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• W2897320609 abstract "Nonulosonic acids (NulOs) are a diverse family of α-keto acid carbohydrates present across all branches of life. Bacteria biosynthesize NulOs among which are several related prokaryotic-specific isomers and one of which, N-acetylneuraminic acid (sialic acid), is common among all vertebrates. Bacteria display various NulO carbohydrates on lipopolysaccharide (LPS), and the identities of these molecules tune host–pathogen recognition mechanisms. The opportunistic bacterial pathogen Vibrio vulnificus possesses the genes for NulO biosynthesis; however, the structures and functions of the V. vulnificus NulO glycan are unknown. Using genetic and chemical approaches, we show here that the major NulO produced by a clinical V. vulnificus strain CMCP6 is 5-N-acetyl-7-N-acetyl-d-alanyl-legionaminic acid (Leg5Ac7AcAla). The CMCP6 strain could catabolize modified legionaminic acid, whereas V. vulnificus strain YJ016 produced but did not catabolize a NulO without the N-acetyl-d-alanyl modification. In silico analysis suggested that Leg5Ac7AcAla biosynthesis follows a noncanonical pathway but appears to be present in several bacterial species. Leg5Ac7AcAla contributed to bacterial outer-membrane integrity, as mutant strains unable to produce or incorporate Leg5Ac7AcAla into the LPS have increased membrane permeability, sensitivity to bile salts and antimicrobial peptides, and defects in biofilm formation. Using the crustacean model, Artemia franciscana, we demonstrate that Leg5Ac7AcAla-deficient bacteria have decreased virulence potential compared with WT. Our data indicate that different V. vulnificus strains produce multiple NulOs and that the modified legionaminic acid Leg5Ac7AcAla plays a critical role in the physiology, survivability, and pathogenicity of V. vulnificus CMCP6. Nonulosonic acids (NulOs) are a diverse family of α-keto acid carbohydrates present across all branches of life. Bacteria biosynthesize NulOs among which are several related prokaryotic-specific isomers and one of which, N-acetylneuraminic acid (sialic acid), is common among all vertebrates. Bacteria display various NulO carbohydrates on lipopolysaccharide (LPS), and the identities of these molecules tune host–pathogen recognition mechanisms. The opportunistic bacterial pathogen Vibrio vulnificus possesses the genes for NulO biosynthesis; however, the structures and functions of the V. vulnificus NulO glycan are unknown. Using genetic and chemical approaches, we show here that the major NulO produced by a clinical V. vulnificus strain CMCP6 is 5-N-acetyl-7-N-acetyl-d-alanyl-legionaminic acid (Leg5Ac7AcAla). The CMCP6 strain could catabolize modified legionaminic acid, whereas V. vulnificus strain YJ016 produced but did not catabolize a NulO without the N-acetyl-d-alanyl modification. In silico analysis suggested that Leg5Ac7AcAla biosynthesis follows a noncanonical pathway but appears to be present in several bacterial species. Leg5Ac7AcAla contributed to bacterial outer-membrane integrity, as mutant strains unable to produce or incorporate Leg5Ac7AcAla into the LPS have increased membrane permeability, sensitivity to bile salts and antimicrobial peptides, and defects in biofilm formation. Using the crustacean model, Artemia franciscana, we demonstrate that Leg5Ac7AcAla-deficient bacteria have decreased virulence potential compared with WT. Our data indicate that different V. vulnificus strains produce multiple NulOs and that the modified legionaminic