Chronic infection with Mycobacterium lepraemurium induces alterations in the hippocampus associated with memory loss.

Murine leprosy, caused by Mycobacterium lepraemurium (MLM), is a chronic disease that closely resembles human leprosy. Even though this disease does not directly involve the nervous system, we investigated a possible effect on working memory during this chronic infection in Balb/c mice. We evaluated alterations in the dorsal region of the hippocampus and measured peripheral levels of cytokines at 40, 80, and 120 days post-infection. To evaluate working memory, we used the T-maze while a morphometric analysis was conducted in the hippocampus regions CA1, CA2, CA3, and dentate gyrus (DG) to measure morphological changes. In addition, a neurochemical analysis was performed by HPLC. Our results show that, at 40 days post-infection, there was an increase in the bacillary load in the liver and spleen associated to increased levels of IL-4, working memory deterioration, and changes in hippocampal morphology, including degeneration in the four subregions analyzed. Also, we found a decrease in neurotransmitter levels at the same time of infection. Although MLM does not directly infect the nervous system, these findings suggest a possible functional link between the immune system and the central nervous system.

Mycobacterium lepraemurium uses TLR-6 and MR, but not lipid rafts or DC-sign, to gain access into mouse macrophages.

OBJECTIVE/BACKGROUND: Mycobacterium lepraemurium (MLM), the etiologic agent of murine leprosy, is an intracellular parasite of macrophages; the mechanism used by this bacterium to enter macrophages is not known. The fate of the MLM phagosome inside macrophages is also unknown. This study was conducted to investigate how MLM enters macrophages and to define the maturation process of MLM phagosome inside macrophages. MATERIALS AND METHODS: Peritoneal macrophages were incubated in the presence of mannan-bovine serum albumin (BSA), and antibodies to known macrophage receptors, including, anti-FcγRIII/RII (anti-CD16/32), anti-CD35 (anti-CR1), anti-TLR2, anti-TLR4, anti-TLR6, anti-CD14, and anti-dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). Then, macrophages were challenged with Iris Fuchsia-stained MLM, at a multiplicity of infection of 50:1. The blocking effect of the antibodies (and mannan-BSA) used was analyzed using direct microscopy and flow cytometry. The maturation process of MLM phagosomes was visualized by their interaction with antibodies to Rab5, Rab7, proton ATPase, and cathepsin D, by confocal microscopy. RESULTS: Only mannan-BSA and anti-TLR6 antibody significantly blocked the entry of MLM into macrophages. None of the other antibodies, including that for DC-SIGN, meaningfully inhibited the endocytic process. We also found that MLM is a fusiogenic mycobacterium. This was deduced from the orderly association of MLM phagosomes with Rab5, Rab7, Proton ATPase, and lysosomes (cathepsin D). CONCLUSION: Fusion of MLM phagosomes with lysosomes seems to be a necessary event for the intracellular multiplication of MLM; similar to Mycobacterium leprae, this microorganism hardly grows on artificial, synthetic, bacteriologic media.

Effect of reactive oxygen intermediaries on the viability and infectivity of Mycobacterium lepraemurium.

Murine leprosy is a natural disease of the mouse, the most popular model animal used in biomedical research; the disease is caused by Mycobacterium lepraemurium (MLM), a successful parasite of macrophages. The aim of the study was to test the hypothesis that MLM survives within macrophages because it highly resists the toxic effects of the reactive oxygen intermediaries produced by these cells in response to infection by the microorganism. MLM cells were incubated in the presence of horseradish peroxidase (HRPO)-H(2)O(2)-halide for several periods of time. The peroxidative effect of this system was investigated by assessing the changes occurred in (a) lipid composition; (b) viability; and (c) infectivity of the microorganism. Changes in the lipid composition of peroxidated- vs. intact-MLM were analysed by thin layer chromatography. The effect of the peroxidative system on the viability and infectivity of MLM was measured by the alamar blue reduction assay and by its ability to produce an infection in the mouse, respectively. Peroxidation of MLM produced drastic changes in the lipid envelope of the microorganism, killed the bacteria and abolished their ability to produce an in vivo infection in the mouse. In vitro, MLM is highly susceptible to the noxious effects of the HRPO-H(2)O(2)-halide system. Although the lipid envelope of MLM might protect the microorganism from the peroxidative substances produced at 'physiological' concentrations in vivo, the success of MLM as a parasite of macrophages might rather obey for other reasons. The ability of MLM to enter macrophages without triggering these cells' oxidative response and the lack of granular MPO in mature macrophages might better explain its success as an intracellular parasite of these cells.

