The secret life of the anthrax agent Bacillus anthracis: bacteriophage-mediated ecological adaptations

Abstract

Ecological and genetic factors that govern the occurrence and persistence of anthrax reservoirs in the environment are obscure. A central tenet, based on limited and often conflicting studies, has long held that growing or vegetative forms of Bacillus anthracis survive poorly outside the mammalian host and must sporulate to survive in the environment. Here, we present evidence of a more dynamic lifecycle, whereby interactions with bacterial viruses, or bacteriophages, elicit phenotypic alterations in B. anthracis and the emergence of infected derivatives, or lysogens, with dramatically altered survival capabilities. Using both laboratory and environmental B. anthracis strains, we show that lysogeny can block or promote sporulation depending on the phage, induce exopolysaccharide expression and biofilm formation, and enable the long-term colonization of both an artificial soil environment and the intestinal tract of the invertebrate redworm, Eisenia fetida. All of the B. anthracis lysogens existed in a pseudolysogenic-like state in both the soil and worm gut, shedding phages that could in turn infect non-lysogenic B. anthracis recipients and confer survival phenotypes in those environments. Finally, the mechanism behind several phenotypic changes was found to require phage-encoded bacterial sigma factors and the expression of at least one host-encoded protein predicted to be involved in the colonization of invertebrate intestines. The results here demonstrate that during its environmental phase, bacteriophages provide B. anthracis with alternatives to sporulation that involve the activation of soil-survival and endosymbiotic capabilities.

Figure 1. The B. anthracis lifecycle.

Solid arrows trace a lifestyle in which dormant spores (the infectious cell-type) are ingested by grazing herbivores and then germinate to produce a vegetative cell-type that causes fulminant disease. After host death, processes like terminal hemorrhage and scavenger action release up to 10⁹ vegetative bacilli per milliliter of blood into the environment , . While the fate of vegetative cells in the soil is unclear , , , , , the long-held model assumes that starvation and sporulation are the only option , . Dashed arrows highlight some alternatives to sporulation implied in studies showing that B. anthracis forms biofilms (a preferred environmental state for soil bacteria) and persists as a vegetative form in a model rhizosphere system . The term “Incubator area” describes the hypothesis of Van Ness that certain soil conditions may favor vegetative growth cycles prior to outbreaks. While it is unknown whether vegetative B. anthracis participates in DNA exchange in the soil, horizontal-gene-transfer is a driving force (in the face of selective pressure) for genetic variability and niche expansion in B. cereus and B. thuringiensis , , .

Figure 2. Transmission electron micrographs of bacteriophages negatively stained with 2% uranyl acetate.

The bacteriophages infecting B. anthracis include, (A) Wβ, (B) Wip1, (C) Wip2, (D) Wip4, (E) Wip5, (F) Frp1, (G) Frp2, (H) Htp1, and (I) Bcp1. An extract from the gut of the earthworm Eisenia fetida is shown (J) with two distinct and uncharacterized phages indicated by arrows. Scale bars represent either 25 nm (A–I) or 50 nm (J).

Figure 3. Certain lysogens of B. anthracis sporulate rapidly.

Total bacterial cells (vegetative cells and spores; dotted lines) and spores (solid lines) in liquid sporulation cultures were determined over time. Values are mean averages (n = 3) and error bars represent standard deviations. Strains include ΔSterne (open squares) and its lysogens obtained with either Wip1 (open diamonds in A, D, G, and J), Wip2 (open triangles in B, E, H, and K), Frp2 (open circles in C, F, I, and L) or Wip4 (cross-hatches in L). A Wip2-cured lysogen (star symbol in K) and a Wip2-cured lysogen re-infected with Wip2 (open circles in K) are included as well. Sporulating cultures were analyzed under the following conditions: (A–C) LD medium incubated at 30°C with agitation at 75 rpm; (D–F) LD medium incubated at 24°C with agitation at 75 rpm; (G–I) LD medium incubated at 24°C with no agitation; and (J–L) soil medium incubated at 24°C with agitation at 75 rpm.

Figure 4. Phenotypic analysis of B. anthracis and its lysogens.

