possess an amino group, which is essential for DHPS inhibition by sulfa drugs. These results suggested that the mechanism of antibacterial action of compounds 16b and 16c is different from that of sulfa drugs. We also confirmed the activity of these compounds against clinical isolates of Gram-positive bacteria.
[Show abstract][Hide abstract] ABSTRACT: The effect of a serine protease (ASP) secreted from Aeromonas sobria on plasma coagulation was investigated. Proteolytically active ASP promoted human plasma coagulation in a dose-dependent manner. Consistent with the preference for a factor Xa-specific oligo-peptide substrate, ASP produced enzymatic activity from human prothrombin but not from factors IX and X. ASP cleaved prothrombin to produce enzymatically active 37 kDa-fragment displaying the same molecular mass as alpha-thrombin. ASP is the first bacterial serine protease that produces alpha-thrombin, through which ASP may contribute to the induction of thrombotic tendency in disseminated intravascular coagulation complicated with sepsis caused by A. sobria infections.
[Show abstract][Hide abstract] ABSTRACT: Gram-negative bacteria possess the outer membrane protein TolC which acts as an exit duct across the outer membrane. However, the region involved in the transport activity of TolC has remained unclear. We analyzed this region by creating chimeric TolCs. First, we expressed the genes for TolCs of Vibrio parahaemolyticus (vp-tolC) and Salmonella typhimurium (sal-tolC) in Escherichia coli. The levels of sequence identity in the mature region of VP-TolC/EC-TolC and Sal-TolC/EC-TolC with maximum matching are 43% and 90%, respectively. We found that the transport activity of VP-TolC was weak compared with that of TolC of E. coli (EC-TolC) although the transport activity of Sal-TolC was similar to that of EC-TolC. A comparison of the sequence of the three tolCs showed that the sequence around the periplasmic region covering Asn-188 to Lys-214 of EC-TolC is lowly identical to that of VP-TolC although the region of EC-TolC is almost identical to that of Sal-TolC. We think, therefore, that the region covering Asn-188 to Lys-214 of EC-TolC may have an important role to express its transport activity in E. coli. To examine the possibility, we divided the region of EC-TolC into three and exchanged the gene for each portion with that of vp-tolC. These mutant ec-tolCs were expressed in E. coli and the activity of each chimeric TolC was measured. The results showed that the portion covering Val-198 to Lys-214 of EC-TolC is deeply involved in the transport activity.
[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: 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. DOI:10.1128/JB.184.24.7058-7061.2002 · 2.69 Impact Factor