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An atypical lipoteichoic acid from Clostridium perfringens elicits a broadly cross-reactive and protective immune response

Open AccessPublished:May 18, 2020DOI:https://doi.org/10.1074/jbc.RA119.009978
      Clostridium perfringens is a leading cause of food-poisoning and causes avian necrotic enteritis, posing a significant problem to both the poultry industry and human health. No effective vaccine against C. perfringens is currently available. Using an antiserum screen of mutants generated from a C. perfringens transposon-mutant library, here we identified an immunoreactive antigen that was lost in a putative glycosyltransferase mutant, suggesting that this antigen is likely a glycoconjugate. Following injection of formalin-fixed whole cells of C. perfringens HN13 (a laboratory strain) and JGS4143 (chicken isolate) intramuscularly into chickens, the HN13-derived antiserum was cross-reactive in immunoblots with all tested 32 field isolates, whereas only 5 of 32 isolates were recognized by JGS4143-derived antiserum. The immunoreactive antigens from both HN13 and JGS4143 were isolated, and structural analysis by MALDI-TOF-MS, GC-MS, and 2D NMR revealed that both were atypical lipoteichoic acids (LTAs) with poly-(β1→4)-ManNAc backbones substituted with phosphoethanolamine. However, although the ManNAc residues in JGS4143 LTA were phosphoethanolamine-modified, a few of these residues were instead modified with phosphoglycerol in the HN13 LTA. The JGS4143 LTA also had a terminal ribose and ManNAc instead of ManN in the core region, suggesting that these differences may contribute to the broadly cross-reactive response elicited by HN13. In a passive-protection chicken experiment, oral challenge with C. perfringens JGS4143 lead to 22% survival, whereas co-gavage with JGS4143 and α-HN13 antiserum resulted in 89% survival. This serum also induced bacterial killing in opsonophagocytosis assays, suggesting that HN13 LTA is an attractive target for future vaccine-development studies.
      Clostridium perfringens is a Gram-positive toxin-producing pathogen that is one of the most common causes of foodborne illness in humans (
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      ) and is also responsible for enteric diseases in numerous species of livestock (
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      ,
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      ). C. perfringens is the second most prevalent cause of bacterial-induced diarrheal disease in the United States (
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      ) and is estimated to cause ∼80,000 cases of foodborne disease annually in the United Kingdom (
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      ). It has also been associated with necrotizing enterocolitis in preterm infants, the most severe and fatal neonatal gastrointestinal emergency worldwide (
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      ,
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      ). In animals, C. perfringens causes a number of serious and economically problematic enteric diseases in livestock, including enterotoxemia in cattle, goats, and sheep (
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      ), equine acute necrotizing enterocolitis in foals (
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      ), and necrotic enteritis (NE) in poultry (
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      ,
      • Hofacre C.L.
      • Smith J.A.
      • Mathis G.F.
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      ). Although not the only causative agent, C. perfringens is the primary cause of avian NE (
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      ,
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      ), which poses a significant problem in the poultry industry. In chickens, the disease can lead to rapid death within 24 h of the onset of acute infection, precluding treatment in most cases (
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      ). Subclinical infections are associated with chronic damage to the intestinal mucosa, leading to reduced weight gain and lower feed efficiency (
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      ). Combined, NE is estimated to be responsible for at least 2 billion U.S. dollars in annual losses worldwide for the poultry industry (
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      ). The European ban on the prophylactic use of antibiotics in livestock (
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      ) has resulted in an increase in NE outbreaks in European countries (
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      ) that has led to a 33% loss in profit for flocks heavily infected with C. perfringens compared with healthy flocks (
      • Lovland A.
      • Kaldhusdal M.
      Severely impaired production performance in broiler flocks with high incidence of Clostridium perfringens–associated hepatitis.
      ), and market demands for poultry raised without the use of antibiotics are expected to further increase the frequency of NE outbreaks and exacerbate losses (
      • Hofacre C.L.
      • Smith J.A.
      • Mathis G.F.
      An optimist’s view on limiting necrotic enteritis and maintaining broiler gut health and performance in today’s marketing, food safety, and regulatory climate.
      ). These losses highlight the need for alternative prevention strategies in place of antibiotic therapy.
      Despite the importance of C. perfringens in a livestock context and the identification of capsular polysaccharide (CPS) as the primary antigenic determinant of the Hobbs typing scheme (
      • Hughes J.
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      Serotyping system for Clostridium welchii (C. perfringens) typea and studies on type-specific antigens.
      ), little is known about carbohydrate structures present on the surface of this organism. Only the CPS structures from C. perfringens Hobbs 5, 9, and 10 have been examined in any detail, with the Hobbs 9 CPS determined to be comprised of Glc, Gal, and galactosamine in a 1:1.6:1.1 ratio in 1977 (
      • Cherniak R.
      • Frederick H.M.
      Capsular polysaccharide of Clostridium perfringens Hobbs 9.
      ) and the complete structures of the Hobbs 5 and Hobbs 10 CPS solved by NMR spectroscopy in 1997 (
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      • van Halbeek H.
      • Morris L.C.
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      Structure of the capsular polysaccharide of Clostridium perfringens Hobbs 5 as determined by NMR spectroscopy.
      ) and 1998 (
      • Sheng S.
      • Cherniak R.
      Structure of the capsular polysaccharide of Clostridium perfringens Hobbs 10 determined by NMR spectroscopy.
      ), respectively.
      In addition to CPS structures, many Gram-positive bacteria produce cell wall teichoic acids and lipoteichoic acids (LTAs), but little information is available about the presence or importance of these or other carbohydrate structures in C. perfringens. Indeed, Richter et al. (
      • Richter S.G.
      • Elli D.
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      Small molecule inhibitor of lipoteichoic acid synthesis is an antibiotic for Gram-positive bacteria.
      ) noted the presence of three homologues of the LTA synthase gene (ltaS) in the genome of C. perfringens SM101 and demonstrated that C. perfringens SM101 was very sensitive to a small molecule inhibitor of LTA synthesis, suggesting the presence and importance of LTA in C. perfringens. However, direct evidence for the presence of LTA in this species has only recently been demonstrated by Vinogradov et al. (
      • Vinogradov E.
      • Aubry A.
      • Logan S.
      Structural characterization of wall and lipidated polysaccharides from Clostridium perfringens ATCC 13124.
      ), who reported that C. perfringens ATCC 13124 produces an LTA with a repeating structure of β-ManNAc6PEtN-(1→4)-[β-ManNAc6PEtN-(1→4)]-β-ManNAc-(1→4)-β-ManNAc6PEtN[3-α-Ribf]-(1→4)-β-ManN-(1→4)-β-Glc-(1→1)-Gro.
      There are no known polysaccharide-based vaccines against C. perfringens. Vaccination strategies to date have centered on the use of protein antigens, such as detoxified versions of toxins produced by C. perfringens (toxoids) and C. perfringens surface and secreted proteins, resulting in varying degrees of protection (
      • Mot D.
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      • Haesebrouck F.
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      ). However, because of the production of more than one toxin by C. perfringens strains causing livestock diseases, including NE in chickens, effective protein vaccine strategies may require multivalent vaccines.
      Commercially available C. perfringens vaccines for poultry (Netvax®) and Clostridium toxoid autovaccine (Vacci-VetTM) are based on α-toxin toxoids, but the toxin NetB has recently been shown to play a more pivotal role in C. perfringens pathology in chickens. Moreover, a recent NE vaccine study found that significant protection levels were only observed when a combination of α-toxin– and NetB–derived antigens were used (
      • Jiang Y.
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      • Roland K.
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      ). One of the major considerations in the development of an NE vaccine is that it must be inexpensive to produce, and multivalent vaccines may prove to be cost-prohibitive for use in poultry because of the low market value of chickens. Therefore, there still remains a need to identify a conserved, immunogenic target molecule from C. perfringens that elicits a broadly cross-reactive immune response to be used as the primary antigen in a safe and effective vaccine against NE in chickens, other livestock diseases, and human food-poisoning caused by C. perfringens.
      In this study, we have identified an atypical LTA produced by C. perfringens HN13 that dominates the immune response of both rabbits and chickens against bacterial whole cells and have demonstrated that serum raised against this LTA is broadly cross-reactive, recognizing all 32 C. perfringens field isolates tested, provides passive protection to chicks, and promotes killing of C. perfringens in an opsonophagocytosis assay. Combined, these results suggest that this LTA is an ideal candidate antigen for glycoconjugate or live-cell surface-display–based NE vaccines.

      Results

      Identification of a nonproteinaceous immunostimulatory surface antigen in C. perfringens

