INTRODUCTION

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Haemophilus influenzae is the cause of several serious human diseases such as meningitis, epiglottitis, septicemia, and otitis media. There are six serotypes of H. influenzae, designated a to f, which are identified by their capsular polysaccharides. H. influenzae type b (Hib) was a major cause of bacterial meningitis until the introduction of several Hib capsular polysaccharide conjugate vaccines in the 1980s (1, 30). The other serotypes of H. influenzae are associated with invasive disease at low frequencies, although there appears to be an increase in disease due to these strains as the incidence of Hib disease declines (18, 25, 34). Nonencapsulated or nontypeable H. influenzae (NTHI) is a major cause of otitis media (middle ear infection) in young children and of respiratory tract infections in adults. NTHI is the second most common bacterial cause of otitis media after Streptococcus pneumoniae and is responsible for about 25% of this disease. Otitis media affects more than 80% of children under 6 years of age, with the peak incidence in infants under the age of two. In the United States, the incidence of disease increased 2.5-fold between 1975 and 1990, and the causative bacteria are becoming increasingly antibiotic resistant (17). Although otitis media is rarely life threatening, there are serious sequelae associated with the disease, including deafness and language or learning deficits, and there is currently no vaccine.

The HtrA protein has been identified as a virulence factor in Salmonella typhimurium, Yersinia enterocolitica and Brucella abortus (8, 16, 20). The HtrA (or DegP) protein of Escherichia coli has been shown to be essential for survival of the organism at temperatures of >42°C. It is a stress response protein belonging to the ^E-dependent family of heat shock proteins (Hsps) (7). It is not related to either the ³²-regulated Hsps such as DnaK or DnaJ or the ⁷⁰-regulated Hsps such as Hsp60, Hsp70, and Hsp90 (21). The S. typhimurium HtrA protein is ~89% identical to E. coli HtrA but is not induced by heat shock, although it is induced by oxidative stress (16).

The E. coli HtrA protein has serine protease activity (22), and two residues of the catalytic triad have been identified by site-directed mutagenesis (31). In this report, we describe the cloning and sequence analysis of the htrA genes from two strains of NTHI and the expression of recombinant HtrA (rHtrA) in E. coli. The rHtrA protein was shown to have serine protease activity, and site-directed mutagenesis was performed on the three residues of the catalytic triad to ablate the endogenous enzyme activity. The rHtrA mutant proteins were expressed at a high yield from E. coli, and the purified proteins were found to be highly immunogenic. Protection studies were performed with two animal models, the passive infant rat model of bacteremia and the active chinchilla model of otitis media.

	MATERIALS AND METHODS

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Recombinant DNA techniques. Restriction endonucleases were purchased from Boehringer Mannheim, New England Biolabs, Bethesda Research Laboratories, and Pharmacia and were used according to the manufacturers' specifications. Oligonucleotides were synthesized on an Applied Biosystems Inc. model 380B DNA synthesizer and purified by chromatography. Plasmid pUC-BgXb is a pUC8-based plasmid with additional BglII and XbaI restriction sites in its multiple-cloning site and was used for construction purposes. Other recombinant DNA methods were performed as described by Sambrook et al. (29).

Bacterial strains and media. NTHI strains 33 and 12 are otitis media clinical isolates kindly provided by S. Barenkamp (St. Louis University, St. Louis, Mo.). NTHI strain LCDC2 was obtained from the Laboratory Centre for Disease Control (Ottawa, Ontario, Canada). Hib strain Eagan is the Pasteur Mérieux Connaught vaccine strain, and Hib strain MinnA was a kind gift from R. Munson (Ohio State University, Columbus). H. influenzae serotype a, c, d, e, and f strains were purchased from the American Type Culture Collection. H. influenzae strains were grown on Mueller-Hinton agar (BBL) supplemented with yeast extract (0.5% [wt/vol]; Sigma), hemin (15 µg ml¹; Difco), and NAD (15 µg ml¹; Difco) or in brain heart infusion (BHI) broth (Difco) as described previously (14). Chocolate agar plates were purchased from Becton Dickinson Microbiology Systems. E. coli strains were grown in NZCYM or YT medium supplemented with 50 µg of ampicillin ml¹ as required.

