Biomedical subjects

W M Shafer

Publications and source records attributed to W M Shafer.

At least 55 records · Page 3Linked to original sources

PubMed1988

Killing of Proteus mirabilis by polymorphonuclear leukocyte granule proteins: evidence for species specificity by antimicrobial proteins.

Low-molecular-weight (Mr, ca. 3,800) polypeptides containing human defensins HNP-1 and HNP-2 (T. Ganz, M. S. Selsted, D. Szlarek, S. L. Harwig, K. Daher, D. F. Bainton, and R. I. Lehrer, J. Clin. Invest. 76:1427-1434, 1985) prepared in our laboratory from acid extracts of human polymorphonuclear granulocyte granules and purified human defensins were found to exert potent bactericidal action against Proteus mirabilis. The antimicrobial action of the extracts of human polymorphonuclear leukocytes granules against P. mirabilis appears to be due to the presence of the defensins. Because P. mirabilis resists the antimicrobial action of other granule proteins, we interpret the present results to mean that the various antimicrobial proteins display species specificity in their microbicidal action.

Blood Bactericidal Activity↗

PubMed1988

Lipopolysaccharide structure determines ionic and hydrophobic binding of a cationic antimicrobial neutrophil granule protein.

Bactericidal activity and binding of a 57,000-dalton cationic antimicrobial neutrophil granule protein (CAP57) are determined by the presence on bacteria of O-antigen polysaccharide chains and the availability of negatively charged groups in the lipid A region, the inner core region, or both regions of lipopolysaccharide. Polymyxin B (PMB)-resistant mutants with well-defined alterations in lipid A structure and charge (pmrA) are also more resistant to CAP57. We used biologically active radioiodinated CAP57 to study the characteristics and kinetics of binding to a sensitive Rb lipopolysaccharide chemotype, Salmonella typhimurium SH9178, and the relatively resistant pmrA mutant strain SH7426. Binding occurred rapidly and was specific and saturable. Because CAP57 appears to be bound in a manner similar to that of PMB, competition binding studies were performed. Excess PMB did compete with CAP57 for binding to SH9178. Nonapeptide, a polycationic derivative of PMB that has lost its hydrophobic portions, demonstrated a marked decrease in ability to compete for binding with CAP57 compared with PMB. This demonstrated the importance of hydrophobic binding in the interaction of CAP57 with the microbial surface. Thus, we have shown that binding of CAP57 to SH9178 is specific, saturable, and similar to binding of PMB. Both hydrophobic and ionic properties of CAP57 appear to be necessary for binding.

Antimicrobial Cationic Peptides↗

PubMed1987

A monoclonal antibody that inhibits the antimicrobial action of a 57 KD cationic protein of human polymorphonuclear leukocytes.

Two monoclonal antibodies (mAb) specific for epitopes of a 57,000 m.w., cationic antimicrobial protein (CAP57) purified from granules of human polymorphonuclear leukocytes (PMN) have been produced. Both were IgG1 mouse antibodies with typical heavy and light chain structure. The mAb reactive with CAP57 failed to react specifically with other heretofore defined PMN or serum proteins as shown by ELISA. Both mAb showed specific reactivity in Western blots with CAP57. One of these mAb (P1G8) inhibited the antimicrobial action of CAP57 by 50% at a ratio of 62.5 micrograms antibody per microgram CAP57. The other mAb, P2A5, had no inhibitory capacity for CAP57. Binding constants of the two mAb for the antigen were determined and were found to be virtually identical. Thus, the greater inhibitory capacity of P1G8 for bacterial killing by CAP57 was not directly related to binding strength of the mAb. Competition experiments showed that unlabeled P1G8 could compete as well against radiolabeled P2A5 as could unlabeled P2A5. In the reverse experiment, it was seen that P1G8 competed with radiolabeled P1G8 for CAP57 better than unlabeled P2A5. These findings could be due to two antibodies that recognize different but adjacent epitopes on CAP57, one of the epitopes (P1G8) being closer to structure(s) of the protein essential to its antimicrobial action. Immunocytochemical studies showed positive staining with both mAb. The reaction was restricted to the cytoplasm of peripheral blood PMN and was of a granular pattern. Other peripheral blood cells (which included red blood cells, eosinophils, monocytes, and lymphocytes) failed to bind either mAb.

