Cloning, expression, purification and ligand binding studies of novel fibrinogen-binding protein FbsB of Streptococcus agalactiae
Introduction
With the emerging bacterial strains resistant to antimicrobial drugs, a major aim in the current field of medicine is to develop strategies to fight against bacterial infections. Attempts are therefore being made to study the diverse population of bacteria that cause diseases as a result of which genome sequencing and protein characterization for a number of bacteria have been done. One among them is of a Group B Streptococcus (GBS), Streptococcus agalactiae. This Gram-positive cocci is a commensal organism which is best known for postpartum infection and neonatal sepsis [1]. Common manifestations of it are urinary tract infections, pneumonia, skin and soft tissue infections, septic arthritis, osteomilitis, meningitis, peritonitis and endo-opthalmitis [1], [2]. In addition, it also colonizes the mammary glands of ruminants causing mastitis leading to major financial losses in the dairy industry [1], [3]. Pathogenesis in Gram-positive bacteria is normally initiated by the adherence of the bacteria to the host via its surface adhesins. A subfamily of adhesins known as MSCRAMMs (Microbial Surface Component Recognizing Adhesive Matrix Molecules) binds specifically to extracellular matrix molecules (ECM) like fibrinogen, fibronectin, collagen, laminin etc. [4]. The MSCRAMM–ECM interaction is one of the good targets for developing new therapeutic agents.
Fibrinogen (Fg)1, an important component of the blood plasma, is often a site of binding among Gram-positive bacteria. Among the well characterized Fg-binding adhesins from Gram-positive bacteria are the ClfA and ClfB from Staphylococcus aureus [5], [6], Fbe from Staphylococcus epidermidis [7] and FgBP from Streptococcus equi subsp. equi, a Group C Streptococcus [8]. In S. agalactiae, two surface proteins that bind to Fg have been characterized. They are Fg-binding surface protein A (FbsA) [9] and Fg-binding surface protein B (FbsB) [10]. FbsA has the LPXTG motif, a specific signal for cell wall anchoring, which is generally seen in the structural organization of MSCRAMMs, however, FbsB is devoid of such a motif [10], [11] suggesting that it is a secreted protein rather than a covalently surface-exposed protein. Although both FbsA and FbsB bind to Fg, there is no sequence homology between them. While FbsA is a repeat protein composed of several repetitive units, each 16 amino acids in length [9], FbsB is a non-repetitive protein. The common feature in both FbsA and FbsB is the number of amino acids that constitute the protein which varies from one strain to another.
FbsB [10] from the human strain S. agalactiae NEM316, a type III GBS, is a 635 amino acid (73 kDa) protein. Its homologous protein in a bovine strain of S. agalactiae was termed as Fgag [3]. The sequence comparison of FbsB/Fgag using MultAlin [12] in various S. agalactiae strains is shown in Fig. 1. FbsB has a conserved N-terminal signal peptide and a C-terminal region of 223 residues. The N-terminal region between the signal peptide and the C-terminal region varies in size from one strain to another and also not conserved. However, this region has been shown to bind to human Fg [10]. In contrast to this observation it has been shown that the conserved C-terminal region in Fgag binds to bovine Fg and not to human Fg [3].
The protein sequence analysis of the C-terminal region of FbsB [FbsB(C), corresponding to 413–635 residues] from S. agalactiae NEM316 using BLAST indicates that other than similarities with strains of the same organism, there is no similarity with any characterized protein. This shows that FbsB(C) is a novel protein. Here we report the cloning, expression, purification, secondary structural analysis and binding studies of FbsB(C) with bovine Fg.
Section snippets
Bacterial strains, plasmids and culture conditions
Freeze-dried culture of Type III S. agalactiae NEM 316 strain was obtained from American Type Culture Collection (ATCC). Escherichia coli DH5α was used for plasmid cloning and Rosetta-gami(DE3)pLysS for protein expression. pET20b(+) from Novagen was used to make the vector-DNA construct. The streptococcal strain was grown in Todd-Hewitt broth (THB) supplemented with 0.1% yeast extract and all other E. coli strains were grown in Luria Bertani (LB) broth and when required 100 μg/ml of ampicillin
Preparation of construct, expression, purification and confirmation of the recombinant protein
fbsB(C) was PCR amplified and fused with pET20b(+) to form the construct pET20b-fbsb(C). The protein expression was observed in two different strains, BL21pLysS and Rosetta-gami(DE3)pLysS. On comparing the expression profile in the two strains at different temperature conditions, it was observed that expression was best in Rosetta-gami(DE3)pLysS at 37 °C. Protein expressed in this strain was therefore purified to near homogeneity as mentioned above (Fig. 2A and B). During the purification, it
Discussion
Pathogenic bacteria find their way into the host tissue via their surface adhesins that recognize ECM as ligands. Adhesins characterized so far are well conserved among different strains and species of bacteria and therefore, adhesin-based vaccines could give protection against a wider range of bacteria. This might overcome the problem of antimicrobial resistance. An important component of the blood plasma, Fg, is found in the ECM and is an important site of bacterial recognition. Two adhesins
Conclusion
The C-terminal region of FbsB or rFbsB408–635 was cloned and expressed for studying its binding activity to bovine Fg which showed the ability of rFbsB(C)408–635 binding to bovine Fg. The purified protein was also used for finding out the secondary structural components by circular dichroism. The secondary structure analysis revealed that it is composed mainly of alpha helices and random coils with very few beta strands. However, it is interesting to note that the crystal structures of
Acknowledgments
We thank the Department of Biotechnology (DBT), Government of India for financially supporting the project. We also thank Dr. Souvik Maiti, Institute of Genomics and Integrative Biology (IGIB), New Delhi, India for allowing us to use their CD facility.
References (23)
A novel family of fibrinogen-binding proteins in Streptococcus agalactiae
Vet. Microbiol.
(2003)- et al.
Localization of a fibrinogen calcium binding site between γ-subunit positions 311 and 366 by terbium fluorescence
J. Biol. Chem.
(1985) - et al.
Genetic relatedness between group B streptococci originating from bovine mastitis and a human group B streptococcus type V cluster displaying an identical pulsed-field gel electrophoresis pattern
Clin. Microbiol. Infect.
(2006) - et al.
The fibrinogen-binding MSCRAMM (clumping factor) of Staphylococcus aureus has a Ca2+-dependent inhibitory site
J. Biol. Chem.
(1998) - et al.
A “dock, lock, and latch” structural model for a Staphylococcal adhesin binding to fibrinogen
Cell
(2003) - C.J. Woods, C.S. Levy, Streptococcus group B infections. Available from...
- et al.
Neonatal group B streptococcal infection
Curr. Paediatr.
(1996) - et al.
MSCRAMM-mediated adherence of microorganisms to host tissues
Annu. Rev. Microbiol.
(1994) - et al.
Identification of the ligand-binding domain of the surface-located fibrinogen receptor (clumping factor) of Staphylococcus aureus
Mol. Microbiol.
(1995) - et al.
Clumping factor B (ClfB), a new surface-located fibrinogen-binding adhesin of Staphylococcus aureus
Mol. Microbiol.
(1998)
A fibrinogen-binding protein of Staphylococcus epidermidis
Infect. Immun.
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