      To identify any immunostimulatory antigens common to C. perfringens, whole-cell lysates of three strains, C. perfringens HN13 (a derivative of strain 13, Table S6), JGS4143, and SM101, were analyzed by Western immunoblotting before and after treatment with lysozyme and/or proteinase K using rabbit antiserum raised against formalin-treated whole cells of C. perfringens strain 13 (Fig. S1). Prior to enzymatic treatment, the lysates for all three strains contained a large immunoreactive “smear,” as well as a few discrete immunoreactive bands, and lysozyme treatment did not alter the profile of the samples. Treatment with proteinase K resulted in the loss of the discreet bands, but the immunoreactive “smear” remained, indicating that the majority of the immune response was directed to a nonproteinaceous antigen.
      Western immunoblotting analysis was subsequently performed on whole-cell lysates of four putative glycosyltransferase mutants isolated from a C. perfringens HN13 transposon library (
      • Liu H.
      • Bouillaut L.
      • Sonenshein A.
      • Melville S.
      Use of a mariner-based transposon mutagenesis system to isolate Clostridium perfringens mutants deficient in gliding motility.
      ), and lysates from the cpe2071 mutant (strain HLL8) no longer showed the proteinase K–resistant antigen observed in the WT strain (Fig. 1) or in negatively stained SDS-polyacrylamide gels (Fig. S2). The Western blot signal was restored by complementation of cpe2071. Conserved Domain Database analysis (
      • Marchler-Bauer A.
      • Anderson J.
      • DeWeese-Scott C.
      • Fedorova N.
      • Geer L.
      • He S.
      • Hurwitz D.
      • Jackson J.
      • Jacobs A.
      • Lanczycki C.
      • Liebert C.
      • Liu C.
      • Madej T.
      • Marchler G.
      • Mazumder R.
      • et al.
      CDD: a curated Entrez database of conserved domain alignments.
      ) (RRID:SCR_002077) of the enzyme encoded by cpe2071 revealed that it shares conserved domains with glycosyltransferases and synthases, including poly-β-1,6-GlcNAc synthase and cellulose synthase (PgaC_IcaA domain [TIGR03937]; BcsA domain [COG1215]), and contains DXD, TED, and QXXRW motifs that are characteristic of GT-2 family enzymes, the latter motif being a defining sequence of membrane processive glycosyltransferases (
      • McNamara J.T.
      • Morgan J.L.
      • Zimmer J.
      A molecular description of cellulose biosynthesis.
      ). PgaC is involved in the biosynthesis of the highly conserved immunogenic carbohydrate poly-β-1,6-GlcNAc expressed by many bacterial, fungal, and eukaryotic pathogens (
      • Skurnik D.
      • Cywes-Bentley C.
      • Pier G.
      The exceptionally broad-based potential of active and passive vaccination targeting the conserved microbial surface polysaccharide PNAG.
      ). These similarities to the cellulose and poly-β-1,6-GlcNAc synthases, which both synthesize glycopolymers (
      • Gerke C.
      • Kraft A.
      • Süssmuth R.
      • Schweitzer O.
      • Götz F.
      Characterization of the N-acetylglucosaminyltransferase activity involved in the biosynthesis of the Staphylococcus epidermidis polysaccharide intercellular adhesin.
      ,
      • Omadjela O.
      • Narahari A.
      • Strumillo J.
      • Melida H.
      • Mazur O.
      • Bulone V.
      • Zimmer J.
      BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis.
      ), indicate that the antigen is likely either a polysaccharide or a polysaccharide–containing glycolipid.
      Figure thumbnail gr1
      Figure 1The primary surface antigen of C. perfringens is a polysaccharide or glycolipid. Left panel, Western immunoblot of whole-cell lysates of C. perfringens HN13 WT and four putative glycosyltransferase mutants isolated from a transposon library of the same strain. Right panel, Western immunoblots of WT C. perfringens HN13, the cpe2071 mutant, and the cpe2071 mutant complemented with a copy of the cpe2071 gene in trans.

      The identified immunoreactive antigen is common to C. perfringens but not related Clostridium spp.

      To determine the level of conservation of the identified immunoreactive antigen among C. perfringens strains, dot-blot analysis was performed on proteinase K–treated whole-cell lysates from 32 C. perfringens field isolates of differing sequence types (
      • Chalmers G.
      • Bruce H.L.
      • Hunter D.B.
      • Parreira V.R.
      • Kulkarni R.R.
      • Jiang Y.F.
      • Prescott J.F.
      • Boerlin P.
      Multilocus sequence typing analysis of Clostridium perfringens isolates from necrotic enteritis outbreaks in broiler chicken populations.
      ), using rabbit α-C. perfringens HN13 antiserum adsorbed against the C. perfringens HN13 cpe2071 mutant. In the resultant dot blot (Fig. S3), all field isolate lysates, as well as the WT strain lysate, were immunoreactive. However, the C. perfringens HN13 cpe2071 mutant lysate was not recognized by the antiserum, indicating that the identified immunoreactive antigen is present in all of the field isolates tested. Additionally, the same immunoreactive antigen was detected in the chicken isolate C. perfringens JGS4143 by Western immunoblotting (Fig. S3). In contrast, lysates from three representative strains of Clostridium cochleatum, Clostridium difficile, and Clostridium symbiosum showed no reactivity by Western immunoblotting analysis with the same antiserum (Fig. S4), indicating that the conserved C. perfringens antigen is not present in these strains and likely not conserved among other Clostridium species.

      Antiserum raised against C. perfringens JGS4143 does not show the broad cross-reactivity observed with α-C. perfringens HN13 antiserum

      To determine whether the conserved C. perfringens antigen dominates the immune response in chickens, antisera against formalin-fixed whole cells of C. perfringens HN13 and C. perfringens JGS4143 were generated, and Western immunoblotting analyses were performed to compare the reactivity of adsorbed rabbit and chicken α-C. perfringens HN13 antisera, as well as the unadsorbed α-C. perfringens JGS4143 antisera against lysates from all 32 C. perfringens field isolates. The JGS4143 and HN13 WT (positive controls) and the HN13 cpe2071 mutant (negative control) strains were also included (Fig. 2). For both the rabbit and chicken antisera raised against C. perfringens HN13, all of the field isolates showed reactivity similar to HN13 and JGS4143, indicating that these strains produce a similar or closely related immunoreactive antigen compared with C. perfringens HN13. Note that reactivity consistent with the glycan of interest was observed in field isolates from both NE and healthy chickens, as well as from equine NE (JP55) and canine hemorrhagic gastroenteritis (JP838) isolates, which indicates that the glycoconjugate is present on isolates of C. perfringens irrespective of the host species or the disease state of the host animals. In contrast, the chicken antiserum raised against C. perfringens JGS4143 was reactive with both the HN13 and JGS4143 lysate controls but reactive with only five of the field isolates, with three isolates (isolates 20, 21, and 149) showing moderate reactivity and a further two field isolates (isolates 10 and 11) only faintly reactive. Thus, it appears the surface polysaccharide antigen from C. perfringens HN13 is either broadly conserved or has one or more epitopes that elicit a broadly cross-reactive immune response, whereas the surface polysaccharide antigen from C. perfringens JGS4143 elicits an immune response that is far less cross-reactive with exemplary field isolates of C. perfringens.
      Figure thumbnail gr2
      Figure 2The immune response to the identified immunoreactive antigen of C. perfringens HN13 is cross-reactive with all C. perfringens strains tested. A–C, Western immunoblots of whole-cell lysates of laboratory strains C. perfringens JGS4143 and HN13, the HN13 cpe2071 mutant (labeled as cpe2071) that lacks the immunoreactive antigen of interest, and 32 C. perfringens field isolates, using α-C. perfringens HN13 antiserum raised in rabbits (A) and in chickens (B), as well as chicken antiserum raised against C. perfringens JGS4143 (C).

      The conserved surface antigens from C. perfringens HN13 and JGS4143 are atypical lipoteichoic acids comprised of a poly-ManNAc backbone modified with phospho-moieties

