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Mycobacterium tuberculosis and other pathogenic mycobacteria export abundant quantities of proteins into their extracellular milieu when growing either axenically or within phagosomes of host cells. One major extracellular protein, the enzyme glutamine synthetase, is of particular interest because of its link to pathogenicity. Pathogenic mycobacteria, but not nonpathogenic mycobacteria, export large amounts of this protein. Interestingly, export of the enzyme is associated with the presence of a poly-L-glutamate/glutamine structure in the mycobacterial cell wall. In this study, we investigated the influence of glutamine synthetase inhibitors on the growth of pathogenic and nonpathogenic mycobacteria and on the poly-L-glutamate/glutamine cell wall structure. The inhibitor L-methionine-S-sulfoximine rapidly inactivated purified M. tuberculosis glutamine synthetase, which was 100-fold more sensitive to this inhibitor than a representative mammalian glutamine synthetase. Added to cultures of pathogenic mycobacteria, L-methionine- S-sulfoximine rapidly inhibited extracellular glutamine synthetase in a concentration-dependent manner but had only a minimal effect on cellular glutamine synthetase, a finding consistent with failure of the drug to cross the mycobacterial cell wall. Remarkably, the inhibitor selectively blocked the growth of pathogenic mycobacteria, all of which release glutamine synthetase extracellularly, but had no effect on nonpathogenic mycobacteria or nonmycobacterial microorganisms, none of which release glutamine synthetase extracellularly. The inhibitor was also bacteriostatic for M. tuberculosis in human mononuclear phagocytes (THP-1 cells), the pathogen's primary host cells. Paralleling and perhaps underlying its bacteriostatic effect, the inhibitor markedly reduced the amount of poly-L-glutamate/glutamine cell wall structure in M. tuberculosis. Although it is possible that glutamine synthetase inhibitors interact with additional extracellular proteins or structures, our findings support the concept that extracellular proteins of M. tuberculosis and other pathogenic mycobacteria are worthy targets for new antibiotics. Such proteins constitute readily accessible targets of these relatively impermeable organisms, which are rapidly developing resistance to conventional antibiotics.

One hypothesis for the etiology of central nervous system (CNS) autoimmune disease is that infection by a virus sharing antigenic epitopes with CNS antigens (molecular mimicry) elicits a virus-specific immune response that also recognizes self-epitopes. To address this hypothesis, transgenic mice were generated that express the nucleoprotein or glycoprotein of lymphocytic choriomeningitis virus (LCMV) as self in oligodendrocytes. Intraperitoneal infection with LCMV strain Armstrong led to infection of tissues in the periphery but not the CNS, and the virus was cleared within 7-14 d. After clearance, a chronic inflammation of the CNS resulted, accompanied by upregulation of CNS expression of MHC class I and II molecules. A second LCMV infection led to enhanced CNS pathology, characterized by loss of myelin and clinical motor dysfunction. Disease enhancement also occurred after a second infection with unrelated viruses that cross-activated LCMV-specific memory T cells. These findings indicate that chronic CNS autoimmune disease may be induced by infection with a virus sharing epitopes with a protein expressed in oligodendrocytes and this disease may be enhanced by a second infection with the same or an unrelated virus. These results may explain the association of several different viruses with some human autoimmune diseases.

We have examined the interaction between virulent egg yolk-grown L. pneumophila, Philadelphia 1 strain, and in vitro-activated human monocytes, under antibiotic-free conditions. Freshly explanted human monocytes activated by incubation with concanavalin A (Con A) and human lymphocytes inhibited the intracellular multiplication of L. pneumophila. Both Con A and lymphocytes were required for activation. Con A was consistently maximally effective at greater than or equal to 4 mug/ml. Monocytes activated by incubation with cell-free filtered supernatant from Con A-sensitized mononuclear cell cultures also inhibited the intracellular multiplication of L. pneumophil a. The most potent supernatant was obtained from mononuclear cell cultures incubated with greater than or equal to 15 mug/ml Con A for 48 h. The degree of monocyte inhibition of L. pneumophila multiplication was proportional to the length of time monocytes were preincubated with supernatant (48 {greater than} 24 {greater than} 12 h) and to the concentration of supernatant added (40 percent {greater than} 20 percent {greater than} 10 percent {greater than} 5 percent). Monocytes treated with supernatant daily were more inhibitory than monocytes treated initially only. With time in culture, monocytes progressively lost a limited degree of spontaneous inhibitory capacity and also lost their capacity to respond to supernatant with inhibition of L. pneumophila multiplication. Supernatant-activated monocytes inhibited L. pneumophila multiplication in two ways. They phagocytosed fewer bacteria, and they slowed the rate of intracellular multiplication of bacteria that were internalized. As was the case with nonactivated monocytes, antibody had no effect on the rate of intracellular multiplication in supernatant-activated monocytes. Neither supernatant-activated nor nonactivated monocytes killed L. pneumophila in the absence of antibody. Both killed a limited proportion of these bacteria in the presence of antibody and complement. We have previously reported that anti-L, pneumophila antibody and complement neither promote effective killing of L. pneumophila by human polymorphonuclear leukocytes and monocytes nor inhibit the rate of L. pneumophila multiplication in monocytes. These findings and our present report that activated monocytes do inhibit L. pneumophila multiplication indicate that cell-mediated immunity plays a major role in host defense against Legionnaires' disease.

We have previously reported that virulent egg yolk-grown Legionella pneumophila, Philadelphia 1 strain, multiplies intracellularly in human blood monocytes. We now report on the interaction between virulent L. pneumophila and human polymorphonuclear leukocytes (PMN), antibody, and complement, in vitro, under antibiotic-free conditions. L. pneumophila in concentrations ranging from 10(3) to 10(6) colony forming units (CFU)/ml are completely resistant to the bactericidal effects of 0-50 percent fresh normal human serum, even in the presence of high concentrations of rabbit or human anti-L. pneumophila antibody. L. pneumophila bacteria fix the third component of complement (C3) to their surfaces, as measured by fluorescence microscopy using rhodamine- conjugated goat anti-human C3 IgG, only when the bacteria are incubated with both specific anti-L. pneumophila antibody and complement. Similarly, L. pneumophila adhere to PMN, as measured by fluorescence microscopy, only in the presence of both specific antibody and complement. Electron microscopy revealed that these opsonized bacteria are phagocytosed by the PMN. PMN require both antibody and complement to kill L. pneumophila; even then, PMN reduced CFU of L. pneumophila by only 0.5 log under conditions in which they reduce CFU of a serum-resistant encapsulated strain of Escherichia coli by 2.5 logs. Separation of PMN-associated and nonassociated CFU of L. pneumophila revealed that the major proportion of the surviving bacteria are PMN associated. Thus, the ineffective killing of opsonized L. pneumophila is a result of a failure of PMN to kill these bacteria after they become PMN- associated. With or without antibody, PMN do not support the growth of L. pneumophila. These findings suggest that PMN, even in conjunction with the humoral immune system, do not play a decisive role in defense against the Legionnaires' disease bacterium.