Early Events of Spore Germination and their Role in Bacillus anthracis Virulence.

Abstract

Bacillus anthracis exists in two forms, the spore and the bacillus. Entry of spores into the host triggers germination and outgrowth into virulent bacilli, causing anthrax. B. anthracis encodes for five distinct germinant receptors, GerH, GerK, GerL, GerS, and GerX, located in the inner membrane of the spore, which initiate germination upon detection of L-amino acid and purines. Using mutant strains lacking one or more of these receptors, I showed that the germinant receptors of B. anthracis are essential for nutrient-mediated germination and have partially redundant specificities. Additionally, the GerH receptor alone is sufficient to mediate a full germination response to inosine based germinants. Germinant receptor mutants were tested in a mouse model of infection, and showed that different routes of infection require different germinant receptors to initiate germination and disease. In an intratracheal infection, any one receptor was sufficient to cause disease, with the exception of GerX. The GerH receptor was necessary and sufficient for full virulence when spores were inoculated subcutaneously. In vivo imaging suggested that spores must germinate at the inoculation site in order to cause disease, suggesting GerH specific germinants at this site. GerH was also essential for germination in both mammalian blood and macrophages. The addition of chemically-defined germination inhibitors to serum or macrophages suggested that they contain both L-amino acids and inosine, explaining the need for the GerH receptor under these conditions. Germination requires that germinants reach their receptors, possibly mediated by the gerPABCDEF operon. gerP mutants exhibited a mild germination delay in nutrient germinants, which was alleviated by removal of the spore coat, suggesting a role in germinant entry. Calcium-dipicolinic acid (Ca-DPA) released from the spore’s core activates lytic enzymes to degrade the cortex, and can be added exogenously to stimulate germination. gerP mutants exhibited a severe defect in Ca-DPA germination. These data implicate the GerP proteins in the early stages of spore germination, possibly by facilitating the interaction between nutrient and non-nutrient germinants and their spore targets. Together these experiments give us a better understanding of the signals required to stimulate germination in a host.