A cytosol derived factor of Group B streptococcus prevent its invasion into human epithelial cells

  • Ohri Manju 
  • Parashar Smriti 
  • Pai Venkatesh S. 
  • Ghosh Sujata 
  • Chakraborti Anuradha 
Original Paper
  • 70 Downloads

Abstract

Group B streptococcus (GBS) or Streptococcus agalactiae, is an opportunistic pathogen causing a wide range of infections like pneumonia, sepsis, and meningitis in newborn, pregnant women and adults. While this bacterium has adapted well to asymptomatic colonization of adult humans, it still remains a potentially devastating pathogen to susceptible infants. Advances in molecular techniques and refinement of in vitro and in vivo model systems have elucidated key elements of the pathogenic process, from initial attachment to the maternal vaginal epithelium to penetration of the newborn blood–brain barrier. Still, the formidable array of GBS virulence factors makes this bacterium at the forefront of neonatal pathogens. The involvement of bacterial components in the host-pathogen interaction of GBS pathogenesis and its related diseases is not clearly understood. In this study we demonstrated the role of a 39 kDa factor from GBS which plays an important role in the process of its invasion. We found a homogeneous 39 kDa factor from the cytosol of GBS after following a combination of sequential purification steps involving molecular sieving and ion exchange chromatography using ACTA-FPLC system. Its N-terminal sequence showed a homology with xenobiotic response element type transcriptional regulator protein, a 40 kDa protein of Streptococcus. This factor leads to inhibition of GBS invasion in HeLa and A549 cells. This protein also showed sensitivity and specific cross reactivity with the antibodies raised against it in New Zealand white rabbits by western immunoblotting. This inhibitory factor was further confirmed tolerant for its cytotoxicity. These results add a novel aspect to bacterial pathogenesis where bacteria’s own intracellular protein component can act as a potential therapeutic candidate by decreasing the severity of disease thus promoting its invasion inhibition.

Keywords

Invasion Group B streptococcus (GBS) Pathogenesis Protein purification Invasion inhibition 

Notes

Acknowledgements

We gratefully acknowledge Indian Council of Medical Research (ICMR) for providing the senior research fellowship to Manju Ohri (MO) to carry out this work. Prof. S. Majumdar is greatly acknowledged for invaluable guidance and PGIMER for Junior Research Fellowship to MO during this study. We would like to thank Dr. Harmeet Kaur who contributed in collection of all serotypes of GBS, Deepak Bhatt (IMTECH) and Central Instrumentation Lab (Panjab University) for assistance in microscopy.

Author contributions

Conceived and designed the experiments: MO, AC. Performed the experiments including invasion assays and protein purification: MO, SP. Microscopy experiments performed by MO. Analyzed the data AC, SG and MO. Wrote the paper MO, VSP, AC.

Compliance with ethical standards

Conflict of interest

The authors have declared that no conflict of interests exists.

Supplementary material

11274_2018_2428_MOESM1_ESM.doc (854 kb)
Supplementary material 1 (DOC 853 KB)