acid Leg5Ac7AcAla plays a critical role in the physiology, survivability, and pathogenicity of V. vulnificus CMCP6. Nonulosonic acids (NulOs), 4The abbreviations used are: NulOnonulosonic acidLeg5Ac7AcAla5-N-acetyl-7-N-acetyl-d-alanyl-legionaminic acidLeglegionaminic acidAceacinetaminic acidFusfusaminic acidPmBpolymyxin BLeg5Ac7Ala5-N-acetyl-7-d-alanyl-legionaminic acidDMB1,2-diamino-4,5-methylene dioxybenzenePGpeptidoglycanLPSlipopolysaccharideCPScapsule polysaccharideHR LCMShigh-resolution LC-MSESIelectrospray ionization. which include the sialic acids, are a family of nine carbon α-keto sugars that serve as critical recognition elements between cells. The most common sugar, N-acetylneuraminic acid (Neu5Ac) (otherwise known as sialic acid) (1Angata T. Varki A. Chemical diversity in the sialic acids and related α-keto acids: an evolutionary perspective.Chem. Rev. 2002; 102 (11841250): 439-46910.1021/cr000407mCrossref PubMed Scopus (1036) Google Scholar), is found in most lineages of metazoans and performs a wide range of functions in eukaryotes such as cell to cell communication (Fig. 1). 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Although sialic acid is found in eukaryotes and some prokaryotes, it is not the predominant NulO produced in bacteria. Nine carbon α-keto sugars bearing a strong resemblance to sialic acid such as derivatives from 5,7-diamino-3,5,7,9-tetradeoxy-non-2-ulosonic acids (Fig. 1; Fig. S1) have been characterized as prokaryote-specific NulOs. Traditionally, these prokaryote-specific NulOs are further categorized into one of four classes of carbohydrates based on their stereochemistry: pseudaminic, legionaminic (Leg), or two epimers of Leg, 4-epi-legionaminic acid and 8-epi-legionaminic acid (25Tsvetkov Y.E. Shashkov A.S. Knirel Y.A. Zähringer U. Synthesis and identification in bacterial lipopolysaccharides of 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto- and -d-glycero-d-talo-non-2-ulosonic acids.Carbohydr. Res. 2001; 331 (11383892): 233-23710.1016/S0008-6215(01)00041-6Crossref PubMed Scopus (58) Google Scholar, 26Knirel Y.A. Vinogradov E.V. L'Vov V.L. Kocharova N.A. Shashkov A.S. 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Hall R.M. 5,7-Di-N-acetyl-acinetaminic acid: a novel non-2-ulosonic acid found in the capsule of an Acinetobacter baumannii isolate.Glycobiology. 2015; 25 (25595948): 644-65410.1093/glycob/cwv007Crossref PubMed Scopus (47) Google Scholar, 29Kenyon J.J. Notaro A. Hsu L.Y. De Castro C. Hall R.M. 5,7-Di-N-acetyl-8-epiacinetaminic acid: a new non-2-ulosonic acid found in the K73 capsule produced by an Acinetobacter baumannii isolate from Singapore.Sci. Rep. 2017; 7 (28900250)1135710.1038/s41598-017-11166-4Crossref PubMed Scopus (24) Google Scholar30Vinogradov E. St Michael F. Cox A.D. The structure of the LPS O-chain of Fusobacterium nucleatum strain 25586 containing two novel monosaccharides, 2-acetamido-2,6-dideoxy-l-altrose and a 5-acetimidoylamino-3,5,9-trideoxy-gluco-non-2-ulosonic acid.Carbohydr. Res. 2017; 440 (28135570): 10-15Crossref PubMed Scopus (22) Google Scholar). The prokaryotic NulOs have been identified in various surface structures, including the LPS and CPS as well as N- and O-linked glycans on flagella and pili (31Thibault P. Logan S.M. Kelly J.F. Brisson J.R. Ewing C.P. Trust T.J. Guerry P. Identification of the carbohydrate moieties and glycosylation motifs in Campylobacter jejuni flagellin.J. Biol. Chem. 2001; 276 (11461915): 34862-3487010.1074/jbc.M104529200Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar32Schirm M. Soo E.C. Aubry A.J. Austin J. Thibault P. Logan S.M. Structural, genetic and functional characterization of the flagellin glycosylation process in Helicobacter pylori.Mol. Microbiol. 