      To determine the nature of the conserved surface antigens from C. perfringens and gain insight into the differences between the antigens from C. perfringens strains HN13 and JGS4143, the conserved antigens from each of these strains were purified from 10-liter fermenter cultures by sequential fractionation using boiling, lysozyme treatment, phenol-hot water extraction, and ultracentrifugation steps. The conserved surface antigens fractionated to the phenol phase and preferentially to the ultracentrifugation pellet of the phenol phase based on dot-blot analysis of the fractions (Fig. S5). These purified antigens were first tested for reactivity with α-HN13 antiserum (Fig. S6), and the purified HN13 antigen was used to affinity purify antibodies directed at that antigen from α-HN13 antiserum; the resultant purified antibodies showed similar reactivity patterns as observed with the adsorbed α-HN13 antiserum, indicating that the observed reactivities with that adsorbed serum correspond to the antigen of interest (Fig. S7). The purified antigens from HN13 and JGS4143 were then structurally characterized through a combination of MALDI-TOF-MS, GC-MS, and 1D/2D NMR experiments.
      The composition of the glycolipids isolated from the two strains was determined by combined GC-MS of per-O-TMS derivatives of the monosaccharide methyl glycosides. Glycosyl composition analysis showed that the HN13 polysaccharide contained Gro, Glc, traces of N-acetylmannosamine (ManNAc) and fatty acids: C20, C18, C16, and C14. The JG4143 polysaccharide contained ribose, Glc, traces of ManNAc, and fatty acids: C20, C18, and C16. As shown and described below, the major glycosyl residue in the glycolipid after treatment with aqueous hydrofluoric acid (HF) is ManNAc, indicating that the ManNAc residues are extensively substituted by phosphate or substituent groups linked to ManNAc via phosphate.
      The HN13 LTA preparation was deacylated (i.e. delipidated) and separated into high- and low-molecular-weight (HMW and LMW) delipidated LTA by Bio-Gel P6 chromatography. Initial structural analysis was performed on the HMW material using 1D/2D NMR spectroscopy; proton, HSQC, COSY, TOCSY, and NOESY experiments. This allowed assignment of the proton and carbon chemical shifts and also determination of their linkages, sugar sequence, as well as identification and determination of substitution positions of substituent groups. The chemical shift assignments for the HMW deacylated LTA are given in Table S1. The 1H NMR spectrum (Fig. 3, top panel) contained a major anomeric signal at δ 4.87 (residue A), which overlapped with minor signals of δ ∼4.85 (residue B) and δ 4.89 (residue B′). Of these minor signals, the one at δ 4.85 seemed to be the more intense. Further assignments for these residues could be made for all protons and carbons of residue A, B1, and B2 protons and carbons, and B′1 proton and carbon. The signals for A and B were consistent with both being β-ManpNAc residues as indicated by their respective downfield H-2 chemical shifts, δ 4.61 and δ 4.58, and C-2 chemical shifts at δ 54.0 and 54.1 (Fig. 3, middle and bottom panels). A high-field signal at δ 2.06 was also observed that is due to the NAc groups attached to C-2 of the ManNAc residues that dominate this polysaccharide (see further description below). Residue B′ was also likely a ManNAc because this is by far the major glycosyl component in this sample (the identities of B and B′ are described further below with regard to the delipidated LMW LTA fraction). The strong intraresidue A1,3 and A1,5 NOE correlations (Fig. 3, middle panel) confirm the β-configuration of the ManNAc residues. Intense signals at δ 4.11 and 3.23 were consistent with PEtN substituents, and the intensity of these signals indicated that PEtN was a major component of the polysaccharide. The NMR experiments also supported the presence or PGro as two primary protons were observed at δ 3.86 and 3.91, which coupled to a secondary proton at δ 3.94 which, in turn was coupled to two primary protons at δ 3.60 and 3.66. The respective corresponding carbon resonances were at δ 67.6, 71.7 and 63.4 (Fig. 3, bottom panel). The intensities of PEtN resonances in the proton spectrum were greater than any of the glycosyl residue or PGro resonances (Fig. 3, top panel), indicating that the polysaccharide contains significantly greater levels of PEtN than PGro. The ∼2/1 ratio of the PEtN H-2 protons to the A1 + B′1 + B1 protons also indicates that the vast majority of the ManNAc residues are substituted by PEtN groups.
      Figure thumbnail gr3
      Figure 3NMR spectra (1H NOESY and 1H-13C HSQC) of the C. perfringens delipidated HMW HN13 LTA. All spectra were recorded in D2O at 30 °C. Chemical shift assignments are listed in . The NAc signals at δH 2.06/δC 23.5 are not shown in these spectra. The NMR spectroscopy data of the deacylated conserved immunoreactive antigen from C. perfringens HN13, confirming the presence of a beta-(1,4)-linked-ManNAc polysaccharide modified with phosphoethanolamine and phosphoglycerol, as illustrated in A. In the NOESY portion of the article, intraresidue contacts are labeled e.g. A3,1 and inter-residue contacts are labeled e.g. A4, A1.
      Sequence information was obtained from the NOESY experiment in which we observed strong A4/A1 NOE contacts (Fig. 3, middle panel). These are likely inter-residue rather than intraresidue contacts because the opposing di-axial positions of the intraresidue A1 and A4 protons in a β-(1,4)-linked ManNAc residue would not be expected to have NOE contacts. The downfield A4 carbon chemical shift of the ManNAc residues (δ 77.9) also supports that they are linked at this position. The remaining chemical shifts for residues B and B′ were not possible to assign because their resonances were of low intensity and likely overlap with those of residue A. An A6/A1 NOE contact was also observed, which is likely due to inter-residue interaction between adjacent A residues because an intraresidue A1,6 NOE would not be expected. Thus, these data indicate that the HMW deacylated LTA consists largely of a β-(1,4)-ManNAc polysaccharide, which, because of the downfield chemical shift of C-6 (δ 65.4), is substituted at that position mainly by PEtN or on occasion by PGro.
      To obtain more information about the structure, the HN13 delipidated HMW LTA was dephosphorylated by dissolving the lyophilized sample in 48% HF and incubating at 4°C for 48 h, followed by evaporation of the sample on ice and lyophilizing once more. The generated product mixture was subjected to size-exclusion chromatography using a Bio-Gel P6 column, and two fractions, denoted F1 and F2, were obtained. F1 eluted in the void volume and was of higher molecular weight than F2. Proton and carbon assignments of the residues in both F1 and F2, as well as the linkage and sequence of these residues, were determined by COSY, TOCSY, NOESY, and HSQC NMR experiments (Table S2). The proton spectra for F1 and F2 are shown in Fig. 4. Both F1 and F2 are devoid of PEtN and PGro signals, indicating that these groups had been removed by the HF treatment. The 1D/2D NMR analysis of F1 shows that it consists of a dephosphorylated linear polysaccharide, containing β-4-linked ManNAc (C) residues (Table S2, Fig. 4A, and Fig. S8). The chemical shift of the C6 carbon is now upfield at δ 61.1, rather than at 65.4 prior to HF treatment, consistent with the removal of PEtN or any possible PGro groups from the 6-position of the ManNAc residues. The 1D/2D NMR analysis of F2 allowed proton and carbon assignment (Table S2 and Fig. S9) and sequence determination of the glycosyl residues. The proton and carbon chemical shifts for residue D were all consistent with it being an unsubstituted (i.e. a terminally linked) β-ManNAc residue (Table S2). The NOESY experiment (Fig. S8) showed a C4/D1 NOE indicating a D1 → 4C sequence. The C4/C1 NOE is consistent with multiple C residues in which each C is linked to an adjacent C at O-4 (multiple C1 → 4C sequences). The E4/C1 NOE showed a C1 → 4E sequence. The chemical shifts for residue E were consistent with a ManN residue. The E2 carbon chemical shift at δ 55.2 showed that it was an amino sugar, and its upfield E2 proton shift at δ 3.91 showed that the attached nitrogen atom was unacetylated; i.e. it was an -NH3+ rather than an -NHAc group. The proton and carbon chemical shifts of residue F were consistent with a →4)-β-Glc-(1→ residue. The downfield shift of the F4 carbon at δ 79.1, as well as the F4/E1 NOE show a E1 → 4F sequence. Lastly, the NOESY experiment showed that residue F is linked from F1 to Gro1 (Fig. S9). The results of the analysis of the F2 fraction show that it has a →4)-β-ManN-(1→4)-β-Glc-(1→1)-Gro linker region at its reducing end followed by a number of β-4-linked ManNAc residues and terminated with a β-ManNAc residue. MALDI-TOF-MS analysis, together with the above NMR data, showed that fraction F2 consisted of a size-heterogeneous mixture of oligosaccharides comprised of the above linker region followed by successive elongation with between three and seven β-4-linked ManNAc residues and terminated by a β-ManNAc residue (Table S3). The NMR results described above are consistent with those reported by Vinogradov et al. (
      • Vinogradov E.
      • Aubry A.
      • Logan S.
      Structural characterization of wall and lipidated polysaccharides from Clostridium perfringens ATCC 13124.
      ).
      Figure thumbnail gr4
      Figure 41H NMR spectrum of the dephosphorylated/delipidated HMW HN13 LTA. Spectra of the high-molecular-weight (F1, A) and low-molecular-weight (F2, B) fractions from size-exclusion chromatography, recorded in D2O at 25 °C. Chemical shift assignments are listed in . The NAc signals at δH 2.06 are not shown in these spectra.
      The fact that HF treatment of the delipidated HMW LTA preparation results in a β-(1,4)-ManNAc polysaccharide (F1) and the F2 oligosaccharide suggests that it likely contained some delipidated LMW LTA; i.e. F1 was derived from HMW LTA and F2 from LMW LTA. Therefore, to further characterize the HN13 LTA structure, the delipidated LMW LTA fraction was analyzed by 1D/2D NMR analysis (Fig. S10). Spectra were taken before and after Superdex Peptide chromatography. Resonances for PGro were present before but absent after chromatography, indicating that the PGro was no longer attached to the LMW LTA polysaccharide and that during the several months of storage after deacylation and prior to this analysis PGro became detached from the LMW LTA polysaccharide. A high intensity resonance at δ 4.87 (ManNAc) and lower intensity resonances for anomeric protons of residues E (ManN), and F (Glc) (δ 5.02 and 4.48, respectively), as described above for fraction F2, and an anomeric proton resonance for another ManNAc, residue X, at δ 4.89 were observed. Two other minor β-ManNAc residues, Y and Z, with anomeric resonances at δ 4.88 and 4.85 were also observed. The down field chemical shift of the Y4 carbon at δ 78.0 indicated Y was a 4-linked residue. The anomeric resonances for X and Z are essentially the same as those unidentified residues B′ and B described above for the delipidated HMW LTA. Chemical shift assignments (both proton and carbon) were made from COSY, TOCSY, HSQC, and heteronuclear multiple bond correlation (HMBC) analysis (Table S4 and Figs. S10 and S11). The X2, Y2, and Z2 proton and carbons have chemical shifts that are the same as those for B2 in the delipidated HMW LTA, showing that residues B and B′ in that sample are likely due to residues X, Y, and Z in this LMW LTA. The proton and carbon chemical shifts of the A residues are consistent with the 6-PEtN-β-(1,4)-ManNAc residues of the HMW preparation shown in Table S1. The chemical shifts for residue X were consistent with a terminally linked 6-PEtN-β-ManNAc residue; its chemical shift values are very similar to those of residue D in fraction F2 (see above and Table S2), except for the X6 carbon and proton, which were downfield because of PEtN substitution. The NOESY experiment (Fig. S10, middle panel) showed a strong NOE between protons at δ 4.87–4.89 with those at δ ∼3.80. This NOE is likely due to several overlapping signals, which makes it challenging to assign. However, it is consistent with an inter-residue A4/A1 contact as well as a A4/X1 contact indicating a X1→(4A1→4A1)n→ sequence. These interactions were also observed in the HMBC analysis (Fig. S11), which showed contacts of the A1 carbon and possibly the X1 carbon with the A4 proton. The reducing end of this polysaccharide is also indicated by the NOESY and HMBC spectra. Both NOESY (Fig. S10, middle panel) and HMBC (Fig. S11) show F1/Gro and F4/E1 contacts and the HMBC shows E4/Y1 and a possible Y4/Z1 contact. This would indicate that the reducing end consists of a Z1 → 4Y1 → 4E1 → 4F1 → Gro sequence. Although we cannot observe a Z4/A1 contact, it would occur at the same region in the NOESY and HMBC spectra as the A4/A1 signals (Figs. S10 and S11). Based on these data we propose the structure of the delipidated LMW LTA is as shown in Fig. S12. If treated with HF, this structure would collapse to the structure shown for fraction F2 produced by HF treatment of the delipidated HMW LTA as shown in Fig. 4 The location of PEtN was confirmed to be at the 6-position of the A and terminal X residues by 1H,31P HMBC spectroscopy (Fig. S13). Unfortunately, we do not know the location of PGro attachment because it was labile and was detached from the polysaccharide. However, because the points of attachment for phosphate-bridged substituents are at the 6-position of the ManNAc residues, there are only two possible points of attachment: residue Y or Z. Given that the polysaccharide backbone consists of β-1,4-linked ManNAc residues, we propose that the PGro had been linked to residue Y and that residue Z, which is attached to Y4, is the initial ManNAc residue of the 4-linked ManNAc polysaccharide portion of the LTA. The loss of PGro also prevented us from determining whether the sn-glycerol-1P or sn-glycerol-3P configuration is the form present in the HN13 LTA.
      The combined results from the analysis of delipidated HMW and LMW LTA preparations support the structure shown in Fig. 5A for the HN13 LTA glycan; namely that it consists of a 4-linked 6-PEtN-β-ManNAc linear polysaccharide of variable length (and possibly containing occasional 4-linked 6-PGro-β-ManNAc residues in the case of HMW LTA) terminated with a 6-PEtN-β-ManNAc residue and that this polysaccharide is in turn joined to the 4-position of a 6-PGro-β-ManNAc, which in turn is 4-linked to the β-ManN-(1→4)-β-Glc-(1→1)-Gro oligosaccharide linker region.
      Figure thumbnail gr5
      Figure 5The major immunoreactive antigens of C. perfringens HN13 and JGS4143 are atypical lipoteichoic acids distinct from LTA types I–V. The polysaccharide region of the broadly cross-reactive LTA from C. perfringens HN13 (A) and the poorly cross-reactive LTA from JGS4143 (B) indicate that both LTAs have a common poly-β-1,4-ManNAc backbone that is heavily substituted at the C6 position of the sugar residues with phospho-moieties, being solely PEtN for JGS4143, and largely PEtN and with a PGro (shaded gray) likely on the proximal ManNAc residue of the polysaccharide in HN13 LTA. For the HN13 LTA structure, residues X, Y, and Z account for residues denoted as B and B′ during the analysis of the delipidated HMW LTA. Note that α-HN13 LTA antiserum recognizes both these structures as well as similar immunoreactive antigens (presumably LTAs) in all field isolates tested but that anti-JGS4143 antiserum only recognizes a small subset of the field isolates tested (see ).
      The 1D and 2D NMR analysis of the JGS4143 polysaccharide, performed as described for the HN13 polysaccharide, allowed complete assignment of the protons and carbons for these residues (Fig. 6 and Table S5). The 1H NMR spectrum of JGS4143 polysaccharide (Fig. 6, top panel) showed the presence of spin systems belonging to: →4)-β-ManNAcPEtN-(1→ (residues A); →4)-β-ManNAc-(1→ (residue C); →4)-β-Glc-(1→ (residue F); →3,4)-β-ManNAcPEtN-(1→ (residue G); t-α-Ribf-(1→ (residue H); and t-β-ManNAcPEtN-(1→ (residue J). The sequence of these residues was deduced from NOESY (Fig. 6, middle panel) and HMBC experiments. For some residues, this was challenging because of the overlap of NMR signals. However, there is a clear NOE connection between J1 and G4 (Fig. 6 and Table S5) showing a J1 → 4G sequence. There is also an NOE connection between the H1 and G3, showing a H1 → 3G sequence. This NOE overlaps with a possible intraresidue H3/H1 interaction; however, an HMBC experiment (not shown) revealed a clear connection between the H1 proton and the G3 carbon, confirming the H1 → 3G sequence. Thus, these data show that the JGS4143 polysaccharide contains a J1 → 4[H1 → 3]G sequence at its nonreducing end. The F4/C1 NOE interaction reveals a C1 → 4F sequence. Residue F, the β-Glc residue, if arranged as in the HN13 polysaccharide, is likely attached to Gro. This would make the “reducing” end of the JGS4143 polysaccharide have a C1 → 4F → Gro sequence. There is a strong NOE signal between the A1 and G1 protons (which overlap) and protons at δ 3.82, which could be the A4 or C4 protons. This indicates possible A4/A1, C4/A1, A4/G1, and C4/G1 interactions. These interactions also all overlapped in the HMBC analysis. The C4/G1 interaction can be ruled out because C is at the reducing end of the oligosaccharide, and G is at the nonreducing end and therefore, separated by multiple A residues. However, the remaining interactions likely all occur. The A4/A1 NOE is an inter-residue interaction between adjacent A residues indicating A1 → 4A sequences. The A4/G1 interaction would be consistent with a G1 → 4A sequence, and the 4C/A1 interaction would be consistent with a A1 → 4C sequence. Combining these results shows that the JGS4143 has the following sequence:
      J14[H13]G1(4A14A1)n4C14F1Gro
      (Sequence 1)


