Aeromonas Sobria a Serine Proteinase from Pressure Lowering Through Kinin Release by Induction of Vascular Leakage and Blood

The Journal of Immunology

Induction of Vascular Leakage and Blood Pressure Lowering through Kinin Release by a Serine Proteinase from Aeromonas sobria1

Takahisa Imamura,2* Hidemoto Kobayashi,‡ Rasel Khan,‡ Hidetoshi Nitta,*† and Keinosuke Okamoto‡

Aeromonas sobria causes septic shock, a condition associated with high mortality. To study the mechanism of septic shock by A. sobria infection, we examined the vascular leakage (VL) activity of A. sobria serine proteinase (ASP), a serine proteinase secreted by this pathogen. Proteolytically active ASP induced VL mainly in a bradykinin (BK) B2 receptor-, and partially in a histamine-H1 receptor-dependent manner in guinea pig skin. The ASP VL activity peaked at 10 min to 1.8-fold of the initial activity with an increased BK B2 receptor dependency, and attenuated almost completely within 30 min. ASP produced VL activity from human plasma apparently through kallikrein/kinin system activation, suggesting that ASP can generate kinin in humans. Consistent with the finding that a major part of the ASP-induced VL was reduced by a potent kallikrein inhibitor, soybean trypsin inhibitor that does not affect ASP enzymatic activity, ASP activated prekallikrein but not factor XII to generate kallikrein in a dose- and incubation time-dependent manner. ASP produced more VL activity directly from human low m.w. kininogen than high m.w. kininogen when both were used at their normal plasma concentrations. Intra- arterial injection of ASP into guinea pigs lowered blood pressure specifically via the BK B2 receptor. These data suggest that ASP induces VL through prekallikrein activation and direct kinin release from kininogens, which is a previously undescribed mechanism of A. sobria virulence and could be associated with the induction of septic shock by infection with this bacterium. ASP-specific inhibitors, and kinin receptor antagonists, might prove useful for the treatment or prevention of this fatal disease. The Journal of Immunology, 2006, 177: 8723–8729.

eromonas species are facultative anaerobic Gram-nega- Aeromonas infections. Understanding the mechanism of Aeromo- tive rods that are ubiquitous, waterborne bacilli (1), most nas septic shock is crucial in the development of therapeutic treat- A commonly implicated as causative agents of gastroenter- ments. Recently, overactivation of the immune system by the ex- itis (2–5). Aeromonas hydrophila, Aeromonas caviae, and Aero- cessive release of proinflammatory cytokines, e.g., TNF-␣, mainly monas sobria recovered predominantly from clinical samples have from monocytes/macrophages stimulated by bacterial lipopolysac- been reported to be involved in a wide range of extraintestinal and charides through Toll-like receptors, a class of pattern recognition systemic infections, including septicemia, wound infections, men- molecules, have been suggested to lead to septic shock in Gram- ingitis, peritonitis, and hepatobiliary disease (6). The majority of negative bacteria infections (11). However, the fact that impair- patients inflicted with Aeromonas septicemia are infants under 2 ment of the host immune system increases the incidence of years of age and immunocompromised adults with multiple un- Aeromonas septic shock (6) may indicate involvement of a mech- derlying medical complications, mostly malignancy, hepatobiliary anism(s) other than overactivation of the immune system in shock disease, or diabetes. Mortality rates for these patients generally induction. range from 25 to 50% (7–9). Patients with severe wound infection The plasma kallikrein/kinin system is initiated by activation of (myonecrosis) caused by this microorganism also develop sepsis, coagulation factor XII on a negatively charged surface, with acti- and Ͼ90% of the patients succumb to their infections (7, 10). vated factor XII converting plasma prekallikrein to kallikrein, re- Septic shock is a major cause of death of these patients, but no leasing bradykinin (BK)3 from high m.w. kininogen (HK) (12). mechanism leading to this fatal complication has been shown in The plasma levels of the kallikrein/kinin system components are low in sepsis patients (13–16), indicating the activation and sub- sequent consumption of these components. BK, the final product, † *Department of Molecular Pathology and Department of Gastroenterological binds to BK-B2 receptor on vascular endothelial cells (17) and Surgery, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, induces vascular leakage (VL), leading to hypotension due to a Kumamoto, Japan; and ‡Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan decrease in circulating blood volume. Indeed, the activation of the plasma kallikrein/kinin system in an animal model causes lethal Received for publication February 28, 2006. Accepted for publication September 28, 2006. hypotension (18, 19). Hence, activation of the kallikrein/kinin sys- The costs of publication of this article were defrayed in part by the payment of page tem, and/or direct kinin release from kininogens, seems to con- charges. This article must therefore be hereby marked advertisement in accordance tribute to septic shock. with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by a grant from the Japanese Ministry of Education and Science (Grant 16590319; to T.I.). 2 Address correspondence and reprint requests to Dr. Takahisa Imamura, Department 3 Abbreviations used in this paper: BK, bradykinin; HK, high m.w. kininogen; VL, of Molecular Pathology, Faculty of Medical and Pharmaceutical Sciences, Kumamoto vascular leakage; LK, low m.w. kininogen; ASP, Aeromonas sobria serine proteinase; University, 1-1-1 Honjo, Kumamoto 860-8556, Japan. E-mail address: taka@kaiju. BP, blood pressure; SBTI, soybean trypsin inhibitor; DFP, diisopropyl fluorophos- medic.kumamoto-u.ac.jp phate; ScpA, staphopain A.

