Haemophilus influenzae immunoglobulin A1 protease genes: cloning by plasmid integration-excision, comparative analyses, and localization of secretion determinants.

Many bacteria which establish infections after invasion at human mucosal surfaces produce enzymes which cleave immunoglobulin A (IgA), the primary immunoglobulin involved with protection at these sites. Bacterial species such as Haemophilus influenzae which produce IgA1 proteases secrete this enzyme into their environment. However, when the gene encoding this protein was isolated from H. influenzae serotype d and introduced into Escherichia coli, the activity was not secreted into the medium but was localized in the periplasmic space. In this study, the IgA1 protease gene (iga) from an H. influenzae serotype c strain was isolated and the gene from the serotype d strain was reisolated. The IgA1 proteases produced in E. coli from these genes were secreted into the growth medium. A sequence linked to the carboxyl terminus of the iga gene but not present in the original clone was shown to be necessary to achieve normal secretion. Tn5 mutagenesis of the additional carboxyl-terminal region was used to define a 75- to 100-kilodalton coding region required for complete secretion of IgA1 protease but nonessential for protease activity. The iga genes were isolated by a plasmid integration-excision procedure. In this method a derivative of plasmid pBR322 containing a portion of the protease gene and the kanamycin resistance determinant of Tn5 was introduced into H. influenzae by transformation. The kanamycin resistance gene was expressed in H. influenzae, but since pBR322 derivatives are unable to replicate in this organism, kanamycin-resistant transformants arose by integration of the plasmid into the Haemophilus chromosome by homologous recombination. The plasmid, together with the adjoining DNA encoding IgA1 protease, was then excised from the chromosome with DNA restriction enzymes, religated, and reintroduced into E. coli. Comparisons between the H. influenzae protease genes were initiated which are useful in locating functional domains of these enzymes.