References

  1. Alenghat E, Esterly JR (1984) Alveolar macrophages in perinatal infants. Pediatrics 74:221–223Google Scholar
  2. Alloush HM, Lopze-Ribot JL, Masetn BJ, Chaffin WL (1997) 3-phosphoglycerate kinase: a glycolytic enzyme protein present in cell wall of Candida albicans. Microbiology 143:321–330CrossRefGoogle Scholar
  3. Baker CJ, Barrett FF (1973) Transmissision of Group B streptococcus among parturient women and the neonates. J Pediatr 83:919–925CrossRefGoogle Scholar
  4. Baker CJ, Edwards MS (1995) Group B Streptococcal infections. In: Remington JS, Klein JO (eds) Infectious diseases of the fetus and newborn infant. W B Saunders, Philadelphia, pp 980–1054Google Scholar
  5. Barragán MJ, Blázquez B, Zamarro MT, Mancheño JM, García JL, Díaz E, Carmona M (2005) BzdR, a repressor that controls the anaerobic catabolism of benzoate in Azoarcus sp. CIB, is the first member of a new subfamily of transcriptional regulators. J Biol Chem 280(11):10683–10694CrossRefGoogle Scholar
  6. Beckman C, Waggoner JD, Harris TO, Tamura GS, Rubens CE (2002) Identification of novel adhesins from Group B Streptococci by use of phage display reveals that C5a peptidase mediates fibronectin binding. Infect Immun 70:2869–2876CrossRefGoogle Scholar
  7. Beischlag TV, Morales JL, Hollingshead BD, Perdew GH (2008) The aryl hydrocarbon receptor complex and the control of gene expression. Crit Rev Eukaryot Gene Expr 18(3):207–250CrossRefGoogle Scholar
  8. Bolduc GR, Baron MJ, Gravecamp C, Lachenauer CS, Madoff LC (2002) The alpha C protein mediates internalization of Group B streptococcus with human cervical epithelial cells. Cell Microbiol 4:751–758CrossRefGoogle Scholar
  9. Boone TJ, Burnham CAD, Tyrrell GJ (2011) Binding of group B Streptococcal phosphoglycerate kinase to plasminogen and actin. Microb Pathog 51:255–261CrossRefGoogle Scholar
  10. Burnham CAD, Shokoples SE, Tyrrell GJ (2005) Phosphoglycerate kinase inhibits epithelial cell invasion by Group B Streptococci. Microbial Pathog 38:189–200CrossRefGoogle Scholar
  11. Carlson SA, Jones BD (1998) Inhibition of Salmonella typhimurium invasion by host cell5 expression of secreted bacterial invasion proteins. Infect Immun 66:5295–5300Google Scholar
  12. Cleary PP, Matsuka YV, Huynh T, Lam H, Hamsted SB (2004) Immunization with C5a peptidase from either Group A or B streptococci enhances clearance of group A streptococci from intranasally infected mice. Vaccine 22:4332–4341CrossRefGoogle Scholar
  13. Dalkara D, Zuber G, Behr JP (2004) Intracytoplasmic delivery of anionic proteins. Mol Ther 9:964–969CrossRefGoogle Scholar
  14. Doran KS, Chang CW, Jennifer VM, Eckmann BL, Nizet V (2002) Group B streptococcal beta-hemolysin/cytolysin promotes invasion of human lung epithelial cells and the release of IL-8. J Infect Dis 185:196–203CrossRefGoogle Scholar
  15. Doran KS, Engelson EJ, Khosrani A, Maisey HC, Fedtke I, Equils O et al (2005) Blood brain barrier invasion by Group B streptococcus depends upon proper cell surface anchoring of the lipotechoic acid. J Clin Investig 115:2499–2507CrossRefGoogle Scholar
  16. Farley MM (2001) Group B Streptococcal disease in non-pregnant adults. Clin Infect Dis 33:556–561CrossRefGoogle Scholar
  17. Farley MM, Harvey RC, Studell T, Smith JD, Schuchat A, Wenger JD et al (1993) A population based assessment of invasive disease due to Group B streptococcus in non-pregnant adults. N Engl J Med 328:1087–1811CrossRefGoogle Scholar
  18. Frieburghans C, Janicke B, Mansson HL, Oredsson SM, Paulson MA (2009) Lactoferrin treatment decreases the rate of cell proliferation. J Dairy Sci 92:2477–2484CrossRefGoogle Scholar
  19. Gibson RL, Lee MK, Soderland C, Chi EY, Rubens CE (1993) Group B Streptococci invade endothelial cells: type III capsular polysaccharide attenuates invasion. Infect Immun 61:478–485Google Scholar
  20. Goldstein E, Lippert W, Warshauer D (1974) Pulmonary alveolar macrophage: defender against bacterial infection of the lung. J Clin Invest 54:519–528CrossRefGoogle Scholar
  21. Gultekin H, Heermann K (1988) The use of polyvinylideneflouride membrane as a general blotting matrix. Ann Biochem 172:320CrossRefGoogle Scholar
