Thrombin-induced platelet microbicidal protein (PMP) is considered to play an important role in preventing streptococcal endocarditis. However, the structural features and functions of PMPs have not been well characterized, and their antibacterial spectra against other common endocarditis pathogens, such as the staphylococci, are not known. Thrombin stimulation of washed rabbit platelets (108/ml) yielded a PMP-rich preparation with a specific activity of-25 U/mg of protein as determined by Bacilus subtilis bioassay. TIwenty-eight clinical and laboratory Staphylococcus aureus isolates, exposed to a standardized PMP preparation (100 U/ml for 2 h at 37°C), exhibited a Poisson-distributed heterogeneity to the bactericidal action of PMP, with approximately one-third designated as PMP resistant. Gel filtration chromatography (Sephadex G-50) identified the bioactive moiety within PMP preparations to be in the major protein elution peak; sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) presumptively identified PMP as a lowmolecular-weight (MW) (8,500) protein present only in such bioactive protein peaks. Both the bioactivity of PMP preparations and the low-MW protein band were removable by specific anionic membranes (e.g., cellulose-acetate/nitrate), as well as by a variety of anionic resins, further corroborating the suspected cationic charge of PMP. In addition, both PMP bioactivity and the low-MW protein band were recoverable by 1.5 M NaCl elution of the anionic membrane filters post-PMP adsorptive removal. Adsorption of bioactive PMP preparations by highly PMP-susceptible B. subtilis (10' CFU/ml, 30 min) resulted in a near-complete loss of residual bioactivity; in contrast, adsorption of bioactive PMP preparations with less PMP-susceptible S. aureus strains failed to reduce bioactivity. Significant lysozyme contamination of PMP-rich preparations was ruled out by determination of differences between bioactive PMP preparations and exogenous lysozyme as regards (i) relative heat stabilities; (ii) differential bactericidal activity versus B. subtilis and Micrococcus luteus; and (iii) SDS-PAGE protein profiles. These data show that the bioactive PMP protein moiety is of low MW, is heat stable, is probably cationic (similar to leukocyte-derived defensins), and possesses potent bactericidal activity against a significant percentage of S. aureus isolates. * Corresponding author. termed thrombodefensins (5); however, their exact relationship to other defensin molecules, such as the leukocyte defensins (12), has not been defined. Thrombin-induced PMP(s) remain poorly characterized, and their structure, function, and microbicidal pathways have not been fully determined. In the present study, we describe the preparation, partial purification, and initial characterization of thrombin-induced PMP. In addition, we utilized Staphylococcus aureus, the most common etiologic agent of intravascular infections (19), to probe the functional microbicidal features of PMP. (A portion of this study was presented at the gen...
The life cycle of Leishmania includes sequential development of invertebratestage promastigotes from a noninfective to an infective stage (1)(2)(3)(4). This can be demonstrated for promastigotes growing both within the phlebotomine midgut and within axenic cultures . Promastigotes grown axenically to log stage are minimally infective for mice and are incapable of establishing intracellular infection within macrophages . In contrast, parasites grown to the stationary phase of growth demonstrate increased infectivity for cells and enhanced virulence for mice . The developmental changes occurring during transition from the noninfective log form to the infective metacyclic stationary phase form of L. major are current areas of study. Sacks et al . (5) have demonstrated that concentrations of peanut agglutinin (PNA)' that agglutinate 100% of organisms from log-phase cultures fail to agglutinate metacyclic promastigotes found within stationary cultures, and that the loss of agglutination with PNA can be used as the basis for purification of infective promastigotes (PNA-) from culture . This difference in PNA agglutinability reflects developmental modification of a prominent surface lipophosphoglycan (LPG) (6) previously shown by Handman et al. (7) to be shed from the parasite . Handman and Goding (8) have also demonstrated that the LPG, also termed excreted factor, is necessary for the attachment of the promastigote to macrophages (8), and that immunization ofmice with this antigen confers protection to subsequent challenge (9) . Studies by Sacks and da Silva (10) have shown that the developmentally regulated modification ofthe LPG involves acquisition of a novel carbohydrate determinant and that this modification appears to be a definitive marker for metacyclogenesis and a prerequisite for parasite infectivity in mice . Nonetheless, the mechanism by which this developmental change renders the parasite infective has not been defined .
The binding of 1251-labeled human myeloma immunoglobulin A (IgA) to four type II strains and one nontypable strain of group B streptococci was measured after streptococcal chains were broken by brief sonication. Some IgA binding was observed with all strains, but specific binding (binding that was inhibited by excess unlabeled IgA, was dose dependent, and was saturable) occurred only with those strains possessing the trypsin-sensitive beta component of the c protein. Similar amounts of binding were observed with myeloma IgA and IgAl purified from normal serum. Specific binding was more rapid at 25°C than at 0 or 37°C and reached a plateau in 6 to 8 h. Binding was drastically reduced (85 to 90%) when streptococci had been heated (90°C for 1 h). Most radioactivity bound to group B streptococci could be displaced (>60% in 3 days) by the addition of excess unlabeled IgA. The binding capacity of one strain (108 streptococci in 1 ml of buffer) was saturated by approximately 24 ,ug of IgA. When transformed for Scatchard analysis, these data indicated that there was a specific binding capacity of 16,000 molecules of monomeric serum IgA per single streptococcal cell. The dissociation constant for IgA binding was 19.3 nM. Since enzyme-linked immunosorbent assay studies showed that the myeloma IgA used for the studies described above was IgAl, our quantitative data apply only to the binding of this subclass to group B streptococci. However, an enzyme-linked immunosorbent-filtration assay showed that both IgAl and IgA2 bound to a type II group B streptococcus bearing the c protein.
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