can you explain why Bacillus anthracis can be pathogenic in a mouse and not be fought off by the immune system?I need help finding the answer in the article and explain in short answer link to article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC106848/ VOL. 180, 199894674330-20-1 2 3 4 5 6 7 8FIG. 6. In vivo expression of the two B. anthracis S-layer components. Im-munoblotting of pellet fractions (odd-numbered lanes) and supernatant fractions(even-numbered lanes) of S-layers from wild-type (CAF10) (lanes 1 and 2), Asap(CBA91) (lanes 3 and 4), Aeag (CSM91) (lanes 5 and 6), and Aeag Asap (CSM11)(lanes 7 and 8) strains was carried out with pooled sera from mice infected withthe CAF10 strain. Molecular masses are indicated in kilodaltons on the left.shown). EA1 and Sap were found to be major surface antigens,showing that both EA1 and Sap can be synthesized by a cap-sulated strain in vivo.DISCUSSIONAlthough various bacteria from natural environments pos-sess both a capsule and an S-layer, their cosynthesis or struc-tural relationship has rarely been studied (15).B. anthracis has a rather unusual capsule: it is composed notof polysaccharide but of poly-y-D-glutamic acid (13). This bac-terium also has an S-layer, previously evidenced only in cap-sule-free strains (10, 16). Another gram-positive bacterium, B.licheniformis, apparently shares these features, namely, a poly-y-D-glutamic acid capsule and an S-layer (9, 33). However, thisB. licheniformis strain, in which the S-layer component is verysimilar to EA1 (20), seems to lack a capsule. We thereforeinvestigated whether these two structures, the capsule and theS-layer, were exclusive. We found that B. anthracis bacilli syn-thesize EA1, Sap, and the capsule both in vivo and in vitro.Furthermore, the capsule and a structured S-layer were foundto be simultaneously present on the bacterial surface, the cap-sule covering the S-layer. Thus, B. anthracis displays a highlycomplex ultrastructural cell wall architecture.The coexistence of the capsule and the S-layer could haveindicated a structural dependence. Such was not the case, asthe S-layer was found in noncapsulated strains and the capsulewas present on the EA1-Sap double deletion mutant. Theseresults further suggest that the capsule is anchored either tothe peptidoglycan-containing sacculus or to the cytoplasmicmembrane, independently of the S-layer. However, the finestructure of the capsule may depend on the presence of theunderlying S-layer: the S-layer may modify the arborescence ofthe poly-y-D-glutamic acid fibers. That these structures can beindependently synthesized and formed does not exclude func-tional interactions.Pathogenic organisms have various strategies to escape hostrecognition. One such strategy, which is widespread, is anti-genic variation of exposed proteins, including S-layer proteins.For example, in Campylobacter fetus, genetic rearrangementsenable the bacterium to change S-layer components (2, 5). Thevariants can therefore multiply before the antibody responsehas developed against the new protein. No gene rearrange-ment between the B. anthracis S-layer genes has been observed(data not shown). The absence of immunolabeling on B. an-thracis whole cells (Fig. 4B and D) in the presence of thecapsule suggests that the cell surface is inaccessible to antibodBACILLUS ANTHRACIS CAPSULE AND S-LAYER 57ies. The presence of anti-EA1 and anti-Sap antibodies in thesera of mice inoculated with strain CAF10 (Fig. 6) indicatesthat these proteins are synthesized in vivo by the capsulatedstrain. The presence of these antibodies could be due to thesynthesis of these proteins prior to the complete coverage ofthe surface by the capsule or to leakage or bacterial lysis.Interestingly, the capsule seems to function as a "one-way"filter. EA1 and Sap are not accessible to antibodies from theoutside, whereas the three toxin components (protective anti-gen, lethal factor, and edema factor) and Sap are found inculture supernatants of capsulated strains, suggesting that