The dissemination of C 10 cysteine protease genes in Bacteroides fragilis species by mobile genetic elements

Background: The C10 family of cysteine proteases includes enzymes that contribute to the virulence of bacterial pathogens, such as SpeB in Streptococcus pyogenes. The presence of homologues of cysteine protease genes in human commensal organisms has not been examined. Bacteroides fragilis is a member of the dominant Bacteroidetes phylum of the human intestinal microbiota, and is a significant opportunistic pathogen. Results: Four homologues of the streptococcal virulence factor SpeB were identified in the B. fragilis genome. These four protease genes, two were directly contiguous to open reading frames predicted to encode staphostatin-like inhibitors, with which the protease genes were co-transcribed. Two of these protease genes are unique to B. fragilis 638R and are associated with two large genomic insertions. Gene annotation indicated that one of these insertions was a conjugative Tn-like element and the other was a prophage-like element, which was shown to be capable of excision. Homologues of the B. fragilis C10 protease genes were present in a panel of clinical isolates, and in DNA extracted from normal human faecal microbiota. Conclusions: This study suggests a mechanism for the evolution and dissemination of an important class of protease in major members of the normal human microbiota. Background Bacteroides fragilis is a Gram-negative member of the normal human gut microbiota. The Bacteroidetes constitutes one of the major bacterial phyla in the healthy human gut [1]. However, B. fragilis is also an important opportunistic pathogen, and it is the most frequently isolated anaerobic bacterium in clinical specimens, including abdominal abscesses and bloodstream infections [2]. Indeed, while B. fragilis accounts for only 4 to 13% of the normal human fecal microbiota, it is responsible for 63 to 80% of Bacteroides infections [3]. Only a few virulence factors have been described for B. fragilis, with the best characterized being the polysaccharide (PS) capsule [4] and a secreted metalloprotease, fragilysin [5]. The capsule, which displays antigenic variation, promotes the formation of abscesses [4], and the reduction of proinflammatory responses to B. fragilis [4,6]. The metalloprotease fragilysin, which has been linked to diarrheal disease [5], has activity against the zonula junctions between cells, and could disrupt tissue integrity [7]. B. fragilis also encodes homologues of C10 proteases [8]. These are members of the CA clan of papain-like proteases. Other C10 proteases include the important virulence factors Streptococcal pyrogenic exotoxin B (SpeB) from Streptococcus pyogenes and Interpain A from Prevotella intermedia. SpeB cleaves a variety of host protein, including immunoglobulin, fibronectin and vitronectin; it also activates IL-1β and releases kinin from kininogen [9]. Interestingly, both SpeB and Interpain A target and inactivate complement factor C3 [10,11]. One further characterized C10 protease is the Periodontain from the oral pathogen Porphyromonas gingivalis, which cleaves α1proteinase inhibitor promoting degradation of connective tissue components [12]. For both SpeB and another well characterized family of cysteine proteases (C47 family) expressed in staphylococci (Staphopain), the protease genes are found juxtaposed to genes encoding specific protease inhibitors, Spi [13] (a propeptide analogue) and Staphostatin [14] (a lipocalin-like entity), respectively. The genomes of Bacteroides spp., including B. fragilis, may include plasmids [15], and typically include multiple prophage remnants, pathogenicity islands and both con* Correspondence: jakki.cooney@ul.ie 1 Department of Life Sciences, University of Limerick, Limerick, Ireland Full list of author information is available at the end of the article Thornton et al. BMC Microbiology 2010, 10:122 http://www.biomedcentral.com/1471-2180/10/122 Page 2 of 15 jugative and non-conjugative transposons (CTn and Tn respectively) [16]. This would facilitate acquisition and dissemination of virulence markers. Indeed, the fragilysin is encoded on a pathogenicity island which has been shown to be mobile [17]. This study centers on the identification and characterization of genes encoding homologues of SpeB, their genetic linkage with putative inhibitors, and the association of these homologous genes with mobile genetic elements. Results The B. fragilis genome harbours four paralogous C10 protease genes A phylogenetic study was undertaken to determine the relatedness of C10 proteases in other members of the Bacteroidetes phylum (Fig. 1). This identified eight-four C10 protease candidates, ranging in size from 269 to 1656 amino acids, in organisms that occupy both human and environmental niches. The larger of these proteins (>600 amino acid residues, average length 803 residues) group together along with SpeB and Interpain A. These larger proteins have additional C-terminal