The Clostridium ramosum IgA Proteinase Represents a Novel Type of Metalloendopeptidase

Abstract

Clostridium ramosum is part of the normal flora in the human intestine. Some strains produce an IgA proteinase that specifically cleaves human IgA1 and the IgA2m(1) allotype. This prolylendopeptidase was purified from a broth culture supernatant, and N-terminal sequences of the native protein and tryptic fragments thereof were determined. A fragment of the iga gene encoding the IgA proteinase was isolated using degenerate primers in PCR, and the complete gene was obtained by inverse PCR. The identity of the iga gene was confirmed by heterologous expression in Escherichia coli. The deduced amino acid sequence indicated a signal peptide of 30 residues and a secreted proteinase of 133,828 Da. A typical Gram-positive cell wall anchor motif was identified in the C terminus. The presence of a putative zinc-binding motif His-Glu-Phe-Gly-His together with inhibition studies indicate that the proteinase belongs to the zinc-dependent metalloproteinases. However, the sequence of the C. ramosum IgA proteinase shows no overall similarity to other proteins except for significant identity around the zinc-binding motif with family M6 of metalloendopeptidases, and the unique sequence of the IgA proteinase in this area presumably establishes a new subfamily. The GC percentage of the iga gene is significantly higher than that for the entire genome of C. ramosum, suggesting that the gene was acquired recently in evolution.

Horizontal transfer of the immunoglobulin A1 protease gene (iga) from Streptococcus to Gemella haemolysans

“…Based on an alignment of 13 published iga gene sequences from Streptococcus species (Gilbert et al, 1991;Poulsen et al, 1996Poulsen et al, , 1998Wani et al, 1996), we designed two degenerate primers, iga-for 59-GGTAAATC-WGGYTWTAACAA-39 and iga-rev 59-ATGSGTCATYTCATGRGT-AT-39, located within conserved regions (corresponding to positions 3949-3968 and 4707-4688, respectively, in the ORF of the S. pneumoniae strain PK81 iga gene, accession number X94909). Inverse PCR was performed as described by Ochman et al (1988) and Kosowska et al (2002). Briefly, restriction enzymes selected on the basis of obtained sequences were used to digest the genomic DNA prior to circularization by self-ligation, and outward-pointing oligonucleotide primers, designed from previous sequences, were used for PCR performed as described above.…”

“…In this way, these bacteria are able to evade the protective functions of this principal mediator of adaptive immunity at mucosal surfaces and may, concomitantly, take advantage of released specific Fab fragments in facilitating adherence and immune disguise (Kilian & Reinholdt, 1987;Kilian et al, 1996;Weiser et al, 2003). The biological significance of these enzymes is indirectly inferred by the facts that nature has developed IgA1-cleaving activity among bacteria colonizing humans along at least five independent evolutionary lineages, and that all three principal bacteria causing meningitis produce an IgA1 protease Kosowska et al, 2002). Metallo-type IgA1 proteases are produced by all members of the species Streptococcus pneumoniae and by some members of the related commensal species Streptococcus sanguinis, Streptococcus oralis, Streptococcus mitis and Streptococcus infantis.…”

Effect of Mutations in the Human Immunoglobulin A1 (IgA1) Hinge on Its Susceptibility to Cleavage by Diverse Bacterial IgA1 Proteases

“…While a few species of bacteria produce enzymes capable of cleaving both human IgA1 and IgA2 (16,38), a smaller number of bacterial species produce enzymes, called IgA1 proteases, capable of cleaving only human IgA1 and not IgA2. The organisms producing IgA1 proteases include the principal causes of bacterial meningitis, Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae, and other bacterial pathogens initiating infection at mucosal surfaces of the genital tract and the oral cavity (13,24,38).…”

Microbial Evasion of IgA Functions

“…As summarized in Figure 1, some of the IgA1 proteases are serine-type proteases, others are metalloproteinases, and yet others are thiol cysteine proteinases. Genetic analyses show that IgA1 protease genes are the result of at least five independent lines of evolutionary events (Figure 1) (Koomey et al, 1982;Bricker et al, 1983;Koomey and Falkow, 1984;Pohlner et al, 1987;Gilbert et al, 1988Gilbert et al, , 1991Poulsen et al, 1988Poulsen et al, , 1996Spooner et al, 1992;Wani et al, 1996;Kosowska et al, 2002;BekThomsen et al, 2012) and are thus a striking example of convergent evolution of a highly specialized enzymatic activity.…”