Reverse vaccinology and subtractive genomics approaches for identifying common therapeutics against Mycobacterium leprae and Mycobacterium lepromatosis

Mycobacterium leprae and Mycobacterium lepromatosis are gram-positive bacterial pathogens and the causative agents of leprosy in humans across the world. The elimination of leprosy cannot be achieved by multidrug therapy alone, and highlights the need for new tools and drugs to prevent the emergence of new resistant strains. Methods In this study, our contribution includes the prediction of vaccine targets and new putative drugs against leprosy, using reverse vaccinology and subtractive genomics. Six strains of Mycobacterium leprae and Mycobacterium lepromatosis (4 and 2 strains, respectively) were used for comparison taking Mycobacterium leprae strain TN as the reference genome. Briefly, we used a combined reverse vaccinology and subtractive genomics approach. Results As a result, we identified 12 common putative antigenic proteins as vaccine targets and three common drug targets against Mycobacterium leprae and Mycobacterium lepromatosis. Furthermore, the docking analysis using 28 natural compounds with three drug targets was done. Conclusions The bis-naphthoquinone compound Diospyrin (CID 308140) obtained from indigenous plant Diospyros spp. showed the most favored binding affinity against predicted drug targets, which can be a candidate therapeutic target in the future against leprosy.(AU)

Induction and treatment of anergy in murine leprosy.

Leprosy is a disease consisting of a spectrum of clinical, bacteriological, histopathological and immunological manifestations. Tuberculoid leprosy is frequently recognized as the benign polar form of the disease, while lepromatous leprosy is regarded as the malignant form. The different forms of leprosy depend on the genetic and immunological characteristics of the patient and on the characteristics of the leprosy bacillus. The malignant manifestations of lepromatous leprosy result from the mycobacterial-specific anergy that develops in this form of the disease. Using murine leprosy as a model of anergy in this study, we first induced the development of anergy to Mycobacterium lepraemurium (MLM) in mice and then attempted to reverse it by the administration of dialysable leucocyte extracts (DLE) prepared from healthy (HLT), BCG-inoculated and MLM-inoculated mice. Mice inoculated with either MLM or BCG developed a robust cell-mediated immune response (CMI) that was temporary in the MLM-inoculated group and long-lasting in the BCG-inoculated group. DLE were prepared from the spleens of MLM- and BCG-inoculated mice at the peak of CMI. Independent MLM intradermally-inoculated groups were treated every other day with HLT-DLE, BCG-DLE or MLM-DLE, and the effect was documented for 98 days. DLE administered at a dose of 1.0 U (1 × 10(6) splenocytes) did not affect the evolution of leprosy, while DLE given at a dose of 0.1 U showed beneficial effects regardless of the DLE source. The dose but not the specificity of DLE was the determining factor for reversing anergy.

The use of the microplate alamar blue assay (MABA) to assess the susceptibility of Mycobacterium lepraemurium to anti-leprosy and other drugs.

Although murine leprosy is no longer a common illness, our understanding of the biology of this disease is incomplete. One particular example of this concerns the etiologic agent Mycobacterium lepraemurium (MLM). MLM is a fastidious microorganism that is difficult to grow in axenic media; in a way, this has hampered attempts to thoroughly study its physiological and metabolic characteristics. MLM is an obligate intracellular bacillus that invades macrophages and replicates profusely with a generation time that oscillates between 0.5 and 11 days. In the present study, we have successfully maintained MLM alive for more than 12 days in vitro, providing us with an opportunity to study its susceptibility to several anti-leprosy agents and other drugs. To achieve this, we used a fluorescence reduction assay of alamar blue (a resazurin) in a microplate format (microplate-alamar-blue-assay; MABA), which is a highly sensitive, practical, and inexpensive method for assaying cell viability. We found that MLM was highly susceptible to clofazimine and rifampicin and was less susceptible to streptomycin, thiacetazone, kanamycin, dapsone, and ethionamide, in that order. MLM was not susceptible to four plant triterpenoids (oleanolic acid, neolignan-c, sitosterol, and ursolic acid) for which bactericidal activity has been reported in M. tuberculosis. Because the MABA has high sensitivity, it can be used to monitor the activity of microorganisms that are difficult to cultivate (such as M. lepraemurium), in response to various drugs, thus offering a method to complement the study of murine leprosy, about which many questions remain unanswered.

La dos especies de micobacterias causantes de la lepra y su emergencia como enfermedad humana / No disponible

Mycobactyerium leprae y Mycobacterium lepromatosis son dos especies de bacterias causantes de la lepra cuya divergencia ocurrió hace varios millones de años. El genoma de M. leprae contiene más de mil pseudogenes que reflejan un fenómeno de inactivación génica masiva probablemente asociado a un cambio en el estilo de vida del ancestro de estas bacterias. El análisis de unos pocos pseudogenes presentes en ambas bacterias indica que este fenómeno tuvo lugar antes de la divergencia de las dos especies. Con el objeto de reconciliar la divergencia antigua entre estas dos especies y la reciente colonización de los continentes por los seres humanos se plantea un posible escenario evolutivo en el que una micobacteria, ancestro de ambas especies, se asoció como patógeno a una especie animal desconocida. En el nuevo ambiente muchos genes se volvieron dispensables lo que permitió su inactivación. Durante la coevolución entre hospedador y patógeno se originaron las dos especies de bacterias de la lepra M. leprae saltó a los seres humanos en África oriental emergiendo como nuevo patógeno humano, mientras M. lepromatosis saltó a los seres humanos en América antes de la llegada de europeos y africanos a partir del siglo XV (AU)

