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Identification of Bacillus anthracis Spore Component Antigens Conserved across Diverse Bacillus cereus sensu lato Strains

2009; Elsevier BV; Volume: 8; Issue: 6 Linguagem: Inglês

10.1074/mcp.m800403-mcp200

ISSN

1535-9484

Autores

Sanghamitra Mukhopadhyay, Arya Akmal, Andrew C. Stewart, Ru‐ching Hsia, Timothy D. Read,

Tópico(s)

Bacterial Genetics and Biotechnology

Resumo

We sought to identify proteins in the Bacillus anthracis spore, conserved in other strains of the closely related Bacillus cereus group, that elicit an immune response in mammals. Two high throughput approaches were used. First, an in silico screening identified 200 conserved putative B. anthracis spore components. A total of 192 of those candidate genes were expressed and purified in vitro, 75 of which reacted with the rabbit immune sera generated against B. anthracis spores. The second approach was to screen for cross-reacting antigens in the spore proteome of 10 diverse B. cereus group strains. Two-dimensional electrophoresis resolved more than 200 protein spots in each spore preparation. About 72% of the protein spots were found in all the strains. 18 of these conserved proteins reacted against anti-B. anthracis spore rabbit immune sera, two of which (alanine racemase, Dal-1 and the methionine transporter, MetN) overlapped the set of proteins identified using the in silico screen. A conserved repeat domain protein (Crd) was the most immunoreactive protein found broadly across B. cereus sensu lato strains. We have established an approach for finding conserved targets across a species using population genomics and proteomics. The results of these screens suggest the possibility of a multiepitope antigen for broad host range diagnostics or therapeutics against Bacillus spore infection. We sought to identify proteins in the Bacillus anthracis spore, conserved in other strains of the closely related Bacillus cereus group, that elicit an immune response in mammals. Two high throughput approaches were used. First, an in silico screening identified 200 conserved putative B. anthracis spore components. A total of 192 of those candidate genes were expressed and purified in vitro, 75 of which reacted with the rabbit immune sera generated against B. anthracis spores. The second approach was to screen for cross-reacting antigens in the spore proteome of 10 diverse B. cereus group strains. Two-dimensional electrophoresis resolved more than 200 protein spots in each spore preparation. About 72% of the protein spots were found in all the strains. 18 of these conserved proteins reacted against anti-B. anthracis spore rabbit immune sera, two of which (alanine racemase, Dal-1 and the methionine transporter, MetN) overlapped the set of proteins identified using the in silico screen. A conserved repeat domain protein (Crd) was the most immunoreactive protein found broadly across B. cereus sensu lato strains. We have established an approach for finding conserved targets across a species using population genomics and proteomics. The results of these screens suggest the possibility of a multiepitope antigen for broad host range diagnostics or therapeutics against Bacillus spore infection. The anthrax causing bacterium Bacillus anthracis is a member of the Bacillus cereus sensu lato (s.l.) 1The abbreviations used are:s.l.sensu lato (translation "in the broad sense")ATCCAmerican Type Culture CollectionBGSCBacillus Genetic Stock CollectionDSMZDeutsche Sammlung von Mikroorganismen und ZellkulturenISinterspace (distance between endospore and exosporium)NCBINational Center for Biotechnology InformationRAESSrabbit anti-exosporium seraRAGSSrabbit anti-GerH whole spore seraRANSrabbit anti-naïve seraRAWSSrabbit anti-whole spore (ungerminated) seraTAPTranscriptionally Active PCRTEMtransmission electron microscopybis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolBLASTbasic local alignment search toolRTSRapid Translational System2Dtwo-dimensionalABCATP-binding cassetteAMUatomic mass unitPAprotective antigen.1The abbreviations used are:s.l.sensu lato (translation "in the broad sense")ATCCAmerican Type Culture CollectionBGSCBacillus Genetic Stock CollectionDSMZDeutsche Sammlung von Mikroorganismen und ZellkulturenISinterspace (distance between endospore and exosporium)NCBINational Center for Biotechnology InformationRAESSrabbit anti-exosporium seraRAGSSrabbit anti-GerH whole spore seraRANSrabbit anti-naïve seraRAWSSrabbit anti-whole spore (ungerminated) seraTAPTranscriptionally Active PCRTEMtransmission electron microscopybis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolBLASTbasic local alignment search toolRTSRapid Translational System2Dtwo-dimensionalABCATP-binding cassetteAMUatomic mass unitPAprotective antigen. group, a term given to the polyphyletic species consisting of Bacillus thuringiensis, Bacillus cereus, Bacillus mycoides, Bacillus weihenstephanensis, and Bacillus pseudomycoides (1Helgason E. 