How to establish a lasting relationship with your host: lessons learned from Mycobacterium spp.
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Mycobacterium spp. enjoy an intracellular lifestyle that is fatal to most microorganisms. Bacilli persist and multiply within mononuclear phagocytes in the face of defences ranging from toxic oxygen and nitrogen radicals, acidic proteases and bactericidal peptides. Uptake of Mycobacterium by phagocytes results in the de novo formation of a phagosome, which is manipulated by the pathogen to accommodate its needs for intracellular survival and replication. The present review describes the intracellular compartment occupied by Mycobacterium spp. and presents current ideas on how mycobacteria may establish this niche, placing special emphasis on the involvement of mycobacterial cell wall lipids. (+info)
Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase.
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In determining the mechanism of neutrophil elastase (NE)-mediated killing of Escherichia coli, we found that NE degraded outer membrane protein A (OmpA), localized on the surface of Gram-negative bacteria. NE killed wild-type, but not OmpA-deficient, E. coli. Also, whereas NE-deficient mice had impaired survival in response to E. coli sepsis, as compared to wild-type mice, the presence or absence of NE had no influence on survival in response to sepsis that had been induced with OmpA-deficient E. coli. These findings define a mechanism of nonoxidative bacterial killing by NE and point to OmpA as a bacterial target in host defense. (+info)
Mycobacterium tuberculosis and Legionella pneumophila phagosomes exhibit arrested maturation despite acquisition of Rab7.
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Rab7 is a small GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. Phagosomes containing inert particles have also been shown to transiently acquire Rab7 as they mature. Disruption in the pathway prior to the acquisition of Rab7 has been suggested as playing a role in the altered maturation of Mycobacterium bovis BCG phagosomes. As a first step to determine whether disruption in the delivery or function of Rab7 could play a role in the altered maturation of Legionella pneumophila and M. tuberculosis phagosomes, we have examined the distribution of wild-type Rab7 and the GTPase-deficient, constitutively active mutant form of Rab7 in HeLa cells infected with L. pneumophila or M. tuberculosis. We have found that the majority of L. pneumophila and M. tuberculosis phagosomes acquire relatively abundant staining for Rab7 and for the constitutively active mutant Rab7 in HeLa cells that overexpress these proteins. Nevertheless, despite acquisition of wild-type or constitutively active Rab7, both the L. pneumophila and the M. tuberculosis phagosomes continue to exhibit altered maturation as manifested by a failure to acquire lysosome-associated membrane glycoprotein 1. These results demonstrate that L. pneumophila and M. tuberculosis phagosomes have receptors for Rab7 and that the altered maturation of these phagosomes is not due to a failure to acquire Rab7. (+info)
Rab5 regulates the kiss and run fusion between phagosomes and endosomes and the acquisition of phagosome leishmanicidal properties in RAW 264.7 macrophages.
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Phagolysosome biogenesis is essential for the killing and degradation of intracellular pathogens. It involves the fusion of phagosomes with various endocytic organelles, a process known to be regulated in part by Rab proteins. We generated RAW 264.7 macrophages expressing an active mutant of Rab5 (Rab5(Q79L)) to determine the role of Rab5 in phagocytosis and phagolysosome biogenesis. Our results indicate that Rab5 stimulates phagocytosis of latex beads but not Fc or C3 receptor-mediated phagocytosis. Rab5 also acts to restrict the complete fusion of phagosomes with endosomes, a phenomenon allowing exchange of solutes from the two compartments without complete intermixing of their membrane (kiss and run). In Rab5(Q79L)-expressing macrophages, uncontrolled fusion events occurred, leading to the appearance of giant phagosomes. These phagosomes could initiate their maturation and acquire LAMP1, but failed to generate the microbicidal conditions needed to kill intracellular parasites. These results identify Rab5 as a key molecule regulating phagosome-endosome fusion and as an essential component in the innate ability of macrophages to restrict the growth of intracellular parasites. (+info)
Tripeptidyl peptidase II promotes maturation of caspase-1 in Shigella flexneri-induced macrophage apoptosis.
