Long-term survival and virulence of Mycobacterium leprae in amoebal cysts.

William H Wheat,Ramanuj Lahiri,Diana L Williams,Gerald E Mcdonnell,Patrick J Brennan,Amy L Casali,John S Spencer,Mary Jackson,Vincent Thomas,Mercedes Gonzalez-Juarrero,Joseph M Vinetz

doi:10.1371/journal.pntd.0003405

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.

Highlights

Human beings have been afflicted by leprosy for over a millennium
Leprosy is a progressive disease of the skin and nervous system caused by the bacillus, Mycobacterium leprae
We demonstrate that M. leprae can survive long-term within cysts of common environmental freeliving amoebae

Summary

Introduction

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]. 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. Wild armadillos are infected with M. leprae.

Methods

Results

Discussion

Conclusion