Genetic and structural identification of an O-acyltransferase gene (oacC) responsible for the 3/4-O-acetylation on rhamnose III in Shigella flexneri serotype 6

BMC Microbiology

Qiangzheng Sun¹  Jianguo Xu¹  Nan Zhang¹  Alexander S Shashkov²  Pengcheng Du¹  Anna M Shpirt²  Ruiting Lan³  Sofya N Senchenkova²  Xia Luo¹  Jianping Wang¹  Yuriy A Knirel² 

[1] State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, China CDC, Changping, Beijing, China;N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation;School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney NSW 2052, Australia

关键词: Anti-O-factor 9 serum;    O-antigen;    oacC;    Acyltransferase;    3/4-O-acetylation;    Shigella flexneri;   

Others  :  1137747
DOI  :  10.1186/s12866-014-0266-7

 received in 2014-06-30, accepted in 2014-10-09,  发布年份 2014

【 摘 要 】

Background

O-antigen (O-polysaccharide) of the lipopolysaccharide is a highly variable cell component of the outer membrane in Shigella flexneri. It defines the serospecificity and plays an important role in the pathogenesis of shigellosis. There are two distinct O-antigen forms for the 19 serotypes of S. flexneri: one for serotypes 1–5, X, Y, 7 (and their subtypes), and the other for serotype 6. Although having different basal O-polysaccharide structures, the two forms share a common disaccharide fragment [→2)-α-L-Rhap^III-(1 → 2)-α-L-Rhap^II]. In serotype 6 and some non-6 serotypes, Rha^III is O-acetylated at position either 3 or 4 (3/4-O-acetylation), conferring to the hosts a novel antigenic determinant named O-factor 9. An acyltransferase gene (oacB) responsible for this modification has been identified in serotypes 1a, 1b, 2a, 5a, and Y, but not in serotype 6.

Results

Using genetic, serological, and chemical approaches, another acyltransferase gene named oacC was demonstrated to be responsible for the 3/4-O-acetylation on Rha^III in the O-antigen of S. flexneri serotype 6. Inactivation of the oacC gene resulted in the loss of the 3/4-O-acetyltion, and the cloned oacC gene restored this modification upon transformation. In accordance with the similarity in the acceptor substrate structure and high sequence homology (72% identity) between oacC and oacB, oacC has the interchangeable function with the oacB gene in mediation of the 3/4-O-acetylation. The oacC gene is located in a prophage on the chromosome and presented in all 77 serotype 6 strains tested.

Conclusions

Identification and functional characterization of the O-acetyltransferase encoding gene, oacC, shows that it is involved in O-antigen modification by 3/4-O-acetylation on Rha^III specific to serotype 6.

