LRRK2 and RAB7L1 coordinately regulate axonal morphology and lysosome integrity in diverse cellular contexts.

Leucine-rich repeat kinase 2 (LRRK2) has been linked to several clinical disorders including Parkinson's disease (PD), Crohn's disease, and leprosy. Furthermore in rodents, LRRK2 deficiency or inhibition leads to lysosomal pathology in kidney and lung. Here we provide evidence that LRRK2 functions together with a second PD-associated gene, RAB7L1, within an evolutionarily conserved genetic module in diverse cellular contexts. In C. elegans neurons, orthologues of LRRK2 and RAB7L1 act coordinately in an ordered genetic pathway to regulate axonal elongation. Further genetic studies implicated the AP-3 complex, which is a known regulator of axonal morphology as well as of intracellular protein trafficking to the lysosome compartment, as a physiological downstream effector of LRRK2 and RAB7L1. Additional cell-based studies implicated LRRK2 in the AP-3 complex-related intracellular trafficking of lysosomal membrane proteins. In mice, deficiency of either RAB7L1 or LRRK2 leads to prominent age-associated lysosomal defects in kidney proximal tubule cells, in the absence of frank CNS pathology. We hypothesize that defects in this evolutionarily conserved genetic pathway underlie the diverse pathologies associated with LRRK2 in humans and in animal models.

ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria.

Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence.

M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells.

M. tuberculosis and M. leprae are considered to be prototypical intracellular pathogens that have evolved strategies to enable growth in the intracellular phagosomes. In contrast, we show that lysosomes rapidly fuse with the virulent M. tuberculosis- and M. leprae-containing phagosomes of human monocyte-derived dendritic cells and macrophages. After 2 days, M. tuberculosis progressively translocates from phagolysosomes into the cytosol in nonapoptotic cells. Cytosolic entry is also observed for M. leprae but not for vaccine strains such as M. bovis BCG or in heat-killed mycobacteria and is dependent upon secretion of the mycobacterial gene products CFP-10 and ESAT-6. The cytosolic bacterial localization and replication are pathogenic features of virulent mycobacteria, causing significant cell death within a week. This may also reveal a mechanism for MHC-based antigen presentation that is lacking in current vaccine strains.

Immunolocalization of cell-wall-deficient forms of Mycobacterium tuberculosis complex in sarcoidosis and in sinus histiocytosis of lymph nodes draining carcinoma.

In sarcoidosis, pleomorphic chromogens (PCs) occur as multivariate pigmented elements within sinusoids of lymph nodes (sinusoidal phase) and as tiny "round bodies" detectable in granulomas (generalized phase). The sinusoidal phase occurs in other conditions as well and characteristically contains yeastlike bodies also known as H-W bodies. To elucidate the antigenic profile of all variant forms, 28 cases of sarcoidosis (series A) and 14 cases of malignancy associated sinus histiocytosis (series B) were studied immunohistochemically with panels of various antibodies, including antimycobacterial MAbs specific for M tuberculosis complex (TB68, TB71), for M. leprae (MMP-I-3C3) and for cross-reactive mycobacterial antigens (F24-2-3 and F116-5, the latter recognizing superoxide dismutase). Results for series A indicate that: 1) PCs are cell-wall-deficient (CWD) mycobacterial forms belonging to M. tuberculosis complex (over 95%); 2) both phases are antigenically identical parts of the L-cycle; 3) "round bodies" of the "infective" phase have an endolysosomal evolution; 4)uncommon vacuolated forms represent a labile spheroplast stage; 5) the yeastlike bodies are specialized sinusoidal large bodies of unknown function. Results for series B show that in roughly two thirds of cases the pigmented forms are also CWD mycobacteria, have the same immunophenotype as sarcoid PCs in 35.7% of cases, have a much higher incidence of labile vacuolated forms and, finally, that malignancy associated "pseudosarcoid" granulomas do not differ antigenically from genuine sarcoid granulomas. Unlike conventional mycobacteria, PCs do not express cytoskeletal proteins consistently. Their general reactivity for HBcAg raises the possibility of phage interactions being responsible for the L-cycle since it may reflect shared epitopes between unrelated virus entities.

Phagosome-lysosome fusions in macrophages infected by Mycobacterium avium: role of mycosides-C and other cells surface components.

The phagosome-lysosome fusions (PLE) were assessed in case of bone-marrow macrophages infected by the opportunistic species Mycobacterium avium, employing the acid-phosphatase (AcPase) electron-cytochemistry. The role of surface components was evaluated by coating the bacteria prior to phagocytosis by specific M. avium antiserum or the anti-mycosides-C serum raised in rabbit. PLF was evaluated under the electron microscope during (2, 4 hours), or after (24 hours) phagocytosis. The preliminary results suggest that although M. avium surface components intervene in PLF inhibition, the role of mycosides-C among these surface components (effectively intervening in PLF inhibition) is questionable.

Resistance of Mycobacterium avium to microbicidal activities in bone-marrow macrophages from naturally-susceptible (C57B1/6) and naturally-resistant (DBA-2) mice.

In this study, the phagosome-lysosome fusion (PLF) inhibition and the presence of the electron-transparent zone (ETZ) around phagocytized Mycobacterium avium both in the naturally-susceptible and naturally-resistant mice was compared. We observed marked differences in case of these two mice strains, as PLF inhibition, as well as the presence of ETZ, was more important in case of naturally-susceptible mice.

