The Bacteroides spp. are a group of Gram-negative anaerobes from the phylum Bacteroidetes. Members of the Bacteroides spp. occupy regions of the terminal ileum and colon, where they are a major component of the normal human gut microbiota. Although they are commensals, Bacteroides can cause opportunistic infections that may be triggered when the integrity of the mucosal wall of the intestine is compromised or breached, commonly leading to abdominal abscesses and bloodstream infections. Conditions that cause such a loss of intestinal barrier function include gastrointestinal surgery, perforated or gangrenous appendicitis, perforated ulcer, diverticulitis, and inflammatory bowel disease (IBD) 1 . Two of the most frequently isolated Bacteroides spp. from anaerobic infections are B. fragilis and B. thetaiotaomicron. Significantly, although B. fragilis accounts for only 4% to 13% of the normal human fecal microbiota it is isolated from 63% to 80% of Bacteroides infections. B. thetaiotaomicron on the other hand accounts for between 15% and 29% of the fecal microbiota but is linked with only 13% to 17% of infection cases 2 . This indicates that B. fragilis may be a more successful opportunistic pathogen then other related Bacteroides spp.

The majority of contemporary molecular studies on Bacteroides spp. focus on the mechanisms of polysaccharide utilization 2 3 4 , with very few virulence mechanisms that contribute to the ability of Bacteroides spp. ability to act as opportunistic pathogens described. Among those that have, cell adherence, lipopolysaccharide production, and the production of neuraminidase, enterotoxin, and proteolytic enzymes have been proposed to play a role in B. fragilis pathogenicity 5 . B. fragilis also has the ability to produce several haemolysins 6 . Haemolysins have been identified as powerful virulence determinants in both Gram-positive and Gram-negative bacteria 7 8 . Recently we identified a large panel of orthologous genes encoding C10 proteases in the phylum Bacteroidetes, including a set of four paralogous genes (called Bfp1-4) in B. fragilis 9 . C10 proteases are papain-like cysteine proteases, and include Streptococcal pyrogenic exotoxin B (SpeB) from Streptococcus pyogenes, and Interpain A from Prevotella intermedia. Both of these enzymes have been implicated in virulence 10 11 12 13 . SpeB has been shown to cleave cytokines 14 , activate the host matrix metalloprotease MMP-9, and to release kinin from kininogen 13 . In this way SpeB contributes to tissue damage and Streptococcus pyogenes invasion of the host 15 . Interpain A contributes to the pathogenesis of P. intermedia infections by inactivating the complement cascade through degradation of the complement factor C3 12 , a property also associated with SpeB 16 . Despite the obvious parallel functions of these orthologues, the activity of Btp proteases and their potential to contribute to virulence has yet to be determined.

SpeB and the Staphopains, papain-like proteases produced by Staphylococcus aureus, have been extensively studied with regard to regulation of gene expression, export and post-translational mechanisms 17 18 19 . These aspects of protease expression have yet to be investigated for papain-like cysteine proteases from members of the Bacteroides spp. The transcriptional coupling of the structural gene for the SpeB protease in S. pyogenes to a gene (spi) encoding a small specific inhibitor of SpeB 20 , is remarkably similar to control of protease activity in some staphylococcal species 21 . The genes for the C47 type cysteine proteases Staphopain A and B, and their cognate inhibitors Staphostatin A and B, respectively, are contiguous and are co-transcribed 22 . Spi and the Staphostatins are thought to inhibit prematurely-activated proteases in the cytoplasm of their respective host cells, and thus prevent toxicity of the protease to the bacterial cell 20 23 24 . Despite the fact that SpeB and the Staphopains have a papain-like fold 10 25 , 26 , the inhibitors Spi and the Staphostatins are not related in sequence and have a different proposed mechanism of protease binding 20 21 . The SpeB-like proteases that we recently described in B. fragilis have Staphostatin-like inhibitors encoded either upstream or downstream of the protease gene, creating an unusual juxtaposition of C10 proteases and C47 protease type inhibitors. The bfp genes encoding the C10 proteases and the bfi genes encoding the inhibitors are co-transcriptionally coupled 9 .