Aeromonas species release a number of putative virulence fac- leased in the solution was measured (fluorescence at 440 nm with ex- tors including hemolysins, enterotoxins, and proteinases (20). We citation at 380 nm) using a fluorescence spectrophotometer (model no. have shown that bacterial proteinases induce VL through activa- 650-40; Hitachi). tion of the plasma kallikrein/kinin system, and/or direct kinin re- Treatment of plasmas and kininogens with ASP lease from HK and low m.w. kininogen (LK), which represents another kinin source in plasma (21–23). A. sobria is predominantly Forty-five microliters of human plasma supplemented with 1 mM 1,10- phenanthroline for kininase inhibition was incubated with 5 ␮lofASP isolated in patient’s blood (24), and is more pathogenic than A. at various concentrations and 37°C for 5 min, followed by the addition hydrophila (25). Therefore, to study the mechanism of septic of 50 ␮l of 10 mM Tris-HCl (pH 7.3) containing 150 mM NaCl (TBS) shock by Aeromonas infection, we examined the A. sobria serine supplemented with 1 mM 1,10-phenanthroline. Ten microliters of ASP ␮ ␮ ␮ proteinase (ASP) for VL and blood pressure (BP)-lowering activ- and 90 l of TBS containing HK (80 g/ml) or LK (130 g/ml) were incubated at 37°C for 5 min. One ␮l of 100 mM diisopropyl fluoro- ities, and investigated an involvement of the plasma kallikrein/ phosphate (DFP) was added to plasma or kininogen samples after in- kinin system in these activities. cubating with ASP to completely inhibit ASP (27). DFP specifically phosphorylates the active-site serine residue of serine proteases, inac- Materials and Methods tivating its enzymatic activity. Materials VL assay Human HK, factor XII, and prekallikrein were purchased from Enzyme This experiment was performed and approved according to the criteria of Research Laboratories, and human LK was obtained from Athens Research animal experiments of the Kumamoto University Animal Experiment Technology. Evans blue was obtained from Merck. Soybean trypsin inhib- Committee. Guinea pigs (350ϳ450 g body weight, both sexes) were anes- itor (SBTI) was purchased from Sigma-Aldrich. BK, benzyloxycarbonyl- thetized with an i.m. injection of ketamine (80 mg/kg body weight). Thirty L-phenylalanyl-L-arginine-4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA), mg/kg body weight of Evans blue (2.5% solution in 0.6% saline) was then and t-butyloxycarbonyl-L-glutaminylglycyl-L-arginine-4-methylcoumaryl- administered i.v., followed by an intradermal injection of 0.1 ml of test 7-amide (Boc-Gln-Gly-Arg-MCA) were obtained from the Peptide Insti- samples (dissolved in TBS) into the clipped flank of the guinea pig. Bluing tute. BK B2 receptor antagonist, HOE140 was obtained from Hoechst. of injection sites starts within a few minutes and terminates by 10 min. Polyclonal anti-human kininogen goat IgG and HRP-conjugated rabbit IgG After 10 min, the guinea pig was euthanized by bleeding under ether an- against goat IgG were purchased from R&D Systems and Nichirei, respec- esthesia and blue dye-leaked skin tissues were excised, and incubated in 3 tively. The ProteoSeek albumin/IgG removal kit was obtained from Pierce. ml of formamide at 60°C for 48 h. VL activity was determined by quan- Other chemicals were purchased from Wako Pure Chemicals. Normal hu- titatively measuring the extracted Evans blue by absorbance at 620 nm as man plasma was prepared by centrifugation of a mixture of 9 vol of freshly described previously (30). Activity was expressed in terms of micrograms drawn blood from healthy volunteers and 1 vol of 3.8% (w/v) sodium of dye extracted. The activity of the buffer was subtracted from the activity citrate. of each sample. In the case of VL activity production from human plasma, the activity of plasma incubated with the buffer, followed by DFP addition, Purification of ASP was subtracted from the activity of each sample. HOE140 (10 nm/kg body ASP was initially purified from the culture supernatant of a clinically iso- weight) or