      Figure thumbnail gr6
      Figure 6NMR spectra (1H, NOESY and 1H-13C HSQC) of the delipidated conserved immunoreactive antigen from C. perfringens JGS4143. All spectra were recorded in D2O at 60 °C. The chemical shift assignments are listed in . The NAc signals at δH 2.06/δC 23.5 are not shown in these spectra. The polysaccharide consists of a poly-ManNAc repeating unit modified with phosphoethanolamine and capped at the nonreducing end with a trisaccharide modified with PEtN. The reducing end is anchored with a disaccharide-glycerol, as illustrated in B.
      This structure is shown in Fig. 5B.
      The main differences between the HN13 and JGS4143 polysaccharides are that the JGS4143 polysaccharide (i) is devoid of PGro, (ii) contains a terminal Ribf residue on the penultimate ManNAcPEtN residue, and (iii) has a ManNAc rather than ManN attached to the proximal Glc residue in the linker region.
      Collectively, these data indicate that C. perfringens strains produce a common class of surface glycolipids best described as an atypical LTA comprised of a 1,4-linked poly-ManNAc fully substituted at the C6 position of sugar residues with phospho-moieties, including PEtN and, occasionally, PGro (Fig. 5).
      It should be noted that no capsular polysaccharide was detected in HN13, but it was discovered that JGS4143 produces a capsule with a structure and composition unrelated to the JGS4143 LTA that surprisingly is not recognized by the α-JGS4143 antiserum raised in this study (Fig. S2). The detailed structure and immunogenicity will be described elsewhere.

      Chicken α-HN13-LTA antiserum provides passive protection against C. perfringens–mediated killing in a chick oral gavage model

      SPF leghorn chicks were challenged at 1 day of age with C. perfringens JGS4143 (a chicken NE strain) in the presence or absence of adsorbed chicken α-HN13-LTA antiserum to evaluate the potential for this serum to provide passive protection against C. perfringens–mediated mortality and/or cause reduction in colonization levels. As demonstrated in Fig. 7A, only 22% survival (2 of 9 birds) was observed in the group gavaged with C. perfringens alone, whereas 89% survival (8 of 9 birds) was observed in the group co-gavaged with C. perfringens and 1:100 α-HN13-LTA antiserum. C. perfringens cecal colonization levels among the surviving birds were not statistically significantly different between groups (C. perfringens alone: median = 1.1 × 107, n = 2; C. perfringens + antiserum: median = 0.64 × 107, n = 8), although it is difficult to make any conclusions with the small number of birds remaining (n = 2) in the untreated group. All control birds that were orally gavaged with PBS alone survived. Post-mortem examination revealed the presence of high levels of bacteria morphologically consistent with C. perfringens in the lung tissues of all birds that did not survive following oral gavage (Fig. 7, D and F), and no abnormalities or tissue damage to intestinal tissues (Fig. 7B). Fluorescent microscopy using adsorbed rabbit α-HN13 antiserum confirmed the presence of C. perfringens and the expression of the LTA antigen in vivo (Fig. 7, C and E). These observations were unexpected and suggested that the presence of these bacteria in the lungs was a result of aspiration.
      Figure thumbnail gr7
      Figure 7Antiserum against C. perfringens HN13 LTA provides passive protection against C. perfringens–mediated mortality in SPF leghorn chicks. A, the percentage of survival of SPF leghorn chicks orally gavaged with either PBS, 1 × 109 C. perfringens JGS4143 cells in PBS, or co-gavaged with 1× 109 C. perfringens JGS4143 cells in 1:100 dilution of adsorbed α-HN13 antiserum in PBS. B, D, and F, histological slides of intestinal tissue at 100× showing normal morphology (B) and lung tissue at 100× (D) and 1000× (F) magnification showing the presence of damaged tissues and rod-shaped bacteria. Green arrows, healthy tissue; red arrows, damaged tissue/bacteria. C and E, fluorescence microscopy of chicken lung tissue using adsorbed rabbit α-HN13 antiserum at (C) 40× and (E) 60× magnification, with bacteria visible in green.

      Chicken α-HN13-LTA antiserum promotes bacterial killing in an opsonophagocytosis assay

      To evaluate the potential of C. perfringens HN13 LTA as a vaccine antigen, we tested the ability of α-HN13-LTA antiserum from seven separate birds to elicit protection through an opsonophagocytosis assay using fresh heparinized chicken blood from four different birds as the source of leukocytes. The percentage of killing for each α-HN13 LTA serum was calculated as the average of values obtained for that serum with each of the heparinized blood samples. Compared with bacteria incubated with naïve chicken antiserum, 26.4% bacterial killing was observed after 2 h of incubation when using day 7 heparinized blood and 25.4% when using day 35 heparinized blood (Fig. 8), indicating that chicken antibodies targeting the C. perfringens HN13 LTA are opsonizing and therefore that this LTA is likely to elicit a protective immune response against C. perfringens if used as an antigen in a glycoconjugate or recombinant live bacterial vaccine.
      Figure thumbnail gr8
      Figure 8Chicken antiserum against C. perfringens HN13 LTA promotes bacterial killing in an opsonophagocytosis assay. The percentage of killing of C. perfringens JGS4143 cells in the presence of adsorbed antiserum from chickens immunized with formalin-fixed C. perfringens HN13 cells versus serum from negative control birds. Individual data points represent each of the negative or α-HN13 LTA sera and represents the average of reactions using that antiserum with each of four heparinized blood samples (used as sources of leukocytes) freshly collected from 7-day-old (A) or 35-day-old (B) birds. Lines represent the means ± S.E. **, p < 0.05; ***, p < 0.005. The mean bacterial killing values were 26.4% for day-7 blood (p = 0.0203) and 25.4% day-35 blood (p = 6.35 × 10−5).

