The Mechanism of Bacillus anthracis Intracellular Germination Requires Multiple and Highly Diverse Genetic Loci

Abstract

In an effort to better understand the mechanisms by which Bacillus anthracis establishes disease, experiments were undertaken to identify the genes essential for intracellular germination. Eighteen diverse genetic loci were identified via an enrichment protocol using a transposon-mutated library of B. anthracis spores, which was screened for mutants delayed in intracellular germination. Fourteen transposon mutants were identified in genes not previously associated with B. anthracis germination and included disruption of factors involved in membrane transport, transcriptional regulation, and intracellular signaling. Four mutants contained transposon insertions in gerHA, gerHB, gerHC, and pagA, respectively, each of which has been previously associated with germination or survival of B. anthracis within macrophages. Strain MIGD101 (named for macrophage intracellular germination defective 101) was of particular interest, since this mutant contained a transposon insertion in an intergenic region between BAs2807 and BAs2808, and was the most highly represented mutant in the enrichment. Analysis of B. anthracis MIGD101 by confocal microscopy and differential heat sensitivity following macrophage infection revealed ungerminated spores within the cell. Moreover, B. anthracis MIGD101 was attenuated in cell killing relative to the parent strain. Further experimental analysis found that B. anthracis MIGD101 was defective in five known B. anthracis germination pathways, supporting a mechanism wherein the intergenic region between BAs2807 and BAs2808 has a global affect on germination of this pathogen. Collectively, these findings provide insight into the mechanisms supporting B. anthracis germination within host cells.Bacillus anthracis spores germinate during early stages of inhalational anthrax, and this is considered to be a critical step in the progression of anthrax disease (4, 5, 7). Indeed, transition from dormant spore to vegetative bacilli is essential for growth, bacteremia, toxin production, and synthesis of a poly-D-glutamic acid capsule, each of which is important to the virulence of B. anthracis.Results from a series of studies indicate B. anthracis spores are engaged by, and can germinate within, alveolar macrophages (13, 15). Sanz et al. recently described the detection of B. anthracis spores in alveolar macrophages under in vivo infection conditions using a mouse model of inhalational anthrax (15). The molecular basis of spore tropism for alveolar macrophages may be due to the collagenlike exosporium protein, BclA, which interacts with the macrophage receptor, Mac-1 (13), while at the same time preventing the interaction of spores with other types of cells. Spores are taken up by Mac-1-dependent phagocytosis into LAMP-1 positive vesicles, where germination is triggered, and the germinated spores become susceptible to killing by the macrophage (10).Very little is known about the bacterial factors that promote, repress, or otherwise regulate germination of B. anthracis spores within macrophages. To date, expe...

Anthrax SET Protein

“…Spores were then diluted 1:200 in germination buffer (10 mM Tris-HCl/ NaCl, 50 mM L-alanine, and 1 mM inosine). At each time point (2,5,10,15,20, and 30 min), 100-l aliquots were removed and incubated at 65°C for 20 min to kill any germinated spores, and then 50-l aliquots were plated on BHI agar and incubated overnight at 30°C. Additionally, a 50-l aliquot at the zero time point was plated without heat treatment to get an initial titter for germinated spores.…”

“…Like all Bacillus species, B. anthracis is a Gram-positive spore-forming organism found in soil environments (12,13). Unlike most other Bacillus species, respiratory, gastrointestinal, or cutaneous entry of B. anthracis spores into mammals can result in a rapid systemic infection and death (14,15). A growing number of studies indicate that B. anthracis uses its toxins to down-regu-late the host innate response.…”

Multigenic Control and Sex Bias in Host Susceptibility to Spore-Induced Pulmonary Anthrax in Mice

“…However, these attenuated strains can cause serious infections in mice. While there is increasing information on the pathogen virulence factors in B. anthracis (3,7,12,34,71), the mechanisms of host defense and pathogenesis are poorly understood for anthrax.Although anthrax primarily affects herbivores, virtually all mammals are susceptible to various degrees. Anthrax susceptibility depends on host species (31), strain (69), and the route of infection (35,46).…”

“…However, these attenuated strains can cause serious infections in mice. While there is increasing information on the pathogen virulence factors in B. anthracis (3,7,12,34,71), the mechanisms of host defense and pathogenesis are poorly understood for anthrax.…”

Testing Nucleoside Analogues as Inhibitors of Bacillus anthracis Spore Germination In Vitro and in Macrophage Cell Culture

“…The first step in the germination process is most commonly the binding of metabolites by germination (Ger) receptors (8,23,38). These receptors are membrane proteins mostly encoded by tricistronic operons.…”