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.
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.
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 Ca2+ concentration and marked leukolysis. Exposure of reputed LKT-nonsusceptible porcine lymphocytes and Raji cells to LKT caused a slightly increased intracellular Ca2+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 μg/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 μg/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 μg of protease K per ml diminished LKT binding to BL3 cells by 75%, with only a 25% decrease in leukolysis. However, pretreatment with 150 μg 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 Ca2+ concentration without leading to leukolysis.
One of the objectives of the National Institutes of Allergy and Infectious Diseases (NIAID) Biodefense Program is to identify or develop broad-spectrum antimicrobials for use against bioterrorism pathogens and emerging infectious agents. As a part of that program, our institution has screened the 10 000-compound MyriaScreen Diversity Collection of high-purity druglike compounds against three NIAID category A and one category B priority pathogens in an effort to identify potential compound classes for further drug development. The effective use of a Clinical and Laboratory Standards Institute–based high-throughput screening (HTS) 96-well–based format allowed for the identification of 49 compounds that had in vitro activity against all four pathogens with minimum inhibitory concentration values of ≤16 μg/mL. Adaptation of the HTS process was necessary to conduct the work in higher-level containment, in this case, biosafety level 3. Examination of chemical scaffolds shared by some of the 49 compounds and assessment of available chemical databases indicates that several may represent broad-spectrum antimicrobials whose activity is based on novel mechanisms of action.
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