2003; 48 (12791140): 1579-159210.1046/j.1365-2958.2003.03527.xCrossref PubMed Scopus (222) Google Scholar, 33Knirel Y.A. Shashkov A.S. Tsvetkov Y.E. Jansson P.E. Zãhringer U. 5,7-Diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids in bacterial glycopolymers: chemistry and biochemistry.Adv. Carbohydr. Chem. Biochem. 2003; 58 (14719362): 371-41710.1016/S0065-2318(03)58007-6Crossref PubMed Scopus (120) Google Scholar, 34MacLean L.L. Vinogradov E. Pagotto F. Perry M.B. Characterization of the lipopolysaccharide O-antigen of Cronobacter turicensis HPB3287 as a polysaccharide containing a 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic acid (legionaminic acid) residue.Carbohydr. Res. 2011; 346 (21963342): 2589-259410.1016/j.carres.2011.09.003Crossref PubMed Scopus (17) Google Scholar, 35Twine S.M. Paul C.J. Vinogradov E. McNally D.J. Brisson J.R. Mullen J.A. McMullin D.R. Jarrell H.C. Austin J.W. Kelly J.F. Logan S.M. Flagellar glycosylation in Clostridium botulinum.FEBS J. 2008; 275 (18671733): 4428-444410.1111/j.1742-4658.2008.06589.xCrossref PubMed Scopus (65) Google Scholar, 36Friedrich V. Janesch B. Windwarder M. Maresch D. Braun M.L. Megson Z.A. Vinogradov E. Goneau M.F. Sharma A. Altmann F. Messner P. Schoenhofen I.C. Schäffer C. Tannerella forsythia strains display different cell-surface nonulosonic acids: biosynthetic pathway characterization and first insight into biological implications.Glycobiology. 2017; 27 (27986835): 342-357PubMed Google Scholar37Kurniyati K. Kelly J.F. Vinogradov E. Robotham A. Tu Y. Wang J. Liu J. Logan S.M. Li C. A novel glycan modifies the flagellar filament proteins of the oral bacterium Treponema denticola.Mol. Microbiol. 2017; 103 (27696564): 67-8510.1111/mmi.13544Crossref PubMed Scopus (20) Google Scholar). Although significant work has investigated the role of sialic acid in molecular mimicry and host evasion, the chemical composition and role of bacteria-specific NulOs in host–pathogen interactions remain under-explored. In addition to the seven isomers of the prokaryote-specific NulOs described, these carbohydrates can display various functionalized N- and O-substitutions such as acetylation. These diverse modifications may further contribute to multiple pathogen–host interaction mechanisms in vivo and in the environment (38Matthew Z. Kiefel M. The occurrence and biological significance of the α-keto-sugars pseudaminic acid and legionaminic acid within pathogenic bacteria.RSC Adv. 2013; 4: 3413-3421Google Scholar). The wide array of structural variants and the newest discoveries Ace and Fus motivated our efforts to discover unique functional groups and novel isomers within the model organism Vibrio vulnificus. Previous work from our group confirmed that strains of V. vulnificus, a marine bacterium and human opportunistic pathogen, produced NulOs; however, the genomic region responsible for the NulO biosynthesis was highly variable within the species (Fig. 2A) (39Lewis A.L. Lubin J.B. Argade S. Naidu N. Choudhury B. Boyd E.F. Genomic and metabolic profiling of nonulosonic acids in Vibrionaceae reveal biochemical phenotypes of allelic divergence in Vibrio vulnificus.Appl. Environ. Microbiol. 