Adhesion Of Two Species Of Pig Origin Streptococcsu To Fibronectin In Vitro

Streptococcus suis is an important pathogen which has been associated with a wide variety of infections in swine such as meningitis, septicemia, arthritis, pneumonia and endorcarditis. Of the 35 official serotypes described to date, serotype 2 is the most virulent and the most commonly isolated from diseased pigs. This miroorganism is also recognized as an agent of zoonosis. Streptococcus equi (Lancefield group C) comprises the two subspecies, S. equi subsp. equi and S. equi subsp. zooepidemicus. Subspecies zooepidemicus is considered an opportunistic commensal; however, after stress or a virus infection, it can cause a secondary infection, which results in strangles-like symptoms, subspecies zooepidemicus also infects a wide range of other animals, such as pigs, dogs, sheep, goats, birds, rabbits, guinea pigs, foxes, cats and cows. Human cases with infection due to subspecies zooepidemicus have also been reported .The attachment of bacteria to surface of the host is considered to be a prerequisite for subsequent colonization or invasion of host. This attachment is mediated by specific interactions between adhesins on the bacterial surface and host cell receptors. For group A streptococcui, fibronectin(Fn) is one of the receptors that is well characterized. Fn is a large glycoprotein composed of two polypeptide chains with a combined molecular mass of 450 kDa. It is found in a soluble form in bodily fluids and in an immobilized form in extracellular matrices and on the surfaces of host cells. Fn has multiple functional domains that interact with diverse substrates, such as fibrin, heparin, collagen, other components of the extracellular matrix, and a wide variety of eukaryotic and prokaryotic cells.Cell surface hydrophobicity of Streptococcus equi subsp . zooepidemicus ATCC35246 and Streptococcus suis type 2 jiangsu isolate HA9801 was determined by "salting out". The interaction of soluble and immobilized fibronectin with two species was investigated by indirect immunofluorescent assay and enzyme-linked immunosorbent assay, respectively. The results showed that ATCC35246 and HA9801 was cell surface hydrophobic and that the former could bind two forms of fibronectin, the late only solube.Aliquots of the Streptococcus equi subsp . zooepidemicus ATCC35246 cells suspended in phosphate buffer solution(PBS) of various pHs were transferred to the ELISA platesprecoated with human fibronectin of various concentrations, and incubated at 37℃ for 1 h. After the buffers and unbound cells were washed out with PBS-Tw,bound cells were detected using strain-specific rabbit antibodies, followed by alkaline phosphatase-labeled second antibody, and colour development with P -nitrophenyl phosphate(lmg/ml) in carbonate buffer(pH9.6 ,with 1 mM MgCl₂). The results showed that adhesion was bacterial cell concentratin-dependent , affected less by fibronectin procoated within concentration range 0. 0625μg/ml¹0μg/ml and was optimal at pH7. 4.fops gene(encoding fibronectin- and fibrinogen-binding protein, FBPS ) was amplified from genomic DNA of Streptococcus suis type 2 Jiangsu isolate HA9801 by polymerase chain reaction(PCR)technique. Recombinant plasmid pMD-T-FBPS was constucted by cloning PCR product into the clone vector pMDT-18. The 1938bp recombinant fragment of pMD-T-FBPS was verified by restriction endonuclease analysis and sequencing. The fops gene nucleotide sequence of Jiangsu isolate was more than 99.99% identical to that of SS2 Netherland strain reported by Greeff and to that of human Germany strain. FBPS displayed 76.0%, 73.0%, 72.4%, 70.1% and 68.8% identity with FbpA of S.gordonii(CAA4628.2), putative fibronectin/fibrinogen-binding protein of S. mutan (AAN59108.1), PavA of S. pneumoniae,(AAK75087.1), adherence and virulence protein A of S. agalactiae 2603V/R(AAN00072.1) and FBP54 of S. pyogenes(AAA57236),respectively. Based on its structural feature , FBPS was speculated to be an anchorless adhesin.Based on the sequence of fops of Streptococcus suis type 2 HA9801, primers were designed for amplification fops gene and its frag...