【 授权许可】

   
2014 Knirel et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Bardhan P, Faruque AS, Naheed A, Sack DA: Decrease in shigellosis-related deaths without Shigella spp.-specific interventions, Asia. Emerg Infect Dis 2010, 16(11):1718-1723.
• [2]Morona R, Daniels C, Van Den Bosch L: Genetic modulation of Shigella flexneri 2a lipopolysaccharide O antigen modal chain length reveals that it has been optimized for virulence. Microbiology 2003, 149(Pt 4):925-939.
• [3]West NP, Sansonetti P, Mounier J, Exley RM, Parsot C, Guadagnini S, Prevost MC, Prochnicka-Chalufour A, Delepierre M, Tanguy M, Tang CM: Optimization of virulence functions through glucosylation of Shigella LPS. Science 2005, 307(5713):1313-1317.
• [4]Phalipon A, Tanguy M, Grandjean C, Guerreiro C, Belot F, Cohen D, Sansonetti PJ, Mulard LA: A synthetic carbohydrate-protein conjugate vaccine candidate against Shigella flexneri 2a infection. J Immunol 2009, 182(4):2241-2247.
• [5]Robbins JB, Kubler-Kielb J, Vinogradov E, Mocca C, Pozsgay V, Shiloach J, Schneerson R: Synthesis, characterization, and immunogenicity in mice of Shigella sonnei O-specific oligosaccharide-core-protein conjugates. Proc Natl Acad Sci U S A 2009, 106(19):7974-7978.
• [6]Kubler-Kielb J, Vinogradov E, Mocca C, Pozsgay V, Coxon B, Robbins JB, Schneerson R: Immunochemical studies of Shigella flexneri 2a and 6, and Shigella dysenteriae type 1 O-specific polysaccharide-core fragments and their protein conjugates as vaccine candidates. Carbohydr Res 2010, 345(11):1600-1608.
• [7]Passwell JH, Ashkenzi S, Banet-Levi Y, Ramon-Saraf R, Farzam N, Lerner-Geva L, Even-Nir H, Yerushalmi B, Chu C, Shiloach J, Robbins JB, Schneerson R: Age-related efficacy of Shigella O-specific polysaccharide conjugates in 1-4-year-old Israeli children. Vaccine 2010, 28(10):2231-2235.
• [8]Kenne L, Lindberg B, Petersson K, Katzenellenbogen E, Romanowska E: Structural studies of Shigella flexneri O-antigens. Eur J Biochem 1978, 91(1):279-284.
• [9]Foster RA, Carlin NI, Majcher M, Tabor H, Ng LK, Widmalm G: Structural elucidation of the O-antigen of the Shigella flexneri provisional serotype 88–893: structural and serological similarities with S. flexneri provisional serotype Y394 (1c). Carbohydr Res 2011, 346(6):872-876.
• [10]Allison GE, Verma NK: Serotype-converting bacteriophages and O-antigen modification in Shigella flexneri. Trends Microbiol 2000, 8(1):17-23.
• [11]Sun Q, Knirel YA, Lan R, Wang J, Senchenkova SN, Jin D, Shashkov AS, Xia S, Perepelov AV, Chen Q, Wang Y, Wang H, Xu J: A novel plasmid-encoded serotype conversion mechanism through addition of phosphoethanolamine to the O-antigen of Shigella flexneri. PLoS One 2012, 7(9):e46095.
• [12]Dmitriev BA, Knirel YA, Sheremet OK, Shashkov AA, Kochetkov NK, Hofman IL: Somatic antigens of Shigella. The structure of the specific polysaccharide of Shigella newcastle (Sh. flexneri type 6) lipopolysaccharide. Eur J Biochem 1979, 98(1):309-316.
• [13]Pupo GM, Lan R, Reeves PR: Multiple independent origins of Shigella clones of Escherichia coli and convergent evolution of many of their characteristics. Proc Natl Acad Sci U S A 2000, 97(19):10567-10572.
• [14]Pupo GM, Karaolis DK, Lan R, Reeves PR: Evolutionary relationships among pathogenic and nonpathogenic Escherichia coli strains inferred from multilocus enzyme electrophoresis and mdh sequence studies. Infect Immun 1997, 65(7):2685-2692.
• [15]Liu B, Knirel YA, Feng L, Perepelov AV, Senchenkova SN, Wang Q, Reeves PR, Wang L: Structure and genetics of Shigella O antigens. FEMS Microbiol Rev 2008, 32(4):627-653.
• [16]Wang J, Knirel YA, Lan R, Senchenkova SN, Luo X, Perepelov AV, Wang Y, Shashkov AS, Xu J, Sun Q: Identification of an O-acyltransferase gene (oacB) that mediates 3- and 4-O-acetylation of rhamnose III in Shigella flexneri O antigens. J Bacteriol 2014, 196(8):1525-1531.
