Effects of Mannheimia haemolytica leukotoxin on apoptosis and oncosis of bovine neutrophils.

OBJECTIVE: To investigate the concentration-dependent effects of Mannheimia haemolytica (formerly Pasteurella haemolytica) leukotoxin (LKT) on apoptosis and oncosis in bovine neutrophils and to examine the role of calcium ions (Ca2+) in LKT-induced apoptosis. SAMPLE POPULATION: Neutrophils isolated from blood samples obtained from healthy calves. PROCEDURE: Neutrophil suspensions were exposed to lytic or sublytic dilutions of LKT and then examined by use of transmission electron microscopy (TEM) or gel electrophoresis. Contribution of extracellular Ca2+ to LKT-induced apoptosis was investigated by incubating neutrophils with LKT or control solutions in buffer containing 1 mM CaCl2 or in Ca2+-free buffer containing 1 mM ethylene glycol-bis (b-aminoethyl ether)-N,N-tetraacetic acid (EGTA) prior to diphenyl amine analysis. RESULTS: Examination by TEM revealed that bovine neutrophils exposed to lytic dilutions of LKT had changes consistent with oncosis, whereas neutrophils exposed to sublytic dilutions of LKT and staurosporin, an inducer of apoptosis, had changes consistent with apoptosis. Effects of sublytic dilutions of LKT on apoptosis were confirmed by gel electrophoresis. Replacement of extracellular Ca2+ with EGTA, a Ca2+ chelator, reduced apoptosis attributable to the calcium ionophore A23187, but it did not have significant effects on apoptosis induced by LKT or staurosporin. CONCLUSIONS AND CLINICAL RELEVANCE: The ability of LKT to cause apoptosis instead of oncosis is concentration-dependent, suggesting that both processes of cell death contribute to an ineffective host-defense response, depending on the LKT concentration in pneumonic lesions. Furthermore, although Ca2+ promotes A23187-induced apoptosis, it is apparently not an essential second messenger for LKT-induced apoptosis.

Ultrastructural characterization of apoptosis in bovine lymphocytes exposed to Pasteurella haemolytica leukotoxin.

OBJECTIVE: To characterize ultrastructural changes of bovine lymphocytes exposed to Pasteurella haemolytica leukotoxin (LKT). SAMPLE POPULATION: Partially purified LKT from a wild type P. haemolytica A1 strain and inactive pro-LKT from an isogeneic mutant Phaemolytica strain. Isolated bovine lymphocytes were obtained from 2 healthy calves. PROCEDURE: Isolated bovine lymphocytes were incubated with various concentrations of LKT and pro-LKT for 3 hours at 37 C and examined by use of transmission electron microscopy. A cytochemical Klenow DNA fragmentation assay was used to examine lymphocytes for DNA fragmentation. RESULTS: Lymphocytes incubated with LKT at a high concentration (1.0 toxic U/ml) had ultrastructural evidence of cytoplasmic and nuclear membrane rupture and swelling or lysis of mitochondria. Low concentrations of leukotoxin (0.1 toxic U/ml) induced DNA fragmentation in 80% of lymphocytes. Ultrastructurally, these cells had nuclear membrane blebbing, cytoplasmic vaculation, chromatin condensation, nuclear fragmentation, and membrane-bound apoptotic bodies. Incubation of lymphocytes with LKT at extremely low concentrations (0.001 toxic U/ml) or with pro-LKT did not alter their ultrastructure. Inclusion of 0.5 mM ZnCl2 in the medium blocked leukotoxin-induced ultrastructural changes in bovine lymphocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Low concentrations of LKT induce apoptosis and high concentrations induce oncotic cell lysis in bovine lymphocytes. The ability of low LKT concentrations to induce apoptosis in host leukocytes may allow bacteria to escape host immune surveillance and colonize the host.

Correlation of Pasteurella haemolytica leukotoxin binding with susceptibility to intoxication of lymphoid cells from various species.

