Role of cell surface proteins and toll-like receptors in the pathogenesis of Streptococcus pneumoniae

Document Type : Original Article


1 Institute of Medical Molecular Biotechnology (IMMB), Faculty of Medicine, Universiti Teknologi MARA (UiTM), Sungai Buloh Campus, Selangor, Malaysia

2 Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, (UPM), Serdang, elangor, Malaysia

3 Department of Medical Microbiology & Parasitology, Faculty of Medicine, Universiti Teknologi MARA (UiTM), Sungai Buloh Campus, Selangor, Malaysia

4 Institute of Pathology, Laboratory and Forensic Medicine (I-PPerForM), Universiti Teknologi MARA (UiTM), Sungai Buloh Campus, Jalan Hospital, Sungai Buloh, Selangor, Malaysia

5 School of Medicine, Taylor’s University Lakeside Campus, Subang Jaya, Selangor, Malaysia

6 Center for Toxicology and Health Risk Studies, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, Kuala Lumpur, Malaysia

7 Department of Medical Microbiology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia


Objective(s): Pneumococcal cell wall (PCW) is an inflammatory component in Streptococcus pneumoniae. The cell surface proteins and the toll-like receptors (TLR) signaling response were investigated in the human lung epithelial (A549) cells inoculated with PCW of different serotypes.
Materials and Methods: The presence of genes encoding these proteins was determined using polymerase chain reaction (PCR). The structure of the cell walls was analyzed by proton nuclear magnetic resonance (1H NMR). The A549 cell line was challenged with PCW extracts of different serotypes. RNA from the infected host cells was extracted and tested against a total of 84 genes associated with TLR signaling pathways (TLR 1-6 and 10) using RT2 Profiler PCR Array.
Results: Cell surface proteins; ply, lytA, nanA, nanB, and cbpD genes were present in all serotypes. The distribution and structure of surface protein genes suggest behavioral changes in the molecules, contributing to the resilience of the strains to antibiotic treatment.
Conclusion: TLR2 showed the highest expression, while serotypes 1, 3, and 5 induced higher TNFα and IL-1α, suggesting to be more immunogenic than the other strains tested.


