Mycobacterium leprae: Morphology, Importance and Growth Factors PDF

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Name: Abhisek Manikonda ID#: 0523735 1. Mycobacterium leprae, belonging to the genus Mycobacterium is a pathogenic bacterium. 2. In the phylum Actinobacteria, the genus Mycobacterium belongs to the order Actinomycetales, and has its own family Mycobacteriaceae (Stackebrandt et al. 1997). The genus M...

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Name: Abhisek Manikonda

ID#: 0523735

1. Mycobacterium leprae, belonging to the genus Mycobacterium is a pathogenic bacterium. 2. In the phylum Actinobacteria, the genus Mycobacterium belongs to the order Actinomycetales, and has its own family Mycobacteriaceae (Stackebrandt et al. 1997). The genus Mycobacterium contains a number of strict and opportunistic pathogens that afflict humans and animals alike. Among the strict pathogens, the principal pathogens of humans include Mycobacterium tuberculosis, the causative agent of tuberculosis, and Mycobacterium leprae, which causes leprosy. In contrast to other pathogens of this genus, M. leprae is unique because of its uncultivatable nature on artificial culture media. a. Morphometry M. leprae is a non-motile, a non-spore forming, a microaerophilic, a rod shaped acid fast bacillus(Parkash & Singh 2012). The cell wall contains arabinose, galactose, and mesodiaminopimelic; the guanine and cytosine (GC) deoxyribonucleic acid (DNA) base ratios is 58% (Rastogi et al. 2001). The cell size (width) of M. leprae was measured to be 0.38 µm(Takade et al. 2003). The cytoplasm was characterized by the presence of widely distributed, clearly identifiable ribosome particles. The DNA fibers were widely distributed in the cytoplasm similar to the ribosome particles; the cell had various sizes of round electron-dense granules of polyphosphate(Toda et al. 1957). Paul & Beveridge (1992) showed how the M. leprae species possessed a multilayer cell envelope which basically consisted of, from inner to outer layer, a plasma membrane (PM), a peptidoglycan layer (PG), an electron-translucent layer (ETL) and an irregular electron-dense outer layer (OL). The width of the PG was measured to be approximately 4-5 nm. The architectures of the cell division septa was similar to the M. tuberculosis cell, PG layer tangentially intruded into the cytoplasm and formed a septal wall together with the PM(Takade et al. 2003). A morphological model by McNeil & Brennan (1991) showed a three layered structure for the cell wall. The layers include a PM, a thin PG layer, an ETL consisting of mycolic acid and glycolipids, and an irregular electron-dense OL from inside to outside. A key feature of M. leprae is its ability to survive and grow inside macrophages. A number of mainly cell surface-associated factors have been recognized as virulence factors of mycobacteria(Frehel & Rastogi 1987). Phenolic glycolipids may scavenge and thus protect the bacterium from toxic forms of oxygen, while lipoarabinomannan is a potent down regulator of several cellular immune functions, such as suppression of T cell proliferation and the prevention of gamma-interferon-triggered macrophage activation(Adams et al. 1993). Most of the studies on the genetic composition of distinct M. leprae isolates have failed to yield evidence for the existence of different strains of M. leprae. Some differences have, however, been discovered in growth rates and in the sequences of the elongation factor genes. Fsihi & Cole (1995) reported variability associated with the polA locus (encoding DNA