Antibodies, Monoclonal↗

PubMed1987

Cleavage of the protein III and major iron-regulated protein of Neisseria gonorrhoeae by lysosomal cathepsin G.

Incubation of either 125I-labelled or unlabelled Neisseria gonorrhoeae with enzymically active preparations of human polymorphonuclear leucocyte lysosomal cathepsin G revealed that surface-exposed outer-membrane proteins were susceptible to proteolytic modification. Electroimmunoblotting experiments confirmed that outer-membrane protein III (PIII) and the major iron-regulated protein (MIRP), two conserved gonococcal proteins, were cleaved by cathepsin G. A direct relationship was observed between susceptibility to the antibacterial properties of cathepsin G and cleavage of PIII among isogenic strains differing in their level of resistance to the bactericidal activity of cathepsin G. Although the antibacterial property of cathepsin G is known to be independent of serine-esterase activity, the data suggest that gonococcal outer-membrane proteins are involved in the binding of cathepsin G, and that variation in the level of resistance reflects the degree to which target outer-membrane proteins such as PIII are exposed.

Autoradiography↗

PubMed1987

Evidence that the serum resistance genetic locus sac-3 of Neisseria gonorrhoeae is involved in lipopolysaccharide structure.

The gonococcal chromosome contains a sequence of closely linked genes (for example, sac-1, sac-3, nmp) known or presumed to affect cell envelope structure and which appear to influence susceptibility of gonococci to killing by normal human sera (NHS). Previous work has shown that the serum-resistant isolate FA19, and FA899, a serum-sensitive transformant of FA19, differ in outer membrane protein I (PI) and at the sac-3 genetic locus. However, the sac-3 locus is separable from changes determined by nmp-3, the gene determining PI species. We found that FA19 and FA899 differ in lipopolysaccharide (LPS) molecular size and in reactivity with a monoclonal antibody which recognizes an LPS (L8) epitope. To address the question of whether the changes in LPS were due to the sac-3 locus, we constructed new transformants of FA19 using donor DNA prepared from FA899. The new transformants could be divided into three groups: (1) those identical to FA19 in serum resistance (greater than 90% survival at 120 min), in LPS molecular size and in expression of the L8 epitope; (2) those identical to FA899 in serum sensitivity (100% killed at 30 min), in LPS molecular size and in lack of expression of the L8 epitope; (3) those significantly killed by 50% NHS at 120 min, whose LPS molecular size was greater than that of FA19 but less than that of FA899 and which did not express the L8 epitope. Except for PI there were no differences in other outer-membrane proteins (e.g. PII, PIII, H.8) among these transformants.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Bactericidal Activity↗

PubMed1987

Nonoxidative antimicrobial effects of human polymorphonuclear leukocyte granule proteins on Chlamydia spp. in vitro.

Proteins from isolated granules of human polymorphonuclear leukocytes were assessed for their nonoxidative microbicidal effect on chlamydiae by two different methods: a radioisotope assay for elementary body integrity and a biological assay for inclusion development. Crude granule extract, which consisted of a mixture of all granule proteins, caused a 20 to 30% decrease in infectivity and a 52% decrease in infectivity when incubated with Chlamydia psittaci CAL-10 and Chlamydia trachomatis serovar E, respectively. To define more specifically the components that were damaging to chlamydiae, crude granule extract was subjected to Sephadex G-75 column chromatography and isolated granule fractions were obtained. Only fractions containing lysozyme as the major component consistently caused reductions in infectivity of C. trachomatis elementary bodies. In contrast, fractions collected after the lysozyme fraction, containing proteins with molecular masses of 13,000 daltons or less, had detrimental effects on C. psittaci infectivity. Additional experiments using highly purified human polymorphonuclear leukocyte lysozyme confirmed its infectivity-reducing action upon C. trachomatis but not upon C. psittaci.

