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. 2012 Mar;78(6):2049-52.
doi: 10.1128/AEM.06965-11. Epub 2012 Jan 13.

Conserved Mechanisms of Mycobacterium Marinum Pathogenesis Within the Environmental Amoeba Acanthamoeba Castellanii

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Free PMC article

Conserved Mechanisms of Mycobacterium Marinum Pathogenesis Within the Environmental Amoeba Acanthamoeba Castellanii

George M Kennedy et al. Appl Environ Microbiol. .
Free PMC article

Abstract

Mycobacterium 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.

Figures

Fig 1
Fig 1
M. marinum pathogenesis within A. castellanii requires the ESX-1 secretion system. (A) M. marinum replicates and persists within A. castellanii, while the M. marinum strain lacking ESX-1 is attenuated. Amoebae were infected (MOI of 1) and treated with gentamicin (150 μg/ml). Amoebae were lysed and bacteria were plated for CFU at the indicated times. Error bars represent standard deviations. Asterisks represent statistically significant differences based on an unpaired tailed Student t test (P < 0.05) (48 hpi, P = 0.035; 72 hpi, P = 0.002). (B) Infection by M. marinum results in a stable percentage of infected amoebae, while the population of infected amoebae decreased over time in the absence of the ESX-1 system. A. castellanii was infected with M. marinum and ΔRD1 M. marinum expressing DsRed at an MOI of 10. Monolayers were imaged using a 20× objective on a Zeiss AxioObserver microscope. The percentage of infected amoebae was established after counting >100 cells in each of five different fields at the indicated time points (12 h, P = 0.005; 24 h, P = 0.010; 48 h, P = 6.547 × 10−5). (C) Infection of A. castellanii results in a relatively constant number of amoebae over time, while infection by M. marinum lacking ESX-1 leads to amoeba growth. At each time point following infection, the amoebae were counted and the number was normalized to the initial number of amoebae plated. See Fig. S3 in the supplemental material for amoeba counts. (D) Infection with M. marinum results in amoeba lysis in an ESX-1-dependent manner. MOI of 5, P = 0.009; MOI of 10, P = 0.001. The control is uninfected amoebae after 72 h of incubation.
Fig 2
Fig 2
Wild-type M. marinum forms actin tails in A. castellanii. Florescence microscopy demonstrating actin tail formation by M. marinum at 22 hpi, MOI of 5. M. marinum is expressing DsRed, and Alexa 488 phalloidin is green. Four representative images are shown. The scale bar is 10 μm. Images were acquired with an Evolution QEi charge-coupled device (CCD) (Media Cybernetics) on a Nikon Eclipse TE300 microscope (60× objective) using IPLab software (Scanalytics). Examples of bacteria bearing tails are indicated with filled arrows. Tails are indicated with asterisks. Examples of bacteria without tails are indicated with open arrows.

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