Macrophage-Mediated Germination of Bacillus anthracis Endospores Requires the gerH Operon

Abstract

The gerHABC operon of Bacillus anthracis, encoding a gerA-like family member of germinant sensors, was shown to be required for endospore germination in the presence of macrophages and in macrophage-conditioned media. The loss of the germination phenotype in macrophage cultures of B. anthracis gerH-null endospores was restored by complementation in trans with a wild-type copy of gerH expressed under the control of its own promoter. Using endospores from both the parental strain B. anthracis Sterne and an isogenic gerH-null strain, we partially characterized germinants secreted by macrophages into the extracellular medium.Bacillus anthracis endospores can persist for long periods in soil but, upon entering a host, germinate quickly into vegetative bacilli that grow and cause disease (4). Endospores germinate inside of or proximal to phagocytes. These cells are believed to be de facto sites of germination and contribute to the establishment of B. anthracis infection, as phagocytes are the primary means of bacterial access to the body in cases of inhalation anthrax (6,8,10,20). Several recent studies examined the fate of B. anthracis endospores in macrophages (M). Guidi-Rontani et al. showed that murine alveolar phagocytes ingest endospores that then germinate and grow into vegetative bacilli but do not replicate (6). Welkos et al. observed intact endospores and recently germinated nascent bacilli within phagosomes in M that are subsequently destroyed by these immune cells (26,27,28). Work by Dixon et al. indicated that newly vegetative bacilli escape from the phagocytic vesicles of the M where they can replicate freely in the host cell cytoplasm and release themselves from the M (3). Though there are important differences among these researchers' observations of the ultimate fates of phagocytosed endospores, it is generally agreed that germinant sensors recognize and bind small-molecule germinants and initiate the germination cascade (17). These sensors belong to the GerA family of germinant receptors, which are best characterized in Bacillus subtilis but which have also been studied in other Bacillus spp. (2,5,15,16,18,19,29,30).Six tricistronic gerA-like chromosomal loci (gerAA, gerAB, gerAC) plus the gerX locus carried on pXO1 exist in B. anthracis; these loci encode germinant recognition proteins (6,10,11,25). The gerS operon in B. anthracis mediates germination in response to aromatic ring structures and is required for germination in the presence of cultured M (11, 12). The gerH operon also mediates germination in response to aromatic ring structures and germination triggered by inosine (Ino) in combination with amino acids (25). The germinants recognized by the gerX operon on pXO1 have not yet been identified, although ⌬Sterne strains lacking pXO1 do not exhibit any gross germination defects in vitro (11,12). Endospores from a gerXnull strain, identified by Guidi-Rontani et al., germinates slightly less well than do Sterne endospores in the presence of M (6, 12). One ger locus has homology to the gerL locus...

Bacillus anthracis spores and lethal toxin induce IL‐1β via functionally distinct signaling pathways

“…Therefore, we next asked whether BA spores, like LPS, could directly induce IL-1b mRNA and caspase-1 activity that processed pro-IL-1b gene product to the mature cytokine. To ensure that we were examining the initial interaction between macrophages and BA spores rather than vegetative bacilli, we used a congenic mutant of the Sterne 34F2 strain, DgerH, that germinates poorly within macrophages [14,16]. Thus, during macrophage cell culture there is limited expression of either LT or edema toxin (ET).…”

Role ofBacillus anthracis Spore Structures in Macrophage Cytokine Responses

“…Realtime PCR allowed an evaluation of cytokine mRNA 1 h after addition of spores to M. Second, we used the ⌬gerH mutant of Sterne 34F2, which germinates poorly in the presence of M (43,44). We measured levels of IL-1␤, TNF-␣, IFN-␤, RANTES, and IL-6 because of their importance in stimulating different components of the immune response, such as phagocytes (TNF-␣, IL-1␤), B cells, and antibody formation (IL-6).…”

“…First, we used the germination-proficient B. anthracis Sterne strain 34F2 (pXO1 ϩ pXO2 Ϫ ) and examined the spore M interaction at 1 h, prior to the spore germination and cytokine production, by employing quantitative PCR to study cytokine gene expression. Second, we measured cytokine protein responses by M by using the congenic germination-deficient ⌬gerH (gerH-null) strain spores (43,44). The tri-cistronic gerHABC operon encodes germinant sensors in the presence of M and M-conditioned media and is required for endospore germination within the M environment (43).…”

“…Second, we measured cytokine protein responses by M by using the congenic germination-deficient ⌬gerH (gerH-null) strain spores (43,44). The tri-cistronic gerHABC operon encodes germinant sensors in the presence of M and M-conditioned media and is required for endospore germination within the M environment (43). Since the germination-deficient (⌬gerH) mutant does not form vegetative bacilli in M cultures, it is a useful tool for dissecting the M response to the B. anthracis spore in the absence of germination and allows us to distinguish the response elicited to the spore form of B. anthracis that does not express toxins or capsule from that elicited to the vegetative form.…”

The Bacillus cereus GerN and GerT Protein Homologs Have Distinct Roles in Spore Germination and Outgrowth, Respectively

“…This did not reveal any GerT function but demonstrated that GerN is important in any circumstances where the GerI receptor is implicated in germination. In B. anthracis, the GerH germinant receptor has been implicated in virulence (26). Apart from sequence differences in the long repeat region near the N termini of GerHA and GerIA, the GerH proteins are almost identical in amino acid sequence to the GerI proteins of B. cereus ATCC 10876.…”