Skip to main content

Advertisement

SpringerLink
Log in

Structural comparison of O-antigen gene clusters of Legionella pneumophila and its application of a serogroup-specific multiplex PCR assay

  • Original Paper
  • Published:
Antonie van Leeuwenhoek Aims and scope Submit manuscript

Abstract

The Legionella pneumophila serogroups O1, O4, O6, O7, O10 and O13 are pathogenic strains associated with pneumonia. The surface O-antigen gene clusters of L. pneumophila serogroups O4, O6, O7, O10 and O13 were sequenced and analyzed, with the function annotated on the basis of homology to that of the genes of L. pneumophila serogroup O1 (L. pneumophila subsp. pneumophila str. Philadelphia 1). The gene locus of the six L. pneumophila serogroups contains genes of yvfE, neuABCD, pseA-like for nucleotide sugar biosynthesis, wecA for sugar transfer, and wzm as well as wzt for O-antigen processing. The detection of O-antigen genes allows the fine differentiation at species and serogroup level without the neccessity of nucleotide sequencing. The O-antigen-processing genes wzm and wzt, which were found to be distinctive for different for different serogroups, have been used as the target genes for the detection and identification of L. pneumophila strains of different O serogroups. In this report, a multiplex PCR assay based on wzm or wzt that diferentiates all the six serogroups by amplicon size was developed with the newly designed specific primer pairs for O1 and O7, and the specific primer pairs for O4, O6, O10, and O13 reported previously. The array was validated by analysis of 34 strains including 15 L. pneumophila O-standard reference strains, eight reference strains of other Legionella non-pneumophila species, six other bacterial species, and five L. pneumophila environmental isolates. The detection sensitivity was one ng genomic DNA. The accurate and sensitive assay is suitable for the identification and detection of strains of these serogroups in environmental and clinical samples.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bastin DA, Reeves PR (1995) Sequence and analysis of the O antigen gene (rfb) cluster of Escherichia coli O111. Gene 164(1):17–23

    Article  CAS  PubMed  Google Scholar 

  • Bateman A, Birney E, Cerruti L, Durbin R, Etwiller L, Eddy SR, Griffiths-Jones S, Howe KL, Marshall M, Sonnhammer EL (2002) The Pfam protein families database. Nucleic Acids Res 30(1):276–280

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, Brown CG, Hall KP, Evers DJ, Barnes CL, Bignell HR, Boutell JM, Bryant J, Carter RJ, Keira Cheetham R, Cox AJ, Ellis DJ, Flatbush MR, Gormley NA, Humphray SJ, Irving LJ, Karbelashvili MS, Kirk SM, Li H, Liu X, Maisinger KS, Murray LJ, Obradovic B, Ost T, Parkinson ML, Pratt MR, Rasolonjatovo IM, Reed MT, Rigatti R, Rodighiero C, Ross MT, Sabot A, Sankar SV, Scally A, Schroth GP, Smith ME, Smith VP, Spiridou A, Torrance PE, Tzonev SS, Vermaas EH, Walter K, Wu X, Zhang L, Alam MD, Anastasi C, Aniebo IC, Bailey DM, Bancarz IR, Banerjee S, Barbour SG, Baybayan PA, Benoit VA, Benson KF, Bevis C, Black PJ, Boodhun A, Brennan JS, Bridgham JA, Brown RC, Brown AA, Buermann DH, Bundu AA, Burrows JC, Carter NP, Castillo N, Chiara E Catenazzi M, Chang S, Neil Cooley R, Crake NR, Dada OO, Diakoumakos KD, Dominguez-Fernandez B, Earnshaw DJ, Egbujor UC, Elmore DW, Etchin SS, Ewan MR, Fedurco M, Fraser LJ, Fuentes Fajardo KV, Scott Furey W, George D, Gietzen KJ, Goddard CP, Golda GS, Granieri PA, Green DE, Gustafson DL, Hansen NF, Harnish K, Haudenschild CD, Heyer NI, Hims MM, Ho JT, Horgan AM, Hoschler K, Hurwitz S, Ivanov DV, Johnson MQ, James T, Huw Jones TA, Kang GD, Kerelska TH, Kersey AD, Khrebtukova I, Kindwall AP, Kingsbury Z, Kokko-Gonzales PI, Kumar A, Laurent MA, Lawley CT, Lee SE, Lee X, Liao AK, Loch JA, Lok M, Luo S, Mammen RM, Martin JW, McCauley PG, McNitt P, Mehta P, Moon KW, Mullens JW, Newington T, Ning Z, Ling Ng B, Novo SM, O’Neill MJ, Osborne MA, Osnowski A, Ostadan O, Paraschos LL, Pickering L, Pike AC, Pike AC, Chris Pinkard D, Pliskin DP, Podhasky J, Quijano VJ, Raczy C, Rae VH, Rawlings SR, Chiva Rodriguez A, Roe PM, Rogers J, Rogert Bacigalupo MC, Romanov N, Romieu A, Roth RK, Rourke NJ, Ruediger ST, Rusman E, Sanches-Kuiper RM, Schenker MR, Seoane JM, Shaw RJ, Shiver MK, Short SW, Sizto NL, Sluis JP, Smith MA, Ernest Sohna Sohna J, Spence EJ, Stevens K, Sutton N, Szajkowski L, Tregidgo CL, Turcatti G, Vandevondele S, Verhovsky Y, Virk SM, Wakelin S, Walcott GC, Wang J, Worsley GJ, Yan J, Yau L, Zuerlein M, Rogers J, Mullikin JC, Hurles ME, McCooke NJ, West JS, Oaks FL, Lundberg PL, Klenerman D, Durbin R, Smith AJ (2008) Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456(7218):53–59

