Role of HtrA in the Virulence and Competence of Streptococcus pneumoniae

Bacterial strains and growth conditions.The bacterial strains used for this study are listed in Table 1. Bacteria were grown on blood agar base number 2 (Oxoid, Basingstoke, United Kingdom) supplemented with 5% (vol/vol) defibrinated horse blood (E&O Laboratories, Bonnybridge, United Kingdom) (BAB) and in brain heart infusion (BHI) broth. E. coli strains DH5α and XL-Gold ultracompetent cells (Stratagene) were grown in Luria-Bertani broth or on Luria-Bertani agar plates. When appropriate, antibiotics were added to the growth media at the following concentrations: ampicillin at 50 μg/ml and spectinomycin at 200 μg/ml for E. coli and 100 μg/ml for S. pneumoniae.

Plasmids and primers.The oligonucleotide primers used for this study are listed in Table 1. The PCR-Script plasmid (Stratagene) was used for cloning according to the manufacturer's recommendations. The pDL278 plasmid (23) was used as a template to amplify the AscI-generated spectinomycin resistance cassette with primers Spec up and Spec dn (Table 1). The pAL2 plasmid (4) was used for the expression of HtrA in S. pneumoniae.

DNA manipulation and transformation.Chromosomal DNAs were prepared as described elsewhere (6). PCRs, restriction endonuclease digestion reactions, DNA ligation, and DNA electrophoresis were performed according to standard protocols (30). Kits from Qiagen were used according to the manufacturer's instructions for DNA purification and plasmid preparations. The transformation of E. coli with plasmid DNA was carried out according to the manufacturer's instructions (Stratagene). The transformation of S. pneumoniae D39 (serotype 2) and S. pneumoniae TIGR4 (serotype 4) was carried out by a modification of the method of Lacks and Hotchkiss (22). Competence-stimulating peptides (CSP-1 for D39 and CSP-2 for TIGR4) were used to induce competence in these different serotypes (29). The transformed cells were selected on BAB plates with appropriate antibiotics.

Construction of ΔhtrA mutant.An isogenic mutant of S. pneumoniae lacking htrA was constructed to study the role played by HtrA in the virulence of this organism. The whole htrA gene was amplified from the chromosomal DNA of S. pneumoniae D39 with primers HtrA for and HtrA rev (Table 1) and was cloned into the PCR-Script plasmid. The internal primers HtrA inv1 and HtrA inv2 (Table 1) were designed to amplify the 5′ and 3′ ends of the gene together with the PCR-Script plasmid and to create an AscI site in the resulting PCR product. This PCR product was self-ligated to produce a plasmid carrying the interrupted gene. To facilitate the selection of transformants, we ligated an AscI-generated spectinomycin resistance cassette into this plasmid after digestion with AscI. The modified version of the gene was amplified with PCR and used to transform S. pneumoniae.

Construction of plasmid expressing htrA in S. pneumoniae. htrA was expressed from pAL2 (4) under the control of the constitutive promoter of the S. pneumoniae aminopterin resistance operon (ami) (1). Primers htrA1 and htrA2 were used to amplify htrA with EcoRI sites and a gram-positive ribosome binding site. This fragment was ligated to EcoRI-digested pAL2 carrying the ami promoter to create the pAL2HtrA plasmid. The proper orientation of the htrA gene in pAL2HtrA was confirmed by a PCR using primers pAL2y1 and pAL2y2, and the htrA gene was sequenced by using the forward primer pAL2y1 and the reverse primer htrA2 for further confirmation.

In vitro stress experiments.To compare the effect of temperature stress on the ΔhtrA mutant to that on the D39 wild type, we used the same number of viable cells (10⁶ CFU/ml) to inoculate BHI broth that was prewarmed at 37 and 40°C. At 1-h intervals, samples were taken for measurements of optical densities at 600 nm (OD₆₀₀) and for viable counts. The sensitivity of cells to H₂O₂ was tested by the exposure of aliquots of cultures grown to an OD of ∼0.3 to 40 mM H₂O₂ for 5, 10, and 15 min at room temperature. Viable cells were counted by plating them onto blood agar plates before and after the exposure to H₂O₂, and the results were expressed as percentages of survival (17). The effect of the redox compound methyl viologen (paraquat) was also studied. A total of 10⁴ cells were exposed to 60 mM paraquat (Sigma) for 2 h. At intervals, samples were taken and the numbers of viable cells were determined (37). To study the role of HtrA in pH tolerance, we used equal numbers of cells of D39 and the ΔhtrA mutant to inoculate BHI broth adjusted to pHs 4, 5, 6, 7, 8, and 9. The growth was observed after an overnight incubation at 37°C (9).