Mycobacterium leprae and Mycobacterium lepromatosis, which are the causative agents of leprosy, are two bacterial species that diverged several million years ago. The genome of M. leprae contains more than 1,000 pesudogenes that reflect a mass gene inactivation event, probably associated with a change in the lifestyle of the ancestor of these bacteria. The analysis of a few shared pseudogenes showed that the divergence of both species took place prior to this inactivation event. With the aim of reconciling the old divergence of these species to the recent colonization of the continents by the human beings, a putative evolutionary scenario is proposed in which a mycobacterial ancestor of both species became associated as a pathogen to an unknown animal species. In the new environment many genes became disposables, leading to their inactivation. During the co-evolution of host and pathogen lineages, the two species, causative agents of leprosy, were originated. M. leprae emerged as a new human pathogen in East Africa, while M. lepromatosis emerged as a new pathogen in America before the arrival of Europeans and Africans in the 15th century (AU)

Contrary to BCG, MLM fails to induce the production of TNF alpha and NO by macrophages.

Pathogenic mycobacteria must possess efficient survival mechanisms to resist the harsh conditions of the intraphagosomal milieu. In this sense, Mycobacterium lepraemurium (MLM) is one of the most evolved intracellular parasites of murine macrophages; this microorganism has developed a series of properties that allows it not only to resist, but also to multiply within the inhospitable environment of the phagolysosome. Inside the macrophages, MLM appears surrounded by a thick lipid-envelope that protects the microorganism from the digestive effect of the phagosomal hydrolases and the acid pH. MLM produces a disease in which the loss of specific cell-mediated immunity ensues, thus preventing activation of macrophages. In vitro, and possibly also in vivo, MLM infects macrophages without triggering the oxidative (respiratory burst) response of these cells, thus preventing the production of the toxic reactive oxygen intermediaries (ROI). Supporting the idea that MLM is within the most evolved pathogenic microorganisms, in the present study we found, that contrary to BCG, M. lepraemurium infects macrophages without stimulating these cells to produce meaningful levels of tumor necrosis factor alpha (TNF alpha) or nitric oxide (NO). Thus, the ability of the microorganisms to stimulate in their cellular hosts, the production of ROI and RNI (reactive nitrogen intermediates), seems to be an inverse correlate of their pathogenicity; the lesser their ability, the greater their pathogenicity.

Propagation of Mycobacterium lepraemurium on supplemented minimal medium and its experimental pathogenesis.

The splenic tissue of a mouse experimentally infected with M. lepraemurium (Hawaiian strain, M-65) and developing 'rat leprosy', yielded a pure culture of an acid - fast bacterium having all the characteristics of M. lepraemurium on mineral salt minimal medium supplemented with simple sources of C and N, e.g., NH4 -salts, liquid paraffin, urea, gelatin etc. This could be maintained, by serial passages in vitro with good growth. Its indefinite propagation with tissue - free washed, small inoculum on complex media including Ogawa medium was difficult, and its serial sub-culture was practically impossible. The in vitro isolate from supplemented minimal medium could produce pathological lesions in mice typical of rat leprosy.

Complemento, daño renal y lepra murina / Complement, renal damage and murine leprosy

A pesar de que la lepra de los ratones es una enfermedad sistémica, el tejido renal de los animales infectados rara vez es invadido por el Mycobacterium lepraemurium. Nuestros resultados indicaron que los animales afectados con el Mycobacterium lepraemurium desarrollaron un defecto en sus niveles de complemento hemolítico que es proporcional al grado de infección. El defecto afecta principalmente la vía clásica de activación del complemento y menor marcadamente la vía alterna. La inactivación de la actividad de complemento, mientras transcurre la infección, disminuye la posibilidad del daño medido por completo y prolonga la vida del huésped murino; ésta es, sin duda, una eficiente estrategia del microorganismo para prolongar también su supervivencia

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.

Impairment of virulence of in vitro subcultures of Mycobacterium lepraemurium.

Mycobacterium lepraemurium was cultivated in vitro on Ogawa egg-yolk medium. The pathogenicity of the third and eighth subcultures for C3H and C57BL mice was compared with that of in vivo grown murine bacilli by evaluating the mean survival time of infected mice. The results strongly suggest that a significant drop of virulence occurs during the in vitro cultivation of M. lepraemurium.

Growth of Mycobacterium leprae in nude mice.

Athymic nude mice were introduced in our laboratories in 1982. In this paper results over one year period of nude mice inoculated with small numbers of M. leprae are described. In this study we showed that 1 X 10(4) M. leprae with low numbers of viable bacilli inoculated into hind foot pads of nude mice housed both in vinyl plastic isolators and "clean room" conditions had the ability to grow and reach remarkable levels. There was dissemination of the infection to other uninoculated foot pads by six months.

The pathogenicity of Mycobacterium avium and related mycobacteria for experimental animals.

The pathogenicity of 40 strains of Mycobacterium avium, M. paratuberculosis, M. intracellulare and M. lepraemurium was investigated in chickens, rabbits, guinea-pigs, mice and calves. Mycobactin dependence and serological type were also determined. There was no evidence that mycobactin dependence was related to pathogenicity. Antigenic similarities were demonstrated between M. avium and M. paratuberculosis, and one isolate had the pathogenic characteristics of both species.