diphenhydramine (30 mg/kg body weight) dissolved in 200 ␮l lated strain of A. sobria. To obtain sufficient ASP for the experiments of TBS was injected s.c. in the thigh at 30 min, or i.p. in the lower abdomen herein, A. sobria T94 was transformed with a plasmid pSA19-5528 con- at 1 h, respectively, before intradermal injection of samples into guinea pigs. taining the ASP gene (26), resulting in the overexpression and secretion of ASP several fold in excess of the initial strain. The transgenic strain was SDS-PAGE of ASP and kininogens incubated with ASP ␮ cultured in Luria broth supplemented with chloramphenicol (50 g/ml), at A portion (20 ␮l) of the purified ASP solution (200 ␮g/ml) was mixed with 37°C with shaking for 20 h. ASP secreted into the culture supernatant was an equal volume of TCA solution (15%) to inactivate ASP and kept at 0°C purified by successive column chromatography (27). The ASP sample was for 15 min. The precipitate collected by the centrifugation at 17,400 ϫ g analyzed by SDS-PAGE (10% polyacrylamide gel) under reducing or non- for 15 min was washed twice with ethanol and dried. The precipitate was reducing conditions. The enzyme preparations proved to be homogenous, then dissolved in SDS-buffer and boiled for 5 min in the presence or ab- showing a single band under both conditions, representing a protein of a sence of 2-ME. Ten microliters of the ASP solution (60 ␮g/ml) was ana- molecular mass of 65 kDa (Fig. 1). lyzed using SDS-PAGE (10% polyacrylamide gel). HK (80 ␮g/ml) and LK ␮ Measurement of activation of prekallikrein or factor XII (130 g/ml) were incubated with 30 nM ASP or TBS at 37°C for 10 min, followed by the addition of 1 mM DFP. Kininogen samples were then by ASP analyzed by SDS-PAGE under reducing conditions using an 8% polyacryl- Ninety microliters of prekallikrein (1 ␮M) or factor XII (1 ␮M) in 0.1 amide gel. Each Gel was silver stained using the Silver Stain II kit (Wako M Tris-HCl buffer (pH 7.6) containing 150 mM NaCl was incubated Pure Chemical Industries). ␮ with 10 l of ASP at 37°C for a range of different time periods, fol- Immunoblot analysis of ASP-treated human plasma lowed by an additional 500 ␮l of the buffer preincubated at 37°C. Then, after the addition of 10 ␮l of 10 mM Z-Phe-Arg-MCA (plasma kal- Ten microliters of citrated human plasma, supplemented with SBTI (0.1 likrein-specific substrate) (28) or Boc-Gln-Gly-Arg-MCA (activated mM) to inhibit kininogen cleavage by kallikrein, was incubated with 1 ␮l factor XII-specific substrate) (29), the 7-amino-4-methyl coumarin re- of ASP (1 ␮M) at 37°C for 30 min, followed by the addition of 0.1 ␮lof DFP (0.1 M) to inactivate ASP. Plasma was then diluted with 60 ␮lof25 mM Tris-HCl buffer (pH 7.5) containing 25 mM NaCl and mixed with 170 ␮l of immobilized Cibacron blue/protein A gel (Pierce) to remove albumin and IgG, which interfere with kininogen immunoblotting. Albumin binds specifically to Cibacron blue and IgG to protein A. As a control, plasma was processed as described above except ASP treatment was used. As further controls, HK (80 ␮g/ml) and LK (130 ␮g/ml) were incubated with 30 nM ASP or TBS at 37°C for 30 min, followed by the addition of 1 mM DFP. Plasma and kininogen samples were analyzed by SDS-PAGE under reducing conditions using an 8% polyacrylamide gel and transferred onto polyvinylidene fluoride membrane. Kininogens were blotted with anti- human kininogen goat IgG (ϫ2500 dilution) and HRP-conjugated anti- goat IgG rabbit IgG (ϫ5000 dilution). The bands were visualized by enhanced chemiluminescence (ECL; Amersham). FIGURE 1. SDS-PAGE of ASP. ASP (0.6 ␮g) was analyzed using a SDS-polyacrylamide gel in the presence (lane 3) or absence of 2-ME Measurement of BP (lanes 1 and 2), and the gel was resolved by silver-staining. Lane 1, Mo- Guinea pigs (350ϳ400 g body weight, both sexes) were anesthetized by an lecular size markers; lanes 2 and 3, ASP. i.m. injection of ketamine (100 mg/kg body weight) and ether inhalation. The Journal of Immunology 8725