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Two of these spore conserved antigens (Crd and MetN) have not been described previously for B. anthracis.DISCUSSIONIn this project we used two complementary approaches to select spore antigens that could produce antibodies cross-reactive against a broad range of B. cereus group spores. For the first method we used in silico filters to produce a list of 200 proteins by their conservation across five genomes. In the second approach we used a comparative proteomics analysis to detect cross-reacting spore antigens (Fig. 6). Both strategies have their drawbacks. Sequence-based analyses alone cannot predict the composition of the Bacillus spore especially considering the likely variability introduced by transcriptional and translational regulation of protein expression. However, any proteomics approach has inherent bias toward identification of abundant proteins. Combining both the approaches produced a relatively small list of potential conserved candidate antigens.The sera we used for challenging the proteins were taken from rabbits injected with B. anthracis exosporium (RAESS) and B. anthracis whole spore germination mutants (RAGSS) (44Weiner M.A. Read T.D. Hanna P.C. Identification and characterization of the gerH operon of Bacillus anthracis endospores: a differential role for purine nucleosides in germination.J. Bacteriol. 2003; 185: 1462-1464Crossref PubMed Scopus (59) Google Scholar). Because B. anthracis has multiple genetic loci dedicated to triggering germination (49Kanda-Nambu K. Yasuda Y. Tochikubo L. Isozymic nature of spore coat-associated alanine racemase of. Bacillus subtilis.Amino Acids. 2000; 18: 375-387Crossref PubMed Scopus (15) Google Scholar, 50Behravan J. Chirakkal H. Masson A. Moir A. Mutations in gerP locus of Bacillus subtilis Bacillus anthracis affect access of germinants to their targets in spores.J. 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Analysis of a novel spore antigen in Bacillus anthracis that contributes to spore opsonization.Microbiology. 2008; 154: 619-632Crossref PubMed Scopus (30) Google Scholar). In the case of the major exosporium component BclA (36Rode L.J. Lewis Jr., C.W. Foster J.W. Electron microscopy of spores of Bacillus megaterium with special reference to the effects of fixation and thin sectioning.J. Cell Biol. 1962; 13: 423-435Crossref PubMed Scopus (12) Google Scholar, 63Brahmbhatt T.N. Janes B.K. Stibitz E.S. Darnell S.C. Sanz P. Rasmussen S.B. O'Brien A.D. Bacillus anthracis exosporium protein BclA affects spore germination, interaction with extracellular matrix proteins, and hydrophobicity.Infect. Immun. 2007; 75: 5233-5239Crossref PubMed Scopus (78) Google Scholar), a lack of trypsin cleavage sites precludes identification.Four of the 18 cross-reacting proteins from the proteome-based screen (AcnA, Eag, DnaK, and FusA), were identified in B. anthracis spore lysates by Liu et al. (29Liu H. Bergman N.H. Thomason B. Shallom S. Hazen A. Crossno J. Rasko D.A. Ravel J. Read T.D. Peterson S.N. Yates J. Hanna P.C. Formation and composition of the Bacillus anthracis endospore.J. Bacteriol. 2004; 186: 164-178Crossref PubMed Scopus (171) Google Scholar). The remaining 18 proteins are not exclusively exosporium or endospore structural components but rather important core metabolic proteins presumably included to facilitate rapid growth upon germination. Translation elongation factors (Tuf and FusA) and chaperones (DnaK and GroEL) are examples of this class of spore proteins. Another class of the proteins listed in Table IV are those that are likely debris from the lysed mother cells adhered onto the surface of the spores. The Eag protein has been shown to be expressed only at the stationary phase of the vegetative cell (64Fouet A. Mesnage S. Bacillus anthracis cell envelope components.Curr. Top. Microbiol. Immunol. 2002; 271: 87-113PubMed Google Scholar). The RhsA protein probably also falls under the category of an incidental spore component. Another cross-reactive protein identified from this screen is the Crd protein. This large protein with 2,358 amino acids (molecular mass/pI, 244.9 kDa/4.17) contains 15 imperfect repeats of a 132-residue domain (PFAM (Protein Family database) accession number PF01345) that is found in three other B. anthracis proteins, GBAA1618 (molecular mass/pI, 523.3 kDa/4.06), GBAA3725 (molecular mass/pI, 219.8 kDa/4.2), and GBAA3721 (molecular mass/pI, 7.8 kDa/5.71). However, only Crd was detected in the protein gels. Genes encoding proteins with this motif are common in the B. cereus group but not found in the outgroup endospore-forming bacteria B. pseudomycoides DSMZ 12442 and B. subtilis. Proteins with multiple PF01345 domains appear in bacteria as diverse as Chlamydia trachomatis and the archaebacterium Methanobacterium. Crd lacks a classic type II signal sequence and LPXTG binding motif suggesting that it is neither cell wall- nor membrane-associated. The broad range of spores of B. cereus that cross-react to Crd suggests a promising candidate, and further investigation on the localization of this protein is necessary.Several other researchers have demonstrated the power of proteomics analysis in a high throughput mode, including combining genome-based bioinformatics approaches (29Liu H. Bergman N.H. Thomason B. Shallom S. Hazen A. Crossno J. Rasko D.A. Ravel J. Read T.D. Peterson S.N. Yates J. Hanna P.C. Formation and composition of the Bacillus anthracis endospore.J. Bacteriol. 2004; 186: 164-178Crossref PubMed Scopus (171) Google Scholar, 37DelVecchio V.G. Connolly J.P. Alefantis T.G. Walz A. Quan M.A. Patra G. Ashton J.M. Whittington J.T. Chafin R.D. Liang X. Grewal P. Khan A.S. Mujer C.V. Proteomic profiling and identification of immunodominant spore a

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