theydiffuse from the cell through the capsule to the extracellularmedium.The S-layer may have a protective role in the absence of thecapsule. It could also be a molecular sieve or could have a stillmore structural role, delimiting the periplasm, as recently de-scribed for gram-positive bacteria (1, 11).ACKNOWLEDGMENTSWe are grateful to A. L. Sonenshein for critical reading of themanuscript. We thank B. Chavinier-Jove and C. Rolin for excellenttechnical assistance with electron microscopy experiments and photo-graphic prints, respectively.S.M. was supported by the Ministère de l'Enseignement Supérieuret de la Recherche.REFERENCES1. Beveridge, T. J. 1995. The periplasmic space and the periplasm in gram-positive and gram-negative bacteria. ASM News 61:125-130.2. Blaser, M. J., E. Wang, M. K. R. Tummuru, R. Washburn, S. Fujimoto, andA. Labigne. 1994. High frequency S-layer protein variation in Campylobacterfetus revealed by sapA mutagenesis. Mol. Microbiol. 14:521-532.Jonas Jon3. Carlemalm, E., R. M. Garavito, and W. Villiger. 1982. Resin developmentfor electron microscopy and an analysis of embedding at low temperature. J.Microsc. 126:123.4. Duguid, J. P. 1951. The demonstration of bacterial capsules and slime.J. Pathol. Bacteriol. 63:673-685.5. Dworkin, J., and M. J. Blaser. 1997. Nested DNA inversion as a paradigm ofprogrammed gene rearrangement. Proc. Natl. Acad. Sci. USA 94:985-990.6. Etienne-Toumelin, I., J.-C. Sirard, E. Duflot, M. Mock, and A. Fouet. 1995.Characterization of the Bacillus anthracis S-layer: cloning and sequencing ofthe structural gene. J. Bacteriol. 177:614-620.7. Farchaus, J. W., W. J. Ribot, M. B. Downs, and J. W. Ezzell. 1995. Purifi-cation and characterization of the major surface array protein from theavirulent Bacillus anthracis Delta Sterne-1. J. Bacteriol. 177:2481-2489.8. Fouet, A., and M. Mock. 1996. Differential influence of the two Bacillusanthracis plasmids on regulation of virulence gene expression. Infect. Im-mun. 64:4928-4932.9. Gardner, J. M., and F. A. Troy. 1979. Chemistry and biosynthesis of the poly(y-D-glutamyl) capsule in Bacillus licheniformis. Activation, racemization,and polymerization of glutamic acid by a membranous polyglutamyl syn-thetase complex. J. Biol. Chem. 254:6262-6269.10. Gerhardt, P. 1967. Cytology of Bacillus anthracis. Fed. Proc. 26:1504-1517.11. Graham, L. L., T. J. Beveridge, and N. Nanninga. 1991. Periplasmic spaceand the concept of periplasm. Trends Biochem. Sci. 16:328-329.12. Green, B. D., L. Battisti, T. M. Koehler, C. B. Thorne, and B. E. Ivins. 1985.Demonstration of a capsule plasmid in Bacillus anthracis. Infect. Immun.49:291-297.T13. Hanby, W. E., and H. N. Rydon. 1946. The capsular substance of Bacillusanthracis. Biochem. J. 40:297-309.tra14. Hayat, M. A. 1981. Fixation for electron microscopy, p. 110-111. AcademicPress, Inc., New York, N.Y.ress,15. Hoiczyk, E., and W. Baumeister. 1995. Envelope structure of four glidingfilamentous cyanobacteria. J. Bacteriol. 177:2387-2395.mant16. Holt, S. C., and E. R. Leadbetter. 1969. Comparative ultrastructure of se-lected aerobic spore-forming bacteria: a freeze-etching study. Bacteriol. Rev.33:346-378.17. Karnovsky, M. J. 1965. A formaldehyde-glutaraldehyde fixative of high os-molarity for use in electron microscopy. J. Cell Biol. 27:137A.18. Keppie, J., H. Smith, and P. W. Harris-Smith. 1953. The chemical basis ofthe virulence of Bacillus anthracis. II. Some biological properties of bacterialproducts. Br. J. Exp. Pathol. 34:486-496.19. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680-685.20. Mesnage, S., E. Tosi-Couture, M. Mock, P. Gounon, and A. Fouet. 