A cytosol derived factor of Group B streptococcus prevent its invasion into human epithelial cells
- 126 Downloads
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.
KeywordsInvasion Group B streptococcus (GBS) Pathogenesis Protein purification Invasion inhibition
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.
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.
- Alenghat E, Esterly JR (1984) Alveolar macrophages in perinatal infants. Pediatrics 74:221–223Google Scholar
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with folin-phenol reagent. J Biol Chem 193:265–275Google Scholar
- Milliotis MD (1991) Acridine orange stain for determining intracellular enteropathogens in HeLa cells. J Clin Microbio 29:830–831Google Scholar
- 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
- 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
- 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
- Schuchat A (1998) Epidemiology of Group B Streptococcal disease in United States: shifting paradigms. Clin Microbiol Rev 11:497–513Google Scholar
- 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
- 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
- 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
- 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
- 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
- Wilson CB, Jacob RF, Smith AL (1982) Cellular antibiotic pharmacology. Semin Perinatol 6:205–213Google Scholar
- Wittington PN (1990) Ch: Clarification and extraction. In: Harris ELV, Angal S (eds) Protein purification methods, a practical approach. Oxford University Press, OxfordGoogle Scholar