ASSAY FOR DEGP PROTEASE INHIBITORS

Abstract: The DegP (HtrA) protease is a multifunctional protein essential for the removal of misfolded and aggregated proteins in the periplasm. The present invention provides an assay for inhibitors of DegP activity, comprising mixing a suspected inhibitor of DegP activity with DegP and a suitable substrate (preferably a native substrate of DegP such as PapA) and detecting changes in DegP activity. DegP has been shown to be essential for virulence in several Gram negative pathogens. Only three natural targets for DegP have been described: colicin A lysis protein (Cal), pilin subunits (K88, K99, Pap) and recently HMW1 and HMW2 from <i>Hemophilus influenzae</i>. <i>In vitro</i>, DegP has shown weak protease activity on casein and several other non-native substrates. The present inventors have identified the major pilin subunit of the Pap pilus, PapA, as a native DegP substrate and demonstrated binding and proteolysis of this substrate <i>in vitro</i>. Using an NH2-terminal affinity tag the present inventors have purified PapA away from the PapD chaperone, in the presence of denaturant, to use as a proteolysis substrate. This finding will allow the identification of the DegP recognition and cleavage sites in substrate proteins, and further, allow the design of small molecule inhibitors of protease function.

Description: ASSAY FOR DEGP PROTEASE INHIBITORS BACKGROUND OF THE INVENTION 1. Field of the Invention The DegP (HtrA) protease is a multifunctional protein essential for the removal of misfolded and aggregated proteins in the periplasm. DegP has been shown to be essential for virulence in several Gram negative pathogens. Only three natural targets for DegP have been described: colicin A lysis protein (Cal), pilin subunits (K88, K99, Pap) and recently HMW1 and HMW2 from Hemophilus influenzae. In vitro, DegP has shown weak protease activity on casein and several other non-native substrates. The present inventors have identified the major pilin subunit of the Pap pilus, PapA, as a native DegP substrate and demonstrated binding and proteolysis of this substrate in vitro. Using an NH 2 -terminal affinity tag the present inventors have purified PapA away from the PapD chaperone, in the presence of denaturant, to use as a proteolysis substrate. This finding will allow the identification of the DegP recognition and cleavage sites in substrate proteins, and further, allow the design of small molecule inhibitors of protease function. 2. Description of the Related Art Proteolysis of misfolded and denatured proteins in the bacterial cytoplasm and periplasm is a crucial housekeeping function and critical for cell viability (Pallen, M.J. and Wren, B.W. (1997) Mol. Microbiol. 26,209-221 ; Miller, C.G. (1996) in Escherichia coli and Salmonella Cellular and Molecular Biology (Neidhardt, F.C., eds) pp. 938-954, ASM Press Washington D.C.). It is becoming increasingly clear that the proteolytic machinery is also an essential component for bacterial pathogenesis (Pallen & Wren, supra). Recently, scientists have uncovered a regulatory system, CpxA/CpxR, that responds to the changing environment of the periplasm; recruiting proteases, chaperones and "foldases" to assist in managing the state of affairs in this bacterial compartment (see, e.g., Danese, P.N., et al. (1995) Genes and Development 9,387-398; Danese, P.N. and Silhavy, T.J. (1997) Genes Dev. 11,1183-1193). As the host often presents a hostile environment to the invading organism it is suggested that the CpxA/CpxR regulatory circuit is "tripped" upon engaging the host defenses. One of the most important proteases in the periplasm, the DegP/HtrA serine protease (Pallen & Wren, supra) is a member of the CpxA/CpxR regulon (Danese, P.N., et al. (1995), supra; Danese and Silhavy (1997) supra). This protein is also a key player in pathogenesis in Salmonella, Brucella, and Yersinia (Pallen & Wren, supra). Specifically, DegP has been shown to be a virulence determinant in Salmonella typhimurium, Brucella abortus and Yersinia enterocolitica. According to the current model of DegP function in pathogenesis, DegP acts to remove misfolded proteins and protein aggregates that result from exposure to reactive oxygen intermediates in the host. In the absence of functional DegP, these protein aggregates compromise the pathogenic process (Pallen & Wren, supra). The DegP (degradation) nomenclature refers to the initial mapping of a mutation in E. coli that allowed the accumulation of unstable fusion proteins in the periplasm (Strauch, K.L., Johnson, K. and Beckwith, J. (1989) J. Bacteriol. 