      Discussion

      The goal of this study was to identify and characterize novel candidate molecules in C. perfringens with potential to be used as target antigens in a vaccine against avian NE and, potentially, other C. perfringens–mediated livestock diseases. Although previous efforts to generate an NE vaccine for chickens have primarily involved targeted approaches focused on the use of C. perfringens NetB– and α-toxin–based toxoids with the goal of neutralizing these toxins (
      • Mot D.
      • Timbermont L.
      • Haesebrouck F.
      • Ducatelle R.
      • Van Immerseel F.
      Progress and problems in vaccination against necrotic enteritis in broiler chickens.
      ,
      • Jiang Y.
      • Mo H.
      • Willingham C.
      • Wang S.
      • Park J.
      • Kong W.
      • Roland K.
      • Curtiss R.
      Protection against necrotic enteritis in broiler chickens by regulated delayed lysis Salmonella vaccines.
      ,
      • Alimolaei M.
      • Golchin M.
      • Abshenas J.
      • Ezatkhah M.
      • Bafti M.S.
      A recombinant probiotic, Lactobacillus casei, expressing the Clostridium perfringens α-toxoid, as an orally vaccine candidate against gas gangrene and necrotic enteritis.
      ,
      • Cooper K.K.
      • Trinh H.T.
      • Songer J.G.
      Immunization with recombinant α toxin partially protects broiler chicks against experimental challenge with Clostridium perfringens.
      ,
      • Keyburn A.
      • Portela R.
      • Ford M.
      • Bannam T.
      • Yan X.
      • Rood J.
      • Moore R.
      Maternal immunization with vaccines containing recombinant NetB toxin partially protects progeny chickens from necrotic enteritis.
      ,
      • Wilde S.
      • Jiang Y.
      • Tafoya A.M.
      • Horsman J.
      • Yousif M.
      • Vazquez L.A.
      • Roland K.L.
      Salmonella-vectored vaccine delivering three Clostridium perfringens antigens protects poultry against necrotic enteritis.
      ,
      • Hunter J.
      • Wilde S.
      • Tafoya A.
      • Horsman J.
      • Yousif M.
      • Diamos A.
      • Roland K.
      • Mason H.
      Evaluation of a toxoid fusion protein vaccine produced in plants to protect poultry against necrotic enteritis.
      ,
      • Lepp D.
      • Ojha S.
      • Gohari I.
      • Chakravarty B.
      • Prescott J.
      • Gong J.
      Immunization with subunits of a novel pilus produced by virulent Clostridium perfringens strains confers partial protection against necrotic enteritis in chickens.
      ), our group took an unbiased approach by identifying and characterizing candidate vaccine antigens that dominate the immune response against C. perfringens whole cells. The discovery that the major antigen recognized by this immune response was a nonproteinaceous antigen suggests that although vaccines targeting C. perfringens toxins may reduce the risk or severity of disease, these and other vaccines targeting protein antigens are unlikely to induce killing or clearing of C. perfringens cells from the intestinal tract. This may explain why several toxoid vaccines and one pilin-based vaccine show reduction in C. perfringens–induced lesion scores in chickens but are unable to fully eliminate lesions in at least half of vaccinated birds (
      • Jiang Y.
      • Mo H.
      • Willingham C.
      • Wang S.
      • Park J.
      • Kong W.
      • Roland K.
      • Curtiss R.
      Protection against necrotic enteritis in broiler chickens by regulated delayed lysis Salmonella vaccines.
      ,
      • Wilde S.
      • Jiang Y.
      • Tafoya A.M.
      • Horsman J.
      • Yousif M.
      • Vazquez L.A.
      • Roland K.L.
      Salmonella-vectored vaccine delivering three Clostridium perfringens antigens protects poultry against necrotic enteritis.
      ,
      • Hunter J.
      • Wilde S.
      • Tafoya A.
      • Horsman J.
      • Yousif M.
      • Diamos A.
      • Roland K.
      • Mason H.
      Evaluation of a toxoid fusion protein vaccine produced in plants to protect poultry against necrotic enteritis.
      ,
      • Lepp D.
      • Ojha S.
      • Gohari I.
      • Chakravarty B.
      • Prescott J.
      • Gong J.
      Immunization with subunits of a novel pilus produced by virulent Clostridium perfringens strains confers partial protection against necrotic enteritis in chickens.
      ).
      The broad conservation of this antigen among C. perfringens, as evidenced by Western immunoblotting analysis with anti-HN13-LTA antiserum, and the absence of a similarly reactive antigen among other Clostridium species is desirable for a potential vaccine target because this supports the possibility that an effective single-antigen vaccine for C. perfringens can be generated and that such a vaccine would have a low risk of causing broader population shifts in the chicken microbiome. In contrast, the antiserum raised in chickens against C. perfringens JGS4143 showed drastically poorer cross-reactivity among C. perfringens field isolates compared with the chicken α-HN13 antiserum. To better understand these differences, the antigens from each of these strains was purified for further characterization.
      Structural analyses of these purified antigens revealed that both were glycolipids consisting of β-1→4-linked poly-ManNAc backbones decorated with phospho-moieties at the C6 position of each sugar residue and that the backbone was linked to a diglycosyl-diacylglycerol (Gly2-DAG) glycolipid anchor. The amphiphilic polymeric nature, high number of phosphodiester linkages, and Gly2-DAG lipid carrier features of these molecules are consistent with key features of LTAs (Fig. 9) (
      • Varki A.
      • Cummings R.D.
      • Aebi M.
      • Packer N.H.
      • Seeberger P.H.
      • Esko J.D.
      • Stanley P.
      • Hart G.
      • Darvill A.
      • Kinoshita T.
      • Prestegard J.J.
      • Schnaar R.L.
      • Freeze H.H.
      • Marth J.D.
      • Bertozzi C.R.
      • et al.
      Symbol nomenclature for graphical representations of glycans.
      ). Furthermore, the polymeric regions of all LTA types described to date are comprised of sugar and/or sugar alcohols linked via O- and/or phosphodiester linkages (
      • Percy M.G.
      • Gründling A.
      Lipoteichoic acid synthesis and function in Gram-positive bacteria.
      ), which is also true of the C. perfringens glycolipids described here. However, despite these shared characteristics, the C. perfringens glycolipids do not fit within any of the existing LTA types described thus far, as summarized by Percy and Gründling (
      • Percy M.G.
      • Gründling A.
      Lipoteichoic acid synthesis and function in Gram-positive bacteria.
      ) (Fig. 9). Type I LTAs embody the canonical unbranched 1,3-linked poly-PGro LTA backbone structure, whereas LTA types II–V contain more complex polymeric regions. LTA types II and III contain different glycosyl-PGro repeating units, type IV LTA has a polymeric region comprised of glycosyl–ribitol–phosphate repeats wherein the C6 positions of all sugar residues are modified with phosphocholine (PCho), and type V LTA displays a polymeric region with a polyglycosyl backbone containing both O- and phosphodiester linkages (
      • Percy M.G.
      • Gründling A.
      Lipoteichoic acid synthesis and function in Gram-positive bacteria.
      ) (Fig. 9). In contrast, the polymeric regions of both LTAs isolated in this study have polyglycosyl backbones that lack phosphodiester linkages and are purely O-linked. Somewhat similar to type IV LTA, all glycosyl residues in this backbone are modified at the C6 position with phospho-moieties; however, in the C. perfringens glycolipids, these modifications are largely PEtN with, in some cases, small nonstoichiometric levels of PGro instead of the PCho groups in type IV LTA. These broader features are also shared with the pPS1 surface polysaccharide from C. perfringens ATCC 13124 that was described by Vinogradov et al. (
      • Vinogradov E.
      • Aubry A.
      • Logan S.
      Structural characterization of wall and lipidated polysaccharides from Clostridium perfringens ATCC 13124.
      ), and we propose that they represent members of a novel LTA type, herein referred to as type VI LTA (Fig. 9). This type VI LTA differs from the other LTA types by polymeric regions consisting of homopolymeric glycans with no backbone phosphodiester bonds while still containing high levels of phosphodiester bonds via PEtN and/or PGro modifications (and potentially other phospholinked modifications) at the C6 positions of the backbone sugar residues. This definition also encompasses the previously described LTA of Bifidobacterium bifidum subsp. Pennsylvanicum DSM20239, which has a structure of H-[5-β-d-Galf(6PGro)-1-]n-[6-β-d-Glc-1-]m-6-Gal-(
      • Grass J.
      • Gould L.
      • Mahon B.
      Epidemiology of foodborne disease outbreaks caused by Clostridium perfringens, United States, 1998–2010.
      ,
      • Songer J.
      Clostridial enteric diseases of domestic animals.
      ,
      • Uzal F.A.
      • Vidal J.E.
      • McClane B.A.
      • Gurjar A.A.
      Toxins involved in mammalian veterinary diseases.
      )-DAG (n = ∼7–10; m = ∼8–15) (
      • Fischer W.
      Lipoteichoic acid of Bifidobacterium bifidum subspecies pennsylvanicum DSM 20239: a lipoglycan with monoglycerophosphate side chains.
      ), and does not correspond to any of the I–V LTA types.
      Figure thumbnail gr9
      Figure 9Schematic of LTA type I–VI structures. The structures were adapted from Ref.
      • Percy M.G.
      • Gründling A.
      Lipoteichoic acid synthesis and function in Gram-positive bacteria.
      and this study, using the Symbol Nomenclature for Glycans (
      • Varki A.
      • Cummings R.D.
      • Aebi M.
      • Packer N.H.
      • Seeberger P.H.
      • Esko J.D.
      • Stanley P.
      • Hart G.
      • Darvill A.
      • Kinoshita T.
      • Prestegard J.J.
      • Schnaar R.L.
      • Freeze H.H.
      • Marth J.D.
      • Bertozzi C.R.
      • et al.
      Symbol nomenclature for graphical representations of glycans.
      ) with a minor adaptation to designate residues that can be either glucosamine/GlcNAc or ManN/ManNAc. Note that the LTA type I–V structures all contain phosphate groups in their main chain, whereas the proposed type VI LTA structure has a linear homopolymeric glycan backbone that lacks phosphates in the main chain but instead has phospho-moiety modification on each of the backbone sugars. *, a minor percentage of residues can be substituted with PGro in place of PEtN (for example, in HN13 the ManNAc residue proximal to the ManN of the Gly2-DAG core is likely substituted with PGro). FucNAc4N, 4-amino-N-acetylfucosamine (2-acetamido-4-amino-2,4,6-trideoxy-galactose); GlcN±Ac, either glucosamine or GlcNAc; GroA, glyceric acid; ManN±Ac, either mannosamine or N-acetylmannosamine; circled P, phosphate.
      Although the biosynthetic pathway for the poly-PGro type I LTA has been fully elucidated (