Cloning and sequencing of htrA genes. The construction of an NTHI strain 33 EMBL 3 chromosomal library has been described in detail elsewhere (23). An oligonucleotide probe (41 nucleotides) was derived from sequence information presented by Weinstein et al. (36) and corresponded to the N terminus of the encoded HtrA protein. The sequence of the probe is shown in Fig. 1. The probe was radiolabelled with [-³²P]dATP, and putative genomic clones were plaque purified three times. A 15.3-kb insert containing the NTHI strain 33 htrA gene was subcloned into pUC-BgXb, and the htrA gene was localized to a 4.7-kb BamHI fragment by restriction mapping and Southern blot analysis. Nested deletion clones were generated with an Erase-a-base kit (Promega), and sequencing was performed on the ABI model 373A DNA sequencer by using dye terminator chemistry. The first strand sequence was obtained with the universal forward primer, and second strand sequence was obtained with 17- to 25-nucleotide custom primers.

View larger version (6K): [in this window] [in a new window]   FIG. 1.   Nucleotide sequence and derived amino acid sequence for the 41-nucleotide probe used in the cloning and sequencing of htrA genes.

View larger version (6K): [in this window] [in a new window]   FIG. 2.   Oligonucleotide primers used for PCR amplification of NTHI strain 12 DNA. The primer sequences are underlined.

Identification of active-site residues. The NTHI HtrA sequence was initially compared with those of the E. coli and S. typhimurium HtrA proteins by using Intelligenetics Suite software. The sequence boundaries of the protease domain of NTHI HtrA were defined both manually and with XALIGN (37). To confirm the assignment of the catalytic triad residues, multiple sequence alignments and three-dimensional superimposition of mammalian and bacterial serine proteases were performed by utilizing XALIGN and the Homology module from BIOSYM Technologies, with crystal structures obtained from the Protein Data Bank (5).

Expression of recombinant HtrA and generation of mutant proteins. The smallest Erase-a-base clone that contained all of the htrA gene was used to construct expression plasmids. By analogy with the E. coli and S. typhimurium HtrA proteins (22), a putative 26-amino-acid signal sequence was identified, and the Thr at position 27 was assumed to represent the start of the mature protein. There is a BspMI site ~72 bp downstream of the start of the coding sequence for the mature HtrA protein and a ClaI site downstream of the end of the htrA gene. Oligonucleotides were synthesized to encode the N terminus of the mature HtrA protein up to the BspMI site (Fig. 3). Plasmid DNA was digested with BspMI and ClaI, and the 1.4-kb fragment was ligated with the NdeI-BspMI oligonucleotides and vector pT7-7, which had been digested with NdeI and ClaI, thus generating pT7-7/htrA. For site-directed mutagenesis, the T7-htrA gene fragment was cloned into M13mp18 and the Amersham oligonucleotide-directed in vitro mutagenesis system was used. The T7-htrA mutant genes were cloned into pT7-7 for expression of the recombinant proteins.

View larger version (8K): [in this window] [in a new window]   FIG. 3.   Oligonucleotides encoding the N terminus of the mature HtrA protein up to the BspM site.

Purification of recombinant HtrA proteins. Cells from a 500-ml culture were harvested by centrifugation at 10,000 × g for 10 min at 4°C, and the pellet was resuspended in 40 ml of 50 mM Tris-HCl (pH 8.0) and disrupted by sonication (three times for 10 min each time). The sonicate was centrifuged at 20,000 × g for 30 min at 4°C, and depending upon the location of the product, the supernatant or pellet was further purified.