171,2689-2696; Strauch, K.L. and Beckwith, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85,1576-1580). The HtrA (heat shock regulated) designation indicates that a transposon insertion in the same gene resulted in a heat sensitive growth phenotype (Lipinska, B., Sharma, S. and Georgopoulos (1988) Nucleic Acids Research 16,10053-10066). Lastly, DegP was also designated protease Do, again as a mutation that conferred a heat sensitive phenotype in E. coli (Seol, J.H., et al. (1991 ) Biochemical and Biophysical Research Communications 176,730-736). DegP exhibited functional protease activity in in vitro assays using casein as a substrate, although its activity on this substrate was weak (Lipinska, B., Zylicz, M. and Georgopoulos, C. (1990) J. Bacteriol. 172,1791-1797). Lipinska et al. demonstrated that the activity on casein was inhibitable by DFP and not by any other known protease inhibitors, suggesting that DegP is a serine protease. Site directed mutagenesis at serine 210 and histidine 105, two components of the serine protease catalytic triad, compromised DegP function in vitro and in vivo; i.e. strains carrying serine 210 or histidine 105 mutant derivatives were sensitive for growth at elevated temperatures (Skorko-Glonek, J., et al. (1995) Gene 163,47-52). The preferred substrate for DegP appears to be proteins that are globally or transiently denatured; suggesting that the role in vivo is to clear misfolded or denatured proteins from the periplasm (Kolmar, H., Waller, P.R.H. and Sauer, R.T. (1996) J. Bacteriol. 178,5925-5929). In support of this finding, Laskowa et al. (Laskowska, E., et al. (1996) Mol. Microbiol. 22,555-571 ) demonstrated in vitro that purified DegP protein would degrade thermally aggregated proteins fractionated from E. coli extracts and that the DnaJ chaperone would antagonize DegP degradation; i.e. the chaperone would aid in refolding the proteins such that they were no longer targets for degradation by DegP. In addition to its weak protease activity, DegP/HtrA has been shown to be a virulence factor for several pathogenic organisms. In Salmonella typhimurium, htrA nulls were found to be avirulent and more susceptible to oxidative stress (Johnson, K., et al. (1991 ) Mol. Microbiol. 5,401-407). The authors of this study suggest that the htrA mutants are less able to withstand oxidative killing within the macrophage. An htrA lesion was found to be useful in attenuating Salmonella typhifor implementation as a vaccine strain. Similarly, Brucella abortus and Brucella melentensis htrA null mutants were attenuated for virulence in goats and found to be significantly more sensitive to oxidative killing by cultured neutrophils in vitro (Elzer, P.H., et al. (1996) Research in Veterinary Science 60,48-50; Elzer, P.H., et al. (1996) Infection and Immunity 64,4838-4841 ; Phillips, R.W., et al. (1997) Research in Veterinary Science 63,165-167). An isogenic pair, wild-type and htrA null mutant, in Yersinia enterocolitica were created and tested in a mouse yersiniosis model. HtrA was found to be essential for virulence and the mutant strain was more sensitive to oxidative stress (Li, S.-R., et al. (1996) Infection and Immunity 64,2088-2094). Finally, Boucher et al. ((1996) J. Bacteriol. 178,511-523) recently demonstrated that Pseudomonas aeruginosa conversion to mucoidy, the so-called CF phenotype involves two HtrA homologs. DegP homologs have been found in Streptococcus pneumoniae (Gasc, A-M et al. (1998) Microbiology 144:433-439), Streptococcus pyogenes, and Staphylococcus aureus. All three homologs share the catalytic triad of the E. coli DegP protein. The first identified in vivo target for DegP was colicin A lysis protein (Cal) (Cavard, D., Lazdunski, C. and Howard, S.P. (1989) J. Bacteriol. 