2011; 77 (21724895): 5782-579310.1128/AEM.00712-11Crossref PubMed Scopus (20) Google Scholar). One study fully elucidated the inner core of the LPS from the V. vulnificus strain ATCC 27562, which was found to have pseudaminic acid as a carbohydrate component (40Vinogradov E. Wilde C. Anderson E.M. Nakhamchik A. Lam J.S. Rowe-Magnus D.A. Structure of the lipopolysaccharide core of Vibrio vulnificus type strain 27562.Carbohydr. Res. 2009; 344 (19185290): 484-49010.1016/j.carres.2008.12.017Crossref PubMed Scopus (22) Google Scholar). Biochemical analysis of V. vulnificus strains with divergent NulO biosynthetic loci revealed that highly variable levels of NulO were produced depending on the genotype; however, no detailed NulO structural information was elucidated (39Lewis A.L. Lubin J.B. Argade S. Naidu N. Choudhury B. Boyd E.F. Genomic and metabolic profiling of nonulosonic acids in Vibrionaceae reveal biochemical phenotypes of allelic divergence in Vibrio vulnificus.Appl. Environ. Microbiol. 2011; 77 (21724895): 5782-579310.1128/AEM.00712-11Crossref PubMed Scopus (20) Google Scholar). Two V. vulnificus strains in particular, CMCP6 and YJ016, were studied in more detail. These two strains are nearly identical; however, the NulO biosynthetic region is unrelated (Fig. 2A). V. vulnificus strains with the CMCP6-like NulO genes produce ∼100-fold more NulO than strains with the YJ016-like genes (41Lubin J.B. Lewis W.G. Gilbert N.M. Weimer C.M. Almagro-Moreno S. Boyd E.F. Lewis A.L. Host-like carbohydrates promote bloodstream survival of Vibrio vulnificus in vivo.Infect. Immun. 2015; 83 (26015477): 3126-313610.1128/IAI.00345-15Crossref PubMed Scopus (18) Google Scholar). Efforts from our laboratory to connect the genetic NulO fingerprint with V. vulnificus pathogenicity revealed that NulO-deficient mutants have significantly increased sensitivity to the bacterial antimicrobial peptide polymyxin B (PmB), defects in swimming and biofilm formation, and are attenuated in a mouse bloodstream model of infection (41Lubin J.B. Lewis W.G. Gilbert N.M. Weimer C.M. Almagro-Moreno S. Boyd E.F. Lewis A.L. Host-like carbohydrates promote bloodstream survival of Vibrio vulnificus in vivo.Infect. Immun. 2015; 83 (26015477): 3126-313610.1128/IAI.00345-15Crossref PubMed Scopus (18) Google Scholar). The extent of bacterial bloodstream survival and dissemination was proportional to the amount of NulO expressed by the WT strain (NulO expression: CMCP6 ≫ YJ016) (41Lubin J.B. Lewis W.G. Gilbert N.M. Weimer C.M. Almagro-Moreno S. Boyd E.F. Lewis A.L. Host-like carbohydrates promote bloodstream survival of Vibrio vulnificus in vivo.Infect. Immun. 2015; 83 (26015477): 3126-313610.1128/IAI.00345-15Crossref PubMed Scopus (18) Google Scholar). In addition, V. vulnificus, mutants in the NulO biosynthetic pathway were defective in virulence (42Yamamoto M. Kashimoto T. Tong P. Xiao J. Sugiyama M. Inoue M. Matsunaga R. Hosohara K. Nakata K. Yokota K. Oguma K. Yamamoto K. Signature-tagged mutagenesis of Vibrio vulnificus.J. Vet. Med. Sci. 2015; 77 (25755021): 823-82810.1292/jvms.14-0655Crossref PubMed Scopus (5) Google Scholar). Given the correlation between NulO display and bacterial pathogenesis, we chemically characterized the carbohydrate biosynthesized in strain CMCP6, which produces higher levels of NulO (39Lewis A.L. Lubin J.B. Argade S. Naidu N. Choudhury B. Boyd E.F. Genomic and metabolic profiling of nonulosonic acids in Vibrionaceae reveal biochemical phenotypes of allelic divergence in Vibrio vulnificus.Appl. Environ. Microbiol. 