(A) Biofilms formed at the liquid-air interface of 3 month BHI cultures grown without aeration at 24°C (scale bars are 0.5 cm). The total number of bacteria (vegetative cells and spores) and spores alone in each adherent biofilm were enumerated and visualized at 200X and 2000X magnification. GFP-PlyG^BD-labeled cells are shown in 2000X fluorescence images taken with 0.3 second exposures. A non-colorized image is shown for the GFP-PlyG^BD-labeled Wip1 lysogen. 2000X fluorescence images (0.3 second exposures) of mid-log phase BHI cultures labeled with GFP-PlyG^BD or WGA^AF are also shown. Relative fluorescence units (RFUs) corresponding to ∼1×10⁸ mid-log phase cells suspended in buffer are shown as averages of three experiments. (B) Lysogeny alters B. anthracis spore architecture. Transmission electron micrographs (TEM) show that the hallmark single-layer exosporium of ΔSterne actually consists of two distinct layers in >30% of Wip1 lysogen spores. Arrows show the surface nape structure for each exosporium. 45,000X magnifications are shown. Phase contrast images (2000X) show the Wip1 lysogen with an unusual surface structure indicated by an arrow. Closer inspection by scanning electron microscopy (SEM) reveals the surface structure to be an extended, grainy matrix (indicated by an arrow). Scale bars represent 1 µm.

Figure 5. Phage-encoded effectors of phenotypic change in B. anthracis.

(A) Tsp509I genomic fragments of Bcp1 (1,948 bp) and Wip4 (1,287 bp) identified in an expression library screen of phage-encoded loci that disrupt the Spo⁺ phenotype of ΔSterne. (B) GFP-PlyG^BD-labeled mid-log-phages cells are shown in 2000X fluorescence images with 0.3 second exposures. ΔSterne/pASD2 derivatives are used with and without the indicated promoter-bearing Bcp1 and Wip4 loci. RFUs corresponding to ∼1×10⁸ mid-log phase cells suspended in buffer are displayed as averages of three experiments. (C) Biofilms formed at liquid-air interfaces of 3 ml LD cultures grown without aeration in 35 mm dishes for 1 week. Average numbers of organisms per ml are shown from three experiments for each condition, indicating total viability (vegetative cells and spores) and spore counts. For each strain, 200X and 2000X images of GFP-PlyG^BD-labeled cells are shown with and without fluorescence emission.

Figure 6. Sporulation phenotypes of natural B. cereus s.l. lysogens.

Kinetic analyses of sporulation were performed in LD medium. All values are mean averages (n = 3) and error bars are standard deviations. (A) Sporulation of environmental B. cereus. Total viability (dashed lines) and spores alone (solid lines) are shown for growth conditions that include 37°C at 180 rpm (RS1045 and ATCC 25621), 30°C at 180 rpm (RS1255) or 24°C at 75 rpm (RS1557). B. anthracis ΔSterne (squares), the indicated environmental isolates (triangles), and phage-cured variants RS1255^CURED and RS1557^CURED (diamonds) are shown. (B) Sporulation for environmental B. anthracis strains. Cultures were grown at 37°C and aerated at 180 rpm. Strains include RS1615 or RS1046 (triangles) and RS1615^CURED or RS1046^CURED (diamonds). An electron micrograph of the inducible phage from RS1615 is shown (scale bar in 50 nm). (C) Sporulation for B. anthracis Sterne. Cultures were grown at 37°C and aerated at 180 rpm. Included are Sterne (circles), Sterne^CURED (diamonds), and the Wip1 or Wip4 lysogens of Sterne (closed circles). An electron micrograph shows the inducible Sterne phage (scale bar is 50 nm).

Figure 7. Exopolysaccharide and biofilm phenotypes of natural B. cereus s.l. lysogens.

(A) Mid-log phase cells from BHI cultures labeled with GFP-PlyG^BD. 2000X fluorescence images are 0.2 second exposures. For ΔSterne, a corresponding phase image is shown. RFUs for ∼1×10⁸ mid-log phase cells suspended in buffer are shown. (B) Biofilms formed by environmental B. cereus (RS1045) and B. anthracis (RS1615) backgrounds. For RS1045 strains, sections at the liquid-air interface of crystal violet-stained biofilms are shown. The RS1045 field is dominated by filamentous forms, while that of the phage-cured for is dominated by spores. Liquid-air interfaces are shown (along with total bacteria ml⁻¹ for each culture) for the RS1615 strains. Additionally, 200X images of GFP-PlyG^BD-stained biofilm (RS1615) or bottom-settled material (RS1615^CURED) are shown.

