A point mutation in cpsE renders Streptococcus pneumoniae nonencapsulated and enhances its growth, adherence and competence

BMC Microbiology

Lucy J Hathaway⁴  Markus Hilty²  Kathrin Mühlemann²  Marianne Küffer⁴  Rémy Bruggmann³  Daniel Wüthrich³  Katherine A Gould¹  Jason Hinds¹  Thierry O Schaffner⁵ 

[1] Bacterial Microarray Group at St George’s (BμG@S), Division of Clinical Sciences, St George’s, University of London, London, United Kingdom;Department of Infectious Diseases, University Hospital of Bern, Bern, Switzerland;Swiss Institute of Bioinformatics, Lausanne, Switzerland;Institute for Infectious Diseases, University of Bern, Friedbühlstrasse 51, Bern, CH-3010, Switzerland;Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland

关键词: SNP;    Nonencapsulated;    Capsule;    cpsE;    Streptococcus pneumoniae;   

Others  :  1140476
DOI  :  10.1186/s12866-014-0210-x

 received in 2014-04-29, accepted in 2014-07-21,  发布年份 2014

【 摘 要 】

Background

The polysaccharide capsule is a major virulence factor of the important human pathogen Streptococcus pneumoniae. However, S. pneumoniae strains lacking capsule do occur.

Results

Here, we report a nasopharyngeal isolate of Streptococcus pneumoniae composed of a mixture of two phenotypes; one encapsulated (serotype 18C) and the other nonencapsulated, determined by serotyping, electron microscopy and fluorescence isothiocyanate dextran exclusion assay.

By whole genome sequencing, we demonstrated that the phenotypes differ by a single nucleotide base pair in capsular gene cpsE (C to G change at gene position 1135) predicted to result in amino acid change from arginine to glycine at position 379, located in the cytoplasmic, enzymatically active, region of this transmembrane protein. This SNP is responsible for loss of capsule production as the phenotype is transferred with the capsule operon. The nonencapsulated variant is superior in growth in vitro and is also 117-fold more adherent to and more invasive into Detroit 562 human epithelial cells than the encapsulated variant.

Expression of six competence pathway genes and one competence-associated gene was 11 to 34-fold higher in the nonencapsulated variant than the encapsulated and transformation frequency was 3.7-fold greater.

Conclusions

We identified a new single point mutation in capsule gene cpsE of a clinical S. pneumoniae serotype 18C isolate sufficient to cause loss of capsule expression resulting in the co-existence of the encapsulated and nonencapsulated phenotype. The mutation caused phenotypic changes in growth, adherence to epithelial cells and transformability. Mutation in capsule gene cpsE may be a way for S. pneumoniae to lose its capsule and increase its colonization potential.