Expression of cyclooxygenase-2, alpha 1-acid-glycoprotein and inducible nitric oxide synthase in the developing lesions of murine leprosy.

Murine leprosy is a chronic disease of the mouse, the most popular animal model used in biomedical investigation, which is caused by Mycobacterium lepraemurium (MLM) whose characteristic lesion is the macrophage-made granuloma. From onset to the end of the disease, the granuloma undergoes changes that gradually transform the environment into a more appropriate milieu for the growth of M. lepraemurium. The mechanisms that participate in the formation and maturation of the murine leprosy granulomas are not completely understood; however, microbial and host-factors are believed to participate in their formation. In this study, we analysed the role of various pro-inflammatory and anti-inflammatory proteins in granulomas of murine leprosy after 21 weeks of infection. We assessed the expression of cyclooxygenase-2 (COX-2), alpha acid-glycoprotein (AGP), and inducible nitric oxide synthase (iNOS) at sequential stages of infection. We also looked for the nitric-oxide nitrosylation product, nitrotyrosine (NT) in the granulomatous lesions of murine leprosy. We found that a pro-inflammatory environment predominates in the early granulomas while an anti-inflammatory environment predominates in late granulomas. No obvious signs of bacillary destruction were observed during the entire period of infection, but nitrosylation products and cell alterations were observed in granulomas in the advanced stages of disease. The change from a pro-inflammatory to an anti-inflammatory environment, which is probably driven by the bacillus itself, results in a more conducive environment for both bacillus replication and the disease progression.

Susceptibility of et, the spontaneously mutating CD-1 derived nude mouse, to infection of M. lepraemurium

We have studied the susceptibility to infection by Mycobacterium lepraemurium (MLM) of a nude, hypothymic, CD1-derived, spontaneous mouse mutant called [quot ]et[quot ] because of its extraterrestrial appearance. We found that despite their hypothymia, et/et mice were not more susceptible to infection by MLM than their euthymic et/+ counterparts. Infection of both et/et and et/+ mice with 50 x 10(6) bacilli by the intraperitoneal route led only to a mild infection with low levels of antimycobacterial antibodies and a small number of lesions. These lesions were indicative of reactive hepatitis and hyaline perisplenitis with lymphoid hyperplasia. Some small bacilliferous granulomas were also observed at the end of the experiment (5 months of infection). CD1 mice behave in a rather [quot ]resistant[quot ] manner to the infection by MLM. It is clear that the nu gene is not necessarily linked to the thymus defect, and it is also clear that the hypothymia of et/et mice does not obviously affect their general cell-mediated immune competence.

Mycobacterium lepraemurium, a well-adapted parasite of macrophages: I. Oxygen metabolites

We measured the release of reactive oxygen intermediaries [ROI (hydrogen peroxide and superoxide anion)] by murine peritoneal macrophages challenged in vitro with Mycobacterium lepraemurium (MLM), complement-opsonized yeast, M. bovis BCG, M. phlei, or phorbol myristate acetate (PMA). We found that except for MLM, all of the other materials provoked the release of significant amounts of hydrogen peroxide and superoxide. MLM entered the macrophages without triggering their oxidative metabolism. Pre-infection of macrophages with MLM did not alter these cells' capacity to release the normal amounts of ROI in response to other microorganisms or PMA. Killing of MLM did not revert the macrophages' failure to release ROI upon ingestion of the microorganism, nor were macrophages able to produce these toxic metabolites when pre-incubated in the presence of murine gamma interferon (IFN-gamma). MLM has several attributes that allow it to survive within macrophages: a) it is a nontoxigenic microorganism (it does not harm its host), b) it resists the harsh conditions of the intraphagolysosomal milieu (a property perhaps dependent on its thick lipidic envelope), and c) it penetrates the macrophages without triggering their oxidative response (thus avoiding the generation of the toxic intermediaries of oxygen). For these attributes (and others discussed in this paper), we recognize MLM as a highly evolved, well-adapted parasite of macrophages. In addition, the results of the present study prompted the analysis of the biochemical pathways used by MLM and M. bovis BCG to penetrate into their cellular hosts, a subject now under investigation in our laboratory.

