Search

Protective immunity against tuberculosis is considered to be essentially cell mediated, and an important role for CD8(+) T lymphocytes has been suggested by several studies of murine and human infections. The present work, using an experimental model of infection with Mycobacterium bovis in cattle, showed that live M. bovis elicits the activation of CD8(+) T cells in vitro. However, a sonic extract prepared from M. bovis (MBSE) and protein purified derivative (PPDb) also induced a considerable degree of activation of the CD8(+) T cells. Analysis of proliferative responses of peripheral blood mononuclear cells, purified CD8(+) T cells, and CD8(+) T-cell clones to M. bovis and to soluble antigenic preparations (MBSE, PPDb) showed that the responses of all three types of cells were always superior for live mycobacteria but that strong responses were also obtained with complex soluble preparations. Furthermore, while cytotoxic capabilities were not investigated, the CD8(+) T cells were found to produce and release gamma interferon in response to antigen (live and soluble), which indicated one possible protective mechanism for these cells in bovine tuberculosis. Finally, it was demonstrated by metabolic inhibition with brefeldin A and cytochalasin D at the clonal level that an endogenous pathway of antigen processing is required for presentation to bovine CD8(+) cells and that presentation is also dependent on phagocytosis of the antigen.

Tuberculosis in cattle remains a major zoonotic and economic problem in many countries. The standard diagnostic assay for bovine tuberculosis, the intradermal tuberculin test, has low accuracy. Therefore, alternative immunodiagnostic methods, such as serological assays, are needed for detection of infected animals. Development of an accurate serodiagnostic test requires a detailed understanding of the humoral immune responses during bovine tuberculosis and, in particular, identification of the key antigens of Mycobacterium bovis involved in antibody production. In this study, we characterized antibody responses in cattle experimentally infected with M. bovis. Sequential serum samples were collected every 3 to 4 weeks for up to 27 months postinfection. Circulating immunoglobulin G antibody levels were measured by an enzyme-linked immunosorbent assay using 12 highly purified recombinant proteins of M. bovis. Six proteins, ESAT-6, 14-kDa protein, MPT63, MPT70, MPT51, and MPT32, were identified as major seroreactive antigens in bovine tuberculosis. A remarkable animal-to-animal variation of antigen recognition by serum antibodies was observed. Kinetic analyses of the antibody production to individual antigens during infection revealed that the heterogeneous antigen recognition profile changed markedly in a given infected animal as disease progressed.

The ability of lysozyme to aggregate and lyse the gram-negative capnophilic periodontal microorganism Capnocytophaga gingivalis 2010 was monitored optically at 540 nm. Both hen egg white and chromatographically purified human lysozymes had significant but similar aggregation potentials for both logarithmic- and stationary-phase bacteria. In general, an increase in enzyme concentration resulted in a graded increase in both the initial and maximum changes in turbidity which occurred during the reaction period. The greatest change in turbidity occurred within the initial minutes of interaction of lysozyme and the cells, and the extent of aggregation paralleled a rapid depletion of lysozyme by the suspensions during the first minute of its incubation with the bacteria. Interestingly, the muramidase inhibitors N-acetyl-D-glucosamine and histamine did not block aggregation, whereas maleylation of lysozyme completely inhibited its aggregating ability. Demaleylation, however, restored aggregation activity comparable to the native enzyme, indicating that maleylated lysozyme retained its integrity and that aggregation was primarily dependent on charge. The addition of up to physiological concentrations of NaHCO3 and NaCl to cell aggregates resulted in varying degrees of deaggregation and lysis. Surprisingly, ultrastructural analysis of lysozyme-treated cells revealed morphological changes with or without the addition of salt. Damage appeared to occur at the blunted polar end of the cells where there was a large spherical outpouching bordered by a damaged cell envelope. Damaged cells uniformly contained dense granular cytoplasmic debris. In effect, the cationic enzyme lysed C. gingivalis 2010, which was not apparent in the spectrophotometric assay. The paradoxical finding that during bacterial aggregation there was lysis may be of significance to the further elucidation of lysozyme's antibacterial role in the gingival sulcus.