Competence assay.Competent cells of the D39 wild type and of the ΔhtrA and ΔhtrA/htrA⁺ mutants were made as described above and stored as 100-μl aliquots in 20% glycerol at −80°C. For a comparison of the abilities of the ΔhtrA and ΔhtrA/htrA⁺ mutants to take up DNA to that of wild-type D39, competent cells were thawed and diluted 1/10, and 100 ng of CSP/ml was added. Pneumococcal genomic DNA conferring kanamycin resistance (from a clpP mutant containing a kanamycin resistance cassette [8]) (0.3 μg) was then added, and the cells were incubated at 37°C for 10 min and then at 30°C for 20 min. Transformed cells were incubated on BAB without antibiotic selection for 2 h at 37°C, 250 μg of kanamycin/ml was then added, and the incubation was continued overnight. The efficiency of transformation was expressed as the percentage of transformants in the total number of cells used.

Preparation of bacterial cell protein and Western immunoblotting.All manipulations were performed at 4°C. Cell pellets of late-log-phase cultures were collected by centrifugation at 5,000 × g for 15 min and were resuspended in 1 ml of phosphate-buffered saline (PBS). Cellular proteins were released by sonication (4 pulses of 30 s each, with 30 s of chilling on ice between pulses). The cell debris was removed by centrifugation at 13,000 × g for 10 min. The total protein concentration of the lysate was determined by the Bradford assay, with bovine serum albumin used as a standard (7). For Western blot analysis, proteins present in culture lysates were separated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis, electroblotted onto nitrocellulose membranes (Amersham), and reacted with specific antisera against pneumolysin, autolysin A, neuraminidase, hyaluronidase, and cbpA according to a standard protocol (30).

Mice and infections.Female outbred MF1 mice (25 to 30 g) were purchased from Harlan Olac, Bicester, United Kingdom. They were used when they were 9 weeks old. For intranasal infections, a group of 30 mice was used for survival studies and a group of 60 mice was used for time course studies. A further group of 15 mice was used to test the effect of complementation of the htrA mutation on the survival of mice. Mice were lightly anesthetized with 1.5% (vol/vol) halothane, and the infectious dose was administered in 50-μl volumes to the nostrils of vertically held mice (19). For intravenous infections, a separate group of 20 mice was used. The infectious dose (10⁵ CFU) was administered in a 50-μl volume injected into the lateral tail vein. Blood was taken from a separate vein immediately after injection to ensure a successful infection.

Bacteriological investigation.At prechosen intervals after infection, groups of mice were sacrificed by cervical dislocation, ensuring intact trachea, and a blood sample was removed via a cardiac puncture. For bronchoalveolar lavage, a 16-gauge nonpyrogenic angiocatheter (F. Baker Scientific, Runcorn, United Kingdom) was inserted into the trachea and the lungs were lavaged with a total volume of 2 ml of sterile PBS. Lavaged lungs were homogenized in 5 ml of PBS with a glass handheld tissue homogenizer (Jencons, Leighton Buzzard, United Kingdom). Viable bacteria in lung and blood samples were counted by plating out serial 10-fold dilutions on BAB (21).

Histological analysis.A group of 10 mice (5 infected with the wild type and 5 infected with the mutant) was used to investigate histological changes at 48 h postinfection. Lungs were inflated with 1 ml of 10% (vol/vol) formal saline prior to their removal. After fixation, the lungs were embedded in paraffin and blocked according to standard histological protocols. Lung blocks were sectioned into 5-mm-thick sections prior to staining with hematoxylin and eosin (BDH Laboratory Supplies, Poole, United Kingdom) (21).