171 ,6316-6322). DegP was found to degrade the acylated precursor form of Cal into two fragments. Mature Cal also accumulated to higher levels in degP mutant strains (Cavard et al.(1989), supra). A second family of DegP targets was identified as bacterial pilins. The K88 and K99 pilin subunits were found to accumulate to higher levels in degP mutant strains (Bakker, D., et al. (1991 ) Mol. Microbiol. 5,875-886). A more detailed study of this phenomenon demonstrated that P pilins, specifically PapA, are substrates for the DegP protease (Jones, C.H., et al. (1997) EMBO J. 16,6394-6406). More recently the H. influenzae non-pilus adhesin proteins HMW1 and HMW2 were found to be in vivo substrates for DegP (St. Geme III, J.W. and Grass, S. (1998) Mol. Microbiol. 27,617-630). The DegP/HtrA sequence was published in 1988 (Lipinska, Sharma, & Georgopoulos, supra). HtrA is one of several dozen proteases in E. coli and is known to have homologs in cyanobacteria, mycobacteria, yeast and man (Pallen & Wren, supra). There are also two homologs of DegP: DegQ and DegS in E. coli (Kolmar et al. (1996), supra; Waller, P.R. and Sauer, R.T. (1996) J. Bacteriol. 178,1146-1153). A new homology region has recently been identified in DegP that is conserved in many eukaryotic proteins (Pallen & Wren, supra). Downstream from the catalytic sequence- 208GNSGGAL214 are two PDZ domains (Levchenko, I., et al. (1997) Cell 91,939-947). These 80-100 amino acid domains are found in nearly 100 proteins, mostly eukaryotic, and probably play roles in protein-protein interactions, either facilitating multimer formation or substrate binding (Levchenko, I., et al. (1997), supra). The PDZ domain homology is maintained in the recently identified Gram-positive DegP homologs. Interestingly, Kolmar et al. (1996, supra) recently demonstrated that DegP forms dodecamers in vitro, although it remains to be seen if the PDZ domains contribute to DegP multimerization. If DegP does function as a multimer in vivo it would be reminiscent of the proteosome machines described in eukaryotic cytosol and ER (Pallen & Wren, supra). Early in vivo data suggested that pilins were DegP substrates (Bakker, D., et al. (1991 ) supra). Expression of pilin subunit proteins in the absence of the chaperone resulted in failure to accumulate subunit in the periplasm and degP mutant strains accumulated more subunit in the periplasm (Bakker, D., et al. (1991 ), supra; Jones, et al. (1997) supra; Hultgren, S.J., Normark, S. and Abraham, S.N. (1991 ) /An/iu. Rev. Microbiol. 45,383-415; Hultgren, S.J., Jones, CH. and Normark, S.N. (1996) in Escherichia coli and Salmonella; Cellular and Molecular Biology (Neidhardt, F.C., eds) pp. 2730-2756, ASM Press Washington DC). Moreover, subunit expression in the degP mutant was highly toxic (Jones et al. (1997), supra). Both the toxicity and accumulation was suppressed by complementation with degP (Jones et al. (1997), supra. A significant obstacle to the study of pilus biogenesis is the inability to purify subunits in the absence of the PapD chaperone. This was overcome by the addition of an affinity tag to the amino-terminus of PapA. This provided for the purification of large quantities of PapA under denaturing conditions. Renaturation of PapA in the presence of the PapD chaperone allowed the formation of the PapD- PapA complex. Moreover, mixing DegP with denatured PapA resulted in affinity purification of a PapA-DegP complex and proteolysis with release of an amino-terminal PapA fragment. SUMMARY OF THE INVENTION Briefly, the present invention provides high through-put screening assays for the identification of inhibitors of DegP protease, an essential virulence factor in several human and non-human pathogens. The compounds identified by the present assay, small molecule inhibitors of DegP protease function, are expected to have high utility in the clinic. With the foregoing and other objects, advantages and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the preferred embodiments of the invention and to the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Expression and purification of PapA-6his2ala and reconstitution of the PapD-PapA complex. A. Accumulation of PapA-6his4ala in the periplasm is dependent on the PapD chaperone. Periplasmic fractions from IPTG