Main Subjects

1. World Health Organization. WHO meeting on maternal and neonatal pneumococcal immunization. Wkly Epidemiol Rec 1998; 73:187–188.
2.    McIntyre PB, O’Brien KL, Greenwood B, Van de Beek D. Effect of vaccines on bacterial meningitis worldwide. Lancet 2012; 380:1703–1711.
3.    World Health Organization (WHO) Priority Pathogens List for RandD of New Antibiotics. 2017
4.    Kim L, McGee L, Tomczyk S, Beall B. Biological and epidemiological features of antibiotic-resistant Streptococcus pneumoniae in pre- and post-conjugate vaccine eras: a United States perspective. Clin Microbiol Rev 2016; 29:525–552.
5.    Flasche S, Van Hoek AJ, Sheasby E, Waight P, Andrews N, Sheppard C, et al. Effect of pneumococcal conjugate vaccination on serotype-specific carriage and invasive disease in England: a cross-sectional study. PLoS Med 2011; 8:1-9.
6.    Golubchik T, Brueggemann AB, Street T, Gertz RE Jr, Spencer CC, Ho T, et al. Pneumococcal genome sequencing tracks a vaccine escape variant formed through a multi-fragment recombination event. Nat Genet 2012; 44:352–355.
7.    Lofling J, Vimberg V, Battig P, Henriques-Normark B. Cellular interactions by LPxTG-anchored pneumococcal adhesins and their streptococcal homologues. Cell Microbiol 2011; 13:186–197.
8.    Hakenbeck R, Madhour A, Denapaite D, Bruckner R. Versatility of choline metabolism and choline-binding proteins in Streptococcus pneumoniae and commensal streptococci. FEMS Microbiol Rev 2009; 33:572–586. 
9.    Mitchell AM, Mitchell TJ. Streptococcus pneumoniae: virulence factors and variation. Clin Microbiol Infect 2010; 16:411–418.
10.    Weiser JN, Ferreira DM, Paton JC. Streptococcus pneumoniae: transmission, colonization and invasion. Nat Rev Microbiol 2018; 16:355–367.
11.    Dave S, Carmicle S, Hammerschmidt S, Pangburn MK, McDaniel LS. Dual roles of PspC, a surface protein of Streptococcus pneumoniae, in binding human secretory IgA and factor H. J Immunol 2004; 173:471–477.
12.    Ren B, McCrory MA, Pass C, Bullard DC, Ballantyne CM, Xu Y, et al. The virulence function of Streptococcus pneumoniae surface protein A involves inhibition of complement activation and impairment of complement receptor-mediated protection. J Immunol 2004; 173:7506–7512.
13.    Gutiérrez-Fernández J, Saleh M, Alcorlo M, Gómez-Mejia A, Pantoja-Uceda D, Trevino MA, et al. Modular architecture and unique teichoic acid recognition features of choline-binding protein L (CbpL) contributing to pneumococcal pathogenesis. Sci Rep 2016; 6:38094-38113.
14.    Yamaguchi M, Goto K, Hirose Y, Yamaguchi Y, Sumitomo T, Nakata M, et al. Identification of evolutionarily conserved virulence factor by selective pressure analysis of Streptococcus pneumoniae. Commun Biol 2019; 2:96-108.
15.    Opitz B, Laak VV, Eitel J, Suttorp N. Innate immune recognition in infectious and noninfectious diseases of the lung. Am J Respir Crit Care Med 2010; 181:1294–1309.
16.    Koppe U, Suttorp N, Opitz B. Recognition of Streptococcus pneumoniae by the innate immune system. Cell Microbiol 2012; 14:460–466.
17.    Bohrer H, Qiu F, Zimmermann T, Zhang Y, Jllmer T, Mannel D, et al. Role of NFκB in the mortality of sepsis. J Clin Invest 1997; 100:972-985.
18.    M23: Development of In Vitro Susceptibility Test Methods, Breakpoints, and Quality Control Parameters, Clinical and Laboratory Institute (CLSI) (2023) 6th Edition. 
19.    Pai R, Gertz RE, Beall B. Sequential multiplex PCR approach for determining capsular serotypes of Streptococcus pneumoniae isolates. J Clin Microbiol 2006; 44:124-131.
20.    Nagai K, Shibasaki Y, Hasegawa K, Davies TA, Jacobs MR, Ubukata K, et al. Evaluation of PCR primers to screen for Streptococcus pneumoniae isolates and β-lactam resistance, and to detect common macrolide resistance determinants. J Antimicrob Chemother 2001; 48:915-918.
21.    Suzuki N, Seki M, Nakano Y, Kiyoura Y, Maeno M, Yamashita Y. Discrimination of Streptococcus pneumoniae from viridans group streptococci by genomic subtractive hybridization. J Clin Microb 2005; 43:4528-4534.