FIGURE 3. Time course of ASP-induced VL. ASP (200 nM) was injected into guinea pig skin 10, 20, or 30 min before Evans blue injection. The 0 time indicates an ASP injection immediately after dye injection. (E), p Ͻ ,ء .(Untreated; (F), HOE140 treated. Values are means Ϯ SD (n ϭ 3 0.01 for time 10 min vs 0 min or 20 min; and for with vs without HOE140 treatment.

of the ASP-induced VL (Fig. 2B), while not affecting ASP enzymatic activity (27). The ASP-induced VL was mostly inhibited by

HOE140, a BK B2 receptor-speciﬁc antagonist (31, 32), and par-

tially by diphenhydramine, a histamine H1 receptor antagonist (Fig. 2B, inset). The sensitivity to SBTI, a potent inhibitor for

plasma kallikrein (33, 34), and the BK B2 receptor dependency of ASP VL activity together suggests an involvement of kinin release FIGURE 2. A, Induction of VL by ASP. One hundred microliters of through activation of the plasma kallikrein/kinin system in ASP- ASP or BK at various concentrations were injected intradermally into a induced VL. The ASP VL activity increased to 1.8-fold 10 min guinea pig that previously received an i.v. injection of Evans blue dye. B, after intradermal ASP injection in comparison with the initial VL VL activity of ASP and the effect of DFP and SBTI. ASP, DFP-treated activity and reduced to the base level almost completely at 30 min ASP, BK, and ASP with SBTI were injected intradermally into guinea pigs (Fig. 3). Through the time course HOE140 strongly inhibited ASP- and the VL activity was measured. (E), ASP; (‚), BK; (m), ASP in the ϳ induced VL, and the BK B2 receptor antagonist abolished 90% presence of 10 ␮M SBTI; (F), ASP treated with DFP (1 mM for 30 min). of the maximal VL at 10 min (Fig. 3). p Ͻ 0.005 for ASP 100 vs 30 or 300 ,ء .(Values are means Ϯ SD (n ϭ 3 nM; for BK 10 vs 100 nM; and for ASP 300 nM in the absence vs in the Production of VL activity from human plasma by ASP ,p Ͻ 0.02 for BK 100 vs 1000 nM. Inset ,ءء .presence of 10 ␮M SBTI Effect of receptor antagonists on ASP-induced VL. ASP, BK, or histamine To study the possibility that ASP can produce VL activity in hu- was injected intradermally to guinea pigs treated with or without a receptor mans, ASP was incubated with human plasma, treated with DFP, antagonist. (Ⅺ), Untreated; (f), HOE140 treated; (u), diphenhydramine and the VL activity of the plasma was measured. ASP produced -p Ͻ 0.01 for with vs without VL activity from human plasma in a dose- and enzymatic activity ,ء .(treated. Values are means Ϯ SD (n ϭ 3 a treatment. dependent manner, with ϳ40% of the ASP VL activity production being inhibited by SBTI (Fig. 4). HOE140 and diphenhydramine