Blood Bactericidal Activity↗

PubMed1987

Antimicrobial binding of a radiolabeled cationic neutrophil granule protein.

A 57,000-dalton protein (CAP57) purified from human polymorphonuclear leukocytes has antimicrobial activity against a number of gram-negative bacteria. We developed a procedure using solid-phase Iodo-gen to radiolabel CAP57 without destroying its antibacterial activity. Iodinated and native CAP57 were electrophoretically identical. Autoradiographs of sodium dodecyl sulfate-polyacrylamide gels revealed greater than 95% of the 125I in a single heavy band in the 57,000-molecular-weight region. The quantity of [125I]CAP57 bound to bacterial test strains was directly proportional to the sensitivity to CAP57.

Antimicrobial Cationic Peptides↗

PubMed1986

A spontaneous mutant of Neisseria gonorrhoeae with decreased resistance to neutrophil granule proteins.

We examined the resistance of Neisseria gonorrhoeae to proteins prepared from the granules of human polymorphonuclear neutrophils (PMNs). We found that nearly isogenic strains differing in lipopolysaccharide subunit molecular weight also differed in levels of resistance to crude granule extracts. N. gonorrhoeae strain WS1 was at least 10-fold less resistant than the parental strain FA 102 to granule extracts. Surprisingly, strain WS1 did not differ from FA 102 in resistance to two isolated antimicrobial proteins obtained previously from extracts of human PMN granules. We used strain WS1 in assays that detected antimicrobial proteins in granule extracts, and we obtained at least two proteins with apparent molecular masses of 24-25.5 kilodaltons that exerted potent in vitro antigonococcal activity. We found that the ED50 (concentration of protein required to kill 50% of gonococci) against the strain WS1 was approximately 0.006 microgram of protein/ml, whereas the ED50 against the parental strain (FA 102) was approximately 0.4 microgram of protein/ml. Accordingly, alterations in lipopolysaccharide structure apparently caused a 66-fold decrease in gonococcal resistance to granule proteins. Our data suggest that gonococcal resistance to oxygen-independent antimicrobial systems of human PMNs may, in part, depend on the availability of certain lipopolysaccharide domains involved in recognition of the antimicrobial granule proteins described in this report.

Antimicrobial Cationic Peptides↗

PubMed1986

Distribution of an antigenically related iron-regulated protein among the Neisseria spp.

Several iron-regulated proteins of Neisseria gonorrhoeae have been reported. One of these, a 37,000-molecular-weight protein (37K protein), appears to be common to all gonococcal isolates. Recently, the occurrence of a similar protein has also been noted in N. meningitidis. The gonococcal 37K protein has been purified and used to produce both rabbit monospecific antiserum and murine monoclonal antibodies. Using these antibody reagents, we analyzed 57 strains from nine species of Neisseria and the closely related organism Branhamella catarrhalis for the presence of proteins antigenically related to the gonococcal 37K protein. Strains grown on medium with low iron content were probed for antigenic reactivity by Western blot techniques and an enzyme-linked immunosorbent assay. Proteins which cross-reacted with the rabbit monospecific antiserum were designated as AgR-37K proteins. The data indicated that the AgR-37K proteins were conserved among the 40 strains of N. gonorrhoeae, N. meningitidis, N. lactamica, and N. cinerea tested. Seventeen strains from other species of Neisseria and Branhamella did not express AgR-37K proteins with the exception of one N. subflava isolate. All AgR-37K proteins appeared to be regulated by the amount of available iron in the growth medium. Murine monoclonal antibodies were used to probe the antigenic heterogeneity of the AgR-37K proteins from different Neisseria spp. Two of seven monoclonal antibodies were broadly cross-reactive, recognizing the AgR-37K proteins from all species examined. The remaining five monoclonal antibodies were more discriminating, recognizing the AgR-37K proteins from certain species. The antigenic conservation of these AgR-37K proteins, particularly among the pathogenic members of the genus Neisseria, may imply that these proteins serve a common function in pathogenicity.

Antibodies, Bacterial↗

PubMed1986

Neisseria gonorrhoeae survive intraleukocytic oxygen-independent antimicrobial capacities of anaerobic and aerobic granulocytes in the presence of pyocin lethal for extracellular gonococci.

