Anti-protective antigen antibody was reported to enhance macrophage killing of ingested Bacillus anthracis spores, but it was unclear whether the antibody-mediated macrophage killing mechanism was directed against the spore itself or the vegetative form emerging from the ingested and germinating spore. To address this question, we compared the killing of germination-proficient (gp) and germination-deficient (⌬gerH) Sterne 34F2 strain spores by murine peritoneal macrophages. While macrophages similarly ingested both spores, only gp Sterne was killed at 5 h (0.37 log kill). Pretreatment of macrophages with gamma interferon (IFN-␥) or opsonization with immunoglobulin G (IgG) isolated from a subject immunized with an anthrax vaccine enhanced the killing of Sterne to 0.49 and 0.73 log, respectively, but the combination of IFN-␥ and IgG was no better than either treatment alone. Under no condition was there killing of ⌬gerH spores. To examine the ability of the exosporium to protect spores from macrophages, we compared the macrophage-mediated killing of nonsonicated (exosporium ؉ ) and sonicated (exosporium ؊ ) Sterne 34F2 spores. More sonicated spores than nonsonicated spores were killed at 5 h (0.98 versus 0.37 log kill, respectively). Pretreatment with IFN-␥ increased the sonicated spore killing to 1.39 log. However, the opsonization with IgG was no better than no treatment or pretreatment with IFN-␥. We conclude that macrophages appear unable to kill the spore form of B. anthracis and that the exosporium may play a role in the protection of spores from macrophages.
We identified a tri-cistronic operon, gerH, in Bacillus anthracis that is important for endospore germination triggered by two distinct germination response pathways termed inosine-His and purine-Ala. Together, the two pathways allow B. anthracis endospores a broader recognition of purines and amino acids that may be important for host-mediated germination.
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...
Enterotoxigenic Escherichia coli (ETEC) is recognized to be a common cause of acute watery diarrhea in children from developing countries. Colonization factors (CFAs) have been identified predominantly in ETEC isolates secreting heat-stable enterotoxin (ST) or cosecreting ST with a heat-labile toxin (LT). We hypothesized that LT-only-secreting ETEC produces unique colonization factors not previously described in ST and LTSTsecreting ETEC. A set of degenerate primers based on nucleotide sequence similarities between the major structural genes of CS20 (csnA), CS18 (fotA), CS12 (cswA), and porcine antigen 987 (fasA) was developed and used to screen a collection of 266 LT-secreting ETEC isolates in which no known CFA was detected. PCRamplified products of different molecular masses were obtained from 49 (18.4%) isolates. Nucleotide sequence analysis of the PCR amplicons followed by GenBank nucleotide BLASTn analysis revealed five novel DNA sequences; translated amino acid BLASTx analysis confirmed sequence similarity to class 1b major structural proteins encoded by csnA, fotA, and fasA. Strains expressing the novel CFAs were phylotyped and analyzed using multilocus sequence typing (MLST; Achtman scheme), and the types detected were compared to those of a collection of archived global E. coli strains. In conclusion, application of the degenerate primer sets to ETEC isolates from surveillance studies increased the total number of ETEC isolates with detectable CFAs by almost 20%. Additionally, MLST analysis suggests that for many CFAs, there may be a requirement for certain genetic backgrounds to acquire and maintain plasmids carrying genes encoding CFAs.
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