Figure 8. Impact of lysogeny on survival in soil and earthworms.

Survival is shown as colony forming units (CFUs) per gram of recovered soil or worm guts (solid lines). Shed phages (dashed lines) are shown as plaque-forming units (PFUs) extracted per gram of soil or worm guts. Total B. anthracis viability (vegetative cells and spores; squares), spore counts (diamonds), and phages (triangles) are shown. Values are mean averages (n = 5) and error bars are standard deviations. (A) ΔSterne/pASD2 (non-lysogen) and its Wip1 and Wip4 lysogens in soil. (B) ΔSterne/pASD2 (non-lysogen) and its Wip1 and Wip4 lysogens in earthworm intestines. (C) Survival of environmental B. anthracis strain RS1615 and its phage-cured derivative (RS1615^CURED) in soil and earthworm intestines. (D) Infection of B. anthracis during co-culture with lysogens or free Wip1 particles in soil or earthworm intestines. Strains ΔSterne/pASD2 and Sterne/pASD2 were either inoculated alone or with strains ΔSterne/Wip1 or RS1615 into each microcosm. At indicated times, ΔSterne/pASD2 and Sterne/pASD2 were recovered and scored by PCR for infection with Wip1 or φ1615. Survival of ΔSterne/pASD2 and Sterne/pASD2 inoculated alone (squares) and derivatives infected with Wip1 (closed circles) or φ1615 (open circles) are shown. For the spiking with Wip1, strain ΔSterne/pASD2 was inoculated alone or with 1×10⁸ Wip1 phages. Free Wip1 (triangles) is shown with total viability (squares) and spore counts (diamonds) for ΔSterne/pASD2 lysogenized with Wip1.

Figure 9. Phage-regulated loci in B. anthracis.

(A) Promoter-gfp fusions expressed on worm agar. Bacteria were plated for 16 hours at 30°C and resulting colonies are shown in 200X phase-contrast and fluorescence images (0.5 second exposures). Strains are ΔSterne or its Wip4 lysogen (“/Wip4”) transformed with pASD2 encoding gfp alone or indicated promoter fusions (P). (B) Expression of P^BA3443-gfp on BHI agar in either the ΔSterne/Wip4 background or ΔSterne co-transformed with the compatible vector pWH1520 (either without and insert or that bearing bcp25,26 or wip48,49). Bacteria were plated for 16 hours at 30°C and colonies are shown in 200X phase-contrast or fluorescence images with 0.5 second exposures. 2000X fluorescence images of representative bacteria are shown with exposure times. RFUs are averages of three experiments for ∼1×10⁸ mid-log phase cells grown in BHI and suspended in buffer. (C) BA3443 and earthworm colonization. One month after colonization, bacteria were recovered, visualized, and enumerated. Strains are ΔSterne/Wip4 transformed with indicated vectors or the ΔSterne/Wip4 mutant ΔBA3443. Hind-guts were examined by phase-contrast or fluorescence microscopy at 200X or 2000X magnification. Exposure times are indicated in 2000X images. Average CFUs per gram of gut is shown from three experiments, indicating total viability (vegetative cells and spores) and spores alone. (D) B. anthracis-encoded loci required in the soil. Bacteria were recovered and enumerated at the indicated times from inoculated soil microcosms. Strains include ΔSterne/Wip4 with pASD2 alone or the indicated mutations. Values are mean averages (n = 3) and error bars are standard deviations.

Figure 10. Alternate lifestyles for B. anthracis in the environment?

Solid arrows trace a lifestyle in which there are alternatives to starvation and sporulation driven by lysogeny. By infecting an otherwise non-lysogenic B. anthracis strain with several distinct phages, we have observed changes in sporulation, exopolysaccharide and biofilm formation, soil survival, and earthworm colonization. Such phenotypic changes may favor saprophytic or endosymbiotic lifestyles in the soil.