A Comprehensive Genetic Study of Streptococcal Immunoglobulin A1 Proteases: Evidence for Recombination within and between Species

An analysis of 13 immunoglobulin A1 (IgA1) protease genes (iga) of strains of Streptococcus pneumoniae, Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguis was carried out to obtain information on the structure, polymorphism, and phylogeny of this specific protease, which enables bacteria to evade functions of the predominant Ig isotype on mucosal surfaces. The analysis included cloning and sequencing of iga genes from S. oralis and S. mitis biovar 1, sequencing of an additional seven iga genes from S. sanguis biovars 1 through 4, and restriction fragment length polymorphism (RFLP) analyses of iga genes of another 10 strains of S. mitis biovar 1 and 6 strains of S. oralis. All 13 genes sequenced had the potential of encoding proteins with molecular masses of approximately 200 kDa containing the sequence motif HEMTH and an E residue 20 amino acids downstream, which are characteristic of Zn metalloproteinases. In addition, all had a typical gram-positive cell wall anchor motif, LPNTG, which, in contrast to such motifs in other known streptococcal and staphylococcal proteins, was located in their N-terminal parts. Repeat structures showing variation in number and sequence were present in all strains and may be of relevance to the immunogenicities of the enzymes. Protease activities in cultures of the streptococcal strains were associated with species of different molecular masses ranging from 130 to 200 kDa, suggesting posttranslational processing possibly as a result of autoproteolysis at post-proline peptide bonds in the N-terminal parts of the molecules. Comparison of deduced amino acid sequences revealed a 94% similarity between S. oralis and S. mitis IgA1 proteases and a 75 to 79% similarity between IgA1 proteases of these species and those of S. pneumoniae and S. sanguis, respectively. Combined with the results of RFLP analyses using different iga gene fragments as probes, the results of nucleotide sequence comparisons provide evidence of horizontal transfer of iga gene sequences among individual strains of S. sanguis as well as among S. mitis and the two species S. pneumoniae and S. oralis. While iga genes of S. sanguis and S. oralis were highly homogeneous, the genes of S. pneumoniae and S. mitis showed extensive polymorphism reflected in different degrees of antigenic diversity.Bacterial immunoglobulin A1 (IgA1) proteases are highly specific endopeptidases that cleave the heavy chain of human IgA1, including its secretory form (S-IgA1), in the hinge region. Such proteases are secreted by a small number of bacteria associated with humans (reviewed in references 20 and 28). IgA1 protease-producing bacteria include the mucosal pathogens Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Streptococcus pneumoniae, and Ureaplasma urealyticum as well as some members of resident oral and pharyngeal microfloras. Among the latter are species of Prevotella and Capnocytophaga, Streptococcus sanguis, Streptococcus oralis, and Streptococcus mitis biovar 1.Three different classes of proteinases are represented among the IgA1 proteases, illustrating that cleavage of human IgA1 is a property that has evolved among bacteria through convergent evolution following at least three independent lines. Molecular characterizations have revealed that the IgA1 proteases of Haemophilus and Neisseria are genetically related serine proteinases (1, 25, 29, 30) and that those of S. sanguis and S. pneumoniae are metalloproteinases (8, 32, 40). Studies using specific inhibitors indicate that the IgA1 protease produced by Prevotella melaninogenica is a cysteine proteinase (26). IgA1 proteases are postproline endopeptidases, and those of streptococcal origin cleave one and the same Pro-Thr peptide bond at positions 227 to 228 in the human IgA1 heavy chain (18, 34).The enzymes have been shown to be active in vivo, and by cleaving human IgA1 they interfere with the protective functions of the principal mediator of specific immunity of the upper respiratory tract (reviewed in reference 20). Consequently, these proteases are thought to be important for the ability of the bacteria to colonize human mucosal surfaces in the presence of specific S-IgA1 antibodies. Furthermore, they may constitute an important factor in the pathogenesis of invasive infections (20). Besides, it has been proposed that increased colonization of the pharynges of infants with IgA1 protease-producing bacteria, in particular S. mitis biovar 1, may compromise S-IgA-mediated protection against allergens and lead to atopic sensitization (17).The iga genes encoding IgA1 protease from S. sanguis and S. pneumoniae have been characterized previously (8, 32, 40). Here we report on the cloning and sequencing of iga genes from the other two Streptococcus species, S. oralis and S. mitis, known to produce IgA1-cleaving activity. Furthermore, we have sequenced iga genes from an additional seven strains of S. sanguis representing the four different biovars of this species.