  • Cao B, Yao F, Liu X, Feng L, Wang L (2013) Development of a DNA microarray method for detection and identification of all 15 distinct O-antigen forms of Legionella pneumophila. Appl Environ Microbiol 79(21):6647–6654

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cao B, Liu X, Yu X, Chen M, Feng L, Wang L (2014) A new oligonucleotide microarray for detection of pathogenic and non-pathogenic Legionella spp. PLoS ONE 9(12):e113863

    Article  PubMed  PubMed Central  Google Scholar 

  • Cazalet C, Rusniok C, Brüggemann H, Zidane N, Magnier A, Ma L, Tichit M, Jarraud S, Bouchier C, Vandenesch F, Kunst F, Etienne J, Glaser P, Buchrieser C (2004) Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity. Nat Genet 36(11):1165–1173

    Article  CAS  PubMed  Google Scholar 

  • Fields BS, Benson RF, Besser RE (2002) Legionella and Legionnaires disease: 25 years of investigation. Clin Microbiol Rev 15(3):506–526

    Article  PubMed  PubMed Central  Google Scholar 

  • Glaze PA, Watson DC, Young NM, Tanner ME (2008) Biosynthesis of CMP-N, N-diacetyllegionaminic acid from UDP-N,N -diacetylbacillosamine in Legionella pneumophila. Biochemistry 47(10):3272–3282

    Article  CAS  PubMed  Google Scholar 

  • Guan W, Xu Y, Chen DL, Xu JN, Tian Y, Chen JP (2012) Application of multilocus sequence analysis (MLSA) for accurate identification of Legionella spp. isolated from municipal fountains in Chengdu, China, based on 16S rRNA, mip, and rpoB genes. J Microbiol 50(1):127–136

    Article  CAS  PubMed  Google Scholar 

  • Hu D, Liu B, Dijkshoorn L, Wang L, Reeves PR (2013) Diversity in the major polysaccharide antigen of Acinetobacter baumannii assessed by DNA sequencing, and development of a molecular serotyping scheme. PLoS ONE 8(7):e70329

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Knirel YA, Rietschel ET, Marre R, Zahringer U (1994) The structure of the O-specific chain of Legionella pneumophila serogroup 1 lipopolysaccharide. Eur J Biochem 221(1):239–245