22.    Sakai F, Talekar SJ, Klugman KP, Vidal JE. Expression of Streptococcus pneumoniae virulence-related genes in the nasopharynx of healthy children. PLoS One 2013; 8:1-9.
23.     Desa MN, Sekaran SD, Vadivelu J, Parasakthi N. Distribution of CBP genes in Streptococcus pneumoniae isolates in relation to vaccine types, penicillin susceptibility and clinical site. Epid Inf 2008; 136:940-942
24.    Gosink KK, Mann ER, Guglielmo C, Tuomanen EI, Masure HR. Role of novel choline binding proteins in virulence of Streptococcus pneumoniae. Inf Imm 2000; 68:5690-5695.
25.    Bui NK, Eberhardt A, Vollmer D, Kern T, Bougault C, Tomasz A, et al.  Isolation and analysis of cell wall components from Streptococcus pneumoniae. Anal Biochem 2012; 421:657–666.
26.    Draing C, Pfitzenmaier M, Zummo S, Mancuso G, Geyer A, Hartung T, et al. Comparison of lipoteichoic acid from different serotypes of Streptococcus pneumoniae. J Biol Chem 2006; 281:33849–33859.
27.    Hausler KG, Prinz M, Nolte C, Weber JR, Schumann RR, Kettenmann H, et al. Interferon-γ differentially modulates the release of cytokines and chemokines in lipopolysaccharide- and pneumococcal cell wall-stimulated mouse microglia and macrophages. Eur J Neurosci 2002; 16:2113–2122.
28.    Kumari NP, Yusof MY, Ong SY, Mansor M, Le CF, Sekaran SD. Variation of sequence of genes encoding the MurMN operon and cell wall composition in Streptococcus pneumoniae strains of different susceptibility levels to penicillin.  J Infect Dis Antimicrob Agents 2009; 26:97-108.
29.    Ezarina AR, Mohd-Zain Z, Hussaini J, Wong EH, Nyein LL, Parasakthi N, et al. Adherence of Streptococcus pneumoniae and expression analysis of neuraminidase gene (NanA and NanB) after interaction of A549 human lung epithelial cells with pneumococcal strains of various serotypes. Malays J Microbiol 2017; 13:210-216.
30.    Jefferies J, Johnston C, Kirkham L, Cowan G, Ross K, Smith A, et al. Presence of nonhemolytic pneumolysin in serotypes of Streptococcus pneumoniae associated with disease outbreaks. J Infect Dis 2007; 196:936-944.
31.    Murdoch C, Read RC, Zhang Q. Choline-binding protein A of Streptococcus pneumoniae elicits chemokine production and expression of intercellular adhesion molecule 1 (CD54) by human alveolar epithelial cells. J Inf Dis 2002; 186:1253-1260.
32.    Fischer H, Tomasz A. Peptidoglycan cross-linking and teichoic acid attachment in Streptococcus pneumoniae. J Bact 1985; 163:46–54.
33.    Giudicelli S, Tomasz A. Attachment of pneumococcal autolysin to wall teichoic acids, an essential step in enzymatic wall degradation. J Bact 1984; 158:1188–1190.
34.    Smith AM, Klugman KP. Alterations in murM, a cell wall muropeptide branching enzyme, increase high-level penicillin and cephalosporin resistance in Streptococcus pneumoniae.  Antimicrob Agents Chemother 2001; 45:2393–2396.
35.    Gehre F, Leib SL, Grandgirard D, Kummer J, Buhlmann A, Simon F, et al. Essential role of choline for pneumococcal virulence in an experimental model of meningitis. J Int Med 2008; 264: 143–154.
36.    Tuomanen E, Liu H, Hengstler B, Zak O, Tomasz A. The induction of meningeal inflammation by components of the pneumococcal cell wall. J Infect Dis 1985; 151:859–868.
37.    Weber JR, Freyer D, Alexander C, Schroder NWJ, Reiss A, Kuster C, et al. Recognition of pneumococcal peptidoglycan: an expanded, pivotal role for LPS binding protein. Immunity 2003; 19:269–279.
38.    Amory-Rivier CF, Mohler J, Bedos JP, Azoulay-Dupuis E, Henin D, Muffat-Joly M, et al. Nuclear factor- κB activation in mouse lung lavage cells in response to Streptococcus pneumoniae pulmonary infection. Crit Care Med 2000; 28:3249–3256.
39.    Heumann D, Barras C, Severin A, Glauser MP, Tomasz A. Gram-positive cell walls stimulate synthesis of tumor necrosis factor alpha and interleukin-6 by human monocytes. Infect Immun 1994; 62:2715–2721.
40.    Kafka D, Ling E, Feldman G, Benharroch D, Voronov E, Givon-Lavi N, et al. Contribution of IL-1 to resistance to Streptococcus pneumoniae infection. Int Immunol 2008; 20:1139–1146.