2011; 77 (21724895): 5782-579310.1128/AEM.00712-11Crossref PubMed Scopus (20) Google Scholar, 41Lubin J.B. Lewis W.G. Gilbert N.M. Weimer C.M. Almagro-Moreno S. Boyd E.F. Lewis A.L. Host-like carbohydrates promote bloodstream survival of Vibrio vulnificus in vivo.Infect. Immun. 2015; 83 (26015477): 3126-313610.1128/IAI.00345-15Crossref PubMed Scopus (18) Google Scholar). We found that a modified legionaminic acid is produced in strain CMCP6, with an N-acetyl-d-alanyl group at C-7 (Leg5Ac7AcAla, Fig. 1). Through gene deletion analysis, we demonstrate that the modified Leg, 5-N-acetyl-7-d-alanyl-legionaminic acid (Leg5Ac7Ala), lacking an acetyl group, is catabolized via the canonical sialic acid pathway. Further analysis of deletion mutants demonstrate that biosynthesis of the acetylated d-alanine carbohydrate is essential for outer membrane integrity, stress survival, and virulence in the brine shrimp model Artemia franciscana. To begin to determine the structure of V. vulnificus NulOs embedded in the sequence and genomic organization of the biosynthesis pathways, we first investigated whether the NulO biosynthesis genomic loci were homologous to previously characterized pathways. The predicted biosynthesis operon of strain CMCP6 was similar in gene arrangement and amino acid composition with the biosynthetic region from Escherichia coli O161 (Fig. 2B). Specifically, the Nab1, Nab2, and Nab3 proteins of CMCP6 shared 54, 81, and 73% amino acid identity with Lea7, Lea4, and Lea5 of E. coli O161. The LPS from E. coli O161 contains a 5-N-acetyl-7-d-alanyl-legionaminic acid (Leg5Ac7Ala) (43Li X. Perepelov A.V. Wang Q. Senchenkova S.N. Liu B. Shevelev S.D. Guo X. Shashkov A.S. Chen W. Wang L. Knirel Y.A. Structural and genetic characterization of the O-antigen of Escherichia coli O161 containing a derivative of a higher acidic diamino sugar, legionaminic acid.Carbohydr. Res. 2010; 345 (20510395): 1581-158710.1016/j.carres.2010.04.008Crossref PubMed Scopus (20) Google Scholar). The Leg5Ac7Ala is modified with a d-alanine at C-7 compared with the typical acetamido group in legionaminic acid (Fig. 2C). Interestingly, the operon of CMCP6 contained a putative N-acetyltransferase (i.e. nab5), which was absent from the E. coli operon (Fig. 2B). We hypothesized that this N-acetyltransferase would acetylate the free amine of the d-alanine, resulting in a NulO with a 7-N-acetyl-d-alanyl modification at C-7 (Fig. 2C). To determine whether CMCP6 does in fact produce this modified Leg, the NulOs were released from CMCP6 via mild acid hydrolysis in 2 n acetic acid, derivatized with the fluorescent molecule 1,2-diamino-4,5-methylene dioxybenzene (DMB), and separated via HPLC as described previously (Fig. 3A) (41Lubin J.B. Lewis W.G. Gilbert N.M. Weimer C.M. Almagro-Moreno S. Boyd E.F. Lewis A.L. Host-like carbohydrates promote bloodstream survival of Vibrio vulnificus in vivo.Infect. Immun. 2015; 83 (26015477): 3126-313610.1128/IAI.00345-15Crossref PubMed Scopus (18) Google Scholar, 44Lewis A.L. Nizet V. Varki A. Discovery and characterization of sialic acid O-acetylation in group B Streptococcus.Proc. Natl. Acad. Sci. U.S.A. 2004; 101 (15263085): 11123-1112810.1073/pnas.0403010101Crossref PubMed Scopus (132) Google Scholar). The major NulO peak eluted at ∼17 min (Fig. 3B). The derivatized NulO was collected and analyzed by electrospray ionization MS (ESI-MS). The [M + H]+ of 522.17 m/z as well as the [M + Na]+ of 544.21 m/z from the NulO peak were consistent with the predicted modification that corresponds to a derivatized NulO modified with acetylated alanine (Leg5Ac7AcAla) (Fig. 3C). To characterize in more detail the native