B. fragilis has been shown to differentially regulate virulence associated genes when occupying environmental niches other then the intestinal lumen. Among adaptive traits are aerotolerance and resistance to reactive oxygen species. These represent physiological adaptation of B. fragilis to its environment that may promote opportunistic infections by enhancing survival in areas outside the strictly anaerobic environment of the intestinal tract 27 . When B. fragilis was exposed to environmental oxygen, as might occur in the blood, a large number of genes for detoxification were induced such as catalase (katB) and superoxide dismutase (sod). Expression of these genes could prevent damage caused by reactive oxygen species 27 . The ferritin (ftnA) gene involved in iron acquisition was expressed at a low constitutive level when B. fragilis was grown under anaerobic conditions, but upon oxygen exposure, the ftnA message increased almost 10-fold in iron-replete medium 28 . This may be important for the ability of the organism to survive in an aerobic environment 28 . It has been proposed that the oxidative stress response regulator OxyR is required for full virulence in B. fragilis 27 . However, it is not known what factors are directly involved in this process. Although strictly anaerobic, P. gingivalis, which is phylogenetically close to B. fragilis, can also survive in the presence of atmospheric oxygen 29 . Significantly, two known virulence factors encoded by this organism, haemolysin (hem) and the cysteine protease gingipain A (rgpA), display elevated expression levels, 3.66-fold and 2-fold respectively, in the presence of atmospheric oxygen 30 . Thus it appears that cysteine protease gene expression in a related Bacteroidetes P. gingivalis is sensitive to environmental cues including oxygen.

This study investigates how the expression of B. fragilis C10 protease genes responds to key changes in environmental stimuli, and thus indicates their potential involvement in pathogenesis and survival in the non-gut environs. In addition, expression analysis data is presented for a set of genes encoding newly identified and described C10 paralogues in B. thetaiotaomicron.

The B. thetaiotaomicron VPI-5482 genome was shown here to harbour genes for four members of the C10 family of papain-like cysteine proteases, three of which are genetically clustered, and associated with two staphostatin-like inhibitors. The fourth unlinked C10 protease gene was also associated with a staphostatin-like protein. Interestingly, the proteins encoded by the clustered genes were more closely related to each other than to BtpA, which had highest sequence identity to Bfp2, a protease in B. fragilis. Although no evidence was found to support the involvement of mobile genetic elements in the acquisition and evolution of these genes by B. thetaiotaomicron, it is nevertheless likely that the current genetic configuration has evolved by two separate horizontal gene transfer events. The first putative event was the acquisition of the btpA locus, and the second involved a single C10 gene insertion which is elsewhere in the genome. This was followed by subsequent gene duplication events yielding btpB, btpC, and btpZ, based on the fact that they share higher residue identity to each other than to btpA.

The btpB and btpC loci are the most closely related across the four paralogues encoding what are predicted to be functional proteases, with 54.3% and 72.5% overall amino acid sequence identity and similarity respectively (Table 1). The characteristic catalytic Cys residue of cysteine proteases is absent from BtpZ, indicating the btpZ gene product is not a functional protease, so the biological role of this molecule is unclear. Since all four B. thetaiotaomicron proteins include both a leader peptide and propeptide, it is likely that these proteins are exported across the inner membrane in a zymogen form, where the proteases would be expected to be anchored by virtue of predicted lipoprotein signal sequences and N-terminal residues. Based on the ‘+2 rule’ for lipoproteins, which relates the final location of a lipoprotein to the amino acid in the +2 position of the secreted protein 32 , the likely cellular location of the Btp zymogens is coupled through a lipid moiety at the post-processing N-terminal Cys residue of the propeptide to the inner leaflet of the outer membrane. They would remain in this inactive form until an activation event occurred. As the proteases would thus have a periplasmic location, for them to contribute to virulence they must come into contact with the host. This could be achieved by a number of mechanisms (1) the presence of protease-specific transporters in the outer membrane, (2) by release of the proteases upon bacterial cell death and lysis, or (3) through vesicle-based transport, as previously described for B. fragilis 33 . In the case of the related organism P. gingivalis these vesicles have been associated with proteolytic activity 34 35 . It is therefore not unlikely that the proteases described in this paper could be exported by vesicles in a similar manner.