Compared with anion-activated cell lysis of oral bacteria damaged with either lysozyme or trypsin, cells which were treated with both of these enzymes showed a far greater degree of lysis. This was true regardless of whether turbidimetric, DNA release, or electron microscopic assays were used to monitor the lytic process. At an acidic pH of 5.2 and an NaHCO3 concentration of 100 mM, the kinetics of lysis for two different serotype c strains of Streptococcus mutans were similar. At 0 to 100 mM bicarbonate, however, differences in the lytic susceptibilities of the two strains were evident. At pH 5.2, NaHCO3, but not NaSCN, NaCl, or NaF, was effective in promoting cell lysis of the oral bacteria. At apparent sublytic concentrations of NaHCO3, lysis was achieved by adding appropriate concentrations of NaSCN, NaCl, or NaF to the lysozyme-protease-damaged cells. In in vivo studies, hamsters given a combination of NaHCO3, NaCl, and NaSCN were found to have significantly reduced levels of S. mutans on their molar teeth compared with that found in controls or animals exposed to any one of the salts alone or to a combination of chloride and thiocyanate only. The results suggest that bicarbonate is an essential anion which, together with the other major salivary inorganic monovalent anions, plays an active role in the lysis and ultimate elimination of cariogenic bacteria.

Growth inhibition and cell viability assays demonstrate that the histidine-rich polypeptides isolated from human parotid saliva are bacteriostatic and bactericidal for strains of Streptococcus mutans belonging to the serotype b and c classifications. Both inhibition of growth and cell division are enhanced by preincubation of bacteria with these polypeptides in low-ionic-strength buffers of acidic and neutral pH before dilution into enriched growth media. With prior exposure at pH 6.8, inhibition by these polypeptides of the serotype c strains, S. mutans GS5 and SB, as well as the serotype b strain, S. mutans BHT, is reversible over time under the experimental conditions selected. With similar exposure at pH 5.2, however, irreversible damage is manifested by complete inhibition of both growth and cell viability. At concentrations of 250 micrograms of the mixture of histidine-rich polypeptides per 5 X 10(5) bacterial cells per ml in the acidic preincubation buffer, bacterial lethality is maintained for a period of 48 h in the enriched growth media. At a 50-micrograms/ml concentration of these salivary agents, approximately 80% killing of S. mutans SB is noted after a 24-h incubation; however, surviving bacteria multiply and reach turbidities of untreated control cells when examined at the 48-h growth point. Similarly, hen egg white lysozyme is also found to be bactericidal for these microorganisms when preincubation is carried out under acidic conditions. However, in contrast to the histidine-rich polypeptides, lysozyme under these experimental conditions does not inhibit growth of S. mutans SB at neutral pH, although it does inhibit growth of both S. mutans BHT and S. mutans GS5 at this pH. Preexposure of S. mutans SB to the peptides in buffer at ionic strengths of 0.025 to 0.125, followed by either viability assays under nongrowing conditions or growth inhibition studies, suggests that there is very little effect of ionic strength on the antibacterial function of these peptides. In contrast to the inhibition of viability noted under growing conditions, lower concentrations of the histidine-rich polypeptides were required to elicit immediate cell death under nongrowing conditions.

Freshly collected parotid saliva collected from human donors were shown by polyacrylamide gel electrophoresis to continuously secrete a group of low-molecular-weight cationic polypeptides. Up to 14 bands could be identified by Coomassie blue staining, and all bands migrated more rapidly than purified human leukemic lysozyme in cationic polyacrylamide gel electrophoresis. These peptides could be isolated as a group relatively free of other salivary components and recovered in high yields from concentrated parotid saliva by Sephadex G-25 chromatography. In sodium dodecyl sulfate gel electrophoresis, the histidine-rich polypeptide bands appeared as just two bands migrating at the tracking dye and ahead of insulin chain B. Amino acid analysis of the mixture revealed an average content of at least 48% cationic residues, of which half were histidine. When stained bands were eluted from electrophoretic gels, hydrolyzed, and subjected to amino acid analyses, they were found to be enriched in histidine. There was also a correlation of the electrophoretic mobility with the content of basic amino acids. Sephadex G-25 chromatography is a convenient, simple method for preparing milligram quantities of the histidine-rich polypeptides for chemical and biochemical studies.

