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

Brézillon C, Haustant M, Dupke S, Corre JP, Lander A, Franz T, Monot M, Couture-Tosi E, Jouvion G, Leendertz FH, Grunow R, Mock ME, Klee SR, Goossens PL - PLoS Negl Trop Dis (2015)

Bottom Line: We explored the respective effect of the virulence factors on colonisation and dissemination of CA within its host by constructing bioluminescent mutants.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.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Pathogénie des Toxi-Infections Bactériennes, Paris, France.

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.

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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.
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pntd.0003455.g002: 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.

Mentions: We visualised the capsules produced by the CA and CI strains through Alcian blue staining. SDS-PAGE with low concentrations of acrylamide revealed two high molecular weight bands in bacterial extracts of these strains (Fig. 2A). The lower band was present in all PDGA-expressing strains, i.e. the PDGA-encapsulated B. anthracis Vollum strain and the CA and CI strains; it was absent in the strains that do not produce a PDGA capsule, i.e. the pBCXO1+ pBCXO2-cured CAR strain and the plasmid-free CAR-R strain. The upper band was present in the CA and CI strains, and in the CAR strain that expresses a HA capsule; it was absent from the CAR-H(ΔhasA) and CAR-R strains, and from the B. anthracis Vollum strain, that do not produce the HA capsule. Furthermore, hyaluronidase enzymatic digestion of the bacterial extracts led to the disappearance of the upper band for the CI, CA and CAR strains (Fig. 2A).


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

Brézillon C, Haustant M, Dupke S, Corre JP, Lander A, Franz T, Monot M, Couture-Tosi E, Jouvion G, Leendertz FH, Grunow R, Mock ME, Klee SR, Goossens PL - PLoS Negl Trop Dis (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4382292&req=5

pntd.0003455.g002: 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.
Mentions: We visualised the capsules produced by the CA and CI strains through Alcian blue staining. SDS-PAGE with low concentrations of acrylamide revealed two high molecular weight bands in bacterial extracts of these strains (Fig. 2A). The lower band was present in all PDGA-expressing strains, i.e. the PDGA-encapsulated B. anthracis Vollum strain and the CA and CI strains; it was absent in the strains that do not produce a PDGA capsule, i.e. the pBCXO1+ pBCXO2-cured CAR strain and the plasmid-free CAR-R strain. The upper band was present in the CA and CI strains, and in the CAR strain that expresses a HA capsule; it was absent from the CAR-H(ΔhasA) and CAR-R strains, and from the B. anthracis Vollum strain, that do not produce the HA capsule. Furthermore, hyaluronidase enzymatic digestion of the bacterial extracts led to the disappearance of the upper band for the CI, CA and CAR strains (Fig. 2A).

Bottom Line: We explored the respective effect of the virulence factors on colonisation and dissemination of CA within its host by constructing bioluminescent mutants.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.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Pathogénie des Toxi-Infections Bactériennes, Paris, France.

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.

Show MeSH
Related in: MedlinePlus