Characterization of wild-type and mutant HtrA proteins. The residual serine protease activities of rHtrA and its analogs were assessed with -casein as the substrate as described by Lipinska et al. (22). To study the kinetics of the protease activity, 5 µg of -casein was mixed with 0.5 µg of HtrA and the mixture was incubated at 37°C. Aliquots were taken at 10, 20, and 30 min and at 1, 2, 4, and 16 h, and the reaction was stopped immediately by the addition of SDS-PAGE sample buffer and heating at 100°C for 5 min. Samples were analyzed by SDS-12.5% PAGE. To determine the residual protease activities of various analogs, 5 µg of -casein was mixed with 0.5 µg of HtrA mutant protein and the mixture was incubated at 37°C for 16 h. Samples were treated and analyzed as described above.

Immunogenicity studies. To study the immunogenicities of rHtrA and mutant proteins H91A, D121A, and S197A, groups of five BALB/c mice (Charles River) were injected subcutaneously (s.c.) on days 1, 29, and 43 with various doses (0.3, 1, 3, and 10 µg) of protein adsorbed to alum. Blood samples were collected on days 0, 14, 28, 42, and 54.

Stress response studies. Two guinea pigs (Charles River, St. Constant, Quebec, Canada) were immunized intramuscularly (i.m.) with 10 µg of rHtrA protein emulsified in complete Freund's adjuvant (Difco). Fourteen and 28 days later, the animals were boosted with the same amount of immunogen emulsified in incomplete Freund's adjuvant. Antisera were collected 2 weeks after the last injection.

1

Antigenic conservation. H. influenzae serotypes a, b (Eagan), c, d, e, and f were grown in BHI medium as described above. Equivalent concentrations of whole-cell lysates were electrophoresed on an SDS-11.5% PAGE gel, electroblotted onto a nitrocellulose membrane, and probed with a 1:1,000 dilution of guinea pig anti-rHtrA antibody. Fc-specific goat anti-guinea pig IgG antibody conjugated to HRP was used as the second antibody, and the blot was visualized with the LumiGlo system.

Protection studies. Chinchilla protection studies were performed as described by Barenkamp (2). Briefly, 1- to 2-year-old chinchillas (Moulton Chinchilla Ranch, Rochester, Minn.) were immunized i.m. with 30 µg of protein adsorbed to alum. Booster immunizations were given on days 14 and 28, and antisera were collected 2 weeks after the last injection. Negative control animals were mock immunized with alum by following the same regimen, and positive controls were convalescent animals that had recovered from previous infection. Animals were challenged with 50 to 350 CFU of live NTHI strain 12 or 200 CFU of live LCDC2 through the bulla. Otoscopy and tympanometry were performed before bacterial inoculation and repeated 4 days postchallenge. Middle ear fluid was collected and immediately mixed with 200 µl of BHI medium. Dilutions of aspirates were made, and 10 µl of undiluted aspirate and 10 µl of 1/10 and 1/100 dilutions were plated onto chocolate agar plates. Bacterial colonies were counted after 24 h.

Nucleotide sequence accession numbers. Nucleotide sequences of NTHI strain 33 htrA and strain 12 htrA have been deposited in GenBank and have accession no. AF018152 and AF018151, respectively.

	RESULTS

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Cloning and sequence analysis of the htrA gene. Weinstein et al. had described the derived amino acid sequence of a 47-kDa H. influenzae protein designated Hin47 and the associated nucleotide sequence (36). An oligonucleotide probe was synthesized based upon the published sequence and the hin47 gene was cloned from a chromosomal library of NTHI strain 33. The hin47 gene was localized on the 15.3-kb chromosomal fragment and sequenced.