The resistance of a piliated, transparent variant of Neisseria gonorrhoeae FA19 to intraleukocytic killing by human polymorphonuclear neutrophils (PMN) was examined. In both aerobic and anaerobic PMN monolayers, approximately 2% of the intracellular gonococci survived for as long as 165 min. Anaerobic PMN were as effective as aerobic PMN in the intracellular killing of gonococci. Hence, O2-independent antimicrobial systems of PMN performed a significant role in the intraleukocytic killing of gonococci were intracellular was supported by the elimination of extracellular bacteria by the addition of pyocin 103 and confirmed by the fluorescent antibody staining of intact gonococci after the PMN were permeabilized to antibody with a Formalin-acetone treatment of PMN monolayers. Our data indicate that while the majority of ingested gonococci are killed by O2-independent antimicrobial systems, a small number (about 2%), survive even when care is taken to eliminate extracellular bacteria.

Aerobiosis↗

PubMed1986

Late intraphagosomal hydrogen ion concentration favors the in vitro antimicrobial capacity of a 37-kilodalton cationic granule protein of human neutrophil granulocytes.

We described previously (W.M. Shafer, L.E. Martin, and J.K. Spitznagel, Infect. Immun. 45:29-35, 1984) the presence of a 37-kilodalton cationic antimicrobial protein (37K CAP) in extracts of granules prepared from human polymorphonuclear granulocytes (PMN). In this investigation, we prepared 37K CAP from PMN granule extracts by sequential ion-exchange and molecular-sieve chromatography and examined its antimicrobial activity against a number of gram-negative and gram-positive bacteria. At concentrations of 5 micrograms/ml or lower, 37K CAP exerted selective antimicrobial activity against gram-negative bacteria. These bacteria included Acinetobacter lwoffii, Escherichia coli, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Pseudomonas cepacia, Salmonella typhi, Salmonella typhimurium, and Shigella sonnei. However, at 5 micrograms of 37K CAP per ml, Proteus mirabilis, Proteus vulgaris, and Serratia marcescens resisted this antimicrobial activity. The bactericidal activity of 37K CAP was greatest in acidic (pH 5.5) as opposed to alkaline (pH 7.5) media. The level of S. typhimurium resistance to 37K CAP correlated with the presence of O antigen in the lipopolysaccharide. In the absence of O antigen repeat units, resistance was proportional to the length of the core oligosaccharide. These results suggest that 37K CAP may contribute significantly to the ability of PMN to kill gram-negative bacteria by nonoxidative means, particularly as the maturing phagolysosome becomes acidified.

Anti-Bacterial Agents↗

PubMed1986

Antigonococcal activity of human neutrophil cathepsin G.

We have shown that lysosomal cathepsin G, prepared from acid extracts of granules derived from human polymorphonuclear granulocytes, exhibits potent in vitro antimicrobial activity against Neisseria gonorrhoeae. An isolated isozyme of cathepsin G was found to exhibit antigonococcal activity by a nonenzymatic mechanism in a time-dependent manner. Moreover, we observed that the antigonococcal activity of cathepsin G was relatively independent of pH and evident over a pH range resembling that invoked for maturing phagolysosomes. Using a number of isogenic strains, we determined that certain mutations known to alter cell envelope structure rendered gonococci more susceptible to cathepsin G. This suggests that the susceptibility of gonococci to cathepsin G, and possibly other antimicrobial proteins derived from PMN granules, is genetically determined and possibly related to the structure of the gonococcal cell envelope.

Antimicrobial Cationic Peptides↗

PubMed1985

Analyses of gonococcal H8 antigen. Surface location, inter- and intrastrain electrophoretic heterogeneity, and unusual two-dimensional electrophoretic characteristics.