domains, the role of which is yet to be determined [12,18]. The Bfp proteases group with proteins <500 amino acid residues in length (average length 435 residues). Although acceptable bootstrap values were obtained for nodes separating deeper phylogenetic levels, the bootstrap values for the shallower divisions were low. This reflects the unstable phylogeny obtained. However, it is noteworthy that all of the candidate protease sequences had a variation on the two active site motifs indicated in Fig 2. Of particular interest was the identification of SpeB homologues in B. fragilis. Analysis of the B. fragilis 638R ftp://ftp.sanger.ac.uk/pub/pathogens/bf/, YCH46 [19] and NCTC9343 [7] genome sequences identified genes encoding a paralogous family of C10 cysteine proteases named Bfp1 (BF638R0104, 45390), Bfp2 (BF638R1641, 56666), Bfp3 (BF638R3679, 47323), Bfp4 (BF638R0223, 48433) for B. fragilis protease, encoded by genes bfp1-4 respectively. The locus identifiers for the unpublished 638R genome, followed by the predicted molecular mass of the preproprotein in Daltons are given in parenthesis. bfp1 and bfp2 were present in all three strains whereas bfp3 and bfp4 were present only in B. fragilis 638R (Table 1). Similarity between the predicted Bfp protein sequences and zymogen SpeB ranges from 33-41.2%, with similarity between the paralogues themselves higher (36.7-46.1%) (Table 2). These low values are not surprising, as it has been established that the overall sequence identity and similarity between the CA clan of Papain-like proteases is low [20]. However, the core of the the protease domains of the C10 proteases SpeB (1DKI) and Interpain (3BBA) [18] are similar in structure (root mean squared deviation of 1.220 Å based on 197 Cα positions), even with only 32.5% sequence identity. Critically, the active site residues (Cys165 and His313, SpeB zymogen numbering [21]) are highly conserved (Fig. 2). It is probable that the bfp genes encode active proteases, and thus, may contribute to the pathogenesis of Bacteroides infections in a manner analogous to the role of SpeB in streptococcal pathogenesis [22]. Bacterial cysteine protease genes have been found coupled to genes encoding specific inhibitors, therefore, the regions both up and downstream of the four bfp genes were analyzed for candidate inhibitors. Three open reading frames encoding small proteins (116-138 amino acids) within 35 base pairs of the proteases were identified. These were named bfi1A (BF638R0103), bfi1B (BF638R0105) and bfi4 (BF638R0222) (for Bacteroides fragilisinhibitor). The encoded proteins showed no significant identity to the propeptides of any known protease, nor to Spi. Surprisingly, they had identity to the C47 cysteine proteases inhibitors, the Staphostatins, ranging from 15.0-23.4% identity and 32.6-45.7% similarity (Table 3). This is in line with identity between Staphostatin A and Staphostatin B with 20.4% identity and 45.0% similarity. Despite low levels of sequence identity, analysis of the predicted secondary structure and the conservation and alignment of a critical glycine residue in these sequences (indicated in Fig. 3) when compared to Staphostatins, suggested that these bfi genes encode specific protease inhibitors. To determine the likely cellular location of Bfp and Bfi proteins, the respective sequences were analyzed using LipPred [23], LipoP [24], SignalP [25] and PSORTb [26]. These analyses suggested that Bfi1A has a typical Sec pathway leader sequence and is likely to be exported to the periplasm. Bfi1B, Bfi4, Bfp1, Bfp2 and Bfp4 have predicted lipoprotein signal sequences and are likely to be tethered to the outer membrane [24,27]. Whilst Bfp3 has a lipoprotein leader sequence it is not clear which membrane it is likely to associate with. It should be noted that maturation of C10 zymogens would release the active protease from the anchoring acyl-lipid into the extracellular milieu. B. fragilis C10 proteases genes, bfp1 and bfp4, are cotranscribed with those for predicted Staphostatin-like inhibitors For both the streptococcal and staphylococcal systems, the proteases and adjacently encoded inhibitors are cotranscribed [13,28]. To determine if this transcriptional coupling of protease and inhibitor genes was also present in B. fragilis, RNA was isolated from broth grown 638R cells, and analysed by reverse transcriptase PCR, using a series of specific primers for the protease and inhibitor Thornton et al. BMC Microbiology 2010, 10:122 http://www.biomedcentral.com/1471-2180/10/122 Page 3 of 15 Figure 1 Phylogenetic tree of the C10 proteases available on the GenBank and NCBI databases. Cluster analysis was based upon the neighbour-joining method. Numbers at branch-points are percentages of 1000 bootstrap re-samplings that support the topology of the tree. The tree was rooted using C47 family cysteine protease sequences (Staphopains). The locus tag identifiers and the organism name are given. SpeB and the Btp proteases are indicated by a red diamond. ! !