The Bti proteins also include predicted leader peptides, and BtiA and BtiB are likely to be lipoproteins, which would also most likely be associated with the outer membrane. BtiZ was not predicted to be a lipoprotein (the signal peptide for BtiZ has a signal peptidase I cleavage site) and it is therefore likely targeted to the periplasm of the Bacteroides cell. Having both membrane associated inhibitor and periplasmic inhibitors may be a strategy for maximizing protection afforded by these inhibitors against the C10 protease activity. Another possibility is that the BtiZ molecule is in the process of accumulating mutations and becoming non-functional in response to loss of BtpZ activity.

We have previously demonstrated the transcriptional coupling of B. fragilis C10 protease genes with those for staphostatin-like inhibitors 9 . In the current study transcriptional coupling was also identified for the B. thetaiotaomicron btp and bti genes by Reverse Transcriptase PCR. The btpA gene was found on the same message as btiA. Furthermore, transcriptional coupling was identified for btpB and btiB, and btpZ and btiZ. The btpC gene appears to be transcribed independently of adjacent btp and bti genes. Although, this study does not preclude that the btpA, btpB and btpZ genes could be transcribed independently of the bti genes, the data indicates a similar genetic linkage of these btp genes with staphostatin-like inhibitors as occurs in B. fragilis. As suggested for effective control of otherwise lethal proteases in streptococcus and staphylococcus, co-transcription with genes for cognate inhibitors ensures immediate availability and precise stoichiometry of the required inhibitor for the respective transcriptionally coupled protease.

B. fragilis and B. thetaiotaomicron are usually commensal components of the normal intestinal microbiota. However, B. fragilis cells adhered to epithelial cells in biopsy samples from IBD patients 36 37 . In addition, release of these organisms into other body sites can result in serious complications and they are associated with a range of extraintestinal infections 5 . Growth of B. fragilis in bile, blood and oxygen has previously been shown to enhance properties associated with increased virulence 6 27 38 . Bile is secreted into the small intestine as a normal part of fat digestion/metabolism. Previous studies on the exposure of B. fragilis to physiological concentrations of bile reported the increase of outer membrane vesicle formation and fimbria-like appendages, and increased expression of genes encoding antibiotic resistance-associated RND-type efflux pumps 38 . The same study showed that the bile salt-treated bacterial cells had increased resistance to a range of antimicrobial agents and as well as increased co-aggregation, biofilm formation, and adhesion to intestinal epithelial cells 38 . Bile is normally associated with small intestinal secretions. In the current study, B. fragilis and B. thetaiotaomicron were grown in the presence of physiological levels of bile (0.15% bile salts approximates to a concentration of 3.7 mM), reflecting concentrations found in the distal ileum (2 mM). These conditions did not alter the expression level of C10 protease genes in either organism. This suggests that in the large intestine, where the bile concentrations are considerably lower (0.09 to 0.9 mM), the production of these proteases is not likely to be responsive to residual levels of bile transiting from the small intestine.

The oxyR gene encodes a redox-sensitive transcriptional regulator of the oxidative stress response in B. fragilis 39 . It has been shown previously that B. fragilis oxyR mutants are attenuated in an intra-abdominal abscess infection model 27 . Thus the ability of B. fragilis to survive in oxygenated environments such as blood is thought to be linked with pathogenesis. Two of the B. fragilis C10 proteases (bfp1 and bfp4) displayed increased expression levels when exposed to oxygen. The expression levels of the other protease genes (bfp2 and bfp3) remained unchanged. Interestingly, genes encoding superoxide dismutase and an oxidoreductase can be found directly upstream of bfp4. These two genes encode proteins involved in the processing of reactive oxygen species and are also likely to be up-regulated in the presence of atmospheric oxygen. Three of the C10 protease genes in B. thetaiotaomicron were up-regulated significantly in the presence of oxygen, while btpA was down-regulated. These findings suggest that as the Bacteroides transit from the anaerobic environment of the gut lumen to a more aerobic environment, the bacterial cells respond and they alter the expression profile of these potential toxins. Oxidative stress is an obvious potential signal to the bacterial cell that it is leaving the anaerobic gut environment. Thus, it is possible that this cue triggers increased production of the C10 proteases as a means to combat the host immune system.