      • Reichmann N.T.
      • Gründling A.
      Location, synthesis and function of glycolipids and polyglycerolphosphate lipoteichoic acid in Gram-positive bacteria of the phylum Firmicutes.
      ), little is known about biosynthesis of the more complex types II–V, and therefore it is difficult to gain insight into the biosynthesis of type VI LTAs. Models for the biosynthesis of LTA types I, II, and V have not yet been proposed, whereas for type IV LTA, whose polymeric region has a repeating-unit structure, a model has been proposed in which individual repeating units are assembled onto undecaprenylphosphate on the cytoplasmic face of the membrane including the addition of PCho modifications. These units are then flipped to the outer face of the membrane, where they are polymerized and transferred either to peptidoglycan as wall teichoic acid or to a Glc-DAG anchor (reviewed in Ref.
      • Percy M.G.
      • Gründling A.
      Lipoteichoic acid synthesis and function in Gram-positive bacteria.
      ) as LTA, in a manner analogous to Wzy-dependent lipopolysaccharide biosynthesis in Gram-negative bacteria (
      • Whitfield C.
      • Trent M.S.
      Biosynthesis and export of bacterial lipopolysaccharides.
      ). However, the homopolymeric backbones in C. perfringens type VI LTA structures and the essential nature of the synthase-like cpe2071 gene for HN13 LTA biosynthesis collectively suggest that the biosynthesis of these LTAs proceeds in a manner analogous to that of other synthase-dependent glycans, in which the polymeric region of the LTA is assembled by processive addition of ManNAc residues to the growing chain on the cytoplasmic face of the membrane and translocated to the outer face of the membrane (
      • Morgan J.L.
      • McNamara J.T.
      • Fischer M.
      • Rich J.
      • Chen H.M.
      • Withers S.G.
      • Zimmer J.
      Observing cellulose biosynthesis and membrane translocation in crystallo.
      ).
      Unlike the C6 PCho modifications in type IV LTA biosynthesis, PEtN and PGro substituents will most likely be added to the translocating backbone on the outer face of the membrane using phosphatidylethanolamine and phosphatidylglycerol as substrate donors, because bacterial PEtN/PGro transferases are typically predicted to be membrane anchored enzymes with their primary soluble domain in the periplasm/outside face of the membrane (
      • Bohin J.P.
      • Kennedy E.P.
      Regulation of the synthesis of membrane-derived oligosaccharides in Escherichia coli: assay of phosphoglycerol transferase I in vivo.
      ,
      • Fage C.D.
      • Brown D.B.
      • Boll J.M.
      • Keatinge-Clay A.T.
      • Trent M.S.
      Crystallographic study of the phosphoethanolamine transferase EptC required for polymyxin resistance and motility in Campylobacter jejuni.
      ,
      • Lequette Y.
      • Lanfroy E.
      • Cogez V.
      • Bohin J.
      • Lacroix J.
      Biosynthesis of osmoregulated periplasmic glucans in Escherichia coli: the membrane-bound and the soluble periplasmic phosphoglycerol transferases are encoded by the same gene.
      ,
      • Wanty C.
      • Anandan A.
      • Piek S.
      • Walshe J.
      • Ganguly J.
      • Carlson R.
      • Stubbs K.
      • Kahler C.
      • Vrielink A.
      The structure of the neisserial lipooligosaccharide phosphoethanolamine transferase A (LptA) required for resistance to polymyxin.
      ). It should be noted, however, that this would only be true for the sn-glycerol-3P configuration of PGro and, because we were unable to determine the stereochemistry of the PGro modification in the HN13 LTA, we cannot rule out the possibility of this modification being sn-glycerol-1-P. Although uncommon in bacteria, this configuration has been recently observed in B. bifidum (
      • Speciale I.
      • Verma R.
      • Di Lorenzo F.
      • Molinaro A.
      • Im S.H.
      • De Castro C.
      Bifidobacterium bifidum presents on the cell surface a complex mixture of glucans and galactans with different immunological properties.
      ), and the presence of this configuration would have significant implications on the donor and location of PGro addition. Because of the high level of substitution with these phospho-moieties, either this transfer will be coupled to export of the polymer or the rate of export would be slower than the rate of PEtN/PGro transfer. It should be noted that identification of additional gene products involved in the biosynthesis of the HN13 LTA will not be straightforward because the cpe2071 gene is not in a cluster, and adjacent genes have no similarity to glycosyltransferase or PEtN/PGro transferase genes. Nonetheless, efforts are underway to identify the remaining enzymes involved in the biosynthesis of the HN13 LTA antigen.
      In addition to structural analyses revealing that the major antigen recognized by the immune response to whole cells of C. perfringens are atypical LTAs, key structural differences between the HN13 LTA versus the JGS4143 and pPS1 LTAs were also revealed, including the identity of the C6-linked phospho-moieties, the presence/absence of a Ribf residue, and the identity of the distal glycosyl residue of the Gly2-DAG anchor region. The difference between HN13 LTA and JGS4143 LTA in their ability to stimulate a broadly cross-reactive response likely stems from their three structural differences: presence/absence of Ribf, ManNAc versus ManN in the Gly2-DAG region, and presence/absence of PGro modifications (note that, like JGS4143 LTA, pPS1 also has Ribf and lacks PGro). However, at present it is unclear which of the three key differences, or possibly which combination of these differences, is responsible for the broad cross-reactivity elicited by the HN13 LTA. Studies are currently underway to purify and structurally characterize LTA molecules from other strains and to identify the genes involved in the biosynthetic pathway of this molecule.
      Vaccine efforts against Gram-positive bacteria have traditionally not considered LTA as a target antigen. However, recent studies evaluating the protective potential of LTA-based vaccines against Staphylococcus aureus (
      • Chen Q.
      • Dintaman J.
      • Lees A.
      • Sen G.
      • Schwartz D.
      • Shirtliff M.E.
      • Park S.
      • Lee J.C.
      • Mond J.J.
      • Snapper C.M.
      Novel synthetic (poly)glycerolphosphate-based antistaphylococcal conjugate vaccine.
      ), Enterococcus spp. (
      • Kodali S.
      • Vinogradov E.
      • Lin F.
      • Khoury N.
      • Hao L.
      • Pavliak V.
      • Jones C.H.
      • Laverde D.
      • Huebner J.
      • Jansen K.U.
      • Anderson A.S.
      • Donald R.G.
      A vaccine approach for the prevention of infections by multidrug-resistant Enterococcus faecium.
      ,
      • Laverde D.
      • Wobser D.
      • Romero-Saavedra F.
      • Hogendorf W.
      • van der Marel G.
      • Berthold M.
      • Kropec A.
      • Codee J.
      • Huebner J.
      Synthetic teichoic acid conjugate vaccine against nosocomial Gram-positive bacteria.
      ), and C. difficile (
      • Broecker F.
      • Martin C.E.
      • Wegner E.
      • Mattner J.
      • Baek J.Y.
      • Pereira C.L.
      • Anish C.
      • Seeberger P.H.
      Synthetic lipoteichoic acid glycans are potential vaccine candidates to protect from Clostridium difficile infections.
      ,
      • Cox A.D.
      • St. Michael F.
      • Aubry A.
      • Cairns C.M.
      • Strong P.C.
      • Hayes A.C.
      • Logan S.M.
      Investigating the candidacy of a lipoteichoic acid-based glycoconjugate as a vaccine to combat Clostridium difficile infection.
      ) have indicated that LTA-based vaccines have significant potential. The broad cross-reactivity of α-HN13 LTA antiserum against C. perfringens strains was encouraging; however, an effective vaccine antigen needs to elicit an immune response that not only recognizes the target antigen but also shows evidence of protection. The co-administration of adsorbed α-HN13 LTA antiserum with the C. perfringens JGS4143 challenge to 1-day-old SPF leghorns was found to significantly improve chick survival compared with birds administered JGS4143 alone, and although the mortalities likely arose because of tissue damage or systemic toxicity from an abundance of bacteria and/or toxins produced in the lungs and not from NE, the results nonetheless demonstrate that the α-HN13 LTA antiserum provided significant passive protection against C. perfringens–mediated mortality. Furthermore, antisera from several birds injected with formalin-fixed C. perfringens HN13 cells were able to stimulate ∼25% killing in opsonophagocytosis assays, providing further evidence that immune responses elicited against HN13 LTA were protective. These levels of killing are consistent with those observed in a similar experiment performed by Goyette-Desjardins et al. (
      • Goyette-Desjardins G.
      • Calzas C.
      • Shiao T.C.
      • Neubauer A.
      • Kempker J.
      • Roy R.
      • Gottschalk M.
      • Segura M.
      Protection against Streptococcus suis serotype 2 infection using a capsular polysaccharide glycoconjugate vaccine.
      ), in which mouse sera raised against Streptococcus suis capsule, which dominates the immune response to the bacterium (
      • Qiu X.
      • Bai X.
      • Lan R.
      • Zheng H.
      • Xu J.
      Novel capsular polysaccharide loci and new diagnostic tools for high-throughput capsular gene typing in Streptococcus suis.
      ), induced ∼20% killing in opsonophagocytosis assays. Collectively, both the passive protection and opsonophagocytosis assay results demonstrate that the immune response against C. perfringens HN13 LTA is protective, further supporting that this molecule is a suitable target antigen for vaccine development.
      In conclusion, we have identified an LTA that is the major antigen recognized by immune response to whole cells of C. perfringens. We have shown that this antigen is an atypical LTA that differs from the existing LTA types and therefore represents a unique type, which we designate type VI, characterized by an exclusively O-linked homopolymeric glycan backbone that is heavily modified at the C6 position of the sugar residues with PEtN and to a lesser degree with PGro. The structure of the LTA from C. perfringens JGS4143 was found to be very similar to the pPS1 of C. perfringens ATCC 13124, and this LTA elicits a poorly cross-reactive immune response in chickens. In contrast, the LTA from C. perfringens HN13 elicits a broadly cross-reactive immune response that recognizes all field isolates tested thus far, and the resultant antiserum provided passive protection against C. perfringens–mediated mortality in 1-day-old chicks and stimulated bacterial killing in opsonophagoctosis assays. These results demonstrate that the LTA from C. perfringens HN13 is an ideal target antigen for vaccines against C. perfringens.