1997.Molecular characterization of the Bacillus anthracis main S-laver compo FIG. 4. Whole-mount noncapsulated (A and C) and capsulated (B and D) CAF10 bacteria immunolabeled with anti-Sap (A and B) and anti-EA1 (C and D)antibodies. Preparations of cultures were incubated with anti-Sap or anti-EA1 antibodies, and binding was revealed with 10-nm gold-conjugated anti-rabbit antibodies.Bar, 1 µm.in conditions inducing capsule synthesis (Fig. 4B and D). La-beling, such as in Fig. 4B, was infrequently observed in prep-arations of capsulated cells and presumably corresponded tothe leakage of S-layer components from the bacteria throughthe capsule. This result indicated that the capsule is distal tothe S-layer components and that it completely masks access ofthe specific antibodies to EA1 and Sap. Capsule and S-layercomponents can therefore coexist, and the S-layer proteins arelocalized between the peptidoglycan and the capsule.Analysis of the state of capsulation of strains with a deletionof S-layer component genes. We determined whether the S-layer proteins were required for normal capsulation of thebacteria. Mutants with deletions of the EA1 gene (CSM91),the Sap gene (CBA91), or both eag and sap (CSM11) wereconstructed as previously described (20) (Table 1). The pelletand supernatant fractions of CAF10 derivatives grown in cap-sule synthesis-inducing conditions were analyzed by polyacryl-amide gel electrophoresis and immunoblotting with anti-EA1and anti-Sap antibodies (data not shown). The results indi-cated that EA1 and Sap are expressed similarly in the presenceand the absence of the capsule and also that, in both cases, Sapis shed into the supernatant.Each strain was grown on CAP plates. The colonies weresmooth, suggesting that a capsule was synthesized. The pres-ence of the capsule around the bacteria was confirmed byoptical microscopy (Fig. 1A and B). Bacilli from wild-type andS-layer mutants were all capsulated, and no obvious differencein the aspect of capsulation could be seen. The capsule wasstudied in more detail by electron microscopy (Fig. 3A to D).The micrographs showed that similar amounts of capsule werefound around all bacteria tested. This result indicated that theS-layer components, EA1 and Sap, are not required for normalcapsulation of B. anthracis bacilli.Coexistence of the capsule and of the structured S-layer. Wetested whether EA1 and Sap are organized in a two-dimen-sional crystalline array when covered by the capsule. Thinsections (Fig. 3A to C) suggested that the S-layer proteins wereorganized in sheaths. The surfaces of strains CAF10 (EA1+Sap), CBA91 (EA1+), CSM91 (Sap), and CSM11 grown incapsule synthesis-inducing conditions were further analyzedfor the presence of structured layers by negative staining (Fig.5). The cells were vortexed in the presence of glass beads (20).This treatment disrupted the bacteria and tore off the capsule,thus unmasking the S-layers. As expected, no crystalline arraywas present on the surface of CSM11 cells (data not shown).Conversely, structured layers were clearly visible on CAF10,CBA91, and CSM91 cells (Fig. 5A, B, and C, respectively).This result suggested that in the presence of the capsule, theS-layer components, EA1 and Sap, were able to form struc-tured surface arrays. However, the lattices on these variousstrains appeared different. The EA1 array was more stablethan the Sap array, which was only observed at glutaraldehydeconcentrations higher than those required for EA1. In addi-tion, this is the first time that a Sap array has been visualized.Our observations are consistent with the previous suggestionthat Sap forms its own, more fragile, structure (20). They alsoshow that the S-layer and capsule structures coexist on thesame cell surface.In vivo production of the capsule and the S-layer. To deter-mine whether both the capsule and the S-layer could be pro-duced in vivo, the presence of anti-EA1 and anti-Sap antibod-ies was tested in sera from mice infected with strain CAF10,which is capsulated in vivo (Fig. 6). Sera from infected micerecognized EA1 and Sap but no other bacterial protein underthe conditions used. The specificities of the antibodies wereconfirmed with sera adsorbed onto either EA1 or Sap (data not

Question