A BP transducer (MIKRO-TIP catheter transducer model SPR-671) con- nected to a transducer ampliﬁer (transducer control unit model TCB-500; Millar Instruments) with a recorder (miniwriter WR7200; Graphtec) was inserted into the right carotid artery. A hundred microliters of ASP diluted with TBS was administered in a single bolus injection into the left ventri- cle. HOE140 (10 nm/kg body weight) dissolved in 200 ␮l of TBS was injected s.c. at 30 min before sample injections into guinea pigs. Repre- sentative results were shown. Statistics Statistical analysis was performed using an unpaired Student’s t test. Val- ues were expressed with means Ϯ SD (n ϭ 3or4). FIGURE 4. Production of VL activity by ASP in human plasma. Hu- Results man plasma in the presence or absence of SBTI was incubated with various concentrations of ASP at 37°C for 5 min. After the inhibition of ASP Induction of VL by ASP activity with 1 mM DFP, VL activity of the plasma was measured in guinea ASP used in this study was proved to be homogenous, showing a pigs treated with or without antagonist administration. (E), Plasma incu- single band under both conditions on a SDS-PAGE gel (Fig. 1). bated with untreated ASP was injected into untreated guinea pigs; (F), First, VL activity of ASP was studied in guinea pigs. ASP induced plasma incubated with DFP (1 mM)-pretreated ASP was injected into un- VL in a dose-dependent manner starting at an enzyme concentra- treated guinea pigs; (m), plasma incubated with untreated ASP in the pres- ␮ छ tion of 30 nM (Fig. 2A). In contrast to a linear increase of VL ence of 10 M SBTI was injected into untreated guinea pigs; ( ), plasma incubated with untreated ASP was injected into guinea pigs treated with caused by exponentially increasing doses of BK, the VL reaction HOE140; (‚), plasma incubated with untreated ASP was injected into triggered by ASP injection increased steeply at higher enzyme guinea pigs treated with diphenhydramine. Values are means Ϯ SD (n ϭ p Ͻ 0.01 for ASP 300 vs 100 or 1000 nM; and for ASP 1000 nM with ,ء .(concentrations (Fig. 2, A and B). Inactivation of ASP by the serine 4 p Ͻ 0.02 for ASP 1000 nM in the absence vs in ,ءء .protease inhibitor DFP resulted in the loss of VL activity (Fig. 2, vs without treatment A and B), suggesting that the proteolytic activity of the enzyme is the presence of 10 ␮M SBTI; and for ASP 1000 nM in the presence of 10 linked to the induction of VL. Interestingly, SBTI reduced Ͼ60% ␮M SBTI vs ASP 1000 nM with HOE140 treatment. 8726 ASP AND VL

FIGURE 6. Production of VL activity from kininogens by ASP. HK (80 ␮g/ml) or LK (130 ␮g/ml) was incubated with various concentrations of ASP at 37°C for 5 min, followed by 1 mM DFP addition. VL activity was then measured in guinea pigs treated with or without HOE140. (E), HK; (‚), LK. Closed symbols indicate the activity in p Ͻ 0.01 ,ء .(HOE140-treated animals. Values are means Ϯ SD (n ϭ 3 p Ͻ 0.02 ,ءء .for HK ϩ ASP3nMvsϩ ASP 30 nM; and for HK vs LK for LK ϩ ASP3nMvsϩ 30 nM.

VL induction. To investigate kininogen cleavage by ASP in FIGURE 5. Activation of prekallikrein by ASP. One micromolar pre- plasma, human plasma was incubated with ASP and analyzed kallikrein (E) or factor XII (‚) was incubated with ASP at 37°C for for kininogen fragments by immunoblotting. HK (110 kDa) and various periods, and the amidolytic activity for a fluorogenic substrate specific for kallikrein or activated factor XII, respectively, was measured. A, LK (64 kDa) were present in the initial plasma samples (Fig. Incubated with various concentrations of ASP for 5 min. B, Incubated with 7A, lane 1), with the two kininogens converted into three frag- 10 nM ASP for various periods. F, Prekallikrein incubated with DFP- ments in ASP-treated plasma (Fig. 7A, lane 2). From the mo- treated ASP. bility of the fragments from ASP-treated kininogens (Fig. 7B, lanes 2 and 4), fragments of 47 and 58 kDa appear to be derived from both kininogens and 32-kDa fragment from HK (Fig. 7). abolished the VL activity by ϳ60 and 40%, respectively (Fig. 4). These kininogen fragment bands detailed in the immunoblotting These results suggest that ASP can induce VL in humans mainly correspond well to those observed under SDS-PAGE analysis through the BK B2 receptor, and partially via the histamine H1 (Fig. 7B). The finding that ASP can cleave kininogens in plasma receptor. supports the VL activity production ability of ASP in humans, which might contribute to the septic shock genesis through ki- Activation of human prekallikrein by ASP nin release.