Pasteurella haemolytica, the causative agent of shipping fever pneumonia in cattle, produces a leukotoxin (LKT) which lyses ruminant leukocytes with high efficiency but is reputed to not affect leukocytes from nonruminant species. In this study, we tested the supposition that LKT binding correlates positively with susceptibility to intoxication of susceptible isolated bovine lymphocytes and lymphoma tissue culture cells (BL3 cells) and negatively with reputed nonsusceptible equine, porcine, and canine lymphocytes and human lymphoid tissue culture cells (Raji cells). Bovine lymphocytes and BL3 cells were highly susceptible to LKT intoxication, exhibiting both substantial increase in intracellular Ca(2+) concentration and marked leukolysis. Exposure of reputed LKT-nonsusceptible porcine lymphocytes and Raji cells to LKT caused a slightly increased intracellular Ca(2+) concentration but no leukolysis. No LKT effect was detected for equine and canine lymphocytes. LKT bound to lymphoid cells from all species tested. Intact 102-kDa LKT was recovered from exposed isolated lymphoid cell membranes. Pro-LKT acylation was not required for LKT binding to BL3 cells. LKT binding was rapid, with maximal binding occurring by 3 min, and was proportional to the LKT concentration in the range 0.04 to 4.0 microg/ml. For this LKT concentration range, BL3 cells bound more LKT than did porcine lymphocytes or Raji cells, suggesting that LKT binds to BL3 cells with higher affinity than to porcine lymphocytes or Raji cells. Above 4.0 microg/ml, LKT demonstrated saturable binding to BL3 cells. Neutralizing anti-LKT monoclonal antibody (MAb) MM601 diminished LKT binding to BL3 by 36% while decreasing leukolysis by 81%. In contrast, MM601 did not diminish LKT binding to Raji cells. Pretreatment of target cells with 120 microg of protease K per ml diminished LKT binding to BL3 cells by 75%, with only a 25% decrease in leukolysis. However, pretreatment with 150 microg of protease K per ml abolished the remaining 25% of LKT binding and 75% leukolysis. Therefore, P. haemolytica LKT binds rapidly to susceptible and to reputed nonsusceptible lymphoid cells. LKT binding resulting in species-specific leukolysis was characterized by high affinity, inhibition by MAb MM601, and relative resistance to protease K pretreatment of lymphoid cells. Two types of LKT binding to lymphoid cells are proposed. High-affinity binding leads to efficient leukolysis. In some lymphoid cells from reputed LKT-nonsusceptible species, low-affinity LKT binding may cause a low-efficiency increase in the intracellular Ca(2+) concentration without leading to leukolysis.

Bovine CD18 identified as a species specific receptor for Pasteurella haemolytica leukotoxin.

Ligand blotting of Pasteurella haemolytica leukotoxin (LKT) susceptible BL3 bovine lymphoma cell membranes with LKT detected two putative receptors with Mr of 95 and 100 kDa, whereas no LKT binding to membrane proteins was detected for LKT non-susceptible human leukemic cells. Anti-bovine CD18 and CD11a/CD18 mAb recognized 95 and 100kDa bands from BL3 cell membranes. CD18 isolated from BL3 cell membranes bound LKT. Pre-incubation of BL3 cells with anti-bovine CD18 or CD11a/CD18 mAb caused partial inhibition of LKT-induced leukolysis. Therefore, we propose that bovine CD18 acts as a species-specific leukocyte receptor for P. haemolytica LKT.

Role of phospholipase D in Pasteurella haemolytica leukotoxin-induced increase in phospholipase A(2) activity in bovine neutrophils.

The effects of Pasteurella haemolytica leukotoxin (LKT) on the activity of phospholipase D (PLD) and the regulatory interaction between PLD and phospholipase A(2) (PLA(2)) were investigated in assays using isolated bovine neutrophils labeled with tritiated phospholipid substrates of the two enzymes. Exposure of [(3)H]lysophosphatidylcholine-labeled neutrophils to LKT caused concentration- and time-dependent production of phosphatidic acid (PA), the product of PLD. LKT-induced generation of PA was dependent on extracellular calcium. Both production of PA and metabolism of [(3)H]-arachidonate ([(3)H]AA)-labeled phospholipids by PLA(2) were inhibited when ethanol was used to promote the alternative PLD-mediated transphosphatidylation reaction, resulting in the production of phosphatidylethanol rather than PA. The role of PA in regulation of PLA(2) activity was then confirmed by means of an add-back experiment, whereby addition of PA in the presence of ethanol restored PLA(2)-mediated release of radioactivity from neutrophil membranes. Considering the involvement of chemotactic phospholipase products in the pathogenesis of pneumonic pasteurellosis, development and use of anti-inflammatory agents that inhibit LKT-induced activation of PLD and PLA(2) may improve therapeutic management of the disease.

Lipopolysaccharide complexes with Pasteurella haemolytica leukotoxin.