can you explain why Bacillus anthracis can be pathogenic in a mouse and not be fought off by the immune system?I need help finding the answer in the article and explain in short answer link to article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC106848/ VOL. 180, 199894674330-20-1 2 3 4 5 6 7 8FIG. 6. In vivo expression of the two B. anthracis S-layer components. Im-munoblotting of pellet fractions (odd-numbered lanes) and supernatant fractions(even-numbered lanes) of S-layers from wild-type (CAF10) (lanes 1 and 2), Asap(CBA91) (lanes 3 and 4), Aeag (CSM91) (lanes 5 and 6), and Aeag Asap (CSM11)(lanes 7 and 8) strains was carried out with pooled sera from mice infected withthe CAF10 strain. Molecular masses are indicated in kilodaltons on the left.shown). EA1 and Sap were found to be major surface antigens,showing that both EA1 and Sap can be synthesized by a cap-sulated strain in vivo.DISCUSSIONAlthough various bacteria from natural environments pos-sess both a capsule and an S-layer, their cosynthesis or struc-tural relationship has rarely been studied (15).B. anthracis has a rather unusual capsule: it is composed notof polysaccharide but of poly-y-D-glutamic acid (13). This bac-terium also has an S-layer, previously evidenced only in cap-sule-free strains (10, 16). Another gram-positive bacterium, B.licheniformis, apparently shares these features, namely, a poly-y-D-glutamic acid capsule and an S-layer (9, 33). However, thisB. licheniformis strain, in which the S-layer component is verysimilar to EA1 (20), seems to lack a capsule. We thereforeinvestigated whether these two structures, the capsule and theS-layer, were exclusive. We found that B. anthracis bacilli syn-thesize EA1, Sap, and the capsule both in vivo and in vitro.Furthermore, the capsule and a structured S-layer were foundto be simultaneously present on the bacterial surface, the cap-sule covering the S-layer. Thus, B. anthracis displays a highlycomplex ultrastructural cell wall architecture.The coexistence of the capsule and the S-layer could haveindicated a structural dependence. Such was not the case, asthe S-layer was found in noncapsulated strains and the capsulewas present on the EA1-Sap double deletion mutant. Theseresults further suggest that the capsule is anchored either tothe peptidoglycan-containing sacculus or to the cytoplasmicmembrane, independently of the S-layer. However, the finestructure of the capsule may depend on the presence of theunderlying S-layer: the S-layer may modify the arborescence ofthe poly-y-D-glutamic acid fibers. That these structures can beindependently synthesized and formed does not exclude func-tional interactions.Pathogenic organisms have various strategies to escape hostrecognition. One such strategy, which is widespread, is anti-genic variation of exposed proteins, including S-layer proteins.For example, in Campylobacter fetus, genetic rearrangementsenable the bacterium to change S-layer components (2, 5). Thevariants can therefore multiply before the antibody responsehas developed against the new protein. No gene rearrange-ment between the B. anthracis S-layer genes has been observed(data not shown). The absence of immunolabeling on B. an-thracis whole cells (Fig. 4B and D) in the presence of thecapsule suggests that the cell surface is inaccessible to antibodBACILLUS ANTHRACIS CAPSULE AND S-LAYER 57ies. The presence of anti-EA1 and anti-Sap antibodies in thesera of mice inoculated with strain CAF10 (Fig. 6) indicatesthat these proteins are synthesized in vivo by the capsulatedstrain. The presence of these antibodies could be due to thesynthesis of these proteins prior to the complete coverage ofthe surface by the capsule or to leakage or bacterial lysis.Interestingly, the capsule seems to function as a &#34;one-way&#34;filter. EA1 and Sap are not accessible to antibodies from theoutside, whereas the three toxin components (protective anti-gen, lethal factor, and edema factor) and Sap are found inculture supernatants of capsulated strains, suggesting that theydiffuse from the cell through the capsule to the extracellularmedium.The S-layer may have a protective role in the absence of thecapsule. It could also be a molecular sieve or