The observation that SBTI inhibited mostly the BK B2 receptor- dependent VL activity of ASP-treated human plasma (Fig. 4), BP lowering by ASP suggests a mediation of activation of human factor XII and/or The kinin generation activity of ASP suggests that this protein- prekallikrein by ASP. To investigate the involvement of this ase can result in the hypotension seen in septic shock patients. pathway, puriﬁed human prekallikrein or factor XII was incu- To study a link between ASP and septic shock further, we in- bated with ASP, and the enzymatic activities were measured vestigated the effect of ASP on BP in guinea pigs. ASP induced with synthetic substrates speciﬁc to each activated form. ASP a drop of BP (by the mean BP 20.3 Ϯ 2.7 mmHg; n ϭ 4), which generated kallikrein activity from human prekallikrein in a peaked 15 s after injection, and returned to initial levels 90 s dose- and incubation time-dependent manner, but no enzymatic later (Fig. 8A). The effect was not induced by enzymatically activity was detected from human factor XII incubated with inactive ASP (by the mean BP 3.0 Ϯ 3.3 mmHg; n ϭ 4; p Ͻ ASP (Fig. 5, A and B). No kallikrein activity was generated 0.0002) (Fig. 8B). Notably, the ASP BP-lowering activity was when ASP was inactivated with DFP (Fig. 5, A and B), and ASP hydrolyzed neither of the synthetic substrates. These results indicate a mechanism of VL induction by ASP, because ASP activates only prekallikrein, and the product, kallikrein, releases BK from HK.

Production of VL activity from human kininogens by ASP

The observation that SBTI did not inhibit BK B2 receptor-dependent VL completely (Fig. 4) prompted us to investigate direct VL activity production from kininogens by ASP. ASP was incubated with either of the puriﬁed human kininogens, and VL FIGURE 7. Kininogen cleavage in plasma by ASP. Human plasma was activity was measured upon the inactivation of ASP with DFP. incubated with ASP and after treating with immobilized Cibacron blue/ ASP produced VL activity from both kininogens; it yielded 2- protein A gel, the plasma was applied to a SDS-polyacrylamide gel under to 3-fold more VL activity from LK than HK when these kini- reducing conditions. Kininogen fragments were then analyzed by immunoblotting (A, lanes 1 and 2). The result of immunoblotting (A, lanes 3–6) nogens were used at their physiological plasma concentrations of ASP-treated kininogens was shown together with the result of SDS- (35) (Fig. 6). Because the kininogen-derived VL activity was PAGE analysis (B, lanes 1–4). A, Immunoblotting: lane 1, plasma; lane 2, completely inhibited by HOE140 (Fig. 6), the activity is medi- ASP-treated plasma; lane 3, HK (0.1 ␮g); lane 4, ASP-treated HK (0.1 ated by kinin generation. The VL activity production from kini- ␮g); lane 5, LK (0.1 ␮g); lane 6, ASP-treated LK (0.1 ␮g). B, SDS-PAGE: nogens by ASP, with a preference for LK rather than HK under lane 1,HK(1␮g); lane 2, ASP-treated HK (1 ␮g); lane 3,LK(1␮g); lane physiological conditions indicates another mechanism of ASP 4, ASP-treated LK (1 ␮g). The Journal of Immunology 8727