【 授权许可】

   
2014 Schaffner et al.; licensee BioMed Central Ltd

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Austrian R: The pneumococcus at the millennium: not down, not out. J Infect Dis 1999, 179(Suppl 2):S338-S341.
• [2]Winkelstein JA, Abramovitz AS, Tomasz A: Activation of C3 via the alternative complement pathway results in fixation of C3b to the pneumococcal cell wall. J Immunol 1980, 124(5):2502-2506.
• [3]Brown EJ, Joiner KA, Cole RM, Berger M: Localization of complement component 3 on Streptococcus pneumoniae: anti-capsular antibody causes complement deposition on the pneumococcal capsule. Infect Immun 1983, 39(1):403-409.
• [4]Abeyta M, Hardy GG, Yother J: Genetic alteration of capsule type but not PspA type affects accessibility of surface-bound complement and surface antigens of Streptococcus pneumoniae. Infect Immun 2003, 71(1):218-225.
• [5]Henrichsen J: Six newly recognized types of Streptococcus pneumoniae. J Clin Microbiol 1995, 33(10):2759-2762.
• [6]Bentley SD, Aanensen DM, Mavroidi A, Saunders D, Rabbinowitsch E, Collins M, Donohoe K, Harris D, Murphy L, Quail MA, Samuel G, Skovsted IC, Kaltoft MS, Barrell B, Reeves PR, Parkhill J, Spratt BG: Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes. PLoS Genet 2006, 2(3):e31.
• [7]Park IH, Park S, Hollingshead SK, Nahm MH: Genetic basis for the new pneumococcal serotype, 6C. Infect Immun 2007, 75(9):4482-4489.
• [8]Jin P, Kong F, Xiao M, Oftadeh S, Zhou F, Liu C, Russell F, Gilbert GL: First report of putative Streptococcus pneumoniae serotype 6D among nasopharyngeal isolates from Fijian children. J Infect Dis 2009, 200(9):1375-1380.
• [9]Bratcher PE, Kim KH, Kang JH, Hong JY, Nahm MH: Identification of natural pneumococcal isolates expressing serotype 6D by genetic, biochemical and serological characterization. Microbiol 2010, 156(Pt 2):555-560.
• [10]Calix JJ, Nahm MH: A new pneumococcal serotype, 11E, has a variably inactivated wcjE gene. J Infect Dis 2010, 202(1):29-38.
• [11]Calix JJ, Porambo RJ, Brady AM, Larson TR, Yother J, Abeygunwardana C, Nahm MH: Biochemical, genetic, and serological characterization of two capsule subtypes among Streptococcus pneumoniae Serotype 20 strains: discovery of a new pneumococcal serotype. J Biol Chem 2012, 287(33):27885-27894.
• [12]Kolkman MA, van der Zeijst BA, Nuijten PJ: Diversity of capsular polysaccharide synthesis gene clusters in Streptococcus pneumoniae. J Biochem 1998, 123(5):937-945.
• [13]Garcia E, Llull D, Munoz R, Mollerach M, Lopez R: Current trends in capsular polysaccharide biosynthesis of Streptococcus pneumoniae. Res Microbiol 2000, 151(6):429-435.
• [14]Morona JK, Paton JC, Miller DC, Morona R: Tyrosine phosphorylation of CpsD negatively regulates capsular polysaccharide biosynthesis in Streptococcus pneumoniae. Mol Microbiol 2000, 35(6):1431-1442.
• [15]Morona JK, Miller DC, Morona R, Paton JC: The effect that mutations in the conserved capsular polysaccharide biosynthesis genes cpsA, cpsB, and cpsD have on virulence of Streptococcus pneumoniae. J Infect Dis 2004, 189(10):1905-1913.
• [16]van der Windt D, Bootsma HJ, Burghout P, van der Gaast-de Jongh CE, Hermans PW, van der Flier M: Nonencapsulated Streptococcus pneumoniae resists extracellular human neutrophil elastase- and cathepsin G-mediated killing. FEMS Immunol Med Microbiol 2012, 66(3):445-448.
• [17]Martin M, Turco JH, Zegans ME, Facklam RR, Sodha S, Elliott JA, Pryor JH, Beall B, Erdman DD, Baumgartner YY, Sanchez PA, Schwartzman JD, Montero J, Schuchat A, Whitney CG: An outbreak of conjunctivitis due to atypical Streptococcus pneumoniae. N Engl J Med 2003, 348(12):1112-1121.
• [18]Crum NF, Barrozo CP, Chapman FA, Ryan MA, Russell KL: An outbreak of conjunctivitis due to a novel unencapsulated Streptococcus pneumoniae among military trainees. Clin Infect Dis 2004, 39(8):1148-1154.