could have a stillmore structural role, delimiting the periplasm, as recently de-scribed for gram-positive bacteria (1, 11).ACKNOWLEDGMENTSWe are grateful to A. L. Sonenshein for critical reading of themanuscript. We thank B. Chavinier-Jove and C. Rolin for excellenttechnical assistance with electron microscopy experiments and photo-graphic prints, respectively.S.M. was supported by the Ministère de l'Enseignement Supérieuret de la Recherche.REFERENCES1. Beveridge, T. J. 1995. The periplasmic space and the periplasm in gram-positive and gram-negative bacteria. ASM News 61:125-130.2. Blaser, M. J., E. Wang, M. K. R. Tummuru, R. Washburn, S. Fujimoto, andA. Labigne. 1994. High frequency S-layer protein variation in Campylobacterfetus revealed by sapA mutagenesis. Mol. Microbiol. 14:521-532.Jonas Jon3. Carlemalm, E., R. M. Garavito, and W. Villiger. 1982. Resin developmentfor electron microscopy and an analysis of embedding at low temperature. J.Microsc. 126:123.4. Duguid, J. P. 1951. The demonstration of bacterial capsules and slime.J. Pathol. Bacteriol. 63:673-685.5. Dworkin, J., and M. J. Blaser. 1997. Nested DNA inversion as a paradigm ofprogrammed gene rearrangement. Proc. Natl. Acad. Sci. USA 94:985-990.6. Etienne-Toumelin, I., J.-C. Sirard, E. Duflot, M. Mock, and A. Fouet. 1995.Characterization of the Bacillus anthracis S-layer: cloning and sequencing ofthe structural gene. J. Bacteriol. 177:614-620.7. Farchaus, J. W., W. J. Ribot, M. B. Downs, and J. W. Ezzell. 1995. Purifi-cation and characterization of the major surface array protein from theavirulent Bacillus anthracis Delta Sterne-1. J. Bacteriol. 177:2481-2489.8. Fouet, A., and M. Mock. 1996. Differential influence of the two Bacillusanthracis plasmids on regulation of virulence gene expression. Infect. Im-mun. 64:4928-4932.9. Gardner, J. M., and F. A. Troy. 1979. Chemistry and biosynthesis of the poly(y-D-glutamyl) capsule in Bacillus licheniformis. Activation, racemization,and polymerization of glutamic acid by a membranous polyglutamyl syn-thetase complex. J. Biol. Chem. 254:6262-6269.10. Gerhardt, P. 1967. Cytology of Bacillus anthracis. Fed. Proc. 26:1504-1517.11. Graham, L. L., T. J. Beveridge, and N. Nanninga. 1991. Periplasmic spaceand the concept of periplasm. Trends Biochem. Sci. 16:328-329.12. Green, B. D., L. Battisti, T. M. Koehler, C. B. Thorne, and B. E. Ivins. 1985.Demonstration of a capsule plasmid in Bacillus anthracis. Infect. Immun.49:291-297.T13. Hanby, W. E., and H. N. Rydon. 1946. The capsular substance of Bacillusanthracis. Biochem. J. 40:297-309.tra14. Hayat, M. A. 1981. Fixation for electron microscopy, p. 110-111. AcademicPress, Inc., New York, N.Y.ress,15. Hoiczyk, E., and W. Baumeister. 1995. Envelope structure of four glidingfilamentous cyanobacteria. J. Bacteriol. 177:2387-2395.mant16. Holt, S. C., and E. R. Leadbetter. 1969. Comparative ultrastructure of se-lected aerobic spore-forming bacteria: a freeze-etching study. Bacteriol. Rev.33:346-378.17. Karnovsky, M. J. 1965. A formaldehyde-glutaraldehyde fixative of high os-molarity for use in electron microscopy. J. Cell Biol. 27:137A.18. Keppie, J., H. Smith, and P. W. Harris-Smith. 1953. The chemical basis ofthe virulence of Bacillus anthracis. II. Some biological properties of bacterialproducts. Br. J. Exp. Pathol. 34:486-496.19. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680-685.20. Mesnage, S., E. Tosi-Couture, M. Mock, P. Gounon, and A. Fouet. 