The presence of lipopolysaccharide (LPS) in gram-negative bacterial repeats-in-toxin (RTX) toxin preparations, as well as the harsh conditions required to remove it, suggests that LPS may complex with RTX toxins. Concentrated culture supernatant (CCS) preparations of the RTX toxin Pasteurella haemolytica leukotoxin (LKT) contained LKT and LPS as the most prominent components, with LKT and LPS constituting approximately 30 and 50% of the density of the silver-stained fraction on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. CCS LKT contained 3.69 +/- 0.46 mg of LPS per mg of protein, which was estimated to indicate an LPS/LKT molar ratio of approximately 60:1. Subjection of the CCS LKT to preparative SDS-PAGE resulted in separation of LPS from LKT as detected by silver-stained analytical SDS-PAGE; however, the LKT fraction (SDS-PAGE LKT) contained significant endotoxin activity as detected by the Limulus amebocyte lysate assay. Subjection of the SDS-PAGE LKT to a second preparative SDS-PAGE run resulted in a reduction of the LPS/LKT molar ratio to 1:20. The target cell specificity of LKT for bovine leukocytic cells was retained by the SDS-PAGE LKT, and isolated LPS at comparable concentrations to that in CCS LKT exhibited no leukolytic activity. Addition of isolated LPS back to SDS-PAGE LKT resulted in reconstitution of an LPS-LKT complex. Immediately following reconstitution of the LPS-LKT complex, there was minimal change in leukolytic activity of the complex, but following 9.5 h at temperatures from -135 to 37 degrees C, the LPS-LKT complex exhibited increased leukolytic activity and thermal stability compared to SDS-PAGE LKT. Therefore, it appears that LPS complexes with LKT, resulting in enhanced and stabilized leukolytic activity.

Pasteurella haemolytica leukotoxin induced apoptosis of bovine lymphocytes involves DNA fragmentation.

It has been reported that Pasteurella haemolytica leukotoxin (LKT) induces morphologic changes in bovine leukocytes consistent with apoptosis in vitro, but DNA fragmentation was not observed. We investigated whether bovine lymphocytes undergo DNA fragmentation during LKT-induced apoptosis. Bovine peripheral blood lymphocytes were isolated by density gradient centrifugation and exposed to LKT or inactive pro-LKT protein from a lktC- mutant strain. After exposure, DNA fragmentation in lymphocytes was quantified colorimetrically by diphenylamine assay and visualized by agarose gel electrophoresis. At high LKT concentrations, bovine lymphocytes were lysed, but at low concentrations, LKT caused DNA fragmentation characteristic of apoptosis. Maximal DNA fragmentation in bovine lymphocytes was induced by 0.1 TU ml(-1) LKT following 3 h exposure, but only background level of DNA fragmentation was observed with the inactive pro-LKT. Equine lymphocytes that are resistant to LKT intoxication did not show DNA fragmentation following exposure to LKT. Preincubation of LKT with a neutralizing anti-LKT monoclonal antibody inhibited LKT-induced DNA fragmentation. Electrophoresis of DNA from bovine lymphocytes treated with 0.1 TU ml(-1) LKT demonstrated the typical 'ladder' pattern of internucleosomal DNA cleavage, the hallmark of apoptosis associated with activation of endonucleases. LKT-induced DNA fragmentation was inhibited by 0.5 mM ZnCl2, an endonuclease inhibitor. The results indicated that LKT at low concentrations induced apoptotic cell death of bovine lymphocytes, which may play a role in initiation and persistence of P. haemolytica infection.

Duration of serum antibody responses following vaccination and revaccination of cattle with non-living commercial Pasteurella haemolytica vaccines.

This study was designed to determine the duration of serum antibody responses to Pasteurella haemolytica whole cells (WC) and leukotoxin (LKT) in weanling beef cattle vaccinated with various non-living P. haemolytica vaccines. Serum antibodies to P. haemolytica antigens were determined periodically through day 140 by enzyme-linked immunosorbent assays. At day 140, cattle were revaccinated, and antibody responses periodically determined through day 196. Three vaccines were used in two experiments (A and B), OneShot, Presponse HP/tK, and Septimune PH-K. In general, all three vaccines between 7 and 14 days induced antibody responses to WC after vaccination. Antibodies to LKT were induced with OneShot and Presponse. Revaccination at days 28 and 140 usually stimulated anamnestic responses. Serum antibodies to the various antigens remained significantly increased for up to 84 days after vaccination or revaccination. The intensity and duration of antibody responses were variable depending on the experiment and vaccines used. Vaccination with OneShot usually stimulated the greatest responses to WC. Vaccination with OneShot or Presponse resulted in equivalent primary anti-LKT responses. In experiment B, spontaneous seroconversion was found in numerous calves on day 112. Revaccination of those cattle at day 140 resulted in markedly variable antibody responses such that several groups had no increase in antibody responses.