• [19]Porat N, Greenberg D, Givon-Lavi N, Shuval DS, Trefler R, Segev O, Hanage WP, Dagan R: The important role of nontypable Streptococcus pneumoniae international clones in acute conjunctivitis. J Infect Dis 2006, 194(5):689-696.
• [20]Beiter K, Wartha F, Hurwitz R, Normark S, Zychlinsky A, Henriques-Normark B: The capsule sensitizes Streptococcus pneumoniae to alpha-defensins human neutrophil proteins 1 to 3. Infect Immun 2008, 76(8):3710-3716.
• [21]Nelson AL, Roche AM, Gould JM, Chim K, Ratner AJ, Weiser JN: Capsule enhances pneumococcal colonization by limiting mucus-mediated clearance. Infect Immun 2007, 75(1):83-90.
• [22]Fernebro J, Andersson I, Sublett J, Morfeldt E, Novak R, Tuomanen E, Normark S, Normark BH: Capsular expression in Streptococcus pneumoniae negatively affects spontaneous and antibiotic-induced lysis and contributes to antibiotic tolerance. J Infect Dis 2004, 189(2):328-338.
• [23]Hathaway LJ, Brugger SD, Morand B, Bangert M, Rotzetter JU, Hauser C, Graber WA, Gore S, Kadioglu A, Muhlemann K: Capsule type of Streptococcus pneumoniae determines growth phenotype. PLoS Pathog 2012, 8(3):e1002574.
• [24]Hammerschmidt S, Wolff S, Hocke A, Rosseau S, Muller E, Rohde M: Illustration of pneumococcal polysaccharide capsule during adherence and invasion of epithelial cells. Infect Immun 2005, 73(8):4653-4667.
• [25]Hathaway LJ, Stutzmann Meier P, Battig P, Aebi S, Muhlemann K: A homologue of aliB is found in the capsule region of nonencapsulated Streptococcus pneumoniae. J Bacteriol 2004, 186(12):3721-3729.
• [26]Salter SJ, Hinds J, Gould KA, Lambertsen L, Hanage WP, Antonio M, Turner P, Hermans PW, Bootsma HJ, O’Brien KL, Bentley SD: Variation at the capsule locus, cps, of mistyped and non-typable Streptococcus pneumoniae isolates. Microbiol 2012, 158(Pt 6):1560-1569.
• [27]Hanage WP, Kaijalainen T, Saukkoriipi A, Rickcord JL, Spratt BG: A successful, diverse disease-associated lineage of nontypeable pneumococci that has lost the capsular biosynthesis locus. J Clin Microbiol 2006, 44(3):743-749.
• [28]Arrecubieta C, Lopez R, Garcia E: Molecular characterization of cap3A, a gene from the operon required for the synthesis of the capsule of Streptococcus pneumoniae type 3: sequencing of mutations responsible for the unencapsulated phenotype and localization of the capsular cluster on the pneumococcal chromosome. J Bacteriol 1994, 176(20):6375-6383.
• [29]Waite RD, Struthers JK, Dowson CG: Spontaneous sequence duplication within an open reading frame of the pneumococcal type 3 capsule locus causes high-frequency phase variation. Mol Microbiol 2001, 42(5):1223-1232.
• [30]Waite RD, Penfold DW, Struthers JK, Dowson CG: Spontaneous sequence duplications within capsule genes cap8E and tts control phase variation in Streptococcus pneumoniae serotypes 8 and 37. Microbiol 2003, 149(Pt 2):497-504.
• [31]McEllistrem MC, Ransford JV, Khan SA: Characterization of in vitro biofilm-associated pneumococcal phase variants of a clinically relevant serotype 3 clone. J Clin Microbiol 2007, 45(1):97-101.
• [32]Allegrucci M, Sauer K: Characterization of colony morphology variants isolated from Streptococcus pneumoniae biofilms. J Bacteriol 2007, 189(5):2030-2038.
• [33]Allegrucci M, Sauer K: Formation of Streptococcus pneumoniae non-phase-variable colony variants is due to increased mutation frequency present under biofilm growth conditions. J Bacteriol 2008, 190(19):6330-6339.
• [34]Xayarath B, Yother J: Mutations blocking side chain assembly, polymerization, or transport of a Wzy-dependent Streptococcus pneumoniae capsule are lethal in the absence of suppressor mutations and can affect polymer transfer to the cell wall. J Bacteriol 2007, 189(9):3369-3381.
