Molecular study of the PE-binding adhesin candidates from Haemophilus influenzae

grantor: University of TorontoIn order to characterize a putative 37 kDa phosphatidylethanolamine (PE)-binding adhesin, its coding sequence was amplified from a nontypeable 'H. influenzae ' (NTHI6564) by PCR and sequenced. Recombinant adhesin was expressed in 'E. coli' as a GST and as a 6XHis tagged fusion protein. Subsequent studies demonstrated that the putative PE-binding adhesin can not bind to PE, but has potent celite binding ability. The celite binding protein has a C-terminal disulfide bond between Cys 254 and Cys308. A Cys308 to Ser mutant was expressed as a GST fusion protein, and a C-terminal truncated mutant was also constructed and expressed as a 6XHis tagged fusion protein. The results indicated that the celite binding protein gene is highly conserved and its protein product is generally expressed in 'H. influenzae' clinical isolates. Subcellular fractionation was used to localize the celite binding protein to the periplasmic region both in NTHI6564 and in a transfected recombinant ' E. coli' strain. Recombinant protein was purified from periplasmic extracts by chromatofocusing and gel filtration chromatography. Direct binding of 65Zn to the recombinant protein was demonstrated by zinc blotting. Thus, this celite binding protein is in fact a periplasmic zinc binding protein (Pzp1). A 'pzp1' deficient mutant was constructed. The growth defect was specifically rescued by supplementing the growth medium with high concentrations of zinc (100 [mu]M). This result indicates that the ' pzp1' gene is essential for growth under aerobic conditions and also is important for anaerobic growth of 'H. influenzae'. Taken together, our results provide direct evidence that Pzp1 is a key protein for zinc uptake in 'H. influenzae'. To identify the real PE-binding adhesin(s) from 'H. influenzae ', whole cell extracts of NTHI6564 were prepared and applied to a PE affinity column. OmpP2 was expressed as a GST-fusion protein (GST-P2). However, only a small portion of GST-P2 could be purified in soluble form. We conclude that OmpP2, and possibly OmpP5 of 'H. influenzae' are PE-binding proteins. (Abstract shortened by UMI.)Ph.D