Serum-free culture of Pasteurella haemolytica optimized for leukotoxin production.

OBJECTIVE: To screen supernatants of Pasteurella haemolytica cultures grown in 4 serum-free culture media for maximal leukotoxin (LKT) production and minimal protein concentration as an optimal source of LKT for purification. SAMPLE POPULATION: One strain of P haemolytica biotype A serotype 1 originally isolated from the pneumonic lung of a calf. PROCEDURE: Pasteurella haemolytica was grown in brain-heart infusion (BHI) broth, yeast-tryptone broth, RPMI-1640 medium, and McCoy's modified 5A medium. Culture biomass and protein concentration, LKT activity, and LKT concentration in culture supernatants were measured. Effects of media pH and supplementation with metal cations and glucose on growth rate of P haemolytica and culture supernatant parameters were evaluated. RESULTS: Pasteurella haemolytica cultivated in BHI broth or RPMI-1640 medium containing 0.1 M phosphate (pH 6.8) produced the highest concentrations of LKT. Supplementation of RPMI-1640 medium with 0.36 mM FeCl3 or 1.0 mM MgSO4 further increased specific activity of LKT in culture supernatant, but addition of 1% glucose did not enhance LKT production. Leukotoxin production in MgSO4-supplemented RPMI-1640 medium was comparable to that in serum protein-supplemented medium. CONCLUSIONS: Although BHI broth was superior to RPMI-1640 medium for P haemolytica growth and LKT production, the higher protein concentration and lower LKT specific activity made BHI broth a less desirable medium, compared with RPMI-1640 medium. Growth rate and LKT production with minimal protein content was optimal in pH 6.8 phosphate-buffered MgSO4-supplemented RPMI-1640 medium. This medium can serve as a source of culture supernatant for purification of LKT.

High-performance liquid chromatography of Pasteurella haemolytica leukotoxin using anion-exchange perfusion columns.

An exotoxin, called leukotoxin (LKT), from Pasteurella haemolytica, which had previously proved difficult to purify, was purified by high-performance liquid chromatography using rigid highly hydrophilic microparticulate anion-exchange columns. These anion-exchange stationary phases were employed to overcome difficulties of the relatively hydrophobic LKT interacting with dextran or styrene-based resins. While a short non-porous DEAE column allowed the partial microscale purification of the leukotoxin at pH 7.0, a high capacity strong anion-exchange column of the perfusion chromatography type permitted the purification of LKT on a much larger scale. The purification of the LKT on the large pore strong anion-exchange perfusion column was best achieved when three consecutive linear gradients at increasing NaCl concentration in 20 mM Tris buffer, pH 8.0, containing 6.0 M urea and 0.25% Tween 20 were used. Under these conditions, a better separation was obtained for the tetrameric and aggregate peaks of LKT from the early eluting contaminant peaks. This separation scheme allowed good recovery of activity and purification of the LKT to near homogeneity.

Serum antibody responses of cattle vaccinated with partially purified native Pasteurella haemolytica leukotoxin.

The objective of these experiments was to study the serum antibody responses of cattle to partially purified, native Pasteurella haemolytica A1 leukotoxin (LKT) formulated with a commercial aluminum hydroxide-DDA-bromide adjuvant. In two experiments, calves received two intramuscular injections 21 days apart and sera were obtained periodically. Serum antibody responses to P. haemolytica outer membrane proteins (OMPs), formalinized P. haemolytica, and LKT were determined. In Experiment A, Holstein calves (140 kg each) were vaccinated with either 10, 1.0 or 0.1 micrograms of LKT, 10(9) c.f.u. of live P. haemolytica, or adjuvanted diluent. In Experiment B, mixed-breed beef calves (200 kg each) were vaccinated with either 100, 50 or 10 micrograms of LKT, 10(9) c.f.u. live P. haemolytica, or adjuvanted diluent. Vaccination of dairy calves with 10 micrograms of partially purified LKT stimulated LKT neutralizing antibody responses similar to those stimulated by vaccination of one calf with live P. haemolytica. In Experiment B, which used larger and different breeds of cattle, two vaccinations 3 weeks apart with 50 micrograms LKT stimulated LKT neutralizing responses equivalent to or greater than those stimulated by vaccination with live P. haemolytica. In both experiments, LKT vaccines stimulated only low antibody responses to formalinized P. haemolytica or to OMPs.

