Research Article: Long-term Survival and Virulence of Mycobacterium leprae in Amoebal Cysts

Date Published: December 18, 2014

Publisher: Public Library of Science

Author(s): William H. Wheat, Amy L. Casali, Vincent Thomas, John S. Spencer, Ramanuj Lahiri, Diana L. Williams, Gerald E. McDonnell, Mercedes Gonzalez-Juarrero, Patrick J. Brennan, Mary Jackson, Joseph M. Vinetz.

Abstract: Leprosy is a curable neglected disease of humans caused by Mycobacterium leprae that affects the skin and peripheral nerves and manifests clinically in various forms ranging from self-resolving, tuberculoid leprosy to lepromatous leprosy having significant pathology with ensuing disfiguration disability and social stigma. Despite the global success of multi-drug therapy (MDT), incidences of clinical leprosy have been observed in individuals with no apparent exposure to other cases, suggestive of possible non-human sources of the bacteria. In this study we show that common free-living amoebae (FLA) can phagocytose M. leprae, and allow the bacillus to remain viable for up to 8 months within amoebic cysts. Viable bacilli were extracted from separate encysted cocultures comprising three common Acanthamoeba spp.: A. lenticulata, A. castellanii, and A. polyphaga and two strains of Hartmannella vermiformis. Trophozoites of these common FLA take up M. leprae by phagocytosis. M. leprae from infected trophozoites induced to encyst for long-term storage of the bacilli emerged viable by assessment of membrane integrity. The majority (80%) of mice that were injected with bacilli extracted from 35 day cocultures of encysted/excysted A. castellanii and A. polyphaga showed lesion development that was similar to mice challenged with fresh M. leprae from passage mice albeit at a slower initial rate. Mice challenged with coculture-extracted bacilli showed evidence of acid-fast bacteria and positive PCR signal for M. leprae. These data support the conclusion that M. leprae can remain viable long-term in environmentally ubiquitous FLA and retain virulence as assessed in the nu/nu mouse model. Additionally, this work supports the idea that M. leprae might be sustained in the environment between hosts in FLA and such residence in FLA may provide a macrophage-like niche contributing to the higher-than-expected rate of leprosy transmission despite a significant decrease in human reservoirs due to MDT.

Partial Text: Human beings have been afflicted by leprosy for over a millennium. Leprosy is a chronic granulomatous infection of skin and peripheral nerves caused by the bacillus Mycobacterium leprae. The bacilli are slow growing obligate intracellular organisms trophic for macrophages, dendritic cells (DC) and Schwann cells in peripheral nerves. The scientific community has reached a generally accepted consensus that M. leprae is principally a parasite of humans and is spread primarily thereby [1]. In addition, there have been autochthonous cases of leprosy among native-born Americans in the southern region of the United States with no prior history of foreign exposure. In the same regions, wild armadillos are infected with M. leprae. A unique M. leprae genotype had been found in the majority of armadillos that was identical to U.S. patients who resided in areas where exposure to armadillo-born M. leprae was possible [2]. This is highly suggestive of the fact that armadillos are a significant natural reservoir for the bacilli and, leprosy might be a zoonosis in the these areas. There has also been a substantial history of studies, anecdotal evidence, rationalizations and opinions that argue in favor of additional non-human sources of the bacillus [3]. What is more intriguing is that, despite many years of using multidrug therapy (MDT) resulting in a significant reduction in disease prevalence, transmission remains stubbornly high implicating among other issues, ineffective detection of early infection, case reporting deficiencies or a lack of a thorough examination of potential environmental sources of the bacillus [3], [4].

The precise manner in which leprosy is transmitted is unknown. Until recently it was widely believed that the disease was transmitted by proximal contact between untreated or asymptomatic cases of leprosy and healthy people. Currently, the possibility of transmission by the respiratory aerosol route has gained considerable interest [39]. Other means such as transmission through insects [40][41] has been considered but there has not been any substantial evidence supporting that claim. The possibility of discharge of M. leprae from the nasal mucosa begs the question of how the discharged organism remains viable in between hosts. Since M. leprae is a fastidiously obligate intracellular bacterium, it would be reasonable to assume that it could find safe refuge in the environment by interaction with ubiquitous free-living organisms with physiological semblance to human phagocytes. Recently, it was shown that M. leprae could be taken up by FLA, survive and remain viable intracellularly in these protozoa for a period of at least 72 hr [42]. In the current study, we demonstrate that M. leprae can survive and remain virulent for at least 35 days within amoebal cysts from both A. castellanii and A. polyphaga as determined by their ability to transfer infection to recipient nu/nu mouse FPs. Furthermore, we show that acid-fast bacilli extracted from M. leprae/amoebae cocultures with A. lenticulata, A. castellanii, A. polyphaga, H. vermiformis str. ATCC 50237 and H. vermiformis str. 172 remain viable for over 8 months in encysted amoebae as determined by viability staining of bacilli in situ within cysts or from the those extracted from the cysts. These data provide a proof of concept that M. leprae can be phagocytized and lysosomally occupy common environmental FLA trophozoites, survive encystment while remaining viable and are fully capable of infectivity under suboptimal conditions endured by the amoebic cyst. Although M. leprae has been shown to be approximately 30% viable in terms of membrane integrity by BacLight after two weeks in optimized medium [36], survival in either amoebae medium described here is very detrimental to axenic M. leprae and necessitates refuge within amoebae. It can be reasonably argued that possible environmental reservoirs for this fastidious bacillus are common FLA.



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