Showing papers by "Vojo Deretic published in 1998"

Effects of cytokines on mycobacterial phagosome maturation

Abstract: One of the major mechanisms permitting intracellular pathogens to parasitize macrophages is their ability to alter maturation of the phagosome or affect its physical integrity. These processes are opposed by the host innate and adaptive immune defenses, and in many instances mononuclear phagocytes can be stimulated with appropriate cytokines to restrict the growth of the microorganisms within the phagosomal compartment. Very little is known about the effects that cytokines have on phagosome maturation. Here we have used green fluorescent protein (GFP)-labeled mycobacteria and a fixable acidotropic probe, LysoTracker Red DND-99, to monitor maturation of the mycobacterial phagosome. The macrophage compartments that stained with the LysoTracker probe were examined first. This dye was found to colocalize preferentially with the late endosomal and lysosomal markers rab7 and Lamp1, and with a fluid phase marker chased into the late endosomal compartments. In contrast, LysoTracker showed only a minor overlap with the early endosomal marker rab5. Pathogenic mycobacteria are believed to reside in nonacidified vacuoles sequestered away from late endosomal compartments as a part of their intracellular survival strategy. We examined the status of mycobacterial phagosomes in macrophages from IL-10 knockout mice, in quiescent cells, and in mononuclear phagocytes stimulated with the macrophage-activating cytokine IFN-(gamma). When macrophages were derived from the bone marrow of transgenic IL-10 mice lacking this major deactivating cytokine, colocalization of GFP-fluorescing mycobacteria with the LysoTracker staining appeared enhanced, suggestive of increased acidification of the mycobacterial phagosome relative to macrophages from normal mice. When bone marrow-derived macrophages from normal mice or a J774 murine macrophage cell line were stimulated with IFN-(gamma) and LPS, this resulted in increased colocalization of mycobacteria and LysoTracker, but no statistically significant enhancement was observed in IL-10 transgenic animals. These studies are consistent with the interpretation that proinflammatory and anti-inflammatory cytokines affect maturation of mycobacterial phagosomes. Although multiple mechanisms are likely to be at work, we propose the existence of a direct link between cytokine effects on the host cell and phagosome maturation in the macrophage.

Microbial Pathogenesis in Cystic Fibrosis: Pulmonary Clearance of Mucoid Pseudomonas aeruginosa and Inflammation in a Mouse Model of Repeated Respiratory Challenge

Abstract: Chronic endobronchiolitis compounded by recurring Pseudomonas aeruginosa infections is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). In this study, a mouse model of repeated respiratory exposure to P. aeruginosa was established to facilitate investigations of factors contributing to P. aeruginosa persistence and associated inflammatory processes in the lung. While a single exposure to P. aeruginosa aerosols resulted in only mild histopathological changes, repeated exposure caused significant lung pathology in C57BL/6J mice. The peak of histopathological changes and inflammation in C57BL/6J mice was characterized by subacute lymphohistiocytic bronchopneumonia and persistent elevation of tumor necrosis factor alpha and macrophage inflammatory protein 2 in the lung but not in the serum. When isogenic nonmucoid (mucA+) and mucoid (mucA22) P. aeruginosa strains were compared, the mucoid cells were cleared several-fold less efficiently than the parental nonmucoid strain during the initial stages of the aerosol exposure regimen. However, the microscopic pathology findings and proinflammatory cytokine levels were similar in mice exposed to nonmucoid and mucoid P. aeruginosa throughout the infection. We also tested lung histopathology and proinflammatory cytokines in interleukin 10 (IL-10)-deficient transgenic (IL-10T) mice. Significant mortality was seen in IL-10T mice on initial challenge with P. aeruginosa, although no histopathological differences could be observed in the lungs of C57BL/6J and surviving IL-10T mice after a single exposure. However, increased pathology was detected in IL-10T mice relative to C57BL/6J after repeated challenge with P. aeruginosa. This observation supports the proposals that anti-inflammatory cytokines may play a role in suppressing P. aeruginosa-induced tissue damage during chronic infection.

Oxidative stress response and characterization of the oxyr-ahpc and fura-katg loci in mycobacterium marinum

Abstract: Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized the oxyR-ahpC and furA-katG loci in the nontuberculous pathogen Mycobacterium marinum. In contrast to Mycobacterium smegmatis and like Mycobacterium tuberculosis and Mycobacterium leprae, M. marinum was shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress response oxyR and its subordinate gene ahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to the oxyR-ahpC promoter region from M. marinum and additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence. M. marinum AhpC levels detected by immunoblotting, were increased upon treatment with H2O2, in keeping with the presence of a functional OxyR and its binding site within the promoter region of ahpC. In contrast, OxyR did not bind to the sequences upstream of the katG structural gene, and katG expression did not follow the pattern seen with ahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream of katG in M. marinum. The furA-katG linkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences in ahpC and katG expression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest that M. marinum will be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.

7.5 Molecular Analysis of Pseudomonas Aeruginosa Virulence

Abstract: Publisher Summary This chapter discusses the molecular analysis of Pseudomonas aeruginosa. The exquisitely broad range of infections caused by the Gram-negative opportunistic pathogen Pseudomonas aeruginosa poses unique strategic and logistic problems when bacterial virulence factors and host contribution to the pathogenesis are investigated. At one end of its wide dynamic range as a pathogen, P. aeruginosa can cause acute and sometimes systemic infections, which can result in fatal sepsis. Several adhesins, toxins, and other virulence factors of P. aeruginosa are characterized at the molecular level. In some cases, these determinants are able to satisfy the molecular Koch's postulates in adequate infection models. In this context, the molecular genetic analyses of P. aeruginosa virulence genes have provided a fair share of contributions to the overall advancement of the field of molecular pathogenesis. The chapter further discusses a recent approach in investigating one particular aspect of P. aeruginosa virulence, the newly discovered extreme stress response, which controls conversion to mucoidy in P. aeruginosa strains, infecting people with cystic fibrosis (CF). Chronic endobronchial infections with P. aeruginosa and the associated host inflammatory response are the major cause of high morbidity and mortality in people with CF.