induction of PapA-6his4ala alone (lane 1 ) or co-induction of PapD chaperone and PapA-6his4ala (lane 2) in E. coli were prepared and loaded on SDS-PAGE. B. Purification of PapA-6his4ala-PapD complex from periplasm (lane 1 , unbound material; lane 2, eluate). Metal affinity resin with bound complex was treated with 8M urea and washed to remove PapD. Eluted PapA-6his4ala is shown in lane 3. Lane 4 shows PapA-6his4ala eluate following dialysis to remove urea. The doublet PapA band in lane 3 is a result of urea treatment and proposed to be an altered form of the protein. C. Reconstitution of chaperone-subunit complex. Denatured PapA-6his2ala (lane 2) was mixed with purified PapD chaperone, allowed to bind to metal affinity resin, washed and eluted with 0.1 M imidazole. The eluted complex is shown in lane 3. Lane 1 contains molecular weight standards. A, B, & C are SDS-PAGE (12.5%) stained with coomassie brilliant blue. Figure 2. DegP protease binding and cleavage of PapA-6his4ala. Denatured PapA-6his4ala linked to metal affinity resin was mixed with control periplasm (lane 1 ) or periplasm enriched with DegP (lane 2). Bound protein was eluted from the metal affinity resin with 0.1 M imidazole. The eluates were then run on SDS-PAGE and stained with coomassie brilliant blue. The 3 novel bands that appear in lane 2 (DegP enriched) and not in lane 1 (control periplasm) are indicated. The round arrow indicates a new band that has not yet been identified. The DegP and PapA-NH2 bands were verified by amino acid sequencing. The sequence for the DegP band was identical to that published by Lipinska et al. (1988; supra) AETSSA. The amino terminal sequence of the 12kDa PapA-NH2 band was AAAHHHH, confirming that signal processing occurred at the correct site; the 4 histidines are part of the 6-histidine tag. Figure 3. Purification of DegP. A. Cation exchange fractionation. Periplasm prepared from 30 grams of cells was applied to a 5ml HiTrap SP column (Amersham-Pharmacia Biotech, Upsalla, Sweden) and eluted with a linear salt gradient. The starting material and flow through fraction are shown in lanes 2 and 3, respectively. The relevant portion of the elution gradient is shown in lanes 4-8. DegP eluted at approximately 100mM NaCI. B. HIC butyl fractionation. Peak fractions from the cation exchange fractionation were pooled and applied to a HiTrap HIC butyl column (Pharmacia). The flow through fraction is shown in lane 2. DegP eluted in approximately 0.3M salt and is shown in lanes 4-8. The small arrows indicate truncated forms of DegP, all of which were identified by amino- terminal sequencing (unpublished data). In both A and B, lane 1 contains high molecular weight markers. Figure 4. DegP activity on casein substrate. A fluorescent casein substrate (Molecular Probes, Eugene, OR) was used to screen for protease activity in the DegP fractions from the HIC purified material. Two separate preparations of DegP (degP-A, degP-B) were tested and shown to have activity comparable to the Trypsin control. Figure 5. In vitro DegP cleavage assay. Reduced and carboxymethylated PapA-6his4ala was mixed with DegP and incubated overnight at 45°C. The reactions were resolved on SDS-PAGE transferred to PVDF membrane and developed with a polyclonal antibody raised against whole P pili. PapA- 6his4ala was incubated in the presence (lane 3-0.25 μg, lane 5-0.5 μg) and absence (lane 2-0.25 μg and lane 4-0.5 μg) of DegP, respectively. Lane 1 contains DegP alone as a control. Incubations were at 37°C for 12 hours. Figure 6. DegP binding ELISA and high-throughput cleavage assay. A. Binding ELISA. PapA-6his4ala (0.8 mg/ml) was captured, using anti-6his antisera, on the wells of a 96 well microtiter plate. Serial five-fold dilutions of the protein preparation were applied in triplicate. DegP (50 pM) was added to the plate and incubated for 60 minutes. The ELISA was developed with anti-DegP antisera followed by alkaline-phosphatase conjugated goat-anti- rabbit secondary. B. Cleavage assay. PapA-6his4ala was captured as above and treated with DegP for two hours. Following washing the assay was developed with polyclonal anti-PapA antisera to detect PapA-6his4ala remaining on the plate. Figure 7. Homology/consensus sequences for DegP-like proteases from gram-negative and gram-positive organisms. Thirty-six gram-negative and five gram-positive DegP homologs were selected, and the sequences for the catalytic domains (Fig. 7A) were separated from the sequences for the PDZ domains (Fig. 7B). Then, the sequences were aligned by hand in order to identify the consensus sequences. This provides a set of hallmarks for defining a DegP-like protease. Figure 8 shows an alignment of E. coli DegP and three Gram-positive homologs. In this alignment, identical residues and conserved changes are shown in bold. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION DegP is a periplasmic protease, present in many if not all Gram- negative bacteria, that is critical to bacterial viability and essential for pathogenesis, as DegP mutants do not survive in the host. The present invention is an in vitro assay for DegP activity. This assay utilizes a natural DegP substrate, the PapA pilin subunit, which is one of only two relevant substrates identified for DegP that are used in vitro, to result in an in vitro assay suitable for high through-put screening. More particularly, the present invention relates to both a new antibiotic target and an assay to identify inhibitors of the target. The assay of the present invention is suitable for use in a high through-put assay for library screens. The assay components are the major pilin subunit of the Pap pilus immobilized on a resin and periplasmic extracts containing the DegP protease. Using the assay, the present inventors affinity-purified a complex containing PapA and DegP as well as a proteolytic amino-terminal fragment of PapA. In a preferred embodiment, all the components of the assay, including DegP protease and PapA target, are homogeneous preparations. Preferably, the target is reduced to a peptide containing the recognition/cleavage site. The peptide is then labeled with a detectable marker to monitor cleavage and/or a to monitor DegP activity. Utilization of this assay to screen inhibitor libraries is useful to identify small molecule compounds that inhibit DegP protease activity; these compounds will then be developed into therapeutically relevant drugs for the clinic. In order to purify the protein to homogeneity, DegP may be cloned into a plasmid under the control of a well-regulated promoter. Using the proteolysis assay the PapA proteolytic products may be identified and sequenced to reveal the DegP cleavage site. Once the site is known a peptide will be designed and tested as a target for the protease. Finally, the peptide will be labeled with a detectable marker or configured for a fluorescence-proximity assay. This substrate will allow rapid analysis of proteolytic function and will be well suited for high-throughput screening ("HTS"). The present inventors have identified a DegP homolog in Streptococcus pyogenes and Staphylococcus aureus; therefore this protein is well represented in Gram-positive organisms, including the recently described homolog in S. pneumoniae (Gasc, A-M. et al. (1998) Microbiology 144:433-439). Peptides may be labeled using any of a variety of labels and methods of labeling. Examples of types of labels which can be used in the present invention include, but are not limited to, enzyme labels, radioisotopic labels, non-radioactive isotopic labels, fluorescent labels, toxin labels, and chemiluminescent labels. Examples of suitable enzyme labels include malate hydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, those phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase, acetylcholine esterase, etc. Examples of suitable radioisotopic labels include 3 H, 125 l, 131 l, 32 P, 35 S, 14 C, 51 Cr, 57 To, 58 Co, 59 Fe, 75 Se, 152 Eu, 90 Y, 67 Cu, 217 Ci, 211 At, 212 Pb, 47 Sc, and 109 Pd. Examples of suitable fluorescent labels include a 152 Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, and allophycocyanin label, an o- phthaldehyde label, an fluorescamine label, etc. Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin. Examples of chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, and imidazole label, and acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, an aequorin label, etc. Those of ordinary skill in the art will know of other suitable labels which may be employed in accordance with the present invention. The binding of these labels to antibodies or fragments thereof can be accomplished using standard techniques commonly known to those of ordinary skill in the art. Typical techniques are described by Kennedy, J.H., et al Clin. Chim. Ada 70:1-31 (1976), and Schurs, A.H.W.M., et al, Clin. Chim. Ada 81 : 1-40 (1977). Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method, all these methods incorporated by reference herein. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. EXAMPLE 1 In the present Example, the inventors have used the assay of the present invention to purify a complex between DegP and PapA and a specific proteolysis fragment of PapA. The products of the assay/reaction are identified using amino acid sequencing. Materials and Methods Constructs. The PapA-6his and PapA-6his4ala were constructed by PCR as previously described (Morrison, H.G. and Desrosiers, R.C. (1993) BioTechniques 14,454-457) using the appropriate primers to create the desired insertions and restriction sites for ease of cloning. The complete PapA open reading frame was sequenced to assure that random mutations were not added during amplification. PapA-6his4ala was sub-cloned into pMMB66 (Furste, J.P., et al. (1986) Gene 48,119-131 ) under control of the IPTG inducible P tac promoter. PapD was expressed from pHJ9203, P ara promoter, as previously described (Jones, C.H., et al. (1997) EMBO J. 16,6394-6406). DegP was produced from pKS17 (see Strauch, K.L. et al. J. Bad. 171 :2689-2696). Expression and purification of PapA-6his4ala. PapA-6his4ala was expressed along with PapD in KS474 (degPr.kari) to provide for maximal translocation and stability in the periplasm. Induction conditions were 1 mM IPTG, 0.5% arabinose, 90 minutes, at A600 = 0.6-0.8. Periplasmic extracts were performed as previously described (Jones et al. (1997), supra) and dialyzed into 20mM Tris, pH=8 to prepare for affinity purification. Talon metal affinity beads (Clontech, Palo Alto, CA) were used in batch per manufacturers instructions to purify PapA-6his4ala in complex with PapD. Elutions were performed by addition of 0.1 M imidazole to the beads, rocking for 10 minutes and collecting supernatant. Three elutions were performed to maximize recovery. The complex, following dialysis, was denatured by addition of urea to 8M and reapplied to Talon resin. After washing in 10mM Tris, pH=8/8M urea, pure PapA-6his4ala was eluted with 0.1 M imidazole in 8M urea. Co-purification of DegP and proteolysis assay DegP was expressed from pKS17 by heat induction of the native heat inducible σE promoter (Lipinska et al., B., Sharma, S. and Georgopoulos (1988) Nucleic Acids Research 16, 10053-10066). Periplasmic extracts were prepared as previously described (Jones et al. (1997), supra) and the periplasm dialyzed into 20mM Tris, pH=8. DegP enriched and control (heat treated HB101 ) periplasm were added to PapA-6his-4ala containing Talon resin (8M urea treated) and incubated at RT for 30 minutes with shaking. The Talon beads were extensively washed and then bound proteins were eluted with 0.1 M imidazole. Results Construction of PapA-histidine tag fusion A six-histidine affinity tagged PapA construct (PapA-6his) was constructed using PCR amplification. It was found that an alanine spacer had to be added between the histidine-tag (his-tag) and the leader peptidase cleavage point in PapA for efficient expression. When the his-tag was too close to the leader peptidase cleavage site PapA remained unprocessed and membrane associated (Jones, C.H., Liu, C. and Hultgren, S.J. (1998), supra ). A two-alanine spacer, making a total of four alanines between the histidine tag and the cleavage site, was placed between the his-tag (PapA-6his-4ala) and the peptidase-processing site to overcome the proposed steric effect of the bulky histidine side chains. The PapA-6his- 4ala derivative was properly leader processed and localized to the periplasm (Figure 1 ). Expression and purification of PapA-6his-4ala derivative Pilin subunit accumulation in the periplasm is highly dependent on the periplasmic chaperone (Jones et