B. fragilis accounts for 55% of bacteraemia in adult patients resulting in systemic blood infections 40 and it is plausible that blood can act as an environmental signal for the expression of virulence factors in Bacteroides cells leaving the intestine. For example, stimulation of virulence gene expression by exposure to blood has been documented for Streptococcus pyogenes 41 . However, the study only sampled for a maximum of 3 hours growth in blood and did not detect an increase in expression of speB, the gene encoding the cysteine protease. SpeB is normally detected in culture supernatant in late-log phase growth. Other studies have suggested a role for SpeB in survival in blood 42 . Thus, the expression of C10 protease genes was also examined when B. fragilis and B. thetaiotaomicron were grown in the presence of blood. Only the expression of btpA from B. thetaiotaomicron increased upon exposure to blood, while the other btp genes were down-regulated. It was recently shown that the Prevotella intermedia Interpain A, a homologue of SpeB, and thus also of BtpA, has a role in the breakdown and release of haeme from haemoglobin 11 . Therefore, it is tempting to speculate that BtpA could carry out a similar function in iron acquisition.

The relatively late transition point in the qPCR for the proteases, combined with the observation that none of the protease genes tested showed differential expression upon exposure to CaCO-2 cells, makes it likely that in the environment of the gut these genes are transcribed at low levels. However, in situations where the bacteria are able to transit to the host tissue or blood stream these bacteria have the ability to produce select combinations of the C10 proteases in response to oxidative stress and the presence of blood, stimuli that would be encountered during transit. Interestingly, while B. fragilis produces four mature proteases that all have a basic (as distinct from acidic) character, the B. thetaiotaomicron proteases have distinct physicochemical properties. The predicted BtpA mature protease is basic in contrast to the predicted acidic character of BtpB, BtpC and BtpZ. This fact, and the mutually exclusive manner in which btpA and the clustered btpB, btpC and btpZ respond to the environment, suggests that these proteases may have very distinct targets and biological functions.

To date extensive attempts by us and others (J. Potempa, personal communication) to express these Bacteroides enzymes in a soluble and/or active format in Escherichia coli have been unsuccessful. There are a number of possible explanations, including the requirement for a chaperone for the correct folding of the proteases. Indeed, Nickerson and colleagues 43 suggest such a role for Staphostatin in the folding of Staphopains. In addition, activation of some bacterial proteases is not autoproteolytic but requires the action of additional proteases. This requirement has also been found in the staphylococcal system where the V8 serine protease is required for the maturation of the cysteine protease, Staphopain B, and in turn aureolysin is required to activate V8 protease 44 . Either of these scenarios would explain the difficulties in expressing active Bacteroides proteases in E. coli. Additional studies to overcome the issues experienced with recombinant protein expression are required, but although technically challenging, the characterization of these proteases at a biochemical level will improve the understanding of their function and potential roles in Bacteroides infections.

The observation that bacterially encoded C10 (SpeB-like) proteases are more commonly co-transcribed with a potential inhibitor is thus established as a norm for cysteine protease systems in Bacteroides spp. The study has also established that these protease genes are expressed in two important members of the Bacteroidetes family, B. fragilis and B. thetaiotaomicron. The distinct expression patterns for each set of paralogs strongly suggest that proteases play diverse roles in the bacterial interaction with the host. In particular the response in gene expression to oxygen and blood exposure imply that the bacteria may alter the expression of these proteases as the bacteria transition from a commensal existence to that of an opportunistic pathogen.

RFT and ECM performed and designed experiments, and interpreted data. TFK designed experiments and interpreted the data. PWOT designed experiments, analyzed data and co-wrote the manuscript. JCC conceived the study, designed the experiments, interpreted the data and co-wrote the manuscript. All authors read and approved the final manuscript.