Ultracytochemical studies of lysosomal function in the macrophages of human leprosy--I.

The present study was undertaken to understand the lysosomal status and function in macrophages across the leprosy spectrum, including the reactional states. Acid phosphatase was used as the marker enzyme to demonstrate lysosomes at the electron microscopic level. It was observed that lysosomal morphology was well maintained in the macrophages of tuberculoid, borderline tuberculoid, and borderline tuberculoid leprosy in reaction, while they lost their cellular morphology and cell membrane integrity in lepromatous leprosy and lepromatous leprosy in reaction. The importance of these findings is discussed.

Intracellular fate of Mycobacterium leprae in normal and activated mouse macrophages.

Mycobacterium leprae replicates within mononuclear phagocytes, reaching enormous numbers in the macrophage-rich granulomas of lepromatous leprosy. To examine the capability of macrophages to digest M. leprae, we studied the intracellular fate of M. leprae organisms in normal and activated mouse macrophages by using the electron-dense secondary lysosome tracer Thoria Sol. Intracellular M. leprae organisms, surrounded by a characteristic electron-transparent zone, were contained within phagosomal vacuoles of macrophages cultured in vitro for 1 to 6 days. In normal macrophages, a majority of phagosomes containing freshly isolated live M. leprae cells resisted fusion with Thoria Sol-labeled lysosomes. The extent of fusion was not significantly affected by pretreatment of M. leprae with human patient serum high in specific immunoglobulin G and M antibodies. In contrast, a majority of phagosomes containing gamma-irradiated M. leprae cells underwent lysosome fusion in normal macrophages. In addition, increased phagolysosome fusion was observed with live M. leprae-containing phagosomes in macrophages activated with gamma interferon. Increased fusion was associated with an increase in the number of fragmented and damaged bacilli, suggesting that increased digestion followed fusion. This study indicates that activated macrophages may have an increased capacity for clearance of normally resistant M. leprae.

Ultrastructural aspects of oral and facial lepromatous lesions.

The ultrastructure of 10 lepromatous lesions in the face and the palatal mucosa after different duration and length of treatment was studied. Biopsies taken from patients who showed erythema nodosum leprosum (ENL) revealed different ultrastructural characteristics from those taken from 'burnt out' and non-medicated cases. Multiple secondary lysosomes were rarely seen in non-ENL cases. The presence of Mycobacterium leprae and an increased lysosomal activity in ENL reactive cases is interpreted as a reaction to the lysis of the cytoplasmic matrix of M. leprae; however, drug-specific (diaminodiphenylsulphone) reactions must also be considered.

Lepromatous leprosy as a model of Schwann cell pathology and lysosomal activity.

A brief illustrated account is presented of the light microscopic pathology, histochemistry of lysosomal enzymes, and fine structural changes in the nerves of patients with untreated or treated lepromatous leprosy. Predominant bacillation of the Schwann cells of unmyelinated fibres, degeneration of their axons, prominence of phagolysosomes, and disappearance of these cells with endoneurial collagenosis were observed on electronmicroscopic examination of the index branch of the radial cutaneous nerve. Although there were changes in the blood vessels and proliferation of perineurium, bacillation of endothelial or perineurial cells was much less conspicuous. Intact and degenerating forms of M. leprae were found in both treated and untreated patients, fragmenting or crumpled forms being more frequent in the treated. Both groups of patients also showed increased lysosomal enzyme activity, evidenced by single or paired paranodal spots of acid phosphatase and beta-glucuronidase in Schwann cells in histochemical preparations of the nerve. There was lesser activity, and activity in fewer cells, in the case of beta-glucuronidase than of acid phosphatase. Diffuse beta-glucuronidase activity was found in the wall of empty-looking oval chambers in the Schwann cells, and acid-fast bacilli were seen in these chambers. In teased fibre preparations, both axonal degeneration and segmental demyelination were found. In semi-thin araldite sections, the myelinated fibre density was either preserved or reduced; large diameter fibres were more frequently depleted, with tall peaks of smaller fibres seen on plotting diameter spectra.

Experimental murine leprosy. 8. Ultrastructural features of the inflammatory exudate and bacterial morphology in C3H and C57BL mice after foot-pad inoculation with Mycobacterium lepraemurium.

Mice of the inbred strains C57BL and C3H were inoculated in the foot-pads with Mycobacterium lepraemurium (MLM) and the inflammatory reaction was studied using light and electron microscopy. In C57BL mice a granulomatous reaction developed 3-4 weeks after inoculation. The inflammatory exudate at this stage showed numerous lymphocytes, monocytes and macrophages. The latter cell type often contained many lysosomes and appeared activated. The bacilli which were all within phagosomes showed extensive electron dense aggregates of the cytoplasm suggesting severe damage. Lymphocytes and macrophages in close contact with each other were often observed. In macrophages which contained damaged bacilli, spherical lipid-like bodies surrounded by granular endoplasmic reticulum were observed. It is suggested that this cell product could be of some significance for the bactericidal function of the macrophage. Contrary to these findings, the cellular infiltrate developing in C3H mice showed no lymphocytes and consisted exclusively of macrophages. These were all heavily loaded with bacilli. The vast majority of bacilli encountered in this strain was morphologically intact and presumably viable. Lipid-like bodies similar to those observed in infected C57BL macrophages were not encountered in C3H mice. It is concluded that unless the infected macrophages become immunologically activited they are unable to cause bacterial damage or to inhibit the growth of MLM.