    Article  CAS  PubMed  Google Scholar 

  • Knirel YA, Senchenkova SN, Kocharova NA, Shashkov AS, Helbig JH, Zähringer U (2001) Identification of a Homopolymer of 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-d-glycero-d-talo-nonulosonic acid in the Lipopolysaccharides of Legionella pneumophila non-1 serogroups. Biochemistry (Mosc) 66(9):1035–1041

    Article  CAS  Google Scholar 

  • Knirel YA, Shashkov AS, Tsvetkov YE, Jansson PE, Zãhringer U (2003) 5,7-Diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids in bacterial glycopolymers: chemistry and biochemistry. Adv Carbohydr Chem Biochem 58:371–417

    Article  CAS  PubMed  Google Scholar 

  • Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305(3):567–580

    Article  CAS  PubMed  Google Scholar 

  • Lehrer J, Vigeant KA, Tatar LD, Valvano MA (2007) Functional characterization and membrane topology of Escherichia coli WecA, a sugar-phosphate transferase initiating the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide. J Bacteriol 189(7):2618–2628

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li Y, Cao B, Liu B, Liu D, Gao Q, Peng X, Wu J, Bastin DA, Feng L, Wang L (2009) Molecular detection of all 34 distinct O-antigen forms of Shigella. J Med Microbiol 58(Pt 1):69–81

    Article  CAS  PubMed  Google Scholar 

  • Li D, Liu B, Chen M, Guo D, Guo X, Liu F, Feng L, Wang L (2010) A multiplex PCR method to detect 14 Escherichia coli serogroups associated with urinary tract infections. J Microbiol Methods 82(1):71–77

    Article  CAS  PubMed  Google Scholar 

  • Lüneberg E, Zetzmann N, Alber D, Knirel YA, Kooistra O, Zähringer U, Frosch M (2000) Cloning and functional characterization of a 30 kb gene locus required for lipopolysaccharide biosynthesis in Legionella pneumophila. Int J Med Microbiol 290(1):37–49

    Article  PubMed  Google Scholar 

  • Marston BJ, Plouffe JF, File TM Jr, Hackman BA, Salstrom SJ, Lipman HB, Kolczak MS, Breiman RF (1997) Incidence of community-acquired pneumonia requiring hospitalization. Results of a population-based active surveillance Study in Ohio. The Community-Based Pneumonia Incidence Study Group. Arch Intern Med 157(15):1709–1718

    Article  CAS  Google Scholar 

  • McNally DJ, Aubry AJ, Hui JPM, Khieu NH, Whitfield D, Ewing CP, Guerry P, Brisson JR, Logan SM, Soo EC (2007) Targeted metabolomics analysis of Campylobacter coli VC167 reveals legionaminic acid derivatives as novel flagellar glycans. J Biol Chem 282(19):14463–14475

    Article  CAS  PubMed  Google Scholar 

  • Muder RR, Yu VL (2002) Infection due to Legionella species other than L. pneumophila. Clin Infect Dis 35(8):990–998

    Article  PubMed  Google Scholar 

  • Newton HJ, Ang DK, van Driel IR, Hartland EL (2010) Molecular pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev 23(2):274–298

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Petzold M, Thürmer A, Menzel S, Mouton JW, Heuner K, Lück C (2013) A structural comparison of lipopolysaccharide biosynthesis loci of Legionella pneumophila serogroup 1 strains. BMC Microbiol 13:198

    Article  PubMed  PubMed Central  Google Scholar 

  • Pilobello KT, Mahal LK (2006) Analyzing the dynamic bacterial glycome with a lectin microarray approach. Nat Chem Biol 2(3):153–157

    Article  PubMed  Google Scholar 

  • Reeves P (1995) Role of O-antigen variation in the immune response. Trends Microbiol 3(10):381–386

    Article  CAS  PubMed  Google Scholar 

  • Ren Y, Liu B, Cheng J, Liu F, Feng L, Wang L (2008) Characterization of Escherichia coli O3 and O21 O antigen gene clusters and development of serogroup-specific PCR assays. J Microbiol Methods 75(2):329–334

    Article  CAS  PubMed  Google Scholar 

  • Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA, Barrell B (2000) Artemis: sequence visualization and annotation. Bioinformatics 16(10):944–945