Hemolytic activity of the Pasteurella haemolytica leukotoxin.

A Pasteurella haemolytica mutant incapable of producing leukotoxin was created by allelic replacement. Concentrated culture supernatants from wild-type P. haemolytica, but not from the mutant, contained the 102-kDa leukotoxin protein and lysed bovine lymphoma cells and sheep erythrocytes. Wild-type P. haemolytica demonstrated the typical beta-hemolytic phenotype on sheep and rabbit blood agar, whereas the mutant did not.

Chaotropic agents cause disaggregation and enhanced activity of Pasteurella haemolytica leukotoxin.

Pasteurella haemolytica biotype A, serotype 1 grown to late logarithmic growth phase in cell culture medium (RPMI 1640) produced highly aggregated leukotoxin. The multimer mass of the highly aggregated leukotoxin in 0.1 M sodium phosphate buffer pH 7.0 as determined by gel filtration chromatography on Sephacryl S400HR was approximately 8000 kDa. Resuspension of leukotoxin in phosphate buffer containing various chaotropic agents resulted in partial disaggregation of leukotoxin and enhanced leukotoxic activity. 3M guanidine disaggregated leukotoxin to a multimer mass of approximately 800 kDa and enhanced leukotoxic activity 3 to 20-fold. In 6 M urea or 1 M sodium thiocyanate, leukotoxin multimers were observed ranging in mass from 8000 kDa to 400 kDa, and activity enhancement was less than that for leukotoxin in 3 M guanidine. Several detergents were tested for enhancement of leukotoxic activity, but only 1% Tween 20 enhanced leukotoxic activity (4-fold), whereas 1.25% octylglucoside, 10 mM CHAPS, and 5 mM deoxycholate diminished and 1% Triton X-100 abolished leukotoxic activity.

Pasteurella haemolytica lipopolysaccharide-induced cytotoxicity in bovine pulmonary artery endothelial monolayers: inhibition by indomethacin.

Exposure of bovine pulmonary artery endothelial cells to Pasteurella haemolytica lipopolysaccharide caused severe morphologic changes. Initially, there was dilatation of the rough endoplasmic reticulum and mitochondrial swelling followed by cell retraction, membrane bleb formation, and cell detachment. The affected endothelial cells had severe membrane damage resulting in the leakage of lactate dehydrogenase. Indomethacin in concentrations of 0.5 mM or greater caused marked decreases in the lipopolysaccharide-induced leakage of lactate dehydrogenase. Indomethacin at 5 mM also caused a marked reduction of the lipopolysaccharide-induced morphologic changes resulting in apparent maintenance of the monolayer integrity for 8 hours versus 1 hour in the lipopolysaccharide-treated control. A marked decrease in the cell and nuclear membrane effects resulted, but the rough endoplasmic reticulum dilatation and mitochondrial changes proceeded. These results indicate that indomethacin does not prevent lipopolysaccharide binding but interferes with later events in lipopolysaccharide-induced cytotoxicity in the bovine pulmonary endothelial cell. The concentration of indomethacin required to produce this inhibition suggests that the primary mechanism is not cyclooxygenase inhibition.

Pasteurella haemolytica leukotoxin-induced synthesis of eicosanoids by bovine neutrophils in vitro.

Exposure of isolated bovine neutrophils to partially purified Pasteurella haemolytica leukotoxin caused increased synthesis of leukotriene B4 (LTB4) but not thromboxane B2 (TXB2) from endogenous arachidonic acid. Synthesis of LTB4 was closely correlated with leukotoxin-induced neutrophil lysis. At low toxin concentrations, LTB4 production lagged behind leukotoxin-induced neutrophil lysis over a 3-h period. The neutralizing monoclonal antileukotoxin antibody MM601 neutralized both leukotoxin-induced neutrophil lysis and LTB4 synthesis. Both leukotoxin-induced neutrophil lysis and LTB4 synthesis were Ca(2+)-dependent. When leukotoxin-induced LTB4 synthesis from exogenous arachidonic acid was examined, significant LTB4 synthesis occurred at 5 min of leukotoxin exposure, which was before leukotoxin-induced lysis developed. Leukotoxin-induced LTB4 synthesis from endogenous arachidonic acid appears to require leukotoxin-induced plasma membrane damage (occurring during neutrophil lysis), whereas LTB4 synthesis from exogenous arachidonic acid is initiated rapidly and occurs in the absence of plasma membrane damage.