• [35]James DB, Gupta K, Hauser JR, Yother J: Biochemical activities of Streptococcus pneumoniae serotype 2 capsular glycosyltransferases and significance of suppressor mutations affecting the initiating glycosyltransferase Cps2E. J Bacteriol 2013, 195(24):5469-5478.
• [36]Cartee RT, Forsee WT, Bender MH, Ambrose KD, Yother J: CpsE from type 2 Streptococcus pneumoniae catalyzes the reversible addition of glucose-1-phosphate to a polyprenyl phosphate acceptor, initiating type 2 capsule repeat unit formation. J Bacteriol 2005, 187(21):7425-7433.
• [37]Kolkman MA, Morrison DA, Van Der Zeijst BA, Nuijten PJ: The capsule polysaccharide synthesis locus of Streptococcus pneumoniae serotype 14: Identification of the glycosyl transferase gene cps14E. J Bacteriol 1996, 178(13):3736-3741.
• [38]Pelosi L, Boumedienne M, Saksouk N, Geiselmann J, Geremia RA: The glucosyl-1-phosphate transferase WchA (Cap8E) primes the capsular polysaccharide repeat unit biosynthesis of Streptococcus pneumoniae serotype 8. Biochem Biophys Res Commun 2005, 327(3):857-865.
• [39]van Selm S, Kolkman MA, van der Zeijst BA, Zwaagstra KA, Gaastra W, van Putten JP: Organization and characterization of the capsule biosynthesis locus of Streptococcus pneumoniae serotype 9 V. Microbiol 2002, 148(Pt 6):1747-1755.
• [40]Jiang SM, Wang L, Reeves PR: Molecular characterization of Streptococcus pneumoniae type 4, 6B, 8, and 18C capsular polysaccharide gene clusters. Infect Immun 2001, 69(3):1244-1255.
• [41]Guidolin A, Morona JK, Morona R, Hansman D, Paton JC: Nucleotide sequence analysis of genes essential for capsular polysaccharide biosynthesis in Streptococcus pneumoniae type 19 F. Infect Immun 1994, 62(12):5384-5396.
• [42]Kronenberg A, Zucs P, Droz S, Muhlemann K: Distribution and invasiveness of Streptococcus pneumoniae serotypes in Switzerland, a country with low antibiotic selection pressure, from 2001 to 2004. J Clin Microbiol 2006, 44(6):2032-2038.
• [43]Hathaway LJ, Brugger S, Martynova A, Aebi S, Muhlemann K: Use of the Agilent 2100 bioanalyzer for rapid and reproducible molecular typing of Streptococcus pneumoniae. J Clin Microbiol 2007, 45(3):803-809.
• [44]Salles C, Creancier L, Claverys JP, Mejean V: The high level streptomycin resistance gene from Streptococcus pneumoniae is a homologue of the ribosomal protein S12 gene from Escherichia coli. Nucleic Acids Res 1992, 20(22):6103.
• [45]Pozzi G, Masala L, Iannelli F, Manganelli R, Havarstein LS, Piccoli L, Simon D, Morrison DA: Competence for genetic transformation in encapsulated strains of Streptococcus pneumoniae: two allelic variants of the peptide pheromone. J Bacteriol 1996, 178(20):6087-6090.
• [46]Meier PS, Utz S, Aebi S, Muhlemann K: Low-level resistance to rifampin in Streptococcus pneumoniae. Antimicrob Agents Chemother 2003, 47(3):863-868.
• [47]Gates MA, Thorkildson P, Kozel TR: Molecular architecture of the Cryptococcus neoformans capsule. Mol Microbiol 2004, 52(1):13-24.
• [48]Weinberger DM, Trzcinski K, Lu YJ, Bogaert D, Brandes A, Galagan J, Anderson PW, Malley R, Lipsitch M: Pneumococcal capsular polysaccharide structure predicts serotype prevalence. PLoS Pathog 2009, 5(6):e1000476.
• [49]Adams MH, Roe AS: A partially defined medium for cultivation of pneumococcus. J Bacteriol 1945, 49(4):401-409.
• [50]Lacks S, Hotchkiss RD: A study of the genetic material determining an enzyme in Pneumococcus. Biochim Biophys Acta 1960, 39:508-518.
• [51]Lacks S: Integration efficiency and genetic recombination in pneumococcal transformation. Genetics 1966, 53(1):207-235.
• [52]Studer D, Graber W, Al-Amoudi A, Eggli P: A new approach for cryofixation by high-pressure freezing. J Microsc 2001, 203(Pt 3):285-294.