• [17]Sun Q, Lan R, Wang Y, Wang J, Xia S, Wang Y, Zhang J, Yu D, Li Z, Jing H, Xu J: Identification of a divergent O-acetyltransferase gene oac1b from Shigella flexneri serotype 1b strains. Emerg Microbes Infect 2012, 1(9):e21.
• [18]Perepelov AV, Shekht ME, Liu B, Shevelev SD, Ledov VA, Senchenkova SN, L’Vov VL, Shashkov AS, Feng L, Aparin PG, Wang L, Knirel YA: Shigella flexneri O-antigens revisited: final elucidation of the O-acetylation profiles and a survey of the O-antigen structure diversity. FEMS Immunol Med Microbiol 2012, 66(2):201-210.
• [19]Sun Q, Knirel YA, Lan R, Wang J, Senchenkova SN, Shashkov AS, Wang Y, Wang Y, Luo X, Xu J: Dissemination and serotype modification potential of pSFxv_2, an O-antigen PEtN modification plasmid in Shigella flexneri. Glycobiology 2014, 24(3):305-313.
• [20]Wang J, Lan R, Knirel YA, Luo X, Senchenkova SN, Shashkov AS, Xu J, Sun Q: Serological identification and prevalence of a novel O-antigen epitope linked to 3- and 4-O-acetylated rhamnose III of lipopolysaccharide in Shigella flexneri. J Clin Microbiol 2014, 52(6):2033-2038.
• [21]Cheah KC, Beger DW, Manning PA: Molecular cloning and genetic analysis of the rfb region from Shigella flexneri type 6 in Escherichia coli K-12. FEMS Microbiol Lett 1991, 67(2):213-218.
• [22]Slauch JM, Lee AA, Mahan MJ, Mekalanos JJ: Molecular characterization of the oafA locus responsible for acetylation of Salmonella typhimurium O-antigen: oafA is a member of a family of integral membrane trans-acylases. J Bacteriol 1996, 178(20):5904-5909.
• [23]Thanweer F, Tahiliani V, Korres H, Verma NK: Topology and identification of critical residues of the O-acetyltransferase of serotype-converting bacteriophage, SF6, of Shigella flexneri. Biochem Biophys Res Commun 2008, 375(4):581-585.
• [24]Simons RW, Houman F, Kleckner N: Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 1987, 53(1):85-96.
• [25]Pradel N, Ye C, Livrelli V, Xu J, Joly B, Wu LF: Contribution of the twin arginine translocation system to the virulence of enterohemorrhagic Escherichia coli O157:H7. Infect Immun 2003, 71(9):4908-4916.
• [26]Datsenko KA, Wanner BL: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 2000, 97(12):6640-6645.
• [27]Hygge Blakeman K, Weintraub A, Widmalm G: Structural determination of the O-antigenic polysaccharide from the enterotoxigenic Escherichia coli O147. Eur J Biochem 1998, 251(1–2):534-537.
• [28]Jansson PE, Kenne L, Schweda E: Nuclear magnetic resonance and conformational studies on monoacetylated methyl D-gluco-and D-galacto-pyranosides. J Chem Soc Perkin Trans 1987, 1(2):377-383.
• [29]Casjens S, Winn-Stapley DA, Gilcrease EB, Morona R, Kuhlewein C, Chua JE, Manning PA, Inwood W, Clark AJ: The chromosome of Shigella flexneri bacteriophage Sf6: complete nucleotide sequence, genetic mosaicism, and DNA packaging. J Mol Biol 2004, 339(2):379-394.
• [30]Sambrook J, Fritsch EF: T. Maniatis: Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York; 1989.
• [31]Morona R, Brown MH, Yeadon J, Heuzenroeder MW, Manning PA: Effect of lipopolysaccharide core synthesis mutations on the production of Vibrio cholerae O-antigen in Escherichia coli K-12. FEMS Microbiol Lett 1991, 66(3):279-285.
• [32]Westphal O, Jann K: Bacterial lipopolysaccharides. Extraction with phenol-water and further applications of the procedure. Meth Carbohydr Chem 1965, 5:83-90.
• [33]Knirel YA, Lan R, Senchenkova SN, Wang J, Shashkov AS, Wang Y, Perepelov AV, Xiong Y, Xu J, Sun Q: O-antigen structure of Shigella flexneri serotype Yv and effect of the lpt-O gene variation on phosphoethanolamine modification of S. flexneri O-antigens. Glycobiology 2013, 23(4):475-485.