Invasion of human microvascular endothelial cells by Mycobacterium leprae through Mce1A protein.

In patients with lepromatous leprosy, Mycobacterium leprae is often observed inside the human microvascular endothelial cells (HMVEC) surrounding Schwann cells (SC) at the site of lesions in the peripheral nerves. Based on this observation, it is considered that the nasal mucous may be the invasion pathway for M. leprae and HMVEC serve as an important reservoir for the bacteria before they invade SC. In light of previous research which revealed that Mce1A protein mediates bacterial invasion into nasal epithelial cells and HMVEC, we conducted a study to determine whether the invasion of M. leprae into HMVEC can be suppressed by blocking the Mce1A protein. In this study, we analyzed bacterial invasive activity by adding recombinant Escherichia coli, which express the active region (InvX:72 a.a.) of Mce1A protein on their external membrane, into cultured HMVEC, using the adhesin involved in the diffuse adherence mechanism. The number of bacteria that invaded into the cells was then measured by a colony counting method. The active region of Mce1A was divided into four sections, and hyperimmune antisera was prepared for each section for analyzing the inhibitory effect against invasion. The invasive activity was suppressed by antibodies against InvX regions 1-24 a.a., 25-46 a.a. and 58-72 a.a. This suggests that the InvX regions 1-24 a.a., 25-46 a.a. and 58-72 a.a. of Mce1A protein play an important role in the invasion of M. leprae into HMVEC and that it may be possible to suppress entry of M. leprae in HMVEC with antibodies against these regions.

The unique tropism of Mycobacterium leprae to the nasal epithelial cells can be explained by the mammalian cell entry protein 1A.

Leprosy is a chronic infection where the skin and peripheral nervous system is invaded by Mycobacterium leprae. The infection mechanism remains unknown in part because culture methods have not been established yet for M. leprae. Mce1A protein (442 aa) is coded by mce1A (1326 bp) of M. leprae. The Mce1A homolog in Mycobacterium tuberculosis is known to be associated with M. tuberculosis epithelial cell entry, and survival and multiplication within macrophages. Studies using recombinant proteins have indicated that Mce1A of M. leprae is also associated with epithelial cell entry. This study is aimed at identifying particular sequences within Mce1A associated with M. leprae epithelial cell entry. Recombinant proteins having N-terminus and C-terminus truncations of the Mce1A region of M. leprae were created in Escherichia coli. Entry activity of latex beads, coated with these truncated proteins (r-lep37 kDa and r-lep27 kDa), into HeLa cells was observed by electron microscopy. The entry activity was preserved even when 315 bp (105 aa) and 922 bp (308 aa) was truncated from the N-terminus and C-terminus, respectively. This 316-921 bp region was divided into three sub-regions: 316-531 bp (InvX), 532-753 bp (InvY), and 754-921 bp (InvZ). Each sub-region was cloned into an AIDA vector and expressed on the surface of E. coli. Entry of these E. coli into monolayer-cultured HeLa and RPMI2650 cells was observed by electron microscopy. Only E. coli harboring the InvX sub-region exhibited cell entry. InvX was further divided into 4 domains, InvXa-InvXd, containing sequences 1-24 aa, 25-46 aa, 47-57 aa, and 58-72 aa, respectively. Recombinant E. coli, expressing each of InvXa-InvXd on the surface, were treated with antibodies against these domains, then added to monolayer cultured RPMI cells. The effectiveness of these antibodies in preventing cell entry was studied by colony counting. Entry activity was suppressed by antibodies against InvXa, InvXb, and InvXd. This suggests that these three InvX domains of Mce1A are important for M. leprae invasion into nasal epithelial cells.

[History for animal model of Hansen's disease and characteristics of leprosy in hypertensive nude rat].

The long search of an animal model for leprosy were carried out as many researchers since the Mycobacterium leprae discovery by Dr. Hansen in 1874. The remarkable results were left after the development of the foot-pad method by Dr. Shepard in 1960. The introduction of the T-R mouse and athymic (nude) mouse for leprosy research, alsospontaneity examples of Hansen's disease was reported to armadillo, chimpanzee and mangabay monkey, and it was confirmed that Hansen's disease was the zoonosis. Although, We have established a congenic hypertensive nude rat, SHR/NCrj-rnu (SHR.F344-Foxn(rnu)), carrying nude (rnu) and hypertension genes. SHR/NCrj-rnu rats obtained showed high susceptibility to M. leprae and showed a characteristic disease with a progressive pattern of leproma formation. Also this hypertensive nude rat strain produce high level of IL-10. Therefore, congenic hypertensive nude rat may be useful for an animal model to leprosy.

Recombinant Mycobacterium leprae protein associated with entry into mammalian cells of respiratory and skin components.

BACKGROUND: The transmission of Mycobacterium leprae, the causative pathogen of leprosy, has been postulated to occur mainly through upper respiratory route rather than skin-to-skin contact via minor injuries. The M. leprae genome contains mce1A gene, which encodes a putative mammalian cell entry protein. However, to date, there have been no functional analyses of the M. leprae mce1A gene product. OBJECTIVE: The aim of this study was to elucidate a possible relationship between this transmission mechanism and the mce1A gene product. METHODS: We analyzed the cell uptake activity in vitro of polystyrene latex beads coated with a purified recombinant (r-) protein expressed by a 849-bp locus within the mce1A gene. RESULTS: The r-protein promoted uptake of the beads into human nasal epithelial cells derived from nasal polyps, human bronchial epithelial cell line, normal human dermal fibroblasts, normal human microvascular endothelial cells and normal human keratinocytes cultured at 0.01 mM extracellular calcium concentration [Ca]; no uptake occurred with keratinocytes cultured at 1.2mM [Ca]. CONCLUSION: These results suggest that the mce1A gene product can mediate M. leprae entry into respiratory epithelial cells as their natural target cells, which may be the main mode of transmission. Endothelial cells, on the other hand, may serve as the reservoir of the bacilli for long-term infection. The M. leprae Mce1A protein has potential important implications for mode of transmission and pathogenesis of leprosy.

Cytokine gene expression in the foot pad and spleen of BALB/cAJcI mice infected with M. leprae

The cytokine mRNAs expressed in the foot pads and spleens of BALB/cAJcl mice infected with Mycobacterium leprae were studied by the reverse transcriptase-polymerase chain reaction (RT-PCR) method using cytokine-specific primers for interleukin-1 alpha (IL-1 alpha), -2, -4, -6, -10, -12-(p40), gamma interferon (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and TNF-beta, and then for CD4 and CD8 markers. The pattern of cytokine gene expression in the foot pad which supports M. leprae growth was different from the expression in the spleen which does not permit M. leprae multiplication in mice. Before BALB/cAjcl mice were infected with M. leprae, IL-1 alpha and TNF-beta mRNAs were expressed physiologically in the foot pad while all of the cytokine genes examined were expressed in the spleen. In the foot pads of mice inoculated with M. leprae, in addition to the physiological appearance of IL-1 alpha and TNF-beta mRNAs, these signals were intensified. TNF-alpha expression was induced by the infection. On the other hand, in the spleens of mice inoculated with M. leprae, CD4 mRNA expression disappeared on day 1 of the infection, which was accompanied by the reduced expression of IL-2, -4, -6, and -12 mRNAs. The recovery of CD4 mRNA expression at a latter stage was accompanied by a corresponding increase of the cytokine mRNA expression. It was suspected that these results might permit restricted growth of M. leprae in the foot pads of normal mice. Furthermore, our study suggests that tissue-specific, local, immunologic characteristics are important in M. leprae growth.