prekallikrein conversion to kallikrein (-Arg-2-Ile-) (37) and for kinin release from kininogens (-Arg-2-Ser-) (38), supporting these ASP abilities. Human prekallikrein activation has been shown for cysteine proteinases (gingipains-R) produced by Por- phyromonas gingivalis (21, 39), the major pathogen of adult periodontitis, and a metalloprotease from Vibrio vulnificus (40). In accordance with the case of ASP (Fig. 2B), SBTI also inhibited VL by these proteases without affecting their enzymatic activities (21, 41). No serine protease of bacteria origin has been reported as a prekallikrein activator; hence, ASP is thus far the only serine proteinase shown to possess this ability. Sta- phopain A (ScpA) from Staphylococcus aureus (23), strep- FIGURE 8. BP-lowering activity of ASP (1.5 nm/100 ␮l/kg body topain (SpeB) from Streptococcus pyogenes (42), 56K-protease weight). A, ASP was injected into an untreated guinea pig; B, DFP-treated from Serratia marcescens (43), and subtilisin from Bacillus ASP was injected into an untreated guinea pig; C, ASP was injected into subtilis (43) were capable of releasing kinin directly from a guinea pig treated with HOE140. human kininogens. The fact that ASP generated VL activity from both kininogens (Fig. 6) is in agreement with the kinin- releasing ability of these proteinases (23, 42, 43), with the inhibited almost completely by HOE140 (by the mean BP 4.1 Ϯ exception of subtilisin, a serine protease, that releases kinin 2.5 mmHg; n ϭ 4; p Ͻ 0.0002) (Fig. 8C). The result suggests only from LK (43). ASP produced more VL activity from kini- that ASP in the bloodstream lowers BP in an enzymatic activ- nogens than ScpA, the protease that generates kinin most effi- ity-dependent manner, and almost all of the activity is mediated ciently of these four listed enzymes (Fig. 6 and Ref. 23). There- by kinin production. fore, ASP would appear to be the most potent in vitro kinin generator of any bacterial protease studied to date. However, Discussion the result that ASP generated less kinin from human plasma An important pathophysiological mechanism of septic shock is than ScpA devoid of prekallikrein activation ability (Fig. 4 and hypovolemic hypotension caused by plasma leakage into the Ref. 23), suggests a strong interference of plasma components, extravascular space. The fact that ASP induced VL at a con- including protease inhibitors, in ASP kinin production when centration as low as 30 nM, 5 min postinjection into guinea pig compared with ScpA. skin (Fig. 2, A and B) indicates that VL induction by this en- The partial inhibition of ASP VL by diphenhydramine (Figs. zyme can occur efficiently in vivo. Moreover, the rapid gener- 2B, inset, and 4) demonstrates the participation of histamine ation of VL activity from human plasma by ASP (Fig. 4) de- release from mast cells. A Vibrio mimicus metalloprotease and creases the likelihood of enzyme clearance from the circulation, a house dust mite serine protease have been shown to cause and suggests that this proteinase may cause septic shock in histamine-dependent VL (44, 45). The mite protease and gin- human cases of severe A. sobria infection. The dependency of gipain-R release anaphylatoxin (C3a and C5a)-like fragments

ASP VL activity on the BK B2 receptor (Figs. 2B, inset,3,and from complement C3 and C5, respectively, which elicit hista- 4) clearly show that this pathogenic activity is exerted by kinin mine release from mast cells (45, 46); and C5a also induces production, which is one of the prominent features of septic neutrophil chemotaxis (46). Indeed, signiﬁcant neutrophil ac- shock (13–16). Because ASP activates prekallikrein (Fig. 5), an cumulation was seen at ASP injection sites, but not DFP-treated upstream component of the proteolytic cascade reaction of the ASP injection sites in guinea pigs (data not shown). Thus, ana- plasma kallikrein/kinin system (12), this facilitates efﬁcient ki- phylatoxin-like molecule production could be a mechanism of nin production by plasma kallikrein, the biological BK liberator H1 receptor-dependent VL by ASP. The histamine-induced VL from HK. Furthermore, because ASP can also act on LK (Figs. terminates within a few minutes, and, once released, histamine 6 and 7), whose plasma molar concentration is 3-fold higher is not restored in mast cells for at least several hours; hence, than that of HK (35), this proteinase also has an opportunity to mast cells cannot continue histamine release, even though ASP interact with more substrates than proteinases that generate BK continues to initiate anaphylatoxin generation. Conversely, be- only from HK. Taken together, these results indicate that VL cause of the continuous supply of prekallikrein and kininogens induction by ASP could be an underlying mechanism of septic to ASP injection sites from plasma due to VL, their local con- shock induction in severe A. sobria infection. This contention is centrations increase to plasma levels until the proteinase is ␣ supported by the BP-lowering effect of ASP, which is depen- completely inactivated; presumably by 2-macroglobulin, dent on both the proteolytic activity of the enzyme and the BK which is the most potent plasma protease inhibitor for bacterial