The H8 protein is a surface-exposed antigen that is found, among members of the Neisseria genus, primarily on pathogenic species. In this study, the surface exposure of H8 was reassessed by four techniques. Results of slide agglutination, indirect fluorescent antibody binding, absorption of sera with whole gonococci, and immune electron microscopy all confirmed the presence of H8 in the outer membrane. The degree to which protein A-gold-labeled monoclonal antibodies bound to H8 was marked, and suggested that this antigen was present in abundant amounts in the outer membrane. Also in this study, the electrophoretic heterogeneity of this common surface antigen was examined. Because H8 stains poorly, electrophoretic mobility was assessed using polyclonal antibodies and a monoclonal antibody that recognizes a common H8 epitope. H8 was analyzed with respect to protein I, lipopolysaccharide (LPS), and pilus and opacity phenotypic variation; results confirmed that heterogeneity of Mr was the rule among strains (21 were examined), however, the variability in Mr was independent of protein I or LPS Mr. In one strain (FA1090), the heterogeneity of H8 was examined among 10 piliation/opacity variants; the H8 (and LPS) Mr was identical in all variants; similar data were generated in strains JS3 and JS1. The electrophoretic mobility of H8 was altered in serum-resistant and neutrophil enzyme-resistant gonococci compared to the sensitive gonococci. Some of the unusual electrophoretic migration characteristics of the antigen were also examined. H8 formed a unique mushroom-shaped band in one-dimensional gels; in a two-dimensional electrophoresis system, the antigen migrated aberrantly, very similarly to LPS. Also seen in the two-dimensional electrophoresis profile were multimers of the H8 antigen; in strain JS3 (Mr 23,500), these migrated at 43,600, 86,000, and greater than 150,000. In other strains, the Mr of the multimers differed depending upon the Mr of the monomer. The two-dimensional migration characteristics (as measured by antigenicity) were completely destroyed by proteinase K digestion. Activity of H8 polyclonal antibodies to the antigens in two-dimensional gels was completely removed by adsorption of formalin-fixed whole cells, but was not affected by adsorption with LPS. These electrophoretic characteristics may reflect the close association of some nonprotein constituent, perhaps lipid or carbohydrate or both.

Animals↗

PubMed1985

Anaerobiosis increases resistance of Neisseria gonorrhoeae to O2-independent antimicrobial proteins from human polymorphonuclear granulocytes.

We investigated the in vitro resistance of Neisseria gonorrhoeae FA19 to the O2-independent antimicrobial systems of human polymorphonuclear leukocytes. Acid extracts of polymorphonuclear leukocyte granules (crude granule extracts) and a purified granule protein (57 kilodaltons) were, at low concentrations, bactericidal for gonococci under aerobic conditions that permitted growth. However, they were less effective under anaerobic conditions that imposed bacteriostasis. We found that adding sodium nitrite to reduced growth media permitted the growth of strain FA19 in an anaerobic environment. Under these conditions with nitrite, anaerobic cultures of strain FA19 were no more resistant to the crude granule extract and the 57-kilodalton protein than aerobic cultures. In contrast, Salmonella typhimurium SL-1004, a facultative anaerobe, was readily killed by both the crude granule extract and the 57-kilodalton antimicrobial protein regardless of the presence or absence of free molecular oxygen. This is the first demonstration that an isolated antimicrobial protein from polymorphonuclear leukocyte granules is active against bacteria under anaerobic conditions. Our results also indicated that the efficacy of human polymorphonuclear leukocyte O2-independent killing of N. gonorrhoeae may, in part, be inhibited by bacteriostatic conditions imposed by hypoxia.

Anaerobiosis↗

PubMed1984

Serum sensitivity of Neisseria gonorrhoeae: the role of lipopolysaccharide.

A lipopolysaccharide (LPS) mutant (FA5100) of a serum-resistant strain of Neisseria gonorrhoeae (FA19) was found to be highly sensitive to the bactericidal activity of normal human serum (NHS). Both strain FA5100 and an unrelated serum-sensitive clinical isolate (F62) were killed by NHS via the classical complement pathway since killing required C2 and Ca++. However, the fact that only strain FA5100 was sensitive to human hypogammaglobulinemic and cord serum suggested that this strain might activate the classical complement pathway in the absence of antibody. Anticomplementary concentrations of LPS from strain FA5100 inhibited the bactericidal activity of NHS against either strain FA5100 or strain F62. However, concentrations of LPS from strain FA5100 that exhibited marginal anticomplementary behavior also inhibited the killing of strain F62 by NHS. The ability of LPS from strain FA5100 to inhibit the bactericidal activity of NHS against strain FA5100 and to activate complement was reduced by treatment with mild alkali. However, alkali-treated LPS from strain FA5100 still inhibited the bactericidal activity of NHS against strain F62.