View larger version (42K): [in this window] [in a new window]   FIG. 4.   Sequence alignment of HtrA proteins from NTHI strains 33 and 12, H. influenzae type d strain Rd, E. coli (Ecoli), and S. typhimurium (Styph). Stop codons are indicated by asterisks. Dots indicate residues identical to those of the NTHI strain 33 HtrA, and dashes have been used to achieve maximum alignment. The residues indicated by arrows are numbered from the putative Thr start of the mature NTHI HtrA proteins. The main numbering scheme refers to the full-length encoded protein from the start methionine of NTHI 33 HtrA. The RGD sequence is underlined in the strain 33 sequence.

H. influenzae HtrA is a stress response protein. Since HtrA is known to function as a stress response protein in E. coli, experiments were performed to determine whether H. influenzae HtrA was also a stress response protein. E. coli and Hib strain Eagan were grown in parallel under standard conditions and under stress-inducing conditions such as high temperature, 6% ethanol, and high salt concentrations. A guinea pig antiserum which recognized HtrA from both organisms was used to assess expression. HtrA was found to be constitutively expressed at low levels for both organisms under all growth conditions (Fig. 5). At 43.5°C or in the presence of 6% ethanol, the expression of a second form of HtrA at a slightly higher apparent molecular weight was induced in both E. coli and H. influenzae, although the induction was significantly better in the medium containing ethanol. No effect was observed at 42°C or in the presence of 0.2 or 0.3 M NaCl (results not shown).

View larger version (43K): [in this window] [in a new window]   FIG. 5.   Stress induction of htrA expression. (A) Immunoblot of E. coli JM109 and Hib strain Eagan grown at 37°C. Lane 1, E. coli at t = 0 min; lane 2, E. coli at t = 20 min; lane 3, E. coli at t = 60 min; lane 4, Hib at t = 0 min; lane 5, Hib at t = 20 min; lane 6, Hib at t = 60 min. (B) Immunoblot of E. coli JM109 and Hib strain Eagan grown at 43.5°C. Lane 1, E. coli at t = 0 min; lane 2, E. coli at t = 20 min; lane 3, E. coli at t = 60 min; lane 4, Hib at t = 0 min; lane 5, Hib at t = 20 min; lane 6, Hib at t = 60 min. (C) Immunoblot of E. coli JM109 and Hib strain Eagan grown in the presence of 6% ethanol. Lane 1, E. coli at t = 0 min; lane 2, E. coli at t = 20 min; lane 3, E. coli at t = 60 min; lane 4, Hib at t = 0 min; lane 5, Hib at t = 20 min; lane 6, Hib at t = 60 min. In all panels, the numbers at the left represent molecular weights in thousands.

H. influenzae HtrA has serine protease activity. The effect of protease inhibitors on rHtrA activity was determined. It was found that two serine protease inhibitors, Pefabloc SC and PMSF, completely inhibited the rHtrA activity at concentrations of 10 mM. In contrast, the thiol protease inhibitor leupeptin did not show any effect even at 500 mM, a concentration which is 250 times higher than that routinely used (27).

Identification of active-site residues. The consensus sequence surrounding the active-site serine of serine proteases is GDSGGPK (6), where the serine is the active-site nucleophile. An examination of the strain 33 HtrA sequence revealed a GNSGGAL motif around Ser197 (numbered from the proposed Thr1 residue). By comparison with other bacterial and mammalian serine proteases, the other members of the catalytic triad were predicted to be His91 and Asp121. Figure 6 shows the alignment of NTHI HtrA with the sequences of serine proteases from the Protein Data Bank.

View larger version (68K): [in this window] [in a new window]   FIG. 6.   Identification of HtrA active-site residues. Multiple sequence alignment of the serine protease domain of NTHI HtrA (residues 57 to 256) with serine proteases from the Protein Data Bank (PDB). Arrows denote the catalytic triad residues. Protease sequences obtained from PDB entries are as follows: TON, pdb1ton.ent (rat tonin); PKA, pdb2pka.ent (porcine kallikrein A); PTN, pdb2ptn.ent (bovine trypsin); CHA, pdb4cha.ent (bovine chymotrypsin); EST, pdb3est.ent (porcine elastase); RP2, pdb3rp2.ent (rat mast cell protease II); SGT, pdb1sgt.ent (Streptomyces griseus trypsin); SGB, pdb3sgb.ent (S. griseus proteinase B); SGA, pdb2sga.ent (S. griseus proteinase A); ALP, pdb2alp.ent (L. enzymogenes alpha-lytic protease).

