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Lysozyme was isolated from Bacillus Calmette-Guerin-elicited rabbit alveolar macrophages by acid extraction and purified to homogeneity by a single-column procedure. Yields of the purified enzyme averaged between 20 and 30 mg per rabbit, values far in excess of those obtained with previously published methods. Rabbit lysozyme has a molecular weight of 14,300 and exhibits optimal lytic activity against Micrococcus lysodeikticus at an ionic strength of 0.04, pH 6.5. Our results indicate that lysozyme and other granule components can be fractionated from elicited alveolar macrophages by using simple techniques, suggesting methods for the bulk purification of lysosomal constituents.

The bactericidal properties of human cell-free lymph have been investigated. Gram-negative and gram-positive bacteria are susceptible to the killing action of lymph fluid. The colony-forming ability of Escherichia coli and Bacillus subtilis was reduced by 3 logs after 20 min of incubation with 10% (vol/vol) lymph. Salmonella and Listeria were less susceptible to killing. The presence of multiple killing factors in lymph showing different specificities for different bacterial cells was demonstrated.

Enteropathogenic strains of Yersinia enterocolitica harbor a virulence plasmid which codes for a series of novel outer membrane proteins. The expression of these proteins on the outer membrane is temperature regulated: when cells are grown at 25 degrees C, these proteins are not exposed on the outer membrane, whereas they occur in high copy number when cells are grown at 37 degrees C. The majority of these proteins are externally exposed on the cell surface as evidenced by their susceptibility to proteolysis by exogenously added proteases. The expression of the plasmid-mediated proteins on the outer membrane does not favor adherence of the bacteria to intestinal epithelial cells in vitro. Cultures grown at 25 degrees C adhered to Henle cell monolayers, whereas those grown at 37 degrees C did so much less effectively. The presence of the proteins on the bacterial surface appears to be involved in rendering the cells resistant to the bactericidal effects of serum, i.e., 37 degrees C-grown cells were resistant to serum killing, and removal of the outer membrane proteins with pronase rendered them sensitive. Evidence is presented which strongly suggests that the plasmid-mediated proteins are synthesized and expressed on the cell surface either during or after transit of the ingested bacteria to the lamina propria. Some properties afforded to the cells by the outer membrane proteins are described.

We purified two microbicidal cationic proteins, MCP-1 and MCP-2, from rabbit alveolar macrophages. MCP-1 was remarkably rich in arginine (25.5 mol%) and half cystine (18.7 mol%) residues and constituted approximately 1.5% of the total protein content of Freund adjuvant-elicited alveolar macrophages. MCP-2 was approximately half as abundant as MCP-1 and contained relatively less arginine (14.9 mol%) and half cystine (9.8 mol%). The amino acid compositions of MCP-1 and MCP-2 resembled those reported for the lysosomal cationic proteins of rabbit granulocytes, but were distinct from those of any known histone. MCP-1 (1 microgram/ml) killed 99.6% of Candida albicans in 20 min, whereas MCP-2 killed approximately 80% under similar conditions. Both proteins rapidly suppressed O2 consumption by C. albicans and induced a rapid loss of intracellular 86Rb+. Although more information is needed about the biological origin, distribution, and roles of macrophage microbicidal proteins, it seems likely that MCP-1 and MCP-2 contribute to the microbicidal efficacy of rabbit alveolar macrophages.

The in vitro bactericidal reaction of human plasma serum against Bacillus subtilis was investigated. Human lysozyme was purified to homogeneity, and antiserum was prepared against the enzyme. The anti-lysozyme immunoglobulin G was used as a specific inhibitor in bactericidal and bacteriolytic reactions. It was found that at low serum concentrations lysozyme was the primary bactericide active against B. subtilis. At appreciably higher serum concentrations, a lysozyme-independent bactericidal activity was also demonstrated.

Resistance to the bactericidal action of normal human serum is one of the characteristics of virulent Yersinia enterocolitica. This property is attributable to the virulence plasmid harbored by pathogenic strains of the species. Serum resistance in Y. enterocolitica is thermoregulated, and its expression correlates well with the presence of virulence plasmid-encoded outer membrane proteins. To further examine the biochemical basis underlying resistance, we cloned a large segment (ca. 30 kilobases) of virulence plasmid DNA and studied the expression of plasmid-encoded outer membrane proteins in a serum-sensitive strain of Escherichia coli. The presence of the 160-kilodalton Y. enterocolitica-derived outer membrane protein 1 on E. coli transformants conferred a high degree of hydrophobicity, autoagglutinability, and resistance to serum killing. All of these properties were thermoregulated in E. coli with fidelity, suggesting that a functional thermoregulatory element was present in the cloned DNA. Elimination of protein 1 from the outer membrane of E. coli transformants by insertional inactivation of the structural gene with a Kanr gene cassette abrogated all of these properties and returned the serum-sensitive phenotype.