Human parotid saliva histidine-rich polypeptides exerted antifungal activity against Candida albicans at concentrations similar to the known antifungal activity of the imidazole antibiotics. Inhibition of both growth and viability could be demonstrated by optical density monitoring and plating assays. Inhibition of growth was observed to be greatest when the histidine-rich polypeptides were added to the inoculum before addition to the growth media. However, complete inhibition by these polypeptides was still noted during active growth at turbidities of C. albicans corresponding to 10(6) CFU/ml. At higher cell densities, growth was delayed but not halted under the experimental conditions investigated. Candidacidal activity was observed with both growing and nongrowing cells. With respect to the latter, reaction of cells in buffer with the histidine-rich polypeptides for a period of 30 min resulted in killing of greater than 90% of two different strains of C. albicans, whereas a third strain was found to be less susceptible. Moreover, the kinetics of loss of cell viability correlated with the loss of potassium from the cells. In addition to the histidine-rich polypeptides, hen egg white lysozyme, poly-L-lysine, and poly-L-histidine affected C. albicans. Both of the polyamino acids completely inhibited the growth of the yeast whereas lysozyme was not as potent. Where delays in growth were observed for all of these agents, including the histidine-rich polypeptides, turbidities reached those of untreated controls after a 24-h period. Enhanced effects were noted if C. albicans was preincubated with these agents in 0.025 2-(N-morpholino)-ethanesulfonic acid buffer, pH 5.2, before growth in the yeast synthetic medium.

Streptococcus mutans GS5 was grown in a synthetic medium containing radioactive thymidine to monitor cell lysis by assay of the release of DNA. Bacteriolysis was achieved by sequential treatment of the cells with either hen egg white lysozyme and sodium thiocyanate or a combination of hen egg white lysozyme and a proteolytic enzyme followed by addition of the thiocyanate. In the absence of sodium thiocyanate, a small percentage of the total macromolecular thymidine was released in control reaction mixtures during incubation. This amount of released DNA more than doubled in trypsin-treated cells, but the inclusion of lysozyme in reaction mixtures prevented assay of the DNA. Lysis was found to be optimal in the late log phase of growth and was dependent on the concentrations of both lysozyme and protease. Concentrations of trypsin or chymotrypsin as low as 0.01 microgram/ml were found to be effective in enhancing the lytic process. The addition of protease to lysozyme-inorganic salt reaction mixtures altered both the pH and ionic strength profiles of cell lysis. At pHs of 5.5 or lower, both the lysozyme-NaSCN and the lysozyme-trypsin-NaSCN systems were inactive in mediating lysis. The loss of insoluble cell wall peptidoglycan by lysozyme treatment was pH independent and did not appear to be affected by the addition of protease. Either diluted whole saliva or neutrophil extracts could replace trypsin to enhance cell lysis further.

Veillonella alcalescens subsp. dispar was grown in a synthetic medium containing either radiolabeled thymidine or uridine to monitor cell lysis by assay of the release of deoxyribonucleic acid or ribonucleic acid (RNA), respectively. Biochemical analyses demonstrated that, although human or hen egg white lysozymes alone did not release deoxyribonucleic acid or RNA, the nucleic acids were liberated in equal amounts from lysozyme-treated cells by the addition of low concentrations of the sodium salts of HCO-3, SCN-, Cl-, and F-, RNA release was dependent on enzyme and anion concentration. Human lysozyme was more potent than hen egg white lysozyme, and bicarbonate was the most effective anion in promoting bacteriolysis. Surprisingly, ultrastructural analyses differed from biochemical results. Lysozyme alone caused lysis in approximately 40% of the cell population. Detailed ultrastructural examination revealed aggregated cytoplasmic components which appeared as small clumps, explaining why nucleic acids were not measurable in the biochemical assays. In reaction mixtures containing lysozyme plus inorganic salts, electron microscopy results were compatible with biochemical data. Ultrastructural studies demonstrated that the addition of inorganic salts to lysozyme-treated cells resulted in the solubilization of the protoplasmic aggregates of lysed cells, presumably freeing the complexed RNA, and in the rapid lysis of the remaining cells (approximately 60%). These data suggest that electron microscopy must be used in conjunction with biochemical assays to assess lytic damage of bacterial cells.