Expression and characterization of recombinant HtrA and mutants. The strain 33 htrA gene was cloned behind the inducible T7 promoter in vector pT7-7 (33), and the recombinant protein was produced at high levels in E. coli BL21(DE3) cells. Nine HtrA analogs were generated at the His91, Asp121, or Ser197 residues of the catalytic triad, and all of the mutant HtrA proteins were expressed at a very high yield (~40 to 50% of total protein) from E. coli. The recombinant proteins were expressed as soluble proteins (wild type and H91A), inclusion bodies (H91R, D121A, D121E, S197A, H91A/S197A, and H91A/D121A/S197A), or a mixture of both forms (S197C and S197T), and all were readily purified (Fig. 7).

View larger version (65K): [in this window] [in a new window]   FIG. 7.   SDS-PAGE analysis of rHtrA and analogs during purification performed on 12.5% polyacrylamide gels. Lanes 1, prestained molecular weight markers; lanes 2, total cellular proteins from E. coli BL21(DE3) expressing rHtrA or mutant proteins; lanes 3, crude extract of recombinant protein; lanes 4, flowthrough fraction from DEAE Sephacel column for soluble rHtrA and H91A; lanes 5, purified protein after the HTP column. The numbers at the left of each row represent molecular weights in thousands.

View larger version (64K): [in this window] [in a new window]   FIG. 8.   Protease activity of rHtrA and its analogs. (A) Wild-type rHtrA (0.5 µg) was mixed with -casein (5 µg) and incubated at 37°C. Samples were taken at various time points and analyzed by SDS-PAGE. Lane 1, molecular weight standards; lane 2, t = 0 min; lane 3, t = 10 min; lane 4, t = 20 min; lane 5, t = 30 min; lane 6, t = 1 h; lane 7, t = 2 h; lane 8, t = 4 h; lane 9, t = 16 h; lane 10, no HtrA added at t = 16 h. (B) Wild-type rHtrA or its analogs (0.5 µg) were mixed with -casein (5 µg) and incubated at 37°C for 16 h. Lane 1, molecular weight standards; lane 2, casein alone; lane 3, rHtrA; lane 4, S197A; lane 5, S197C; lane 6, S197T; lane 7, H91A; lane 8, H91R; lane 9, D121A; lane 10, D121E. For both panels, the numbers at the left represent molecular weights in thousands.

Immunogenicity and protection studies. Purified rHtrA, H91A, D121A, and S197A were used to immunize BALB/c mice, and after three immunizations, all four proteins were found to be very immunogenic at doses as low as 0.3 µg when administered in alum (Table 1). The antibody isotype profiles were determined for rHtrA, H91A, and S197A and were found to be equivalent for the three proteins, with a dominant IgG1 response (Table 2). Guinea pig anti-NTHI rHtrA recognized putative HtrA from H. influenzae serotype a, b, c, d, e, and f strains, although the reactivity with serotype f was very weak (Fig. 9).

View larger version (70K): [in this window] [in a new window]   FIG. 9.   Antigenic conservation of HtrA in H. influenzae strains. Lane 1, serotype a; lane 2, serotype b (Eagan); lane 3, serotype c; lane 4, serotype d; lane 5, serotype e; lane 6, serotype f. Guinea pig anti-NTHI rHtrA was used to probe the blots. The numbers at the left represent molecular weights in thousands.

