An atypical lipoteichoic acid from Clostridium perfringens elicits a broadly cross-reactive and protective immune response
2020; Elsevier BV; Volume: 295; Issue: 28 Linguagem: Inglês
10.1074/jbc.ra119.009978
ISSN1083-351X
AutoresCory Q. Wenzel, Dominic C. Mills, Justyna M. Dobruchowska, J. Vlach, Harald Nothaft, Patrick N. Nation, Parastoo Azadi, Stephen B. Melville, Russell W. Carlson, Mario F. Feldman, Christine M. Szymanski,
Tópico(s)Veterinary medicine and infectious diseases
ResumoClostridium 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 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. 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Sci. 2018; 97 (29762789): 1929-193310.3382/ps/pey082Crossref PubMed Scopus (32) Google Scholar). 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 (23Hughes J. Turnbull P. Stringer M. Serotyping system for Clostridium welchii (C. perfringens) typea and studies on type-specific antigens.J. Med. Microbiol. 1976; 9 (63553): 475-48510.1099/00222615-9-4-475Crossref PubMed Scopus (20) Google Scholar), 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 (24Cherniak R. Frederick H.M. Capsular polysaccharide of Clostridium perfringens Hobbs 9.Infect. Immun. 1977; 15 (192674): 765-77110.1128/IAI.15.3.765-771.1977Crossref PubMed Google Scholar) and the complete structures of the Hobbs 5 and Hobbs 10 CPS solved by NMR spectroscopy in 1997 (25Kalelkar S. Glushka J. van Halbeek H. Morris L.C. Cherniak R. Structure of the capsular polysaccharide of Clostridium perfringens Hobbs 5 as determined by NMR spectroscopy.Carbohydr. Res. 1997; 299 (9163894): 119-12810.1016/S0008-6215(97)00010-4Crossref PubMed Scopus (17) Google Scholar) and 1998 (26Sheng S. Cherniak R. Structure of the capsular polysaccharide of Clostridium perfringens Hobbs 10 determined by NMR spectroscopy.Carbohydr. Res. 1997; 305 (9534227): 65-7210.1016/S0008-6215(97)00280-2Crossref PubMed Scopus (15) Google Scholar), 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. (27Richter S.G. Elli D. Kim H.K. Hendrickx A.P. Sorg J.A. Schneewind O. Missiakas D. Small molecule inhibitor of lipoteichoic acid synthesis is an antibiotic for Gram-positive bacteria.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23401520): 3531-353610.1073/pnas.1217337110Crossref PubMed Scopus (67) Google Scholar) 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. (28Vinogradov E. Aubry A. Logan S. Structural characterization of wall and lipidated polysaccharides from Clostridium perfringens ATCC 13124.Carbohydr. Res. 2017; 448 (28628892): 88-9410.1016/j.carres.2017.06.003Crossref PubMed Scopus (5) Google Scholar), 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 (29Mot D. Timbermont L. Haesebrouck F. Ducatelle R. Van Immerseel F. Progress and problems in vaccination against necrotic enteritis in broiler chickens.Avian Pathol. 2014; 43 (24980518): 290-30010.1080/03079457.2014.939942Crossref PubMed Scopus (49) Google Scholar). 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 (30Jiang 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.Avian Dis. 2015; 59 (26629620): 475-48510.1637/11094-041715-RegCrossref PubMed Scopus (29) Google Scholar). 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. 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 (31Liu H. Bouillaut L. Sonenshein A. Melville S. Use of a mariner-based transposon mutagenesis system to isolate Clostridium perfringens mutants deficient in gliding motility.J. Bacteriol. 2013; 195 (23204460): 629-63610.1128/JB.01288-12Crossref PubMed Scopus (28) Google Scholar), 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 (32Marchler-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.Nucleic Acids Res. 2003; 31 (12520028): 383-38710.1093/nar/gkg087Crossref PubMed Scopus (650) Google Scholar) (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 (33McNamara J.T. Morgan J.L. Zimmer J. A molecular description of cellulose biosynthesis.Annu. Rev. Biochem. 2015; 84 (26034894): 895-92110.1146/annurev-biochem-060614-033930Crossref PubMed Scopus (165) Google Scholar). 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 (34Skurnik D. Cywes-Bentley C. Pier G. The exceptionally broad-based potential of active and passive vaccination targeting the conserved microbial surface polysaccharide PNAG.Exp.Rev. Vaccines. 2016; 15 (26918288): 1041-105310.1586/14760584.2016.1159135Crossref PubMed Scopus (32) Google Scholar). These similarities to the cellulose and poly-β-1,6-GlcNAc synthases, which both synthesize glycopolymers (35Gerke 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.J. Biol. Chem. 1998; 273 (9660830): 18586-1859310.1074/jbc.273.29.18586Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 36Omadjela 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.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (24127606): 17856-1786110.1073/pnas.1314063110Crossref PubMed Scopus (153) Google Scholar), indicate that the antigen is likely either a polysaccharide or a polysaccharide–containing glycolipid. 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 (37Chalmers 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.J. Clin. Microbiol. 2008; 46 (18945840): 3957-396410.1128/JCM.01548-08Crossref PubMed Scopus (99) Google Scholar), 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. 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. 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. 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 c
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