B2 receptor (Fig. 8). proteases (47, 48), and is found to increase at VL sites (49). The dominant inhibitory effect of HOE140 on ASP VL (Figs. These observations may explain the result that histamine was 2B, inset, 3, and 4) indicates that kinin release contributes initially involved for only a short period, and afterward the mainly to the VL. VL by ASP and by ASP-treated human kallikrein/kinin system contributed almost all to ASP-induced plasma was inhibited by SBTI more effectively than by diphen- VL (Fig. 3). To induce histamine release, anaphylatoxins, when hydramine, and the VL inhibition ratios of SBTI vs those of produced in the blood stream by ASP, must permeate to the HOE140 were Ͼ60% (Figs. 2B, inset, and 4). It is likely that extravascular space where mast cells reside. Due to dilution in most of the ASP B2 receptor-dependent VL is caused by BK blood, and diffusion in the extravascular space, the anaphyla- liberated by kallikrein converted from plasma prekallikrein toxins are probably unable to keep a concentration sufficient to (Fig. 5) with the rest mediated by direct kinin release from elicit mast cell histamine release. Kinins generated in the blood kininogens (Fig. 6). The substrate specificity of ASP, which stream can bind directly to B2 receptor on endothelial cells and demonstrates a strong preference for basic amino acid at the P1 cause BP reduction via vasodilation through the production of position (36), is consistent with the cleavage sites requisite for NO and prostacyclin (50), in addition to by VL. As shown by 8728 ASP AND VL the finding that the BP-lowering activity of ASP was com- 18. Pixley, R. A., R. A. DeLa Cadena, J. D. Page, N. Kaufman, E. G. Wyshock, pletely inhibited by HOE140 (Fig. 8), it is clear that the kal- W. R. Colman, A. Chang, and F. B. Taylor, Jr. 1992. Activation of the contact system in lethal hypotensive bacteremia in a baboon model. Am. J. Pathol. 140: likrein/kinin system plays a major role in the effect of ASP on 897–906. blood vessels in contrast to the marginal contribution of the 19. Pixley, R. A., R. A. DeLa Cadena, J. D. Page, N. Kaufman, E. G. Wyshock, A. Chang, F. B. Taylor, Jr., and R. W. Colman. 1993. The contact system con- histamine pathway. tributes to hypotension but not disseminated intravascular coagulation in lethal We have conclusively demonstrated that ASP, secreted from bacteremia: in vivo use of a monoclonal anti-factor XII antibody to block contact A. sobria at infection sites or in the circulation, is likely to activation in baboons. J. Clin. Invest. 91: 61–68. 20. Janda, J. M. 1991. Recent advances in the study of the taxonomy, pathogenicity, produce kinin, possibly contributing to septic shock through its and infectious syndromes associated with the genus Aeromonas. Clin. Microbiol. VL and vasodilation activities. ASP may, therefore, constitute Rev. 4: 397–410. an important therapeutic target for A. sobria septic shock, and 21. Imamura, T., R. N. Pike, J. Potempa, and J. Travis. 1994. Pathogenesis of periodontitis: a major arginine-specific cysteine proteinase from Porphyromonas gin- inhibitors of this enzyme together with BK B2 receptor antag- givalis induces vascular permeability enhancement through activation of the kal- onists could be developed as drugs. The benefit of such treat- likrein/kinin pathway. J. Clin. Invest. 94: 361–367. ments is further compounded by the observation that other 22. Imamura, T., J. Potempa, R. N. Pike, and J. Travis. 1995. Dependence of vascular permeability enhancement on cysteine proteinases in vesicles of Porphyromonas Aeromonas spp. also secrete serine proteinases similar to ASP gingivalis. Infect. Immun. 63: 1999–2003. (51–53). Thus, any such inhibitors of these proteolytic enzymes 23. Imamura, T., S. Tanase, G. Szmyd, A. Kozik, J. Travis, and J. Potempa. 2005. may prove widely successful in the treatment of the diseases Induction of vascular leakage through release of bradykinin and a novel kinin by cysteine proteinases from Staphylococcus aureus. J. Exp. Med. 201: caused by aeromonads infection. 1669–1676. 24. Janda, J. M., M. Reitano, and E. J. Bottone. 1984. Biotyping of Aeromonas isolates as a correlate to delineating a species-associated disease spectrum. Acknowledgments J. Clin. Microbiol. 19: 44–47. We thank Dr. Ivette Revollo for reviewing the paper. We also thank 25. Daily, O. P., S. W. Joseph, J. C. Coolbaugh, R. I. Walker, B. R. 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