The roles of AtxA orthologs in virulence of anthrax‐like Bacillus cereus G9241

Abstract

AtxA is a critical transcriptional regulator of plasmid-encoded virulence genes in Bacillus anthracis. Bacillus cereus G9241, which caused an anthrax-like infection, has two virulence plasmids, pBCXO1 and pBC210, that each harbor toxin genes and a capsule locus. G9241 also produces two orthologs of AtxA: AtxA1, encoded on pBCXO1, and AtxA2, encoded on pBC210. The amino acid sequence of AtxA1 is identical to that of AtxA from B. anthracis, while the sequences of AtxA1 and AtxA2 are 79% identical and 91% similar to one another. We found by qRT-PCR that AtxA1 and AtxA2 function as positive regulators of toxin (AtxA1) and capsule operon (both) transcription in G9241 and that a ΔatxA1 mutant produced lower levels of the anthrax toxins and no hyaluronic acid capsule. Deletion of atxA1 or atxA2 decreased the virulence of spores administered intranasally or subcutaneously to C57BL/6 mice but not to A/J mice, and deletion of both genes rendered spores avirulent in A/J mice. In addition, unlike AtxA1, AtxA2 did not form stable homomultimers in vitro, although AtxA1 and AtxA2 formed heterodimers. Our data show that AtxA1 is the primary regulator of G9241 virulence factor expression and that AtxA1 and AtxA2 are both required for full virulence.

Assembly and Function of the Bacillus anthracis S-Layer

“…cereus exo- p oly s accharide), which requires the expression of the bpsXABCDEFGH genes located on the pBC210 virulence plasmid (104) (Figure 1). B. cereus G9241 and B. cereus biovar anthracis CI express atxA and carry an inactivating mutation in their chromosomal plcR gene that is distinct from that of B. anthracis (6, 16, 125). pBC210 carries an atxA -like gene ( atxA2 ) that contributes to the pathogenesis of anthrax-like disease in B. cereus G9241 (125).…”

“…B. cereus G9241 and B. cereus biovar anthracis CI express atxA and carry an inactivating mutation in their chromosomal plcR gene that is distinct from that of B. anthracis (6, 16, 125). pBC210 carries an atxA -like gene ( atxA2 ) that contributes to the pathogenesis of anthrax-like disease in B. cereus G9241 (125). pBC210 also carries pag2 (60% identity with pag ) and lef 2 (36% identity with lef ) (Figure 1); the lef 2 product shows the presence of a putative PA binding domain; however, the metalloprotease domain of LF is replaced with an ADP-ribosyltransferase domain (41, 143).…”

Certhrax Is an Antivirulence Factor for the Anthrax-Like Organism Bacillus cereus Strain G9241

“…G9241 spores were prepared as described by Scarff et al (35). Briefly, G9241 was grown overnight in 50 ml of brain heart infusion (BHI) broth (BD Biosciences) at 37°C with shaking at 225 rpm.…”

“…Construction of B. cereus mutant strains and recombinant plasmids. B. cereus deletion mutations were generated through homologous recombination with a temperature-sensitive plasmid as described by Scarff et al (35). Plasmid pJMS1 was derived from pLM4 (19).…”

“…S3 and S4 in the supplemental material) with digoxigenin (DIG)-labeled gene-specific probes as per the manufacturer's guidelines (Roche, Pleasanton, CA). Furthermore, each strain was characterized as follows (data not shown): expression of PA, LF, EF, and PA2 was confirmed by Western blot analyses with specific antibodies, growth rates in comparison to that of wild-type G9241 were measured in LB broth, expression of both capsules was visualized through Maneval staining with and without hyaluronidase treatment (35), and in vitro germination of spores was assessed in the presence of L-alanine and inosine (18). Only mutant strains that showed no defects in any of these additional phenotypic assays were utilized in this study.…”