  22. Harrison LH, Elliot JA, Dwyer DM et al (1998) Group B streptococcal serotype distribution of isolates from colonized pregnant women at time of delivery in United Arab Emirates. Ped Infect Dis 17:499–503CrossRefGoogle Scholar
  23. Henneke P, Takeuchi O, Strijp JA, Guttormsen HK, Smith JA, Schromm AB et al (2001) Novel engagement of CD14 and multiple toll like receptors by Group B Streptococci. J Immunol 167:7069–7076CrossRefGoogle Scholar
  24. Henneke P, Morath S, Uematsu S, Weichert S, Pfitzenmaier M, Takeuchi O, Muller A et al (2005) Role of lipotechoic acid in phagocyte response to GBS. J Immunol 174:6449–6455CrossRefGoogle Scholar
  25. Holst CM, Oredsson SM (2005) Comparison of three cytotoxicity tests in the evaluation of cytotoxicity of a spermine analogue on human breast cancer cell lines. Toxicol In Vitro 19:379–387CrossRefGoogle Scholar
  26. Huang G, Elferink CJ (2012) A novel nonconsensus xenobiotic response element capable of mediating aryl hydrocarbon receptor-dependent gene expression. Mol Pharmacol 81(3):338–347CrossRefGoogle Scholar
  27. Hughes MJH, Moore JC, Lane JD, Wilson R, Pribul PK, Younes ZN, Dobson RJ, Everest P, Reason AJ, Redfern JM, Greer FM, Paxton T, Panico M, Morris HR, Feldman RG, Santengelo JD (2002) Identification of major outer membrane proteins of Streptococcus agalactiae. Infect Immun 10:1254–1259CrossRefGoogle Scholar
  28. Hulse ML, Smith S, Chi EY, Pham A, Rubens CE (1993) Effect of Type III Group B Streptococcal capsular polysaccharide on invasion of respiratory epithelial cells. Infect Immun 61:4835–4841Google Scholar
  29. Johri AK, Paoletti LC, Glaser P, Dua M, Sharma PK, Grandi G, Rappuoli R (2006) Group B streptococcus: global incidence and vaccine development. Nature 4:932–942Google Scholar
  30. Kaur H, Kumar P, Ray P, Chakraborti A (2009) Biofilm formation in clinical isolates of Group B Streptococci from North India. Microb Pathog 46:321–327CrossRefGoogle Scholar
  31. Keung WL, Salaby KA, Thankur A, Medhat AM, Karim AM, Overde PT (1995) Cloning of the gene for phosphoglyceratekinase from Schistosoma mansoni and characterization of its gene products. Mol Biochem Parasitol 71:221–231CrossRefGoogle Scholar
  32. Laemmeli UK (1970) Cleavage of structural proteins during the assembly of bacteriophages T4. Nature 227:680–685CrossRefGoogle Scholar
  33. Lee KW, Thakur A, Karim AM, Lo Verde PT (1995) Immune response to Schistosoma mansoni phosphoglycerate kinase during natural and experimental infection: identification of a schistosome specific B cell epitope. Infect Immun 63:4307–4311Google Scholar
  34. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with folin-phenol reagent. J Biol Chem 193:265–275Google Scholar
  35. Manning PA (1995) Use of confocal microscopy in studying bacterial adhesion and invasion. Methods Enzymol 253:159CrossRefGoogle Scholar
  36. Memami B, Bouttier M, Dimitrov V, Ouellette M, Behr MA, Fritz JH, White JH (2015) Engagement of the aryl hydrocarbon receptor in Mycobacterium tuberculosis-infected macrophages has pleiotropic effects on innate immune signaling. J Immunol 195(9):4479–4491CrossRefGoogle Scholar
  37. Mikamo H, Johri AK, Paoletti LC, Madoff LC, Onderdonk AC (2004) Adherence to invasion by and cytokine production in response to serotype VIII Group B Streptococci. Infect Immun 72:4716–4722CrossRefGoogle Scholar
  38. Milliotis MD (1991) Acridine orange stain for determining intracellular enteropathogens in HeLa cells. J Clin Microbio 29:830–831Google Scholar
  39. Moyo SR, Maeland JA (2003) Antibodies raised in animals against S.agalactiae protein C-alpha and R4 and normal human serum antibodies target distinct epitopes. J Med Microbiol 52:379–383CrossRefGoogle Scholar
  40. Nizet V, Kim KS, Stins M, Jonas M, Chi EY, Nguyen D, Rubens CE (1977) Invasion of brain microvascular endothelial cells by Group B Streptococci. Infect Immun 65:5074–5081Google Scholar
  41. Ozeri V, Rosenshine I, Ben-Ze’ev A, Bokoch GM, Jou T, Heinski E (2001) De novo formation of focal complexes like structures in host cells by invading streptococci. Mol Microbiol 41:561–573CrossRefGoogle Scholar
