Interaction of Bordetella pertussis filamentous hemagglutinin with human TLR2: identification of the TLR2-binding domain.

Filamentous hemagglutinin (FHA) is a major adhesion and virulence factor of Bordetella pertussis and also a main component of acellular pertussis vaccines. Interaction of FHA with different receptors on human epithelial and immune cells facilitates entrance and colonization of bacteria as well as immunomodulation of the host immune response. Three overlapping segments of the FHA gene were cloned in a prokaryotic expression vector and the recombinant proteins were purified. These recombinant fragments along with the native FHA protein were employed to assess their potential Toll-like receptor (TLR) stimulatory effects and to localize the TLR binding region. TLR stimulation was monitored by applying HEK293-Blue cell lines cotransfected with TLR2, 4, or 5 and a NF-κB reporter gene. Culture supernatants were checked for secretion of the reporter gene product and IL-8 as indicators of TLR stimulation. Native FHA was found to strongly stimulate TLR2, but not TLR4 or TLR5 transfected cells. Among recombinant FHA fragments only the fragment spanning amino acid residues 1544-1917 was able to exhibit the TLR2 stimulating property of FHA. Interaction of FHA with TLR2 suggests its involvement in induction of the innate immune system against Bordetella pertussis. The TLR2-binding domain of FHA may contribute to immunoprotection against pertussis infection.

Expression, Purification and Characterization of Three Overlapping Immunodominant Recombinant Fragments from Bordetella pertussis Filamentous Hemagglutinin.

BACKGROUND: Filamentous hemagglutinin (FHA) is one of the most important immunoprotective antigens of Bordetella pertussis (B. pertussis) and a major component of the acellular pertussis vaccine. In the present study, three overlapping recombinant fragments from the immunodominant region of FHA were produced and their immunogenicity was investigated. METHODS: Three overlapping coding sequences of FHA antigen were amplified from B. pertussis genomic DNA by PCR. Amplified fragments were expressed in Escherichia coli (E. coli) BL21(DE3) strain and purified through His-tag using Nickel-based chromatography. Purified fragments were characterized by SDS-PAGE and Western blotting techniques. In vitro peripheral blood mononuclear cells (PBMC) proliferation and IFN-γ production were assessed in a limited number of healthy adults vaccinated with a commercial acellular pertussis vaccine in response to all purified FHA fragments by H3-Thymidine incorporation and ELISA, respectively. RESULTS: Recombinant FHA segments were successfully cloned and produced at high levels in E. coli BL21(DE3). SDS-PAGE and Western blot analyses confirmed their purity and reactivity. All three recombinant fragments together with a commercial native FHA were able to induce in vitro PBMC proliferation and IFN-γ production. CONCLUSION: Our preliminary results suggest that these overlapping recombinant FHA fragments are immunogenic and may prove to be immuno-protective.

Importance of ATM gene as a susceptible trait: predisposition role of D1853N polymorphism in breast cancer.

The involvement of ATM gene and specifically, the important role of D1853N polymorphism, as a three-hit hypothesis has been previously reported in an Iranian proband affected with brain tumor and this polymorphism could be screened in her relatives as well. The aim of present study was to investigate the involvement of D1853N polymorphism as a predisposition factor in 129 Iranian patients affected with primary breast cancer and 248 sex- and age-matched healthy controls. Mutant allele-specific PCR amplification (MASA) assay was performed to analyze the D1853N polymorphism in the ATM gene. The frequency of D1853N polymorphism in cases, internal and external controls was 31.0% (40/129), 26.9% (28/104) and 12.5% (18/144), respectively. The frequency of D1853N in total control groups, including normal external control and pedigree internal control, was 18.6% (46/248). The odds ratio was calculated with the logistic regression test, with an estimated relative risk of 2.579 (P=0.005). The significant difference was observed between the patient-carriers of this alteration and external controls (P=0.001). The number of controls harboring D1853N polymorphism was higher in internal control compared to external controls, and the difference was statistically significant (P=0.004). The significant difference was observed between the patient-carriers and external controls and could be considered as a predisposing and diagnostic marker in the population and specifically in the cancer-prone pedigrees.