Capsules, toxins and AtxA as virulence factors of emerging Bacillus cereus biovar anthracis

Abstract

Emerging B. cereus strains that cause anthrax-like disease have been isolated in Cameroon (CA strain) and Côte d'Ivoire (CI strain). These strains are unusual, because their genomic characterisation shows that they belong to the B. cereus species, although they harbour two plasmids, pBCXO1 and pBCXO2, that are highly similar to the pXO1 and pXO2 plasmids of B. anthracis that encode the toxins and the polyglutamate capsule respectively. The virulence factors implicated in the pathogenicity of these B. cereus bv anthracis strains remain to be characterised. We tested their virulence by cutaneous and intranasal delivery in mice and guinea pigs; they were as virulent as wild-type B. anthracis. Unlike as described for pXO2-cured B. anthracis, the CA strain cured of the pBCXO2 plasmid was still highly virulent, showing the existence of other virulence factors. Indeed, these strains concomitantly expressed a hyaluronic acid (HA) capsule and the B. anthracis polyglutamate (PDGA) capsule. The HA capsule was encoded by the hasACB operon on pBCXO1, and its expression was regulated by the global transcription regulator AtxA, which controls anthrax toxins and PDGA capsule in B. anthracis. Thus, the HA and PDGA capsules and toxins were co-regulated by AtxA. We explored the respective effect of the virulence factors on colonisation and dissemination of CA within its host by constructing bioluminescent mutants. Expression of the HA capsule by itself led to local multiplication and, during intranasal infection, to local dissemination to the adjacent brain tissue. Co-expression of either toxins or PDGA capsule with HA capsule enabled systemic dissemination, thus providing a clear evolutionary advantage. Protection against infection by B. cereus bv anthracis required the same vaccination formulation as that used against B. anthracis. Thus, these strains, at the frontier between B. anthracis and B. cereus, provide insight into how the monomorphic B. anthracis may have emerged.

Fig 1. The B. cereus bv anthracis CA strain expresses a PDGA and a HA capsule, and toxins.

(A) Capsule expression in the CAP(ΔpagA), the CAR and the CAR-H(ΔhasA) strains in inducing conditions; the polyglutamate (PDGA) and hyaluronic acid (HA) capsule was visualised by immunofluorescence with a polyclonal anti-PDGA immune serum or by India ink staining; degradation of the HA capsule was achieved by incubation with hyaluronidase as described in the Materials and Methods section. (B) The production of toxin components PA and LF in overnight bacterial culture supernatants was determined by western blot with or without CO₂/bicarbonate as described in the Materials and Methods section.

Fig 2. Coexpression of a PDGA and a HA capsule by the B. cereus bv anthracis strains.

(A) Alcian Blue staining was performed on filtrates of colony lysates from various strains grown in CO₂/bicarbonate conditions: these were the Vollum strain (wild-type B. anthracis), the B. cereus bv anthracis CI and CA strains, and the CA-derived strains devoid of pBCXO2 (CAR) and further deleted in the hasA gene (CAR-H) or having lost pBCXO1 (CAR-R); hyaluronidase treatment was performed before PAGE, as described in the Materials and Methods section. (B) mRNA of the hasA gene (involved in synthesis of the HA capsule) and the capB gene (involved in synthesis of the PDGA capsule) was assessed in the strains described in (A) grown under CO₂/bicarbonate (CO₂) or aerobic (O₂) culture conditions as described in the Materials and Methods section; gyrB gene expression was used as reference.

Fig 3. Ultrastructural analysis of the capsules of the B. cereus bv anthracis CA strain and its derivatives.

Bacterial cells from the PDGA and HA capsule-expressing CA strain and its derivatives expressing a HA capsule (CAR), a PDGA capsule (CA-H(ΔhasA)) or no capsule (CAR-H(ΔhasA)) were prepared for Transmission Electron Microscopy as described in the Materials and Methods section. Scale bar: 500nm

Fig 4. Expression of the HA capsule is regulated by AtxA.

Complementation of the CAR20 strain with the pDACatxA plasmid restores capsule and toxin expression after IPTG induction as observed by (A) India ink staining, (B) Alcian Blue staining of the bacterial culture supernatants, and (C) PA and LF toxin component production as described in Figs. 1 and 2 and in the Materials and Methods section. Hyaluronidase treatment confirms the HA nature of the capsule (A,B).

Fig 5. In vivo dissemination of the B. cereus bv anthracis CA strain during cutaneous infection in mice.

Mice were inoculated into the ear pinna with spores of (A) CARP-lux (9 mice, inoculum 1 x 10⁷; all mice survived), (B) CAR-lux (8 mice, inoculum 1 x 10⁵; all mice died), or (C) CAP-lux (11 mice, inoculum 1 x 10⁵; all mice died) strains and bioluminescence was analysed at the indicated times after infection (D: days). These image series show a representative dorsal and ventral view of the same mouse for the various strains. Black and white photographs are overlaid with false-colour representation of luminescence intensity expressed in photons s -1 cm² sr -1.

Fig 6. Local role of the HA capsule and in vivo dissemination of the B. cereus bv anthracis CA strain during intranasal infection in mice.

Mice were inoculated intranasally with spores of (A) CARP-lux (13 mice, inoculum 1 x 10⁸), (B) CAR-lux (16 mice, inoculum 1 x 10⁶; all mice died), or (C) CAP-lux (14 mice, inoculum 1 x 10⁶; all mice died) strains and bioluminescence was analysed at the indicated times after infection as in Fig. 5 (D: days). (Ab-e) Histological characterisation of the infected brain tissue shown in Aa, bottom panel; (Ab) Diffuse inflammatory lesion centred on leptomeninges (LM), multifocally extending to the brain parenchyma (star); (Ac) high density of bacteria in the leptomeninges and Virchow-Robin spaces; (Ad) inflammatory infiltrates consisting of neutrophils, haemorrhages and oedema provoking a marked distension of leptomeninges and (Ae), at higher magnification, extending to the cerebral parenchyma with the presence of bacteria in the neuropil (arrowhead) highly suggestive of a rupture of the blood-brain barrier. (Ab & d): HE staining; (Ac & e): Gram staining.

Fig 7. FIS+PA vaccine provides protection against subcutaneous challenge with the CA and CI strains in mice.

Mice were immunised subcutaneously with rPA (10μg) and formaldehyde inactivated spores (FIS; 1 x 10⁸) at day 0 and 15, and were challenged with spores of the B. cereus bv anthracis CA (460 spores), CI (1150 spores), CAR (400 spores) or the B. anthracis 9602 (480 spores) strain as described in Materials and Methods. Survival was followed over a 20-day period. Results are representative of at least two independent experiments that gave similar results (six mice per group). Statistical significance was calculated by the log-rank test with GraphPad Prism software (p < 0.001; comparisons were made between the group immunised with PA+FIS versus that immunised with Al₂O₃ for each strain).