Agammaglobulinemia↗

PubMed1984

Identification of an envelope mutation (env-10) resulting in increased antibiotic susceptibility and pyocin resistance in a clinical isolate of Neisseria gonorrhoeae.

A mutation (env-10) conferring increased susceptibility to drugs, dyes, and detergents was detected in a clinical isolate of Neisseria gonorrhoeae. In certain strains, env-10 also affected susceptibility to pyocins. This mutation was phenotypically similar to but genotypically distinct from previously described env mutations.

Anti-Bacterial Agents↗

PubMed1984

Lipid A and resistance of Salmonella typhimurium to antimicrobial granule proteins of human neutrophil granulocytes.

Granule extracts from human polymorphonuclear leukocytes were prepared and fractionated by chromatography on Sephadex G75-SF. One fraction exhibited potent antimicrobial activity against an Rd1 lipopolysaccharide (LPS) mutant of Salmonella typhimurium. Susceptibility of the mutant to antimicrobial activity appeared to be due to binding of granule proteins to lipid A because isolated native LPS succeeded in blocking the antimicrobial activity of granule extracts whereas base-hydrolyzed LPS failed to do so. Centrifugation of control and base-hydrolyzed LPS-protein mixtures in cesium chloride gradients suggested that only control LPS formed complexes with antimicrobial proteins. Further evidence that bactericidal proteins from polymorphonuclear leukocyte granules interact with lipid A was that sublethal concentrations of polymyxin B (an antibiotic known to bind to lipid A) rendered target bacteria phenotypically resistant to granule proteins. Moreover, a mutant of S. typhimurium which synthesized a lipid A with decreased electronegativity due to increased 4-amino-4-deoxy-L-arabinosylation at the 4'-phosphate exhibited increased resistance to both polymyxin B and granule proteins. These results suggest that polymyxin B and antimicrobial proteins derived from polymorphonuclear leukocyte granules interact with lipid A in an analogous manner.

Antimicrobial Cationic Peptides↗

PubMed1984

Cationic antimicrobial proteins isolated from human neutrophil granulocytes in the presence of diisopropyl fluorophosphate.

Acid (0.2 M sodium acetate, pH 4.0) extracts of granules recovered from disrupted human polymorphonuclear granulocytes (PMNs) exhibited in vitro antimicrobial activity against Salmonella typhimurium. To minimize proteolytic destruction or modification of antimicrobial proteins derived from these granules, we pretreated the PMNs with the serine protease inhibitor diisopropyl fluorophosphate. Fractionation of such extracts by carboxymethyl Sephadex and Sephadex G-75 chromatography resulted in the recovery of at least two antimicrobial, cationic proteins. These proteins differed substantially in antimicrobial activity, amino acid composition, and molecular weight (Mr, 37,000 and 57,000). As we have shown before (Shafer et al., Infect. Immun. 43:834-858), with unfractionated proteins, these two proteins exhibited diminished activity against a polymyxin B-resistant (PBr) mutant of S. typhimurium compared with their activity against the isogenic parental polymyxin B-sensitive (PBs) strain. Expression of the relevant mutation (prmA) in the PBr mutant decreases the electronegativity of lipid A, owing to increased 4-amino-4-deoxy-L-arabinosylation at the 4' phosphate residue (Vaara et al., FEBS Lett. 129:145-149). The data suggest that at least two different cationic proteins account for the antimicrobial capacity of extracts from human PMN granules. Moreover, the availability of anionic charges in the outer membrane of S. typhimurium due to free lipid A phosphates apparently dictates phenotypic levels of resistance to both of the cationic proteins extracted from human PMN granules.

Amino Acids↗