Cytolytic T lymphocytes (CTLs) kill intracellular pathogens by a granule-dependent mechanism. Granulysin, a protein found in granules of CTLs, reduced the viability of a broad spectrum of pathogenic bacteria, fungi, and parasites in vitro. Granulysin directly killed extracellular Mycobacterium tuberculosis, altering the membrane integrity of the bacillus, and, in combination with perforin, decreased the viability of intracellular M. tuberculosis. The ability of CTLs to kill intracellular M. tuberculosis was dependent on the presence of granulysin in cytotoxic granules, defining a mechanism by which T cells directly contribute to immunity against intracellular pathogens.

Murine mAbs reactive with the surface of Mycobacterium tuberculosis were assayed for their ability to affect the course of infection in mice challenged with virulent organisms. An IgG3 mAb (9d8) specific for arabinomannan and reactive with purified antigen from a clinical isolate of M. tuberculosis conferred partial protection on mice after respiratory challenge (30-60% survival >75 days; P < 0.05). Control mice pretreated with an irrelevant mAb of the same isotype succumbed to tuberculosis within 30 days. Mice with gene disruptions in interferon ␥ and major histocompatibility complex Class II also were partially protected from challenge. The protective mAb was neither bactericidal nor inhibitory of infection or bacterial replication. Nevertheless, it profoundly altered the nature of the granulomas in the infected lungs. Mice treated with mAb 9d8 and challenged with M. tuberculosis localized the pathogen within granuloma centers, suggesting that the mAb conferred protection by enhancing a cellular immune response.

Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four α helical bundle motif, with the α helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the α helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic β-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.

Infectious diseases and malnutrition represent major burdens aff licting millions of people in developing countries. Both conditions affect individuals in industrialized nations, particularly the aged, the HIV-infected, and people with chronic diseases. While malnutrition is known to induce a state of immunodeficiency, the mechanisms responsible for compromised antimicrobial resistance in malnourished hosts remain obscure. In the present study, mice fed a 2% protein diet and developing protein calorie malnutrition, in contrast to well-nourished controls receiving a 20% protein diet, rapidly succumbed to infection with Mycobacterium tuberculosis. Malnourished mice exhibited a tissue-specific diminution in the expression of interferon ␥, tumor necrosis factor ␣, and the inducible form of nitric oxide synthase in the lungs, but not the liver. The expression of these molecules critical to the production of mycobactericidal nitrogen oxides was depressed in malnourished animals in the lungs specifically at early times (<14 days) after infection. At later times, levels of expression became comparable to those in well-nourished controls, although the bacillary burden in the malnourished animals continued to rise. Nevertheless, urinary and serum nitrate contents, an index of total nitric oxide (NO) production in vivo, were not detectably diminished in malnourished, mycobacteria-infected mice. In contrast to the selective and early reduction of lymphokines and the inducible form of nitric oxide synthase in the lung, a marked diminution of the granulomatous reaction was observed in malnourished mice throughout the entire course of infection in all tissues examined (lungs, liver, and spleen). Remarkably, the progressively fatal course of tuberculosis observed in the malnourished mice could be reversed by restoring a full protein (20%) diet. The results indicate that protein calorie malnutrition selectively compromises several components of the cellular immune response that are important for containing and restricting tuberculous infection, and suggest that malnutritioninduced susceptibility to some infectious diseases can be reversed or ameliorated by nutritional intervention.

C ell-mediated immunity plays a major role in host defense against mycobacterial infections. Presentation of mycobacterial antigens by both the MHC class I and class II families is critical to protection because adoptive transfer of the T cells restricted by these molecules enhances survival (1). Additionally, mice genetically deficient in ␤ 2 -microglobulin, resulting in a lack of MHC class I and class I-like molecule expression (2), and mice depleted of CD8 T cells (1) more readily succumb to infection with Mycobacterium tuberculosis (M. tb).Soluble antigens and phagocytosed bacteria, including mycobacteria, ultimately localize within membrane-bound endocytic vesicles (3, 4), where antigens are processed for association with MHC class II molecules and presented to CD4 ϩ T cells (5). Peptides presented through the MHC class I pathway generally derive from within the cytoplasm, where they are transported to the lumen of the endoplasmic reticulum by the transporter protein, TAP, for association with MHC class I molecules (5). Because mycobacteria remain within a vacuolar compartment, the mechanism of presentation of mycobacterial antigens through MHC class I molecules is highly problematic. Previously, it has been shown that infection of macrophages with bacille Calmette-Guerin (BCG) or M. tb facilitated presentation of soluble antigens (6), by a pathway that is TAPdependent. Thus, mycobacterial infection facilitated peptide access to the cytoplasm for presentation on MHC class I, despite its presumed intracellular localization within membrane-bound vacuoles.The current studies were undertaken to establish whether vacuoles containing mycobacteria become permeable to macromolecules located in the cytosol. Using confocal microscopy, we assessed colocalization of fluorescent signals from labeled mycobacteria within infected macrophages microinjected intracytoplasmically with tagged molecules of varying sizes. Our results indicate that viable mycobacteria have the ability to facilitate transit of macromolecules between the cytosolic and vacuolar compartments of infected cells, which has implications for both pathogenicity and immunity. Materials and MethodsCell Lines. The C57BL͞6-derived murine bone marrow macrophage cell line BMA3.1A7 (7) was a kind gift from Kenneth Rock (University of Masachusetts Medical School). Cells were maintained as described (6).Construction of pYUB 921. The FACS-optimized mutant 2 of the green fluorescent protein (GFP) gene from Aqueorea victoria (8) was kindly provided by S. Falcow (Stanford University). GFP was amplified by the PCR with primers: 5Ј-TTGCGGATCCAAGTA-AAGGAGAAGAACTTTTCA CT-3Ј and 5Ј-CGGAATTC-CTATTTGTATAGTTCATCCATCCATGCCATG-3Ј. The reaction product was digested with BamHI and EcoRI and was cloned into the appropriate sites within the mycobacteria͞E. coli shuttle vector PMV-261 (9).Preparation of Mycobacterial Strains. BCG (Connaught Laboratories) was transformed with pYUB 921 at room temperature as described (10). Transformants, designated ''BCG-GFP'' were plated on Middlebro...