al. (1997), supra). Therefore, the PapD chaperone was co-expressed with the affinity-tagged PapA. Figure 1A illustrates that accumulation of PapA-6his4ala in the periplasm is dependent on PapD. Purification of PapA-6his-4ala by affinity-chromatography on Talon metal-affinity resin (Clontech) resulted in co-purification of PapD in roughly equal molar amounts (Figure 1 B, lane 2). In order to remove the PapD from the PapA-6his4ala, the Talon resin with bound complex was treated with 8 molar urea. The urea-washes contained virtually all of the PapD and little to none of the PapA-6his4ala, due to the fact that the metal affinity binding to poly-histidine is stable in 8M urea. Purified, denatured PapA-6his-4ala could be purified by simple elution with 0.1 M imidazole (Figure 1 B, lane 3). Chaperone-subunit complexes, PapD-PapA-6his4ala could be reformed in vitro by dilution of the resin-containing denatured PapA-6his4ala into either periplasm containing PapD or purified PapD. (Fig.lC). Purification of a PapA-6his4ala-DegP complex Addition of DegP containing periplasm to the denatured PapA- 6his4ala resin, mixing 30 minutes at room temperature and elution of bound material revealed several new bands in addition to PapA-6his4ala, one of which is approximately 48kDa (Figure 2, lane 2). Amino-terminal sequencing identified the 48kDa protein as DegP, suggesting that the protease bound to the denatured PapA-6his4ala. In addition to the PapA-6his4ala and DegP proteins, two other proteins were eluted from the PapA-6his4ala affinity resin. The approximately 12kDa band was found to have the 6his amino terminus of the PapA-6his4ala, suggesting that it represents an amino- terminal cleavage product of full length PapA-6his4ala (Figure 2, lane 3). Purification of DegP DegP protease was purified from whole periplasm prepared as previously described (Jones et al. (1997)). It was noted that increased yields of DegP were obtained from an overnight (saturated) culture as opposed to a transient heat shock (45°C) of a mid-log culture. The periplasmic extract was dialyzed into 33 mM Mes, 33 mM HEPES, 33 mM acetate, pH=5.9 and applied to a HiTrapS, cation exchange, column (Pharmacia, Upsalla, Sweden). DegP eluted at approximately 100 mM NaCI in a linear gradient (Figure 3A). The peak fractions from the S column were dialyzed against 20 mM Tris, pH = 7.0/0.5M AmSO4 and applied to a HIC butyl column (Pharmacia, Upsalla, Sweden). DegP eluted from the HIC column in approximately 40% Buffer B (0.3M AmSO4). The Deg P appears to be >98% pure with the only contaminants being two DegP specific truncates that occur due to auto-cleavage. Protease assay - casein substrate We used a commercial protease assay (EnzChek) to test for DegP activity on casein, which was previously observed (Lipinska et al., 1990). Heavily fluoresceinated (BODIPY) casein (Molecular Probes, Eugene, OR) that is intramolecularly quenched is used as substrate and is a good target for detecting many types of proteases. Trypsin and DegP both cleave the casein substrate (Figure 4). This assay was used to verify that the DegP that was eluted from each column purification step was active. Soluble PapA cleavage assay Purified DegP was tested on the PapA-6his4ala substrate under denaturing conditions. 20 μl of PapA-6his4ala (8M urea) was added to DegP (fraction 5 and 6 from the S column) in a final volume of 50 μl. The reaction was allwed to proceed overnight at 37°C. The experiment shown in figure 5 reveals two activities for DegP on the PapA substrate. In lanes 1-4 PapA has undergone limited degradation, as can be seen by the appearance of two novel bands around 12 kDa, similar to that seen in figure 2, and the decrease in the intensity of the full-length band when compared to lanes 5 and 6. The second activity appears to be blocking aggregate formation that appears only at pH-8. The reaction run in lanes 1 , 2, and 5 were performed at pH=5.9, whereas the reactions run in lanes 3, 4, and 6 were performed at pH=8. DegP will also degrade PapA that has been denatured, reduced, carboxymethylated and dialyzed to remove urea as efficiently as the denatured substrate. DegP binding ELISA and cleavage assay. Taking advantage of the 6-his affinity tagged PapA