View larger version (15K): [in this window] [in a new window]   FIG. 10.   Protection studies in the chinchilla intrabulla challenge model of otitis media. Chinchillas (1 to 2 years old) were immunized three times (i.m.) with 30 µg of antigen adsorbed to alum. Two weeks after the last injection, animals were challenged with live NTHI strain 12 (50 to 350 CFU) (A and B) or LCDC2 (200 CFU) (C) through the epitympanic bulla. Middle ear fluid was collected 4 days postchallenge. Each data point represents the bacteria recovered from a single chinchilla.

	DISCUSSION

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The sequences of the NTHI HtrA proteins and the Rd HtrA (HI1259) sequence (11) were found to be highly conserved, with differences at only five positions among the strains. One of these amino acid changes is in an Arg-Gly-Asp (RGD) motif found at the carboxy terminus of HtrA. The RGD motif has been identified as a cell attachment site for mammalian adhesion proteins (28). Of the 13 H. influenzae htrA genes that we studied, five encoded an RGN motif and the rest encoded RGD. E. coli expresses two homologs of HtrA (or DegP), which are called DegQ (HhoA) and DegS (HhoB) (3, 35). The DegP and DegQ proteins have 60% identical and 75% similar sequences and are serine proteases. One noticeable sequence difference is that DegP contains an RGD sequence and DegQ contains an RGN sequence (35). In a recent paper by Li et al. (20), an alignment of the HtrA proteins from Y. enterocolitica, S. typhimurium, E. coli, B. abortus, Rochalimaea hensalae, Helicobacter pylori, and Campylobacter jejuni demonstrated that only the first three of these organisms contain the carboxy-terminal RGD sequence. Since HtrA is a periplasmic protein, it is unclear what function the RGD or RGN sequence might play, but the fact that it is at least semiconserved among different species is intriguing. In addition, if it is surface expressed under stress conditions, the RGD sequence may play an attachment function.

The E. coli HtrA protein has been shown to be inducible by temperatures of >42°C, but the S. typhimurium HtrA protein is not temperature inducible (16). By immunoblot analysis, we demonstrated low-level constitutive expression of HtrA in both E. coli and H. influenzae under normal culture conditions, with an increased expression of a slightly larger protein under stress conditions. The amount of protein expressed was too small to allow N-terminal sequence determination, so we can only speculate as to the nature of the second protein. It is possible that it is the unprocessed precursor protein or a conformational variant of HtrA, although the latter possibility seems unlikely based upon the results of Skorko-Glonek et al. (32), who showed that E. coli HtrA was relatively thermostable up to 70°C. Since DegQ is not heat inducible, the second protein band is not likely to be DegQ (35).

The proteolytic activity of H. influenzae rHtrA was blocked by serine protease inhibitors, indicating that like the E. coli HtrA, it is a serine protease. Alignment of the NTHI, E. coli, and S. typhimurium HtrA protein sequences showed that there is a consensus sequence of GNSGGAL surrounding the putative Ser197 active-site residue. With alignments anchored on this residue, the other residues of the catalytic triad were identified from alignments with other bacterial and mammalian serine proteases of known structure. In general, bacterial trypsin-like serine proteases such as alpha-lytic protease from Lysobacter enzymogenes and proteases A and B from Streptomyces griseus are significantly smaller than mammalian ones (4). However, because the active sites and cores of these proteases can be reliably aligned to mammalian serine proteases, they provide an indication of where sequence variability, including the locations of insertions and deletions, can be expected (12, 13). Thus, the active-site histidine could be assigned to His91 of NTHI HtrA despite the presence of only a low level of local sequence homology and the absence of a conserved disulfide bond immediately following this residue. Asp121 was assigned on the basis of its local sequence alignment with a combination of identical or similar residues in other proteases. To confirm these residues, a three-dimensional model of the HtrA site was constructed by using L. enzymogenes alpha-lytic protease as a template (12) and various serine protease structures from the Protein Data Bank (5). The catalytic triad could be readily accommodated within this model in comparison to the crystal structures of mammalian and bacterial serine proteases. Using site-directed mutagenesis, Skorko-Glonek et al. (31) demonstrated that the Ser210 and His105 residues of the mature E. coli HtrA protein were part of the catalytic triad. The serine residue was identified by the homology of the sequences of the surrounding amino acids to the consensus sequence for serine proteases, and there is only one histidine residue in the molecule. Li et al. (20) and Waller and Sauer (35) predicted that the Asp136 residue of E. coli HtrA is part of the catalytic residue, a prediction which correlates very well with our finding of Asp121 in the NTHI proteins. Mutations of the His91, Asp121, and Ser197 codons were found to abolish the protease activity. The D121E analog still retained its enzymatic activity, although D121A did not, suggesting that the negative charge is important for activity.

