Conserved Mechanisms of Mycobacterium marinum Pathogenesis within the Environmental Amoeba Acanthamoeba castellanii

Abstract

bMycobacterium marinum is a waterborne mycobacterial pathogen. Due to their common niche, protozoa likely represent natural hosts for M. marinum. We demonstrate that the ESX-1 secretion system is required for M. marinum pathogenesis and that M. marinum utilizes actin-based motility in amoebae. Therefore, at least two virulence pathways used by M. marinum in macrophages are conserved during M. marinum infection of amoebae. Mycobacterial pathogens are responsible for some of the leading causes of death by infectious disease. The majority of these deaths are caused by mycobacterial species within the Mycobacterium tuberculosis complex (MTC) (3). However, mycobacterial species in the environment, including atypical or nontuberculous mycobacteria (NTM), pose an emerging disease threat (19). Little is understood about the basic molecular virulence mechanisms employed by environmental mycobacterial pathogens.Mycobacterium marinum is a waterborne pathogen that causes a tuberculosis (TB)-like infection in ectotherms and is an occasional opportunistic human pathogen (21). M. marinum is related to M. tuberculosis and is used to model aspects of MTC pathogenesis (4,7,21,23,27). Free-living amoebae (FLA), including Acanthamoeba castellanii, are professional phagocytes (12). Pathogenic bacteria, including M. marinum and other NTM, have been recovered from samples of water colonized by free-living amoebae (8,26). Several mycobacterial species are established amoebaresistant bacteria (ARB) and resist destruction by FLA (1,6,12,17,18). It has been posited that protozoa serve as a reservoir for mycobacteria in the environment (17).It is probable that M. marinum naturally interacts with protozoa, including A. castellanii, that share an environmental niche. It was demonstrated that M. marinum are pathogenic to A. castellanii (6, 17, 20, 28). The molecular mechanisms underlying this interaction have not been well established.We hypothesized that virulence mechanisms required for infection of macrophages by M. marinum would also be required for pathogenesis of A. castellanii. The ESX-1 protein secretion system is required by mycobacteria and other Gram-positive pathogens to cause disease (2,11,13,16,24). Amoebae were infected (multiplicity of infection [MOI] of 1) with either the wild-type (WT) strain or an attenuated RD1 deletion (⌬RD1) strain of M. marinum, which bears a deletion in components and substrates of the ESX-1 system ( Fig. 1A) (27). M. marinum replicated roughly three logs in A. castellanii over the 72-h experiment, with an average generation time of 7 h. The ⌬RD1 strain replicated approximately two logs in A. castellanii over the 72-h experiment, with an average generation time of 12 h. Following an early rapid growth phase for both strains, the bacteria were maintained at relatively constant levels throughout the experiment. However, the ⌬RD1 strain failed to reach the levels of growth achieved by the wild-type strain (Fig. 1A). Significant differences in growth between the WT and ⌬RD1 strains were observed at 48 (P Ͻ...

Taxonomy, Physiology, and Natural Products of Actinobacteria

“…In their protozoan hosts, "hidden" mycobacteria might find easier opportunities to infect vertebrate end hosts, multiplying within protozoans to escape immune reactions (480). This ability to persist within amoebae has been widely documented (481)(482)(483). The internalization of infectious agents inside other parasites represents an evolutionary strategy for survival that may sometimes enhance pathogenesis or transmissibility (480).…”

A Novel ESX-1 Locus Reveals that Surface-Associated ESX-1 Substrates Mediate Virulence in Mycobacterium marinum

“…marinum infection of amoebae and macrophage-like cells. Acanthamoeba castellanii amoebae were cultured and maintained at room temperature in PYG-712 medium as previously described (40). M. marinum strains were cultured in vitro as described above.…”

“…In M. marinum the extended RD1 locus (MMAR_5439 through MMAR_5459, based on homology to the genes in M. tuberculosis) is the only genomic region reported to be required for ESX-1 export (18,32,34,(36)(37)(38)(39). Transposon insertions within or targeted deletion of individual genes in the extended RD1 locus prevents the export of ESX-1 substrates, including EsxA (ESAT-6) and EsxB (CFP-10) (EsxA/B) in vitro and attenuates M. marinum in macrophage, amoeba, and zebrafish models of infection (32,40,41).In general, genetic manipulations to the ESX-1 system which result in loss of substrate secretion into the culture supernatant in vitro lead to loss of virulence in infection models (initially described in references 3 and 5). Recent work from Chen et al reported variants of EspA, an ESX-1 substrate in M. tuberculosis, which led to strains that were deficient for ESX-1 secretion as measured by Western blot analysis of culture filtrate proteins (42).…”

“…In M. marinum the extended RD1 locus (MMAR_5439 through MMAR_5459, based on homology to the genes in M. tuberculosis) is the only genomic region reported to be required for ESX-1 export (18,32,34,(36)(37)(38)(39). Transposon insertions within or targeted deletion of individual genes in the extended RD1 locus prevents the export of ESX-1 substrates, including EsxA (ESAT-6) and EsxB (CFP-10) (EsxA/B) in vitro and attenuates M. marinum in macrophage, amoeba, and zebrafish models of infection (32,40,41).…”

DisconnectingIn VitroESX-1 Secretion from Mycobacterial Virulence

“…The ESX-1 secretion system causes cytotoxicity in cell-based ex vivo models of infection, including macrophages and amoebae (12,27,30). Following lysis of the phagosomal membrane, the bacteria induce cytotoxicity, which leads to cell death.…”