1997.Molecular characterization of the Bacillus anthracis main S-laver compo FIG. 4. Whole-mount noncapsulated (A and C) and capsulated (B and D) CAF10 bacteria immunolabeled with anti-Sap (A and B) and anti-EA1 (C and D)antibodies. Preparations of cultures were incubated with anti-Sap or anti-EA1 antibodies, and binding was revealed with 10-nm gold-conjugated anti-rabbit antibodies.Bar, 1 µm.in conditions inducing capsule synthesis (Fig. 4B and D). La-beling, such as in Fig. 4B, was infrequently observed in prep-arations of capsulated cells and presumably corresponded tothe leakage of S-layer components from the bacteria throughthe capsule. This result indicated that the capsule is distal tothe S-layer components and that it completely masks access ofthe specific antibodies to EA1 and Sap. Capsule and S-layercomponents can therefore coexist, and the S-layer proteins arelocalized between the peptidoglycan and the capsule.Analysis of the state of capsulation of strains with a deletionof S-layer component genes. We determined whether the S-layer proteins were required for normal capsulation of thebacteria. Mutants with deletions of the EA1 gene (CSM91),the Sap gene (CBA91), or both eag and sap (CSM11) wereconstructed as previously described (20) (Table 1). The pelletand supernatant fractions of CAF10 derivatives grown in cap-sule synthesis-inducing conditions were analyzed by polyacryl-amide gel electrophoresis and immunoblotting with anti-EA1and anti-Sap antibodies (data not shown). The results indi-cated that EA1 and Sap are expressed similarly in the presenceand the absence of the capsule and also that, in both cases, Sapis shed into the supernatant.Each strain was grown on CAP plates. The colonies weresmooth, suggesting that a capsule was synthesized. The pres-ence of the capsule around the bacteria was confirmed byoptical microscopy (Fig. 1A and B). Bacilli from wild-type andS-layer mutants were all capsulated, and no obvious differencein the aspect of capsulation could be seen. The capsule wasstudied in more detail by electron microscopy (Fig. 3A to D).The micrographs showed that similar amounts of capsule werefound around all bacteria tested. This result indicated that theS-layer components, EA1 and Sap, are not required for normalcapsulation of B. anthracis bacilli.Coexistence of the capsule and of the structured S-layer. Wetested whether EA1 and Sap are organized in a two-dimen-sional crystalline array when covered by the capsule. Thinsections (Fig. 3A to C) suggested that the S-layer proteins wereorganized in sheaths. The surfaces of strains CAF10 (EA1+Sap), CBA91 (EA1+), CSM91 (Sap), and CSM11 grown incapsule synthesis-inducing conditions were further analyzedfor the presence of structured layers by negative staining (Fig.5). The cells were vortexed in the presence of glass beads (20).This treatment disrupted the bacteria and tore off the capsule,thus unmasking the S-layers. As expected, no crystalline arraywas present on the surface of CSM11 cells (data not shown).Conversely, structured layers were clearly visible on CAF10,CBA91, and CSM91 cells (Fig. 5A, B, and C, respectively).This result suggested that in the presence of the capsule, theS-layer components, EA1 and Sap, were able to form struc-tured surface arrays. However, the lattices on these variousstrains appeared different. The EA1 array was more stablethan the Sap array, which was only observed at glutaraldehydeconcentrations higher than those required for EA1. In addi-tion, this is the first time that a Sap array has been visualized.Our observations are consistent with the previous suggestionthat Sap forms its own, more fragile, structure (20). They alsoshow that the S-layer and capsule structures coexist on thesame cell surface.In vivo production of the capsule and the S-layer. To deter-mine whether both the capsule and the S-layer could be pro-duced in vivo, the presence of anti-EA1 and anti-Sap antibod-ies was tested in sera from mice infected with strain CAF10,which is capsulated in vivo (Fig. 6). Sera from infected micerecognized EA1 and Sap but no other bacterial protein underthe conditions used. The specificities of the antibodies wereconfirmed with sera adsorbed onto either EA1 or Sap (data not

Accepted Answer

Anthrax, a severe infection originating from the bacterium Bacillus anthracis, is a matter of…

can you explain why Bacillus anthracis can be pathogenic in a mouse and not be fought off by the immune system? I need help finding the answer in the article and explain in short answer link to article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC106848/