Cloning, Expression, and Immunogenicity Analysis of the CpsE Protein from Group B Streptococcus Isolated from Tilapia (Oreochromis Niloticus)

Group B Streptococcus (GBS) is a major cause of serious bacterial infection in numerous animal species. The production of capsular polysaccharide(CPs) is vital to GBS to evade host immunity. One of the genes that required for production of CPs, cpsE, has been determined to be well conserved in capsule gene cluster (cps).This study cloned the cpsE gene from Tilapia of GBS clinical isolate (serotype Ia) and expressed this gene with aid of pET-32a(+) in Escherichia coli BL21(DE3) competent cells to obtain high levels of the recombinant protein for further study about CpsE in fish and examination of its immunogenicity. The optimization of induction conditions (IPTG concentration, temperature and time) in E.coli was accomplished and let us to perform the recombinant protein induction at 37℃ for 3h,with 0.2mM IPTG in Luria Bertani (LB) medium. At the optimal conditions, recombinant protein was expressed in an insoluble form (inclusion bodies) and accounted for approximately 23% of the total protein. Purification by affinity chromatography yielded about 480mg fusion protein per liter culture.

[1]  E. Moxon,et al.  The role of bacterial polysaccharide capsules as virulence factors. , 1990, Current topics in microbiology and immunology.

[2]  A. Cross,et al.  The biologic significance of bacterial encapsulation. , 1990, Current topics in microbiology and immunology.

[3]  H. Yim,et al.  The Serotype of Type Ia and III Group B Streptococci Is Determined by the Polymerase Gene within the Polycistronic Capsule Operon , 2000, Journal of bacteriology.

[4]  G. Keefe,et al.  Streptococcus agalactiae mastitis: a review. , 1997, The Canadian veterinary journal = La revue veterinaire canadienne.

[5]  B. Mollet,et al.  Identification and characterization of the eps (Exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6 , 1996, Journal of bacteriology.

[6]  A. Lusiastuti,et al.  Streptococcus agalactiae INFECTION ON TILAPIA (Oreochromis niloticus) IN CIRATA RESERVOIR, WEST JAVA , 2009 .

[7]  J. C. Freitas,et al.  Isolation and characterization of Streptococcus spp. group B in Nile tilapias (Oreochromis niloticus) reared in hapas nets and earth nurseries in the northern region of Parana State, Brazil , 2005 .

[8]  C. Rubens,et al.  Sialylation of Group B Streptococcal Capsular Polysaccharide Is Mediated by cpsK and Is Required for Optimal Capsule Polymerization and Expression , 2005, Journal of bacteriology.

[9]  A. Eldar,et al.  Streptococcus shiloi andStreptococcus difficile: Two new streptococcal species causing a meningoencephalitis in fish , 1994, Current Microbiology.

[10]  F. Kong,et al.  Occurrence of rare genotypes of Streptococcus agalactiae in cultured red tilapia Oreochromis sp. and Nile tilapia O. niloticus in Thailand—Relationship to human isolates? , 2008 .

[11]  P. Ferrieri,et al.  Changing Epidemiology of Group B Streptococcal Colonization , 1999, Pediatrics.

[12]  F. Kong,et al.  Serotype IX, a Proposed New Streptococcus agalactiae Serotype , 2007, Journal of Clinical Microbiology.

[13]  A. L. Larentis,et al.  Cloning and optimization of induction conditions for mature PsaA (pneumococcal surface adhesin A) expression in Escherichia coli and recombinant protein stability during long-term storage. , 2011, Protein expression and purification.

[14]  R. Lukacovic,et al.  Association of Streptococcus sp. with fish mortalities in the Chesapeake Bay and its tributaries , 1990 .

[15]  R. Morona,et al.  Nucleotide sequence analysis of genes essential for capsular polysaccharide biosynthesis in Streptococcus pneumoniae type 19F , 1994, Infection and immunity.

[16]  M. Wagner,et al.  JM9 strains, a new type of group B streptococci from Japan. , 1994, Zentralblatt fur Bakteriologie : international journal of medical microbiology.

[17]  P. Klesius,et al.  Characterization of β‐haemolytic Group B Streptococcus agalactiae in cultured seabream, Sparus auratus L., and wild mullet, Liza klunzingeri (Day), in Kuwait , 2002 .

[18]  P. Vandamme,et al.  Streptococcus difficile is a nonhemolytic group B, type Ib Streptococcus. , 1997, International journal of systematic bacteriology.

[19]  J. Qasem,et al.  Isolation of Streptococcus agalactiae from cultured silver pomfret, Pampus argenteus (Euphrasen), in Kuwait. , 2004, Journal of fish diseases.

[20]  M. Wessels,et al.  Identification of cpsD, a gene essential for type III capsule expression in group B streptococci , 1993, Molecular microbiology.

[21]  D. Kasper,et al.  Functional Analysis in Type Ia Group B Streptococcusof a Cluster of Genes Involved in Extracellular Polysaccharide Production by Diverse Species of Streptococci* , 2001, The Journal of Biological Chemistry.

[22]  R. Morona,et al.  Tyrosine phosphorylation of CpsD negatively regulates capsular polysaccharide biosynthesis in Streptococcus pneumoniae , 2000, Molecular microbiology.

[23]  A. Schuchat,et al.  A population-based comparison of strategies to prevent early-onset group B streptococcal disease in neonates. , 2002, The New England journal of medicine.

[24]  C. Baker,et al.  Group B streptococcal infections in elderly adults. , 2005, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[25]  P. Larsson,et al.  Serotypes and clinical manifestations of invasive group B streptococcal infections in western Sweden 1998-2001. , 2004, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[26]  W. Schaffner,et al.  Increasing burden of invasive group B streptococcal disease in nonpregnant adults, 1990-2007. , 2009, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[27]  S. Iijima,et al.  Molecular Characterization of Type-Specific Capsular Polysaccharide Biosynthesis Genes of Streptococcus agalactiae Type Ia , 1999, Journal of bacteriology.

[28]  G. Orefici,et al.  A multiplex PCR assay for the direct identification of the capsular type (Ia to IX) of Streptococcus agalactiae. , 2010, Journal of microbiological methods.