Mycobacterium lepraemurium, a well-adapted parasite of macrophages: I. Oxygen metabolites.

We measured the release of reactive oxygen intermediaries [ROI (hydrogen peroxide and superoxide anion)] by murine peritoneal macrophages challenged in vitro with Mycobacterium lepraemurium (MLM), complement-opsonized yeast, M. bovis BCG, M. phlei, or phorbol myristate acetate (PMA). We found that except for MLM, all of the other materials provoked the release of significant amounts of hydrogen peroxide and superoxide. MLM entered the macrophages without triggering their oxidative metabolism. Pre-infection of macrophages with MLM did not alter these cells' capacity to release the normal amounts of ROI in response to other microorganisms or PMA. Killing of MLM did not revert the macrophages' failure to release ROI upon ingestion of the microorganism, nor were macrophages able to produce these toxic metabolites when pre-incubated in the presence of murine gamma interferon (IFN-gamma). MLM has several attributes that allow it to survive within macrophages: a) it is a nontoxigenic microorganism (it does not harm its host), b) it resists the harsh conditions of the intraphagolysosomal milieu (a property perhaps dependent on its thick lipidic envelope), and c) it penetrates the macrophages without triggering their oxidative response (thus avoiding the generation of the toxic intermediaries of oxygen). For these attributes (and others discussed in this paper), we recognize MLM as a highly evolved, well-adapted parasite of macrophages. In addition, the results of the present study prompted the analysis of the biochemical pathways used by MLM and M. bovis BCG to penetrate into their cellular hosts, a subject now under investigation in our laboratory.

Identification of cat leprosy bacillus grown in mice.

Cat leprosy bacilli passaged in mice could be isolated on 1% Ogawa yolk medium. The isolated cat leprosy bacilli which were cultivated successively four times on 1% Ogawa yolk medium produced a leproma in mice. All characteristics of the isolated cat leprosy bacillus were the same as isolated murine leprosy bacillus, as follows: slow grower, light yellowish-white rough colony, production of much coproporphyrin on the medium, heat-resistant catalase negative, heat-resistant phosphatase negative, arylsulfatase negative, niacin negative, hydrolysis of Tween 80 negative, urease negative, nicotinamidase positive, pyrazinamidase positive, cytochrome b1 at 560 nm positive, cytochrome a2 at 630 nm positive, and cytochrome c at 550 nm negative. Cats are susceptible to both cat and murine leprosy bacilli; the bacilli produced a leproma in a newborn cat at 3 to 4 months and in an adult cat at 2 months after inoculation. Many globi of acid-fast bacilli (AFB) were observed in the histopathological sections and the smear preparations of the newborn cat's lepromas, especially in the necrotic areas of the lepromas. Many AFB and polymorphonuclear leukocytes were seen in the histopathological sections and the smear preparations of the adult cat's lepromas. These lepromas formed ulcers by autolysis and healed or absorbed without ulcer formation over the course of months. Large lepromas remained for a long time without ulcer formation and caseation in some cats. Secondary infections with cat and murine leprosy bacilli were done respectively to the right and left femoral subcutaneous regions of newborn cats carrying primary lepromas. After one month, granulomas in which many AFB were observed were produced in both infection sites. Cats are susceptible to infection with cat and murine leprosy bacilli; however, the bacilli did not invade progressively to internal organs or other subcutaneous areas. Cat leprosy bacilli which were passaged in the mouse are identical to murine leprosy bacilli.

Restored pathogenicity of attenuated Mycobacterium lepraemurium in mice.