polymerase I) and preliminary evidence suggests that this may provide a basis for distinguishing M. leprae strains. b. Metabolism Pathogenic mycobacteria have complex lipoidal cell walls. Most of them secrete further lipids which appear as a layer around intracellular organisms. This lipoidal exterior may protect mycobacteria inside macrophages from attempts that those host cells make to kill them. Such protection could be especially important in M. leprae which unusually lacks catalase, an important ‘self-defence’ enzyme. Intracellular mycobacteria must obtain key nutrients from the host. Wheeler & Ratledge (1988) discussed the role of mycobactin and exochelin in acquiring iron, the carbon and nitrogen sources, including metabolic intermediates. M. leprae depends on the host for purines (precursors of nucleic acids), and maybe other intermediates. Pathogenic mycobacteria grow slowly, and the possibilities that permeability of the envelope to nutrients, catabolic or anabolic (particularly DNA, RNA synthesis) reactions are limiting to growth are considered. c. Nutrition Throughout the living world, iron is contained in the active centres of most redox enzymes. Because iron occurs in the insoluble Fe3+ form under oxic conditions (10−9 M Fe3+ in soil and water) (Ratledge & Dover 2000), proteins and siderophores with high binding affinity are required to make Fe3+biologically available. M. leprae produces salicylate-containing siderophores named mycobactins. The more polar form (carboxymycobactin) is released into the medium, whereas the less polar form (mycobactin) remains cell-associated (Niederweis 2008). Upon binding by siderophores, Fe3+is transported into the bacterium and released from the siderophore, possibly by reduction. In most bacteria, Fe3+–siderophore complexes bind to specific receptor proteins on the cell surface and are actively transported into the cytoplasm by specialized proteins that belong to the family of ABC transporters(Braun & Killmann 1999). The ABC transporter IrtAB is required byM. leprae to replicate in iron-deficient medium and to use Fe3+–carboxymycobactin as an iron source, indicating that IrtAB is involved in the transport of Fe3+ carboxymycobactin(Rodriguez & Smith 2006). Deletion of the irtAB genes also reduced the ability of M. leprae to survive in macrophages. d. Environmental growth factors Franzblau & Harris (1988) studied the metabolic response of M. leprae to biophysical parameters. Quantitation of intracellular ATP and the rate of [U-14C]palmitic acid(PA) incorporation into phenolic glycolipid I (PGL-I) were used as metabolic indicators under various biophysical conditions(Ramasesh et al. 1987). The rate of incorporation of [14C]PA into PGL-1 was clearly temperature dependent. Cellular ATP content at 5 days postincubation was relatively insensitive to temperatures of =37°C showed significantly lower ATP levels(Khanolkar et al. 1981). Whereas the pH optimum of 5.6 (Franzblau & Harris 1988) for PGL-I synthesis appears to be somewhat lower than that for ATP maintenance, the cessation of the former was observed

at pH 5.0 to 5.5 after approximately 1 week, whereas at slightly higher pH values, PGL-I synthesis, although slower, continued in a linear fashion for up to 2 weeks. Ishaque et al. (1977) reported a pH optimum of 5.8 for respiration in M. leprae. Viable M. leprae may indeed prevent phagosome-lysosome fusion in nonactivated macrophages(Sibley et al. 1987). The marked difference in long-term ATP maintenance between pHs 5.1 and 5.6 suggests that the organisms are sensitive to compartment acidification. Descriptions of an intact tricarboxylic acid cycle(Wheeler 1984), acytochrome system(Ishaque et al. 1977) , and oxidation of glucose, glycerol, and succinate to carbon dioxide, with oxygen serving as the terminal electron acceptor in M. leprae(P. R. Wheeler & Ratledge 1988), are all indicative of a requirement for molecular oxygen. This is consistent with the observations of Kvach et al. (1986) on temporal ATP synthesis and longer-term decay rates. M. leprae reportedly possesses superoxide dismutase but not catalase(Wheeler & Gregory 1980), which suggests microaerophily. Franzblau & Harris (1988) indicate that oxygen rapidly becomes a limiting factor in ATP maintenance and further suggest that an actual (maintained) oxygen concentration of approximately 5% is optimal. e. Transmission It is assumed that M. leprae is not very pathogenic and that most infections do not result in symptoms. Early symptoms of leprosy can be self-limiting and skin lesions can heal spontaneously(Fine 1982). Individuals who suffer from the disease, particularly those with multibacillary (MB) leprosy, are sources for spread of the infection. The most important port of entry and exit of M. leprae is the respiratory system, particularly the nose; its dissemination through skin lesions seems to be less important. There is increasing evidence from nasal polymerase chain reaction (PCR) studies of temporary carriage or even subclinical infection(Visschedijk et al. 2000; Beers et al. 1996) and that infected persons may go through a transient period of nasal excretion, indicating that the mycobacterium is highly infective(Hatta et al. 1995). Apart from being a portal of exit, the entry of the bacteria is also likely to take place through the nose and this entry may be facilitated by small injuries in the nasal mucosa. It might be worthwhile to further explore the role and condition of nasal mucosa in the transmission of and the susceptibility to leprosy. Impairment of the nasal function may enhance the development of disease as is shown in other infectious diseases. The integrity of the nasal mucosa can be influenced by climatic conditions, for example, low humidity can make people infected with meningococci more prone to systemic disease, probably because meningococci can penetrate more easily through desiccated mucosa. The nasal mucosa can be damaged by concomitant respiratory infections, for example bacterial pneumonia can establish after a viral infection. Also, it has been shown that viral respiratory infections can convert non-disseminating nasal carriers of staphylococci to highly infectious carriers. Silva et al. (2013) suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans and shed light on the potentially critical adhesions involved in M. leprae-epithelial cell interaction. Several glycolytic and other cystosolic enzymes have been shown to localize on the M. leprae surface and mediate the pathogen colonization of