  42. Pulskal MG, Przekop MB, Kavonian MR, Vecoli C, Hicks DA (1986) Immobilion PVDF transfer membrane: a new membrane substrate for Western blotting of proteins. Biotechniques 4:272Google Scholar
  43. Rubens CE, Raff HV, Jackson JC, Chi EY, Bielitzki JT, Hillier SL (1991) Pathophysiology and histopathology of Group B Streptococcal sepsis in Macaca nemestrina primates induced after intra amniotic inoculation: evidence for bacterial cellular invasion. J Infect Dis 164:320–330CrossRefGoogle Scholar
  44. Rubens CE, Smith S, Hulse M, Chi EY, Belle GV (1992) Respiratory epithelial cell invasion by Group B Streptococci. Infect Immun 60:5157–5163Google Scholar
  45. Samen U, Eikmanns BJ, Reinscheid DJ, Borges F (2007) The surface protein Srr-1 binds human keratin 4 and promotes adherence to epithelial Hep-2 cells. Infect Immun 75:5405–5414CrossRefGoogle Scholar
  46. Schuchat A (1998) Epidemiology of Group B Streptococcal disease in United States: shifting paradigms. Clin Microbiol Rev 11:497–513Google Scholar
  47. Schuchat A, Wenger JD (1994) Epidemiology of Group B streptococcal disease: risk factors, prevention strategies and vaccine development. Epidemiol Rev 16:372–402CrossRefGoogle Scholar
  48. Schuchat A, Robinson K, Wegner JD, Harrison LH, Farley M, Reingold AL, Lefkowitz L, Perkin BA (1997) Bacterial meningitis in the United States in 1995. New Engl J Med 337:970–976CrossRefGoogle Scholar
  49. Sherman MP, Lehrer RI (1992) Oxidative metabolism of neonatal and adult rabbit lung macrophages stimulated with opsonized group B Streptococci. Infect Immun 47:26–30Google Scholar
  50. Spector DL (2011) Immunoflorescence localization of nuclear proteins. Cold Spring Harb Protoc.  https://doi.org/10.1101/pdb.prot065755 Google Scholar
  51. Spellerberg B, Rozdinski E, Martin S, Weber-Heynemann J, Schnitzler N, Lutticken R, Podbielski (1999) A Lmb, a protein with similarities to the Lra I adhesion family, mediates attachment of S. agalactiae to human laminin. Infect Immun 67:871–878Google Scholar
  52. Tamura GS, Kuypers JM, Smith S, Raff H, Rubens CE (1994) Adherence of Group B Sterptococci to cultured epithelial cells: role of environmental factors and bacterial surface components. Infect Immun 62:2450–2458Google Scholar
  53. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets:procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354CrossRefGoogle Scholar
  54. Tyrell GJ, Kennedy A, Shokoples SE, Sherburne RK (2002) Binding and invasion of HeLa and MRC-5 cells by Streptococcus agalactiae. Microbiology 148:3921–3931CrossRefGoogle Scholar
  55. Valenti P, Antonini G (2005) Lactoferrin: an important host defence against microbial and viral attack. Cell Mol Life Sci 62:1–12CrossRefGoogle Scholar
  56. Vogel CF, Matsumura F (2009) A new cross-talk between the aryl hydrocarbon receptor and RelB, a member of the NF-kappaB family. Biochem Pharmacol 77(4):734–745CrossRefGoogle Scholar
  57. Wang C, Ye F, Kumar V, Gao YG, Zhang LH (2014) BswR controls bacterial motility and biofilm formation in Pseudomonas aeruginosa through modulation of the small RNA rsmZ. Nucleic Acids Res 42(7):4563–4576CrossRefGoogle Scholar
  58. Weigand PV, Chattwal GS (1995) Correlation of epithelial cell invasiveness of GBS with clinical source of isolation. Microb Pathog 19:83–91CrossRefGoogle Scholar
  59. Weigand PV, Benkel P, Rohde M, Chhatwal GS (1996) Entry and intracellular survival of Group B Streptoccoci in J774 macrophages. Infect Immun 64:2467–2473Google Scholar
  60. Wessels MR, Paoletti LC, Rodewald AK, Michon F, Difabio J, Jennings HJ, Kasper DL (1993) Stimulation of protective antibody against type Ia and Ib GBS by a Type Ia polysaccharide-tetanus toxoid conjugate vaccine. Infect Immun; 61:4760–4766Google Scholar
  61. Wilson CB, Jacob RF, Smith AL (1982) Cellular antibiotic pharmacology. Semin Perinatol 6:205–213Google Scholar
  62. Wittington PN (1990) Ch: Clarification and extraction. In: Harris ELV, Angal S (eds) Protein purification methods, a practical approach. Oxford University Press, OxfordGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Post Graduate Institute of Medical Education and ResearchChandigarhIndia
  2. 2.All India Institute of Medical SciencesRishikeshIndia

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