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.
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 ). 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
]. In the case of the related organism P. gingivalis
these vesicles have been associated with proteolytic activity [34
]. 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
genes by Reverse Transcriptase PCR. The btpA
gene was found on the same message as btiA
. Furthermore, transcriptional coupling was identified for btpB
, and btpZ
. The btpC
gene appears to be transcribed independently of adjacent btp
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.
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
]. 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
]. 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.
gene encodes a redox-sensitive transcriptional regulator of the oxidative stress response in B. fragilis
]. 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
) displayed increased expression levels when exposed to oxygen. The expression levels of the other protease genes (bfp2
) 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.
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
]. 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.