      Experimental procedures

      Reagents, strains, plasmids, and growth conditions

      Unless otherwise specified, all reagents were obtained from Sigma–Aldrich Canada, and all media were obtained from Difco Laboratories (Detroit, MI, USA). All Clostridium strains (Table S6) were grown at 37 °C under anaerobic conditions in either a Whitley DG250 anaerobic work station (Don Whitley Scientific, Frederick, MD, USA) supplied with 5% hydrogen, 5% CO2, 90% N2) or in an anaerobic jar with 3.5-liter AnaeroGen sachets (Oxoid Company, Nepean, Canada) unless otherwise specified and routinely propagated in PGY broth (3% proteose peptone #3, 2% dextrose, 1% yeast extract, 0.1% sodium thioglycolate) without agitation or on PGY agar (PGY broth containing 1.5% agar). For NMR experiments, Clostridium strains were grown in PGY broth at 37 °C with agitation at 50 rpm in a BioFlo 115 Fermenter (Eppendorf, Mississauga, Canada) supplied with N2 at a flow rate of 1 liter/min, with the media prewarmed and conditioned with N2 for 1 h prior to inoculation with a 40-ml overnight liquid culture. Where appropriate, the media were supplemented with 30 µg ml−1 erythromycin.

      Animal studies

      For all animal studies, all procedures involving animals were approved by the Biosciences Animal Care and Use Committee at the University of Alberta. The animals were maintained and used in accordance with the recommendations of the Canadian Council on Animal Care. The chickens were obtained from the Poultry Research Facility, Department of Agricultural, Food and Nutritional Sciences, University of Alberta.

      Generation of whole-cell lysates of C. perfringens and analysis by Western immunoblotting and dot-blot analyses

      To prepare whole-cell lysates of C. perfringens for SDS-PAGE and Western immunoblotting analysis, the strains were streaked from −80 °C stocks onto PGY agar plates (with antibiotics as appropriate) and grown overnight. For each strain, a single colony was used to inoculate 10 ml of PGY broth, allowed to grow for 6 h, harvested by centrifugation (13,000 × g, 10 min), washed with PBS, and resuspended in PBS to OD600 = 2.0. Cells from 1 ml were harvested by centrifugation as above, resuspended in 100 μl of PBS, and incubated with 2 mg ml−1 lysozyme at 37 °C for 1 h. Each sample was combined with 67 μl of 4× SDS-PAGE sample buffer, heated to 95 °C for 10 min, allowed to cool, and then either analyzed by SDS-PAGE according to the method of Laemmli (
      • Laemmli U.
      Cleavage of structural proteins during assembly of head of bacteriophage-T4.
      ) or incubated with 0.5 mg ml−1 proteinase K at 55 °C for 1 h prior to SDS-PAGE analysis. Following electrophoresis, the samples were transferred to 0.2 μm nitrocellulose membrane (Bio-Rad) and subjected to Western immunoblotting analysis according to the method of Burnette (
      • Burnette W.N.
      Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate–polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.
      ), using polyclonal rabbit antiserum raised against whole cells of C. perfringens strain 13 (
      • O'Brien D.K.
      • Melville S.B.
      The anaerobic pathogen Clostridium perfringens can escape the phagosome of macrophages under aerobic conditions.
      ) (the parent strain of HN13 (
      • Nariya H.
      • Miyata S.
      • Suzuki M.
      • Tamai E.
      • Okabe A.
      Development and application of a method for counterselectable in-frame deletion in Clostridium perfringens.
      )) at a 1:1000 dilution or polyclonal chicken antiserum raised against whole cells of C. perfringens strains (this study; 1:200–1:1000 dilution) as the primary and either IRDye 680LT goat anti-rabbit IgG or IRDye 680 donkey anti-chicken IgG (as appropriate; LI-COR Biosciences, Lincoln, NE, USA) for the secondary antibody (1:15,000). The membranes were visualized on a LI-COR Odyssey IR imaging system (LI-COR Biosciences). Dot-blot analyses were performed similarly, except 3-μl aliquots of proteinase K–treated whole-cell lysates were spotted directly onto nitrocellulose membranes instead of being separated by SDS-PAGE and transferred to nitrocellulose.

      Generation of chicken antiserum against C. perfringens HN13 and JGS4143 strains

      Formalin-fixed C. perfringens HN13 and JGS4143 cells were prepared as follows for intramuscular (IM) injection into chickens. The cells were grown overnight on PGY agar plates as described above. The cells from one plate each were harvested and resuspended in 10 ml of PBS, pelleted by centrifugation, resuspended in 10 ml of PBS containing 1% (v/v) formalin, and incubated at 4°C for 2 h. The cells were washed four times in 2 ml of PBS to remove formalin and resuspended in PBS to an OD600 of 1.0. The cell suspension was mixed 1:1 with either Freund’s complete adjuvant (primary injection) or Freund’s incomplete adjuvant (boost injection). Primary injections (150 μl × 2, IM in the breast muscle) were given to broilers at 7 days of age, followed by boost injections (150 μl × 2, IM in the breast muscle) at 21 days of age. Chickens were culled on day 35 and exsanguinated. Blood was allowed to clot at room temperature overnight, and the next day the samples were centrifuged at 13,000 × g, and the serum was aspirated by pipette and stored at 4°C.

      Adsorption of polyclonal antisera against the LTA-deficient C. perfringens HN13 cpe2071 mutant to generate α-HN13-LTA antiserum

      To remove undesirable signals from antigens other than the glycan of interest, the rabbit and chicken antisera raised against C. perfringens HN13 were adsorbed against whole cells of the C. perfringens HN13 cpe2071 mutant (strain HLL8 (31)), which does not make the glycan of interest. The chicken antiserum raised against C. perfringens JGS4143 was used without any adsorption step because no glycan-minus mutant was available in that background. The adsorption was performed in the following manner: C. perfringens HN13 cpe2071 was grown as described for whole-cell lysates, washed with PBS, and adjusted to OD600 = 1.0 in PBS, and four 1-ml aliquots were pelleted by centrifugation as described above. The first aliquot was resuspended in 100 μl of either rabbit or chicken α-C. perfringens HN13 antiserum, allowed to incubate at room temperature for 1 h, and pelleted by centrifugation, and the supernatant was decanted. This process was repeated sequentially for each of the three remaining cell aliquots using the supernatant from the previous round to resuspend the cells. This adsorbed antiserum was used as the primary antibody, and either IRDye 680LT goat anti-rabbit IgG or IRDye 680RD donkey anti-chicken IgG were used as the secondary antibody, as described above.

      Zinc–imidazole negative staining

      To examine all contents in proteinase K–treated whole-cell lysates, rather than only those that react with the antisera, the samples were separate by SDS-PAGE, followed by negative staining of the gel using the lipopolysaccharide/LOS zinc-imidazole negative staining method of Castellanos-Serra and Hardy (
      • Castellanos-Serra L.
      • Hardy E.
      Negative detection of biomolecules separated in polyacrylamide electrophoresis gels.
      ). Briefly, after separating samples by SDS-PAGE, the gels were washed three times with boiling water (15 min/wash), followed by incubation with 10 mm ZnSO4 for a further 15 min. The gels were then washed with water for 30 s with vigorous shaking and incubated with 100 mm imidazole until fully developed (∼5 min). The gels were visualized and imaged against a black background.

      Extraction and purification of the conserved polysaccharide from C. perfringens

      The glycans of interest were extracted and purified from 10-liter fermenter cultures of C. perfringens as follows: the cultures were inoculated with a 40-ml overnight culture and allowed to grow 16 h before harvesting by centrifugation (13,000 × g, 30 min). The cells were washed once with PBS, resuspended in 400 ml of water, and boiled for 30 min with stirring on a hot plate. The mixture was cooled, the cells were pelleted by centrifugation (as above), the supernatant was removed, and the pellet was subjected to phenol:hot water extraction according to the method of Westphal and Jann (
      • Westphal O.
      • Jann K.
      Bacterial lipopolysaccharides: extraction with phenol water and further applications of the procedure.
      ) with modifications. The pellet was resuspended in 200 ml of saline (125 mm NaCl) and combined with 200 ml of liquified phenol preheated in a 70 °C water bath, and the mixture was incubated with stirring for 1 h. The mixture was cooled on ice and centrifuged (13,000 × g for 30 min) to separate the aqueous and phenol phases, and the phenol phase was dialyzed against tap water for 5 days and then lyophilized. The lyophilized sample was resuspended in 100 ml of water, subjected to centrifugation at 13,000 × g for 30 min, and then placed in an ultracentrifuge for 16 h. After removing the supernatant, the clear pellet was resuspended again in water and repelleted by ultracentrifugation (as above) to remove residual traces of the supernatant, resuspended in 20 ml of water, and lyophilized. The isolated compounds used for NMR were compared with the proteinase K–resistant antigenic molecules as observed in Western immunoblots.

      Affinity purification of antibodies against the purified conserved HN13 polysaccharide from chicken α-HN13 antiserum

      Antibodies specifically targeting the conserved polysaccharide from C. perfringens HN13 were affinity-purified from unadsorbed chicken α-HN13 antiserum against the purified polysaccharide from that strain according to the method of Salamitou et al. (
      • Salamitou S.
      • Lemaire M.
      • Fujino T.
      • Ohayon H.
      • Gounon P.
      • Béguin P.
      • Aubert J.P.
      Subcellular localization of Clostridium thermocellum ORF3p, a protein carrying a receptor for the docking sequence borne by the catalytic components of the cellulosome.
      ) with minor modifications. Briefly, the purified conserved HN13 polysaccharide was separated by SDS-PAGE, transferred electrophoretically to nitrocellulose, and blocked overnight with InterceptTM blocking buffer (LI-COR). The membrane was trimmed to the region containing the purified polysaccharide (as determined by Western immunoblotting analysis of thin strips trimmed from both the left and right sides of the membrane), for use as the affinity substratum. Antiserum raised against whole cells of C. perfringens HN13 were incubated (1:100 in PBST) with the trimmed membrane for 4 h, washed extensively with PBST, and incubated with 0.2 m glycine, pH 2.2, to elute bound antibodies. After 2 min, the eluate was removed, neutralized with 1 m K2HPO4, and desalted on a PD-10 into Intercept:PBST (1:1) and then used in immunoblotting as previously described.