    Article  CAS  PubMed  Google Scholar 

  • Schoenhofen IC, McNally DJ, Vinogradov E, Whitfield D, Young NM, Dick S, Wakarchuk WW, Brisson JR, Logan SM (2006) Functional characterization of dehydratase/aminotransferase pairs from Helicobacter and Campylobacter: enzymes distinguishing the pseudaminic acid and bacillosamine biosynthetic pathways. J Biol Chem 281(2):723–732

    Article  CAS  PubMed  Google Scholar 

  • Schoenhofen IC, Vinogradov E, Whitfield DM, Brisson JR, Logan SM (2009) The CMP-legionaminic acid pathway in Campylobacter: biosynthesis involving novel GDP-linked precursors. Glycobiology 19(7):715–725

    Article  CAS  PubMed  Google Scholar 

  • Severi E, Hood DW, Thomas GH (2007) Sialic acid utilization by bacterial pathogens. Microbiology 153(Pt 9):2817–2822

    Article  CAS  PubMed  Google Scholar 

  • Su HP, Tung SK, Tseng LR, Tsai WC, Chung TC, Chang TC (2009) Identification of Legionella species by use of an oligonucleotide array. J Clin Microbiol 47(5):1386–1392

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22(22):4673–4680

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tsvetkov YE, Shashkov AS, Knirel YA, Zähringer U (2001) 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 331(3):233–237

    Article  CAS  PubMed  Google Scholar 

  • Wang L, Reeves PR (1998) Organization of Escherichia coli O157 O antigen gene cluster and identification of its specific genes. Infect Immun 66(8):3545–3551

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wang L, Liu B, Kong Q, Steinrück H, Krause G, Beutin L, Feng L (2005) Molecular markers for detection of pathogenic Escherichia coli strains belonging to serogroups O138 and O139. Vet Microbiol 111(3–4):181–190

    Article  CAS  PubMed  Google Scholar 

  • Zhou G, Cao B, Dou Y, Liu Y, Feng L, Wang L (2011) PCR methods for the rapid detection and identification of four pathogenic Legionella spp. and two Legionella pneumophila subspecies based on the gene amplification of gyrB. Appl Microbiol Biotechnol 91(3):777–787

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the National 863 program of China (2012AA020103); the National Natural Science Foundation of China (NSFC) Program (31170094); the Tianjin Research Program of Application Foundation and Advanced Technology (13JCQNJC09400).

Author information

Authors and Affiliations

  1. Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, TEDA College, Nankai University, Tianjin, 300457, People’s Republic of China

    Boyang Cao, Zhenyang Tian, Suwei Wang, Lu Feng & Lei Wang

  2. TEDA Institue of Biological Sciences and Biotechnology, Nankai University, Tianjin, 300457, People’s Republic of China

    Boyang Cao, Zhenyang Tian, Suwei Wang, Lu Feng & Lei Wang

  3. Tianjin Research Center for Functional Genomics and Biochips, TEDA College, Nankai University, Tianjin, People’s Republic of China

    Boyang Cao, Zhenyang Tian, Suwei Wang, Yamin Sun, Lu Feng & Lei Wang

  4. Tianjin Key Laboratory of Microbial Functional Genomics, TEDA College, Nankai University, Tianjin, People’s Republic of China

    Boyang Cao, Zhenyang Tian, Suwei Wang, Yamin Sun, Lu Feng & Lei Wang

  5. Basic Medical College, Tianjin Medical University, Tianjin, 300070, People’s Republic of China

    Zhiyan Zhu

Authors
  1. Boyang Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhenyang Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Suwei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhiyan Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yamin Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lu Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Boyang Cao or Lei Wang.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 1855 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cao, B., Tian, Z., Wang, S. et al. Structural comparison of O-antigen gene clusters of Legionella pneumophila and its application of a serogroup-specific multiplex PCR assay. Antonie van Leeuwenhoek 108, 1405–1423 (2015). https://doi.org/10.1007/s10482-015-0594-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10482-015-0594-0

Keywords

Search

Navigation