The ability of Mycobacterium lepraemurium (Mlm) to adhere to A31 cells in culture decreased with the number of passages of the bacilli on Ogawa egg-yolk medium. Pathogenic Mlm consistently grew in tissue culture cells but growth was not seen with attenuated Mlm isolated from a smooth colony. After prolonged incubation, attenuated Mlm became adapted to tissue culture growth. The pathogenicity of the attenuated bacilli was restored partially by the adaptation to tissue culture cells and restored almost completely by passage in mice. After restoration of pathogenicity by these methods, the Mlm formed rough-type colonies on Ogawa egg-yolk medium although the colonies were not completely of the rough type. Attenuated Mlm did not interfere with the growth of in vivo-derived Mlm in tissue culture or in mice.

Survival of Mycobacterium lepraemurium in vitro for thirty years by lyophilization.

It was demonstrated that the virulence of Mycobacterium lepraemurium could be maintained in vitro for 30 years when the bacilli from the infected subcutaneous mouse tissue were suspended in 10% bovine serum-water, frozen, dried, and stored in a refrigerator. However, it was noted that a complete loss of virulence occurred when the bacilli were suspended in saline. Thus, the selection of the suspending solution is of the utmost importance in maintaining bacterial virulence by lyophilization.

Genetic resistance of mice to persistent infection with Mycobacterium lepraemurium in vitro: association with macrophage bactericidal responsiveness to lymphokines and dissociation from production of hydrogen peroxide by macrophages.

Peritoneal and splenic adherent macrophages (SAC) from M. lepraemurium susceptible (C3H/HeJ) and resistant (C57B1/6J) mice were studied for their abilities to generate H2O2 in vitro. Unexpectedly, SAC from the susceptible C3H/HeJ strain produced more H2O2 than those of the resistant C57BL/6J. In vivo sensitization with M. bovis (BCG), or C. parvum increased production of H2O2 by SAC from both strains, whereas in vivo sensitization with M. lepraemurium enhanced H2O2 production only in the C3H/HeJ susceptible strains. In vitro addition of a crude lymphokine enhanced H2O2 production by C3H/HeJ SAC more than by C57BL/6J SAC. In vitro addition of M. lepraemurium caused an inhibition of H2O2 production by SAC from both strains but the inhibition was greater for the resistant C57BL/6J strain. M. lepraemurium phagocytosed in vitro by untreated peritoneal macrophages of both mouse strains were morphologically altered to the same extent. However, the addition of lymphokine dramatically increased the degree of bacterial lysis in only the C57BL/6J strain. These results, support the view that H2O2 plays a limited, if any, role in the protection of the host from M. lepraemurium and may even contribute to susceptibility by inhibiting the host's immune response.

Stimulation of growth of Mycobacterium lepraemurium in cell-free liquid medium by DL-aspartic acid.

The growth of M. lepraemurium in cell-free liquid medium was strongly stimulated by addition of DL-aspartic acid at a final concentration of 0.01% to 0.02% to ND and ND-5 media containing dextran and liposome. No effect of DL-aspartic acid was observed when it was added to ND and ND-5 media without dextran and liposome. Optimal pH of the culture medium is critical for the stimulating effect of DL-aspartic acid, and it varies with the composition of the medium; the optimal pH was 6.0 in ND medium containing dextran and liposome (NDLA), and was 6.6 in NDLA medium supplemented with alpha-ketoglutarate, 1-cysteine HCl, hemin, and cytochrome c (NDLA-5). A possible mechanism of the effect of aspartic acid is discussed.

Pedigreed stocks of Mycobacterium lepraemurium for cultivation and metabolic studies.

Experience has shown that a prime requirement for investigations with Mycobacterium lepraemurium is pedigreed stocks of cells which provide constant baselines over a long period of time. Biochemical indicator, ATP, was employed to devise a method for preservation of metabolic pools of M. lepraemurium during storage. ATP assays were made by the luciferin-luciferase bioluminescent method. Several cryoprotective agents were compared at -76 and 4 degrees C. The essential steps have been found to be (i) for prolonged storage and constant supply of material, to freeze the intracellular bacteria within infected cells containing 28% proteins, i.e., to freeze the infected tissue; and (ii) when a large number of diverse experiments are to be undertaken on a single suspension, to stabilize working stocks of refrigerated cells for 12-16 weeks by using bovine serum albumin, fraction V, and Difco yeast supplement B to compensate for the leaching of intracellular cofactors, metabolites, nucleotides, etc.