Can S-layer proteins be detected by immunolabelling when a capsule is present? How do you know?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/ Capsule observation. The aspect and homogeneity of capsulation werechecked by India ink exclusion (4).Electron microscopy. (i) Thin sections. Cells were fixed with 2% formaldehyde(made freshly from paraformaldehyde) and 2.5% glutaraldehyde in 0.1 M caco-dylate buffer (pH 7.2) containing 5 mM CaCl₂ (14, 17). After being washed, thecells were postfixed for 2 h with 2% OsO4 in the same buffer. The pelletedbacteria were embedded in 2% low-melting-point agar (type IX; Sigma) (36).The samples were then treated for 16 h with 0.5% uranyl acetate in water. Afterextensive washing, small blocks were dehydrated with alcohol and embedded inSpurr's medium (Ladd Inc.) (32). Thin sections were stained conventionally andobserved with a Philips CM12 electron microscope.(ii) Immunocytochemistry with thin sections. B. anthracis cells were fixed with2% formaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate-buffered saline(14 mM Na₂HPO4, 7 mM NaH₂PO4, 150 mM NaCl) (PBS) (pH 7.4) for 1 h,rinsed in the same buffer, and embedded in 2% low-melting-point agar (36).Small blocks containing bacteria were embedded in Lowicryl HM20 (Poly-sciences Ltd.) at -50°C following the progressively lower temperatures protocolof Carlemalm et al. (3) as described by Newman and Hobot (25). Thin sectionswere collected onto Formvar-carbon-coated nickel grids and incubated succes-sively at room temperature with the following solutions: PBS-50 mM NH4Cl for10 min; PBS-1% bovine serum albumin (BSA)-1% normal goat serum-0.1%Tween 20 for 10 min; specific anti-EA1 or anti-Sap antibodies diluted 1/50 inPBS-1% BSA-1% normal goat serum-0.1% Tween 20 for 1 h; PBS-0.1% BSAthree times for 5 min each time; goat immunoglobulin G (heavy and light chains)anti-rabbit immunoglobulin-gold conjugate diluted 1/20 in PBS-0.01% gelatinfor 1 h; PBS three times for 5 min each time; PBS-1% glutaraldehyde for 5 min;and five times with water. The thin sections were then stained by incubation with2% uranyl acetate in water for 35 min and then in lead tartrate for 2 min (23).(iii) Immunocytochemistry with whole-mount cells. Immunocytochemistrywith whole-mount cells was carried out as previously described (20).(iv) Negative staining experiments. B. anthracis cells were resuspended in a1/10 volume of 25 mM Tris-HCl (pH 8.0)–10 mM MgCl₂ with 0.25 or 0.5%glutaraldehyde for EA1 or Sap, respectively, in the presence of approximately 30μl of 425- to 600-µm glass beads (Sigma) and disrupted by vortexing for 30 s. Thistreatment disintegrated the capsule. Negative staining was performed as previ-ously described (20). Micrographs were recorded with a Philips CM12 electronmicroscope under low-dose (17 electrons/Å/s) transmission electron microscopyconditions.RESULTSCosynthesis and respective localization of the capsule andthe S-layer components. All reported data on the B. anthracisS-layer is from noncapsulated strains (6, 7, 10, 16, 20). Wetherefore investigated whether the capsule and the S-layercomponents, EA1 and Sap, could all be simultaneouslypresent. The genes for EA1 and Sap are chromosomal andhave been well characterized for the plasmid-free strain 9131S0ABFIG. 1. Homogeneity of the capsulation state of B. anthracis cells. Cultures of CAF10 (A) or of its derivative, CSM11, with deletions of both S-layer genes (B), grownin capsule synthesis-inducing conditions were incubated in the presence of India ink. The capsule appears as a bright halo surrounding the cells under the light 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

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Can S-layer proteins be detected by immunolabelling when a capsule is present? How do you know? I need help finding the answer in the article and explain in short answer