MPB70 and MPB80 (MPB70/80) and MPB83 are closely related antigens which are highly expressed in Mycobacterium bovis. MPB70/80 are soluble secreted antigens, while MPB83 is an exported lipoprotein associated with the bacterial surface. In the present study, these antigens had different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. These differences may be explained by the fact that MPB70 and MPB83 both have two internal cysteine residues which would create ring structures by disulfide bonding. We analyzed the structures of MPB70/80 and MPB83 by using monoclonal antibodies (MAbs) raised against bovine purified protein derivative or whole M. bovis cells. MAb 1-5C reacted specifically with MPB70 and MPB80, and MAb MBS43 reacted specifically with MPB83, while the other antibodies, including several previously described MAbs, bound all three antigens. MAbs and polyclonal antibodies reacted strongly with reduced protein and less well with nonreduced protein, indicating involvement of linear epitopes. Epitopes of MAbs Bov-1, 2-6B, 1-5C, and 1-1D were mapped by using synthetic peptides of MPB70. Sequence comparison showed the peptide with the 1-5C-reactive epitope to have three residues different from those in the homologous region of MPB83. Exchanges of A for S in position 112 or Q for E in position 116 abolished the reactivity of MAb 1-5C. Polyclonal rabbit antibodies to native purified MPB70 reacted strongly with peptides 6, 7, and 8 of the N-terminal half of mature MPB70. Cattle sera of experimentally M. bovis-infected animals recognized a broader spectrum of peptides. These findings indicate that there is diagnostic potential for these proteins and that there is also a possible role for antibodies in elucidation of the host-mycobacterium relationship involving a surface-bound and exposed lipoprotein, MPB83, and its highly homologous soluble secreted MPB70/80 counterparts.

The ability of both human and hen egg white lysozymes to lyse Actinobacillus actinomycetemcomitans Y4 was investigated. Lysis was followed optically at 540 nm by measuring the percent reduction in turbidity of freshly harvested log-phase cells suspended in Tris-maleate buffers within a wide range of pH (5.2 to 8.5) and molarity (0.01 to 0.2 M) and containing various amounts of enzyme and EDTA. In several instances, treated microorganisms were subsequently examined in thin sections by electron microscopy. Reductions in turbidity and clearing of suspensions occurred with small amounts of lysozyme (less than 1 microgram) under relatively alkaline conditions and at low ionic strength and in the presence of small amounts of EDTA (greater than 0.01 mM). Under the most alkaline conditions, EDTA alone effected turbidity reductions similar to those observed in the presence of lysozyme, which suggested that EDTA not only increased outer membrane permeability but also caused cell lysis. Ultrastructural analysis did not always correspond to turbidimetric observations. Cell lysis was virtually complete in suspensions containing both lysozyme and EDTA. However, in contrast to turbidimetric findings, a significant percentage of cells (greater than 25%) was lysed in the presence of lysozyme alone. Furthermore, significant damage occurred in the presence of EDTA alone. Spheroplast-like cell ghosts were present which surrounded condensed cytoplasm or relatively clear spaces. These findings further support the concept of the requirement for electron microscopy to assess lytic damage in addition to turbidimetric and biochemical methods. Our results are the first to demonstrate the remarkable sensitivity of A. actinomycetemcomitans Y4 to lysozyme and to show that EDTA not only affects outer membrane permeability but effects cell lysis, possibly through activation of autolytic enzymes at the cytoplasmic membrane. The exquisite sensitivity of A. actinomycetemcomitans Y4 to lysis could be an important mechanism by which lysozyme participates in the regulation of this suspected periodontal pathogen.