The assignment of the catalytic triad residues provided a basis for delineating the boundaries of the protease domain, which comprises about 45% of the HtrA sequence, i.e., between residues 54 and 255. The rHtrA protein was found to contain a major degradation product that started with residue Asp54. By homology with other proteases, residues 1 to 53 may be cleaved from a zymogen form of HtrA in order for it to become active. E. coli cells overproducing wild-type recombinant E. coli HtrA were found to contain two major ~43-kDa HtrA degradation products, beginning at Cys69 and Gln82 (31). It was speculated that these products were autocatalytic in nature since the mutant HtrA proteins did not contain them.

Antisera raised to NTHI strain 33-derived rHtrA, H91A, or S197A were all equally protective in the passive infant rat model of bacteremia (24), wherein 6 of 10 animals had no or minimal bacteremia. These data indicated that the three proteins were inducing functional antibodies. In this model, the animals are challenged with live Hib organisms, and the partial protection demonstrates that the anti-NTHI rHtrA antibodies recognize the HtrA protein in Hib. Immunoblot analysis showed that anti-NTHI rHtrA antisera recognize ~46-kDa proteins in H. influenzae serotypes a, b, c, d, e, and f (weak), demonstrating the antigenic conservation of the HtrA proteins. In the chinchilla model of otitis media, animals were immunized with NTHI strain 33-derived rHtrA or its H91A or S197A analogs and were challenged with live NTHI strain 12 or LCDC2 organisms. The rHtrA and H91A proteins induced partial protection, but the S197A protein was not protective. The IgG subtype profiles elicited by immunization with the three immunogens were determined with mice, and it was found that all three were identical, with a predominant IgG1 response, a small IgG2b response, and no IgG2a or IgG3 response. One explanation for the observed difference in protection between H91A and S197A could be that they have different conformations after purification as soluble or inclusion body-derived proteins; however, we were unable to demonstrate any difference by circular dichroism analysis (data not shown). It is also unclear why this difference is evident only in the chinchilla model and not the infant rat model.

The exact role of the HtrA protein in the pathogenesis of H. influenzae remains to be determined; however, if HtrA serves as a target for bactericidal antibodies or opsonic activity, it must be accessible to antibodies. Since HtrA is an apparently periplasmic protein, it is not evident how such antigen-antibody interactions can occur. It is possible that during infection, some surface expression of HtrA is induced as part of a stress response mechanism, e.g., fever. Other intracellular Hsps have been shown to become surface expressed under physiological stress conditions and have been implicated as adhesion factors (9, 10, 15).

In these studies, we have cloned, sequenced, and expressed the NTHI htrA gene and have shown that the H. influenzae HtrA protein has serine protease activity. This activity can be abolished by site-directed mutagenesis of the htrA gene, and the resultant H91A and S197A HtrA analogs induce antibodies which are partially protective in a passive model of bacteremia. In the chinchilla model of otitis media, H91A was also partially protective, indicating that this antigen may be suitable for inclusion in a multicomponent otitis media vaccine. A phase I clinical trial of the H91A HtrA protein is in progress.