Functional Analysis Of Tracheal Colonization Factor (TcfA) In

Whooping cough or pertussis is an acute respiratory tract infection caused byBordetella pertussis, mostly happened among infants. The pathogenesis involvesseries of processes, including adherence, invasion and toxin-producing.Many membraneassociated proteins, such as filamentous hemagglutinin, pertactin and Trachealcolonization factor participate in this process. Bordetella pertussis tracheal colonizationfactor is an membrane protein existed outside the bacterial. Previous study showed that itmay be an important adhesion factor, but its function has not identified. So we take therecombinant technique to express the protein in the Escherichia coli(BL21;DE3),and thenpurify the protein. Next we investigate the protein though various adherent experiments,including cell adhesive assay of recombinant bacteria, purified proteinimmunofluorescence and competitive inhibition. On the basis of this,we next prove thefunction of the RGD tripetide located on the site of317-319and then try to find thepotential receptor which may interact with TcfA. Last but not east, we compare theadhesive ability between the FHA and PRN gene deleted bacteria and the non-deletedbacteria.We successfully express the TcfA protein though the recombinant technique andpurify the protein at the level of85%.Immunoblot shows that the recombinant proteincan react with the serum obtained from the vaccine producing bacteria(CS), illustratingthat the purified protein has a good ability of immunoreactivity and antigen specificity.Slide agglutination test and V plate agglutination assay prove that the TcfA proteinexpresses on the surface of the bacteria. Experiments of fluorescence adherencedemonstrate that there is obvious difference in the adherent ability between the TcfAexpressed bacteria and empty vector containing bacteria. Adherent ELISA also showsthat statistical difference exists between the two bacteria. The fluorescence resultsindirectly show the interaction between the TcfA protein and the receptor. The TcfAprotein can competitively inhibit the adherence of the TcfA expressed bacteria, behavinga protein concentration dependent manner. Antibody inhibition assay reveals that thelevel of adherence exerts great reduction when the TcfA polyclonal antibody blocks theTcfA protein site on the bacteria, suggesting that the TcfA protein plays a specific role.RGD may be an important site though which the TcfA protein behaves its function. Whenwe use RGD and RGE short peptide to inhibit the bacteria adherence, the result is asexpected. The gentamicin killing assay is one of effective experiments to test whether thepathogen has the ability of invading into the mammalian cells, aiming to identify whether the adherent molecule mediates the invasion. Our study shows that TcfA protein does nothelp the bacteria invade the Hela cell. Compared with the wild strain, the FHA and PRNgene-delete strain shows less adherence to the epithelial cells after it contacts with theanti-TcfA polyclonal antibody, but the difference is not significant, This may beconcluded that, as a membrane associated adhesion factor, TcfA may play an importantrole in mediating the adherence.The GST pull down assay proves that the protein myosin-9may be the potentialreceptor though which the TcfA interacts with the HeLa cells,but the result needs furtherconfirmation.