M. tuberculosis accesses the terminal lung and is phagocytosed by alveolar macrophages. Utilizing a mouse intratracheal challenge model, we demonstrate that M. tuberculosis rapidly enters through M cells as well. From there, bacilli are deposited within associated intraepithelial leukocytes and subsequently conveyed to the draining lymph nodes early after infection. Osteopetrotic (Csfm(op)/Csfm(op)) mice, null mutants for macrophage colony-stimulating factor, possess diminished numbers of circulating monocytes and tissue macrophages. Csfm(op)/Csfm(op) mice were highly susceptible to challenge with M. tuberculosis. In contrast to controls, tubercle bacilli were not conveyed to draining lymph nodes early after infection but were instead retained within the mucosa. These results indicate that M cells represent an alternate portal of entry for M. tuberculosis, which may contribute to the rapid development of protective lung immune responses.

The L-arginine-dependent generation of reactive nitrogen intermediates (RNI) has been identified as a key intracellular antimicrobial mechanism of activated mouse macrophages. To determine the role of this mechanism in the activity of human mononuclear phagocytes, monocyte-derived macrophages activated in vitro by interferon (IFN)-gamma and monocytes from patients receiving IFN-gamma as therapy were treated with NG-monomethyl-L-arginine (NMA) or arginase. Neither competitive inhibition of L-arginine metabolism (NMA) nor depletion of L-arginine (arginase) altered intracellular antimicrobial activity against Toxoplasma gondii, Chlamydia psittaci, or Leishmania donovani. In contrast, NMA and arginase readily reversed the antimicrobial effect of mouse peritoneal macrophages stimulated either in vitro or in vivo by IFN-gamma, and activated mouse but not human cells could be induced to release enhanced levels of nitrite. These results suggest that the L-arginine-dependent generation of RNI is a species-restricted macrophage mechanism unlikely to participate in the intracellular antimicrobial activity of IFN-gamma-stimulated human mononuclear phagocytes.

Tuberculosis is responsible for the deaths of more people each year than any other single infectious disease, with greater than 7 million new cases and 2 million deaths annually. It remains the largest attributable cause of death in HIV-infected individuals, responsible for 32% of deaths of HIV-infected individuals in Africa. The only currently available vaccine for tuberculosis, bacille Calmette-Guerin (BCG) is the most widely used vaccine in the world, being administered to approximately 100 million children each year. Although untoward effects were not seen in several studies of HIV-seropositive children, the safety of live attenuated BCG vaccine in HIV-positive adults remains unknown and a matter of some concern. To obviate potential adverse affects of BCG vaccines in immunodeficient individuals, we have studied five auxotrophic strains of BCG produced by insertional mutagenesis for safety in administration to mice with severe combined immunodeficiency disease (SCID), and for protection in a susceptible strain of mice. The results indicate that viable BCG could no longer be detected in mice receiving the auxotrophs after 16-32 weeks, and that infected SCID mice survived for at least 230 days. In contrast, all SCID mice succumbed within eight weeks to conventional BCG vaccine. When susceptible BALB/c mice were immunized with auxotrophs and subsequently challenged with virulent Mycobacterium tuberculosis, several of the auxotrophs produced comparable protection against intravenous and intratracheal challenge with M. tuberculosis relative to conventional BCG. These results suggest that auxotrophic strains of BCG represent a potentially safe and useful vaccine against tuberculosis for populations at risk for HIV.