host cells and facilitate the establishment of infection(Chhatwal 2002). Among the many identified adhesin candidates on the surface of M. leprae, Soares de Lima et al. (2005) have shown the in vivo expression of the two proteins, Hlp and HBHA by M. leprae. The skin has also been raised as a possible port of exit and entry of leprosy bacteria, most likely due to the prominent role it plays in disease manifestation. Untreated lepromatous patients may shed large numbers of bacteria from their ulcers or otherwise injured skin. However, only accidental inoculations with M. leprae favour the skin being a possible port of entry. A number of organism, host and environmental-related factors may be incriminated in the dynamic process of the development of leprosy disease, but still many characteristics of the epidemiology of leprosy remain to be deciphered. This is a prerequisite for effective control of the disease. Continued recording of the descriptive aspects of the epidemiology of leprosy is thus much needed; the additional use of modem molecular and immunological tools will also be important. 3. Adams, L.B., Fukutomi, Y. & Krahenbuhl, J.L., 1993. Regulation of murine macrophage effector functions by lipoarabinomannan from mycobacterial strains with different degrees of virulence. Infect. Immun., 61(10), pp.4173–4181. Beers, S.M., Wit, M.Y.L. & Klatser, P.R., 1996. The epidemiology of mycobacterium leprae : Recent insight. FEMS Microbiology Letters, 136(3), pp.221–230. Braun, V. & Killmann, H., 1999. Bacterial solutions to the iron-supply problem. Trends in Biochemical Sciences, 24(3), pp.104–109. Chhatwal, G.S., 2002. Anchorless adhesins and invasins of Gram-positive bacteria: a new class of virulence factors. Trends in microbiology, 10(5), pp.205–8. Fine, P.E., 1982. Leprosy: the epidemiology of a slow bacterium. Epidemiologic reviews, 4, pp.161–88. Franzblau, S. & Harris, E., 1988. Biophysical optima for metabolism of Mycobacterium leprae. Journal of clinical microbiology, 26(6). Frehel, C. & Rastogi, N., 1987. Mycobacterium leprae surface components intervene in the early phagosome-lysosome fusion inhibition event. Infect. Immun., 55(12), pp.2916–2921. Fsihi, H. & Cole, S.T., 1995. The Mycobacterium leprae genome: systematic sequence analysis identifies key catabolic enzymes, ATP-dependent transport systems and a novel polA locus associated with genomic variability. Molecular Microbiology, 16(5), pp.909–919.

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