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. 2012 Nov;78(22):8075-81.
doi: 10.1128/AEM.02034-12. Epub 2012 Sep 14.

Germination and amplification of anthrax spores by soil-dwelling amoebas

Rafik Dey  1 , Paul S Hoffman, Ian J Glomski
Affiliations

Germination and amplification of anthrax spores by soil-dwelling amoebas

Rafik Dey et al. Appl Environ Microbiol. 2012 Nov.

Abstract

While anthrax is typically associated with bioterrorism, in many parts of the world the anthrax bacillus (Bacillus anthracis) is endemic in soils, where it causes sporadic disease in livestock. These soils are typically rich in organic matter and calcium that promote survival of resilient B. anthracis spores. Outbreaks of anthrax tend to occur in warm weather following rains that are believed to concentrate spores in low-lying areas where runoff collects. It has been concluded that elevated spore concentrations are not the result of vegetative growth as B. anthracis competes poorly against indigenous bacteria. Here, we test an alternative hypothesis in which amoebas, common in moist soils and pools of standing water, serve as amplifiers of B. anthracis spores by enabling germination and intracellular multiplication. Under simulated environmental conditions, we show that B. anthracis germinates and multiplies within Acanthamoeba castellanii. The growth kinetics of a fully virulent B. anthracis Ames strain (containing both the pX01 and pX02 virulence plasmids) and vaccine strain Sterne (containing only pX01) inoculated as spores in coculture with A. castellanii showed a nearly 50-fold increase in spore numbers after 72 h. In contrast, the plasmidless strain 9131 showed little growth, demonstrating that plasmid pX01 is essential for growth within A. castellanii. Electron and time-lapse fluorescence microscopy revealed that spores germinate within amoebal phagosomes, vegetative bacilli undergo multiplication, and, following demise of the amoebas, bacilli sporulate in the extracellular milieu. This analysis supports our hypothesis that amoebas contribute to the persistence and amplification of B. anthracis in natural environments.

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Figures

Fig 1
Fig 1
Intracellular germination and growth of B. anthracis in coculture with A. castellanii. (A) Micrograph (magnification, ×100) of A. castellanii infected with RFP-expressing Sterne 7702 spores (red) after 6 h at 37°C. rp, replicative phagosome containing vegetative B. anthracis; sp, spores; vf, vegetative form; n, nucleus. (B) Growth of B. anthracis Sterne 7702, TKO, and 9131 with or without (spores only) A. castellanii (Ac) at the indicated times at 37°C. Data are the means ± standard errors of the means (n = 4 to 6; performed in triplicate). Statistical differences were determined by Student's t test comparing the indicated strain to the Sterne strain cultured in the absence of amoebas at 72 h (**; P1 = 0.000285; P2 = 0.00105). (C) Phase-contrast micrograph (magnification, ×100) after 6 h of culture at 37°C of A. castellanii with fluorescently labeled strain 9131(red spores). p, phagosome containing B. anthracis spores; sp, spores; n, nucleus. (D) Similar to the experiment described for panel B but with B. anthracis Sterne 34F2 as the parental strain control for mutants with either GerX or GerH eliminated. Data are the means ± standard errors of the means (n = 4). Statistical differences were determined by a Student's t test (**, P < 0.001). (E) Similar to the experiment described for panel B but with the B. anthracis Ames strain with or without A. castellanii. Data are the means ± standard errors of the means (n = 4). Statistical differences were determined by a Student's t test (**, P = 0.000356).
Fig 2
Fig 2
Quantification of B. anthracis spores in coculture with A. castellanii. Spore quantification was determined by heating samples from the indicated times to 65°C for 20 min and then plating for CFU. Ac, A. castellanii. Open squares, Sterne spores only. Data are the means ± standard errors of the means (n = 4 to 6; performed in triplicate). Statistical differences were determined by a Student's t test (**, P < 0.001).
Fig 3
Fig 3
Effect of B. anthracis on amoeba viability. (A) Representative phase-contrast images (Axio Imager; 63× objective) of A. castellanii cultured with or without the indicated B. anthracis strains for 24 h at 37°C. Insets are ×10 enlargements of a subsection of the larger photograph (dashed box). (B) Representative phase-contrast image (Axio Imager; 63× objective) of B. anthracis filaments undergoing sporulation after 12 h of coculture with A. castellanii at 37°C. r, spores being released from the mother cell. (C) The number of viable A. castellanii amoebas cocultured without bacteria (amoeba only) or with Sterne 7702, TKO, or 9131 at the indicated times at 37°C was quantified using a hemacytometer and trypan blue staining.
Fig 4
Fig 4
Intracellular germination and growth of B. anthracis in coculture with A. castellanii at 30°C and in CWM+. (A) Growth of B. anthracis Sterne 7702, TKO, and 9131 with or without A. castellanii (Ac) (spores only) at the indicated times at 30°C in standard CWM. Data are the means ± standard errors of the means for experiments performed in triplicate. (B) Growth of B. anthracis Ames with or without A. castellanii (Ac) at the indicated times at 30°C in standard CWM. Data are the means ± standard errors of the means of experiments performed in triplicate. (C) Growth of B. anthracis Sterne 7702, TKO, and 9131 with or without A. castellanii (Ac) (spores only) at the indicated times at 30°C in CWM+. Data are the means ± standard errors of the means of experiments performed in triplicate. (D) Growth of B. anthracis Ames with or without (spores only) A. castellanii (Ac) at the indicated times at 30°C in CWM+. Data are the means ± standard errors of the means of experiments performed in triplicate.
Fig 5
Fig 5
Transmission electron microscopy analysis of B. anthracis-A. castellanii interactions. (A and B) Micrographs show spores of strain 9131 contained in A. castellanii phagosomes (open arrowheads) after 6 and 12 h of coculture at 37°C, respectively. (C) A vegetative Sterne spore within an A. castellanii trophozoite in a phagosome after 6 h of coculture. (D) Vegetative forms of Sterne inside and outside amoebas after 12 h of infection (black arrows). N, nucleus; vf, vegetative form.
Fig 6
Fig 6
Model for the B. anthracis amoebal life cycle illustrated with time-lapse phase-contrast micrographs. The following stages are shown: spore uptake within the first 2 h when cells were cocultured at 37°C (sp, spores) (frame i); germination and replication of B. anthracis with subsequent destruction of amoebas after ≈6 h (frame ii) (g, germinated spores); sporulation of B. anthracis with release of spores (r) from the mother cell after 12 h (frame iii).
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