we designed a capture ELISA to detect DegP binding to PapA. The ELISA was modified to determine if, following incubation with DegP, a loss of PapA epitopes could be detected. Purified PapA6his4Ala was bound to the ELISA plate overnight. The plate was blocked with 3% BSA /1 *PBS for 2 hours. DegP was then added to the plate for 1 hour and incubated at room temperature. The plate was washed in 1 *PBS and developed with anti-DegP antisera. As shown in Figure 6A, DegP bound efficiently to native PapA-6his4ala. Next, native PapA-6his4ala was captured via the 6-his affinity tag and exposed to DegP for two hours. The assay was otherwise set up as described above. The ELISA was then developed with anti-PapA polyclonal antisera. Clearly, as shown in figure 6B, DegP proteolyzed a significant amount of PapA. EXAMPLE 2: Identification of Homologous Sequences A BLAST (Basic Local Alignment Search Tool) search was conducted to look for DegP/HtrA homologs. The E. coli DegP was used as the query sequence in the search and each subject sequence found was aligned with E. coli DegP. Except for the Ricksettia sequences, all of the bacterial homologs contain the catalytic triad (Fig. 7A) and at least one PDZ domain (Fig. 7B). Many of the eucaryotic homologs have the triad and PDZ domains, although some share homology only through the PDZ domain. Figure 8 shows an alignment of E. coli DegP and three Gram-positive homologs. In this alignment, identical residues and conserved changes are shown in bold. The important residues in this alignment that are conserved are histidine 105 (E. coli residue 137-numbering includes signal sequence and starts with the longer S. pneumoniae sequence), aspartic acid 135 (167) and serine 210 (252) which make up the catalytic triad of the serine protease. The first PDZ domain starts at residue 304 (methionine) and continues to glycine 401. E. coli Deg. P contains a second PDZ domain that runs from glutamine 417 to aspartic acid 496. Given that this family of proteins is very large, these homologies are well preserved and fitted to structural motifs. While the invention has been described and illustrated herein by references to various specific material, procedures and examples, it is understood that the invention is not restricted to the particular material, combinations of material, and procedures selected for that purpose. Numerous variations of such details can be implied and will be appreciated by those skilled in the art.

Claims: WHAT IS CLAIMED IS: 1. An assay for inhibitors of the DegP protease, comprising the steps of providing the major pilin subunit of the Pap pilus, the DegP protease, and a target peptide comprising the DegP recognition/cleavage site; - mixing DegP and a suitable substrate in the presence of a suspected inhibitor of DegP activity; detecting the enzymatic activity of the DegP protease in the presence of the suspected inhibitor; and comparing the DegP activity in the presence of the inhibitor to the activity in the absence of the inhibitor. 2. An assay according to claim 1 , wherein the major pilin subunit of the Pap pilus is immobilized on a solid substrate. 3. An assay according to claim 1 , wherein the DegP protease is provided in the form of a periplasmic extract comprising the DegP protease. 4. An assay according to claim 1 , wherein the suitable substrate of DegP is a natural substrate of DegP. 5. An assay according to claim 4, wherein said natural substrate of DegP is PapA pilin. 6. An assay according to claim 1 , wherein all the components of the assay, including DegP protease and PapA target, are homogeneous preparations. 7. An assay according to claim 1 , wherein the peptide containing the recognition/cleavage site is conjugated to a detectable label. 8. An assay according to claim 7, wherein the detectable label is a fluorescent dye.

Inventors: EVANS AMY K, FRANKE CHRISTINE A, HRUBY DENNIS E, HULTGREN SCOTT J, JONES HAL C, LIU CHRISTOPHER, EVANS, AMY, K., FRANKE, CHRISTINE, A., HRUBY, DENNIS, E., HULTGREN, SCOTT, J., JONES, HAL, C., LIU, CHRISTOPHER
Applicants: SIGA TECHNOLOGIES INC, UNIV WASHINGTON, SIGA TECHNOLOGIES, INC., WASHINGTON UNIVERSITY
Application year: 2000
Main classification: G01N33
IPC codes: C12Q1, G01N33