Interaction of fimbriated and nonfimbriated strains of unencapsulated Haemophilus influenzae with human respiratory tract mucus in vitro

Adherence to mucus may influence bacterial colonization of the respiratory tract. Clinical isolates of nontypable Haemophilus influenzae (NTHi) from the respiratory tract are often fimbriated. We wondered whether fimbriated strains have a different adherence from related nonfimbriated strains. A microtitre plate assay has been developed to study adherence of nontypable H. influenzae to mucus. Wells were coated by incubation either with sol phase of sterile mucoid secretions or with purified preparations of mucins. Two laboratory pairs of fimbriated (F+) and nonfimbriated (F-) nontypable H. influenzae, and six fresh clinical isolates of fimbriated nontypable H. influenzae each with nonfimbriated partners derived by serial passage on agar, were cultured to mid-log phase, washed, and then added to the wells. They were then incubated at 37 degrees C for 30 min before washing to remove unbound bacteria. Adherent bacteria were desorbed by agitation with 0.5% Tween 80 and a viable count performed. The two fimbriated laboratory strains (n = 12 and n = 17), and 5 of the 6 fimbriated clinical isolates were more adherent to sol phase than their respective nonfimbriated partners. Two nonfimbriated clinical isolates were more adherent to plastic than their fimbriated partners. A fimbriated laboratory strain was more adherent than its nonfimbriated partner both to a purified preparation of high molecular mass mucin and to the glycopeptide fraction of the same. We conclude that fimbriated strains of nontypable H. influenzae have increased adherence to sol phase of mucus and purified human respiratory tract mucin. The interactions of fimbriae with mucus are likely to be complex, and may involve both nonspecific and specific interactions.</p

Toxiques et analyses biologiques en toxicologie hospitalière

peer reviewe

Characterisation of novel defective thiopurine S-methyltransferase allelic variants.

Human thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) is a key enzyme in the detoxification of thiopurine drugs widely used in the treatment of various diseases, such as inflammatory bowel diseases, acute lymphoblastic leukaemia and rheumatic diseases. The TPMT gene is genetically polymorphic and the inverse relationship between TPMT activity and the risk of developing severe hematopoietic toxicity is well known. In this study, the entire coding sequence of TPMT, together with its 5'-flanking promoter region, was analysed in patients with an intermediate phenotype for thiopurine drug methylation. Four polymorphisms were identified, two previously described, c.356A>C (p.Lys(119)Thr, TPMT*9) and c.205C>G (p.Leu(69)Val, TPMT*21), and two novel missense mutations, c.537G>T (p.Gln(179)His, TPMT*24) and c.634T>C (p.Cys(212)Arg, TPMT*25). Structural investigations, using molecular modeling, were undertaken in an attempt to explain the potential impact of the amino acid substitutions on the structure and activity of the variant proteins. Additionally, in order to determine kinetic parameters (K(m) and V(max)) of 6-thioguanine (6-TG) methylation, the four variants were expressed in a recombinant yeast expression system. Assays were performed by HPLC and the results were compared with those of wild-type TPMT. The p.Leu(69)Val and the p.Cys(212)Arg substitutions encode recombinant enzymes with a significantly decreased intrinsic clearance compared to that of the wild-type protein, and, consequently, characterise non-functional alleles of TPMT. The p.Lys(119)Thr and the p.Gln(179)His substitutions do not affect significantly the catalytic activity of the corresponding variant proteins, which prevents to unambiguously describe these latter alleles as defective TPMT variants