Enhancement of Pasteurella haemolytica leukotoxic activity by bovine serum albumin.

Growth of Pasteurella haemolytica A1 in RPMI 1640 medium containing 0.5% bovine serum albumin (BSA) for 2.5 hours enhanced culture supernatant leukotoxic activity (30,700 +/- 12,900 toxic units/ml, compared with leukotoxic activity of culture supernatants produced in RPMI 1640 medium alone (120 +/- 40 toxic units/ml). Gel filtration chromatography of the leukotoxic activity from RPMI 1640 medium supernatants in buffer containing 50 mM NaCl indicated a single leukotoxic activity peak (peak I) eluting near the gel resin molecular mass exclusion limit (estimated molecular mass of approx 8,000 kd). In contrast, culture supernatants produced in RPMI 1640 plus bovine serum albumin medium (RPMI + BSA) had peak I and 2 additional leukotoxic activity peaks (peaks II and III) with estimated molecular mass of approximately 80 and < 30 kd, respectively. All leukotoxic activity peaks were composed of approximately 100-kd molecular mass leukotoxin protomer, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody against leukotoxin. Subjecting culture supernatant leukotoxic activity produced in RPMI + BSA to gel filtration chromatography in buffer containing 500 mM NaCl or 6M urea resulted in detection of only a single leukotoxic activity peak with estimated approximate molecular mass of 250 and 800 kd, respectively. These findings suggest that P haemolytica exists as a high molecular mass aggregate with low leukotoxic activity which, in the presence of BSA, partially disaggregates to multiple toxin forms with enhanced leukotoxic activity. Some of these leukotoxin forms interact with dextran-based gel resins at low ionic strength.

Lysis of bovine platelets by Pasteurella haemolytica leukotoxin.

Pasteurella haemolytica A1 culture supernatants caused rapid cytolysis (less than 5 minutes) of isolated bovine platelets as measured by leakage of the cytoplasmic enzyme lactate dehydrogenase (LD). The platelet lytic factor had several features similar to P haemolytica leukotoxin. Like P haemolytica leukotoxin, the platelet lytic factor was produced by P haemolytica during logarithmic growth phase, was heat-labile, and was active against target cells (platelets) from ruminant species (cattle and sheep), but not from non-ruminant species (horses, pigs, and human beings). Additionally, the platelet lytic factor was neutralized with antileukotoxin rabbit serum. The amount of LD leaked by a fixed concentration of bovine platelets was proportional to the amount of toxin added at low toxic doses and became maximal at 88 +/- 11% of the total platelet LD activity for high doses of toxin. When a fixed dose of toxin was used and the platelet concentration was varied, LD leakage was initially proportional to the platelet concentration, but plateaued at higher platelet concentrations. The platelet lytic factor required Ca2+ and was inhibited by addition of the Ca2+ chelator ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid. Toxin-mediated platelet damage may be important in thrombi formation and fibrin exudation typically associated with P haemolytica pleuropneumonia of cattle.

Mechanism of action of Moraxella bovis hemolysin.

Bovine erythrocytes (RBCs) exposed to Moraxella bovis culture supernatants exhibited rapid leakage of intracellular K+ (95% in 10 min), slower cell swelling (1.20-fold increase in mean corpuscular volume in 20 min), and subsequent lysis (76% leakage of hemoglobin in 25 min). Incubation media made hypertonic by the addition of 75 mM carbohydrates with molecular diameters of 0.72 to 1.32 nm prevented hemolysin-induced RBC swelling, but incubation media made hypertonic by the addition of carbohydrates with molecular diameters of less than 0.72 nm did not protect against hemolysin-induced RBC swelling. Raffinose (75 mM; molecular diameter, 1.14 nm) did not block hemolysin-induced K+ leakage but did block hemolysis. These findings support the hypothesis that hemolysin-induced lysis occurs by colloid-osmotic swelling and are compatible with M. bovis hemolysin acting as a pore-forming cytolysin. Assuming that M. bovis hemolysin acts as a transmembrane molecular sieve, then the functional size of the hemolysin transmembrane pores in bovine RBCs is approximately 0.9 nm, the molecular size of sucrose. Hemolytic activity was inhibited by the Ca2+ chelator ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), but hemolysin-induced K+ leakage was not affected by EGTA.