Outer membrane preparations of Bdellovibrio bacteriovorus grown intraperiplasmically on Escherichia coli containing OmpF were prepared by the Triton X-100 procedure of Schnaitman (J. Bacteriol. 108:545-552, 1971). They contained a protein that migrated to almost the same position as E. coli OmpF in sodium dodecyl sulfate-acrylamide gradient gel electrophoresis and to the same position as E. coli OmpF when urea was incorporated into the gel. The mobility of this protein increased relative to that of OmpC in urea-containing gels as does E. coli OmpF. However, the same protein was also produced during axenic growth and during intraperiplasmic growth on prey lacking OmpF. The peptide profile generated by partial proteolysis of this protein showed no homology to that produced from E. coli OmpF. We conclude that B. bacteriovorus synthesizes an OmpF-like protein. Previous claims that the bdellovibrio incorporates an intact E. coli OmpF are not consistent with these observations.

Measurements of the sucrose-permeable and -impermeable volumes during Bdellovibrio bacteriovorus attack on Escherichia coli or Pseudomonas putida showed that the volume of the bdelloplast increased over that of the substrate cell. Although the pattern of the increase differed with the two organisms, the volumes reached maximum at about 60 min into the bdellovibrio growth cycle. By this time, the cytoplasmic membranes of the attacked cells were completely permeable to sucrose. The kinetics of increase in sucrosepermeable volumes were similar to the kinetics of attachment and penetration (Varon and Shilo, J. Bacteriol. 95:744-753, 1968). These data show that the original cytoplasmic and periplasmic compartmentalization of the substrate cell ceases to exist with respect to small hydrophilic molecules during bdellovibrio attack. In contrast, the effective pore size of the outer membrane of the substrate cell to small oligosaccharides remains unaltered during bdelloplast formation as was shown by direct measurements of its exclusion limits. The major porin protein of E. coli, OmpF, was recoverable from the bdelloplast outer membrane fraction until the onset of lysis. The Braun lipoprotein was removed from the bdelloplast wall early, and OmpA was lost in the terminal part of the bdellovibrio growth cycle.

Rabbit alveolar macrophages contain two highly cationic microbicidal proteins. These were shown to be distinct from histones and not to arise from granulocyte contamination. The macrophage proteins were especially active against Candida albicans and Candida parapsilosis. Gram-positive bacteria (Bacillus subtilis, Listeria monocytogenes, and Streptococcus faecalis) were also susceptible, whereas Escherichia coli and Salmonella typhimurium appeared more resistant. The proteins may be present in lysosomes, based on their solubilization by dilute acids and their distribution with lysosomal markers on sucrose density gradients. Such microbicidal proteins have not previously been demonstrated in any mammalian macrophage. They may play a significant role in the host-defense functions of the rabbit lung.

Several metabolic parameters indicative of Escherichia coli function and integrity were kinetically examined in response to treatment with normal human serum in the presence and absence of functional human lysozyme. Specific inhibition of this enzyme in bacteriolytic and bactericidal reactions was accomplished by using purified rabbit anti-human lysozyme immunoglobulin G. Initiation of the complement-mediated alterations of cytoplasmic membrane integrity, as judged by the leakage of 86Rb from prelabeled cells or the hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by a cryptic strain, was found to be independent of lysozyme action. Furthermore, inhibition of macromolecular synthesis by E. coli in response to serum treatment occurred at the same time regardless of the functional state of lysozyme. Although the rate and extent of bacteriolysis were reduced in the absence of lysozyme, the bactericidal kinetics was unaffected. These results demonstrate that the lethal events associated with the action of antibody and complement on gram-negative bacteria are independent of lysozyme, suggesting an accessory role for this enzyme in immune reactions. A possible temporal sequence of complement-induced effects occurring at the cell surface is presented.

Martinez, R. J. (University of California, Los Angeles), and E. Z. Gordee. Formation of bacterial flagella. I. Demonstration of a functional flagellin pool in Spirillum serpensand Bacillus subtilis. J. Bacteriol. 91:870–875. 1966—Exponentially growing cultures of Spirillum serpensand Bacillus subtilisregained motility and flagella within one generation after mechanical deflagellation. Regeneration of flagella occurred in both cultures in the presence of chloramphenicol at concentrations shown to inhibit flagellin synthesis. Cells labeled with C14-amino acids regenerated radioactive flagella in the presence of chloramphenicol. A conditional mutant of S. serpens(T-45) was isolated. This strain did not produce flagella when grown at 45 C, but formed the organelles upon temperature shift to 30 C, even in the presence of chloramphenicol. A reduction of intracellular antibody-precipitable flagellin counts in labeled S. serpensT-45 occurred concomitant with the generation of flagella at 30 C. The data suggest that the flagella of S. serpensand B. subtilisare formed from a pool of intracellular flagellin proteins.