      Composition analysis of the purified surface polysaccharides from C. perfringens HN13 and JGS4143

      The composition of the glycolipids isolated from these two strains (as described above) was determined by combined GC-MS of per-O-TMS derivatives of the monosaccharide methyl glycosides and fatty acid methyl esters produced by acid methanolysis of the samples as described by Merkle and Poppe (1994) (
      • Merkle R.K.
      • Poppe I.
      Carbohydrate composition analysis of glycoconjugates by gas-liquid chromatography/mass spectrometry.
      ). Briefly, lyophilized HN13 and JGS4143 glycolipids were heated with methanolic 1 m HCl in a sealed screw-top glass test tube for 18 h at 80 °C. After cooling and removal of the solvent under a stream of nitrogen, the samples were treated with a mixture of methanol, pyridine, and acetic anhydride for 30 min. The solvents were evaporated, and the samples were derivatized with Tri-Sil® (Pierce) at 80 °C for 30 min. GC-MS analysis of the TMS methyl glycosides was performed on an Agilent 7890A GC interfaced to a 5975 °C MSD, using a Supelco Equity-1 fused silica capillary column (30-m × 0.25-mm inner diameter).

      NMR spectroscopy of purified C. perfringens polysaccharide

      To prepare samples for NMR spectroscopy, all purified glycolipids were deacylated as follows: lyophilized samples were dissolved in concentrated NH4OH, incubated at 80 °C for 1 h, allowed to cool, and lyophilized. The lyophilized material was dissolved in distilled water and fractionated on a BioGel P6 column using deionized water as the eluent. Fractions were collected based on response from a refractive index detector, lyophilized, and then washed three times with dichloromethane to completely remove free fatty acids from the samples. Delipidated polysaccharide was analyzed by 1D/2D NMR spectroscopy; proton, HSQC, COSY, TOCSY (mixing time was 50, 80, or 150 ms), NOESY (mixing time 100 or 300 ms), 1H-13C HMBC, and 1H-31P HMBC spectra were collected. These experiments allowed assignment of the proton, carbon, and phosphorus chemical shifts of each residue and also the determination of their linkages, sequence, and the substitution positions of the PEtN and PGro substituents.
      To get more information about the structure, the HN13 polysaccharide was dephosphorylated by dissolving the lyophilized delipidated sample in 48% HF and incubating at 4 °C for 48 h, followed by evaporation of the sample on ice and lyophilized once more. The generated product mixture was subjected to size-exclusion chromatography on a Bio-Gel P6 column, as described above, prior to 1D/2D NMR analyses.
      For all NMR experiments, lyophilized samples were dissolved in 0.17–0.2 ml of D2O, and transferred into a 3-mm NMR tube. 1H and 13C NMR data were recorded on a Varian Inova spectrometer (1H, 599.73 MHz) equipped with a 3-mm cold probe (Varian Inova, Palo Alto, CA) or a Bruker Avance III spectrometer (1H, 600.13 MHz) equipped with a 5-mm cryoprobe (Bruker Biospin, Billerica, MA). 31P NMR data were recorded on a Varian Inova spectrometer (1H, 500 MHz) equipped with a standard 3-mm HX probe. 1D proton spectra were acquired at 25, 30, and 60 °C with presaturation solvent signal suppression. All spectra were acquired with standard pulse sequences included in the spectrometer software. The NMR acquisitions were processed using MNova software (Mestrelab Research, Spain). The spectra were referenced relative to the 4,4-dimethyl-4-silapentane-1-sulfonic acid signal (δH = 0 ppm; δC = 0 ppm) and to external phosphoric acid (δP = 0 ppm).

      MALDI-TOF MS analysis of C. perfringens polysaccharide

      For MALDI-TOF MS analysis, the samples were dissolved in water, mixed 1:1 (v/v) with 0.5 m 2,4,6-trihydroxyacetophenone monohydrate matrix in methanol, and spotted onto a MALDI plate. MALDI-TOF MS spectra were acquired on an Applied Biosystems AB SCIEX TOF/TOF 5800 system in the positive mode and processed with Data Explorer (Applied Biosystems).

      Passive protection of chickens against C. perfringens–mediated killing

      For passive protection experiments, SPF leghorn chicks were challenged at 1 day of age with C. perfringens in the presence and absence of chicken α-C. perfringens antiserum as follows. To prepare the oral gavage solutions, the chicken NE strain C. perfringens JGS4143 was streaked on PGY agar the day before gavage (day 0) and grown overnight as described above. On the day of gavage (day 1), the cells were harvested in PBS, pelleted by centrifugation at 13,000 × g for 30 min, and washed twice with PBS. The washed cell pellet was resuspended to ∼3.7 × 109 cells/ml in PBS, and separately a 1/10 dilution of the highly cross-reactive chicken α-C. perfringens HN13 antiserum in PBS was prepared. The C. perfringens JGS4143 cell suspension was then mixed 9:1 with either PBS or the diluted chicken α-C. perfringens antiserum immediately prior to gavage, as appropriate. In total, nine birds were orally gavaged with 300 μl of the C. perfringens/PBS mixture without antiserum (1 × 109 cells), nine birds were orally gavaged with 300 μl of the C. perfringens/PBS mixture containing antiserum (1 × 109 cells), and five birds were orally gavaged with PBS alone as a control, and bird mortality was monitored over 7 days.

      Histology and fluorescent microscopy

      During tissue collection, a set of organs was removed from each bird according to the research protocol. The removed tissues were immediately fixed in 10% neutral buffered formalin for a minimum of 24 h. Following fixation, the tissues were trimmed with a scalpel to a thickness of 2–3 mm, and a section of each organ was placed in a tissue cassette. Tissues in cassettes were processed into paraffin, embedded in a paraffin block, sectioned on a microtome to a thickness of 5 μm, placed on a microscope slide, and stained with hematoxylin and eosin, with all procedures following standard histology techniques. The slides were examined by a board-certified veterinary pathologist. Either each section of tissue was recorded as normal, or a description was made of any abnormalities. Photomicrographs were taken with a Moticam 10 digital camera mounted on a Nikon Eclipse 80i microscope. Immunofluorescent staining of paraffin-embedded tissue sections was performed as described previously (
      • Patry R.T..
      • Stahl M.
      • Perez-Munoz M.E.
      • Nothaft H.
      • Wenzel C.Q.
      • Sacher J.C.
      • Coros C.
      • Walter J.
      • Vallance B.A.
      • Szymanski C.M.
      Bacterial AB(5) toxins inhibit the growth of gut bacteria by targeting ganglioside-like glycoconjugates.
      ). After deparaffinization and antigen retrieval, the slides were probed with adsorbed rabbit α-C. perfringens HN13 antiserum (1:1000 in PBS) and Alexa Fluor 488–conjugated α-rabbit antiserum (1:500 in PBS; Invitrogen). Incubations with antibodies were done at room temperature for 10–15 min, and the slides were washed three times (3 min each) with PBS after each antibody incubation step. The slides were counterstained with 4′,6-diamidino-2-phenylindole (1 μg ml − 1, included in the third washing step) and analyzed on a DeltaVision fluorescent microscope equipped with an scientific complementary metal–oxide–semiconductor camera.

      Opsonophagocytosis assay using fresh heparinized chicken blood

      For opsonophagocytosis assays, C. perfringens JGS4143 cells were incubated with heparinized chicken blood and either naïve chicken serum or α-C. perfringens HN13 antiserum according to the method previously described by (
      • Goyette-Desjardins G.
      • Calzas C.
      • Shiao T.C.
      • Neubauer A.
      • Kempker J.
      • Roy R.
      • Gottschalk M.
      • Segura M.
      Protection against Streptococcus suis serotype 2 infection using a capsular polysaccharide glycoconjugate vaccine.
      ) with modifications, as follows. To prepare the bacterial cells for this assay, the chicken NE strain C. perfringens JGS4143 was streaked on PGY agar the day before the cull of four 1-week-old broiler chickens (day 6) and four 5-week-old broiler chickens (day 34) as sources of fresh chicken blood and grown overnight as described above. On the cull and blood collection days (days 7 and 35), the cells were harvested in PBS, pelleted by centrifugation at 13,000 × g for 30 min, and washed twice with PBS. The washed cell pellet was resuspended to ∼2.9 × 105 cells/ml in RPMI 1640 medium supplemented with 5% heat-inactivated chicken serum, 10 mm HEPES, 2 mm l-glutamine, and 50 μM β-mercaptoethanol, and blood from each of the culled chickens was collected in a heparin-coated tube to prevent coagulation. The heparinized blood samples were diluted 1/3 in the supplemented RPMI 1640 listed above and used in reactions with sera from seven birds that were immunized with formalin-fixed C. perfringens HN13. For the reactions, each of the diluted blood samples (50 μl) was combined with 40 μl of either naïve chicken serum or one of the chicken α-C. perfringens HN13 antisera, followed by the addition of 10 μl of the C. perfringens JGS4143 suspension, resulting in an approximate multiplicity of infection of 0.015 based on 2.9 × 103 bacterial cells in the reaction and a calculated content of 1.9 × 105 leukocytes based on literature values of leukocytes in the blood of broiler chickens (Orawan and Aengwanich (2007) (
      • Orawan C.
      • Aengwanich W.
      Blood cell characteristics, hematological values and average daily gained weight of Thai indigenous crossbred and broiler chickens.
      )). The tops of the tubes were pierced using a sterile 25-gauge needle and then placed in a 5% CO2 incubator at 37 °C for 2 h, after which each reaction was combined with 80% sterile glycerol and incubated at −80 °C until ready to be plated. To enumerate the cells in each reaction, the samples were thawed on ice, and 100-μl aliquots of 10-fold serial dilutions were plated in triplicate on PGY agar and incubated under anaerobic conditions for 18 h. The percentage of bacterial killing values were calculated using the following formula: % bacteria killed = [(# of cells in naïve chicken serum reaction − # of cells recovered in the reaction of interest)/(# of cells in naïve chicken serum reaction)] × 100. Individual data points represent the % killing values for each serum calculated as the average of the bacterial killing values obtained for that antiserum with each of the four different day 7 or 35 heparinized blood samples.

      Data availability

      The source data for Fig. 8 are provided in the supporting information. The remaining spectral data for Figs. 3, 4, and 6, Figs. S8–11 and S13, and Tables S1–S5 are available from the corresponding authors. All the rest of the data are contained within the article.

      Acknowledgments

      We thank John Prescott for the C. perfringens field isolates, Hualan Liu for creating strain HLL8 (C. perfringens HN13 cpe2071), Jochen Zimmer for helpful discussions about synthases, and Arlene Oatway for processing of the chicken samples for microscopy.

      Supplementary Material

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