NulO structure, the LPS of WT CMCP6 was purified via the hot water/phenol method (45Westphal O. Jann K. Extraction with Phenol-Water and Further Applications of the Procedure.in: BeMiller J.N. Methods in Carbohydrate Chemistry. Academic Press, New York1965: 83-91Google Scholar), and the NulO was released from purified LPS of CMCP6 in 6% acetic acid for 4 h at 100 °C. The hydrolysate was separated by size-exclusion chromatography. The fractions containing NulOs, as indicated by staining with p-anisaldehyde via thin-layer LC (TLC), were collected and analyzed via high-resolution LC–MS (HR LCMS) to confirm the presence and identity of the native carbohydrate (Fig. 4). The expected ionization pattern for the native NulO (Leg5Ac7AcAla) was confirmed via HR LCMS to include the dehydrated mass [M − 18]+ of 388 m/z, [M + H]+ of 406 m/z, and [M + Na]+ of 428 m/z (Fig. 4). Detailed structural 1H NMR and 13C NMR analyses were conducted on the native NulO (Leg5Ac7AcAla) fractions. 460 mg of LPS was purified from 45 liters of culture, and the LPS was hydrolyzed in acetic acid to release the NulOs, which were further purified for NMR characterization in D2O (Fig. S2, A–D). The NMR analysis revealed the presence of the corresponding CH3 of the alanine functionality upfield (∼1.5 ppm) from hydrogens attached to α-carbon downfield (∼3.9 ppm) as indicated via 2D 1H NMR analysis (Fig. S2, C and D). These signature chemical shifts confirmed that the NulO produced by V. vulnificus CMCP6 contained an alanyl functionality. In tandem with the MS data and genomic analysis, the NulO structure was determined to be 5-N-acetyl-7-N-acetyl-d-alanyl-legionaminic acid (Leg5Ac7AcAla) (Fig. 2C). Our previous studies demonstrated that there are variable amounts of NulO produced depending on the genotype of V. vulnificus strains. In particular, strain CMCP6 produces 100-fold more NulO than YJ016 under the same culture conditions (Fig. 5, A and B) (41Lubin J.B. Lewis W.G. Gilbert N.M. Weimer C.M. Almagro-Moreno S. Boyd E.F. Lewis A.L. Host-like carbohydrates promote bloodstream survival of Vibrio vulnificus in vivo.Infect. Immun. 2015; 83 (26015477): 3126-313610.1128/IAI.00345-15Crossref PubMed Scopus (18) Google Scholar). When characterizing the biosynthetic loci of these strains, the Δnab1 deletion resulted in the opposite phenotype in which YJ016 produced more NulO than CMCP6. The Nab1 enzyme is a cytidine monophosphate (CMP)–NulO synthetase and catalyzes the transfer of CMP from CTP to activate the NulO for use by a downstream glycosyltransferase, ultimately linking the sugar to LPS (46Schoenhofen I.C. Vinogradov E. Whitfield D.M. Brisson J.R. Logan S.M. The CMP-legionaminic acid pathway in campylobacter: biosynthesis involving novel GDP-linked precursors.Glycobiology. 2009; 19 (19282391): 715-72510.1093/glycob/cwp039Crossref PubMed Scopus (100) Google Scholar). No detectable NulO was observed in the HPLC analysis of DMB-derivatized total cellular fractions of the CMCP6 Δnab1 mutant (Fig. 5C), suggesting that the Leg5Ac7AcAla is catabolized by this strain. Conversely, in the YJ016 Δnab1 we observe accumulation of a NulO eluting at ∼14 min, which suggests that this carbohydrate is not catabolized (Fig. 5D). Mass spectrometry analysis of the" @default.
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• W2897320609 title "Structural and functional characterization of a modified legionaminic acid involved in glycosylation of a bacterial lipopolysaccharide" @default.
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