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

In an effort to better understand the mechanisms by whichBacillus 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 ofB. anthracis spores, which was screened for mutants delayed in intracellular germination. Fourteen transposon mutants were identified in genes not previously associated withB. anthracis germination and included disruption of factors involved in membrane transport, transcriptional regulation, and intracellular signaling. Four mutants contained transposon insertions ingerHA, gerHB, gerHC , andpagA , respectively, each of which has been previously associated with germination or survival ofB. 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 ofB. 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 thatB. anthracis MIGD101 was defective in five knownB. 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 supportingB. anthracis germination within host cells.