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The invasive enteropathogenic bacterium Shigella flexneri activates apoptosis in macrophages. Shigella-induced apoptosis requires caspase-1. We demonstrate here that tripeptidyl peptidase II (TPPII), a cytoplasmic, high-molecular-weight protease, participates in the apoptotic pathway triggered by Shigella. The TPPII inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk) and clasto-lactacystin beta-lactone (lactacystin), an inhibitor of both TPPII and the proteasome, protected macrophages from Shigella-induced apoptosis. AAF-cmk was more potent than lactacystin and irreversibly blocked Shigella-induced apoptosis by 95% at a concentration of 1 microM. Conversely, peptide aldehyde and peptide vinylsulfone proteasome inhibitors had little effect on Shigella-mediated cytotoxicity. Both AAF-cmk and lactacystin prevented the maturation of pro-caspase-1 and its substrate pro-interleukin 1beta in Shigella-infected macrophages, indicating that TPPII is upstream of caspase-1. Neither of these compounds directly inhibited caspase-1. AAF-cmk and lactacystin did not impair macrophage phagocytosis or the ability of Shigella to escape the macrophage phagosome. TPPII was also found to be involved in apoptosis induced by ATP and the protein kinase inhibitor staurosporine. We propose that TPPII participates in apoptotic pathways. (+info)
Interaction of Mycobacterium avium with human monocyte-derived dendritic cells.
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The mechanism by which mycobacteria elicit class I-restricted T-cell responses remains undefined because these organisms have been shown to reside exclusively within membrane-bound vesicles in macrophages (Mphi), their primary host cells. We studied the interaction of M. avium with dendritic cells (DC) because they are the most potent antigen-presenting cells and are abundant at M. avium infection sites. We observed that both DC and Mphi, generated from human peripheral blood monocytes by short-term culture, internalized M. avium. The onset of programmed cell death and the percentage of apoptotic cells in infected DC and Mphi were comparable. However, following infection, DC secreted significantly larger amounts of interleukin-12, but not interleukin-1beta, than infected autologous Mphi. Further analysis of infected cells showed that while phagosomes failed to acidify in both M. avium-infected DC and Mphi, bacilli grew more slowly in DC. Electron microscopy studies revealed that M. avium resided within endocytic vacuoles in both cell types. The vacuolar membrane surrounding some bacilli in approximately 10% of the vacuoles in DC possessed several breaks. The importance of this finding will have to be addressed in future studies. (+info)
Cathepsin D deficiency induces lysosomal storage with ceroid lipofuscin in mouse CNS neurons.
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Cathepsin D-deficient (CD-/-) mice have been shown to manifest seizures and become blind near the terminal stage [approximately postnatal day (P) 26]. We therefore examined the morphological, immunocytochemical, and biochemical features of CNS tissues of these mice. By electron microscopy, autophagosome/autolysosome-like bodies containing part of the cytoplasm, granular osmiophilic deposits, and fingerprint profiles were demonstrated in the neuronal perikarya of CD-/- mouse brains after P20. Autophagosomes and granular osmiophilic deposits were detected in neurons at P0 but were few in number, whereas they increased in the neuronal perikarya within days after birth. Some large-sized neurons having autophagosome/autolysosome-like bodies in the perikarya appeared in the CNS tissues, especially in the thalamic region and the cerebral cortex, at P17. These lysosomal bodies occupied the perikarya of almost all neurons in CD-/- mouse brains obtained from P23 until the terminal stage. Because these neurons exhibited autofluorescence, it was considered that ceroid lipofuscin may accumulate in lysosomal structures of CD-/- neurons. Subunit c of mitochondrial ATP synthase was found to accumulate in the lysosomes of neurons, although the activity of tripeptidyl peptidase-I significantly increased in the brain. Moreover, neurons near the terminal stage were often shrunken and possessed irregular nuclei through which small dense chromatin masses were scattered. These results suggest that the CNS neurons in CD-/- mice show a new form of lysosomal accumulation disease with a phenotype resembling neuronal ceroid lipofuscinosis. (+info)
Altered membrane trafficking in activated bone marrow-derived macrophages.
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Activation of macrophages with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) leads to increased intracellular resistance to microbes and increased major histocompatibility complex class II-restricted antigen presentation, processes that both use the vacuolar compartment. Despite the requirement of the macrophage vacuolar compartment for microbicidal activities and antigen processing, the rates of endocytosis and membrane trafficking in activated macrophages are not clearly defined. In this study, vacuolar compartment dynamics were analyzed in murine bone marrow-derived macrophages activated with LPS and/or IFN-gamma, conditions that increased macrophage nitric oxide production and resistance to infection by Listeria monocytogenes. Relative to nonactivated cells, activated macrophages showed diminished rates of fluid-phase pinocytosis and phagocytosis and delayed progression of macropinosomes and phagosomes to late endosomes and lysosomes. In contrast to the slowing of membrane trafficking, rates of macropinosome acidification were similar between activated and nonactivated cells. One consequence of this slowed membrane trafficking in activated macrophages was a prolonged exposure of incoming molecules to an acidic nonlysosomal compartment, a condition which may facilitate microbicidal chemistries or antigen processing. (+info)