• [53]Hunziker EB, Graber W: Differential extraction of proteoglycans from cartilage tissue matrix compartments in isotonic buffer salt solutions and commercial tissue-culture media. J Histochem Cytochem 1986, 34(9):1149-1153.
• [54]van de Rijn I, Kessler RE: Growth characteristics of group A streptococci in a new chemically defined medium. Infect Immun 1980, 27(2):444-448.
• [55]Luer S, Troller R, Jetter M, Spaniol V, Aebi C: Topical curcumin can inhibit deleterious effects of upper respiratory tract bacteria on human oropharyngeal cells in vitro: potential role for patients with cancer therapy induced mucositis? Support Care Cancer 2011, 19(6):799-806.
• [56]Spaniol V, Heiniger N, Troller R, Aebi C: Outer membrane protein UspA1 and lipooligosaccharide are involved in invasion of human epithelial cells by Moraxella catarrhalis. Microbes Infect 2008, 10(1):3-11.
• [57]Brugger SD, Baumberger C, Jost M, Jenni W, Brugger U, Muhlemann K: Automated counting of bacterial colony forming units on agar plates. PLoS One 2012, 7(3):e33695.
• [58]Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA: SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 2012, 19(5):455-477.
• [59]Langmead B, Salzberg SL: Fast gapped-read alignment with Bowtie 2. Nat Methods 2012, 9(4):357-359.
• [60]Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R: The sequence alignment/Map format and SAMtools. Bioinform 2009, 25(16):2078-2079.
• [61]Hathaway LJ, Battig P, Muhlemann K: In vitro expression of the first capsule gene of Streptococcus pneumoniae, cpsA, is associated with serotype-specific colonization prevalence and invasiveness. Microbiol 2007, 153(Pt 8):2465-2471.
• [62]Melchiorre S, Camilli R, Pietrantoni A, Moschioni M, Berti F, Del Grosso M, Superti F, Barocchi MA, Pantosti A: Point mutations in wchA are responsible for the non-typability of two invasive Streptococcus pneumoniae isolates. Microbiol 2012, 158(Pt 2):338-344.
• [63]Iannelli F, Pearce BJ, Pozzi G: The type 2 capsule locus of Streptococcus pneumoniae. J Bacteriol 1999, 181(8):2652-2654.
• [64]Morona JK, Morona R, Paton JC: Analysis of the 5′ portion of the type 19A capsule locus identifies two classes of cpsC, cpsD, and cpsE genes in Streptococcus pneumoniae. J Bacteriol 1999, 181(11):3599-3605.
• [65]Weiser JN, Austrian R, Sreenivasan PK, Masure HR: Phase variation in pneumococcal opacity: relationship between colonial morphology and nasopharyngeal colonization. Infect Immun 1994, 62(6):2582-2589.
• [66]Li-Korotky HS, Lo CY, Banks JM: Interaction of pneumococcal phase variation, host and pressure/gas composition: virulence expression of NanA, HylA, PspA and CbpA in simulated otitis media. Microb Pathog 2010, 49(4):204-210.
• [67]Cundell DR, Weiser JN, Shen J, Young A, Tuomanen EI: Relationship between colonial morphology and adherence of Streptococcus pneumoniae. Infect Immun 1995, 63(3):757-761.
• [68]Ottolenghi-Nightingale E: Competence of pneumococcal isolates and bacterial transformations in man. Infect Immun 1972, 6(5):785-792.
• [69]Weiser JN, Kapoor M: Effect of intrastrain variation in the amount of capsular polysaccharide on genetic transformation of Streptococcus pneumoniae: implications for virulence studies of encapsulated strains. Infect Immun 1999, 67(7):3690-3692.
• [70]Peterson SN, Sung CK, Cline R, Desai BV, Snesrud EC, Luo P, Walling J, Li H, Mintz M, Tsegaye G, Burr PC, Do Y, Ahn S, Gilbert J, Fleischmann RD, Morrison DA: Identification of competence pheromone responsive genes in Streptococcus pneumoniae by use of DNA microarrays. Mol Microbiol 2004, 51(4):1051-1070.
• [71]Oggioni MR, Trappetti C, Kadioglu A, Cassone M, Iannelli F, Ricci S, Andrew PW, Pozzi G: Switch from planktonic to sessile life: a major event in pneumococcal pathogenesis. Mol Microbiol 2006, 61(5):1196-1210.