Measurement of immune modulators.A group of 30 mice was used to study the production of immune modulators at three time points postinfection. Bronchoalveolar lavage was done as described above, but the fluid was snap-frozen by immersion in liquid nitrogen. Lungs were then removed, wrapped in aluminum foil, and snap-frozen. Samples were then stored at −80°C until further processing. Upon thawing, the whole lungs were homogenized as described above. Homogenates were centrifuged at 1,600 × g for 30 min at 4°C; the supernatants were then centrifuged at 5,000 × g at 4°C, filter sterilized (0.2-μm-pore-size filter) (Gelman Sciences, Northampton, United Kingdom), and stored at −80°C. Upon thawing, the lavage fluids were centrifuged at 17,900 × g for 3 min. Interleukin-6 (IL-6) was measured by an enzyme-linked immunosorbent assay utilizing a commercially available antibody pair (clones MP5-20F3 and MP5-32C11; Pharmingen and Becton Dickinson). Tumor necrosis factor alpha (TNF-α) levels were measured by using an enzyme-linked immunosorbent assay kit for TNF-α, the OptEIA mouse TNF-α set (Pharmingen).

Statistical analysis.Statistical analyses were performed with StatView 4.1 (Abacus Concept). Survival times and comparisons of cytokine levels were analyzed by a nonparametric Mann-Whitney U analysis. Cytokine levels are expressed as medians ± median absolute deviations (21). Bacteriology results are expressed as geometric means ± standard errors of the means (SEM). Comparisons of bacterial loads in the time course bacteriology experiment were done with an unpaired t test. We controlled for intergroup variation by ensuring that there were no statistical differences in any parameter between wild-type and ΔhtrA-infected mice immediately after infection. For all analyses, a P value of <0.05 was considered statistically significant.

Construction of ΔhtrA mutation in S. pneumoniae.The htrA gene of S. pneumoniae strains D39 and TIGR4 was disrupted by the introduction of a spectinomycin resistance cassette into the gene. The mutation was confirmed by using diagnostic primers htrA C1 and htrA C2 (Table 1), flanking htrA, to amplify a PCR fragment of 1.7 kb from the chromosomal DNA of transformed cells grown on spectinomycin and a 1.2-kb fragment of the wild-type copy of htrA. The mutated PCR fragment was sequenced to further confirm that the mutation occurred at the correct position in the pneumococcal chromosome (data not shown).

In vitro phenotype of ΔhtrA mutant of strain D39.The ΔhtrA mutant was found to grow at a rate similar to that of the wild type at 37°C (data not shown). However, the mutant grew more slowly than the D39 wild type at 40°C (Fig. 1). The ΔhtrA mutant was also more sensitive to hydrogen peroxide than the wild type (Fig. 2). There was no difference in the effect of paraquat and no significant differences in growth were found at different pHs (data not shown). The amounts of the toxin pneumolysin and the major autolytic enzyme LytA expressed at 37 and 40°C were also similar (data not shown). We also examined the distribution of three known surface proteins of the pneumococcus. We found no difference in the levels of CbpA, hyaluronidase, or neuraminidase associated with the different cell fractions, as determined by Western blotting (data not shown). There were no differences in the distribution of enzymatic activity of hyaluronidase or neuraminidase between the mutant and wild-type organisms. We also compared the transformation efficiencies of the strains. The disruption of the htrA gene caused a decrease in transformation efficiency of approximately 32-fold. This reduction could be reversed by complementation with a plasmid containing the htrA gene (Table 2).

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FIG. 1.

Representative graph of in vitro growth of D39 wild type and ΔhtrA mutant at 40°C. BHI broth prewarmed at 40°C was inoculated with either bacterial strain to 10⁶ CFU/ml. Samples were taken at 1-h intervals for measurement of the OD₆₀₀. The growth of the ΔhtrA mutant was much slower than that of the wild type, but it eventually reached a maximum OD similar to that of the wild type.

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FIG. 2.

H₂O₂ sensitivity assay for D39 wild type and ΔhtrA mutant. H₂O₂ (40 mM) was added to 1-ml aliquots of culture grown to an OD₆₀₀ of ∼0.3. Viable counts were performed on BAB plates before and after the addition of peroxide, and the survival rates were calculated. Values are expressed as the means plus SEM of three independent experiments. *, P < 0.05 for lower survival rate of ΔhtrA mutant than that of the wild type.

ΔhtrA mutant is attenuated in a murine model of pneumonia.We examined the contribution of HtrA to the virulence of the pneumococcus. Groups of mice (five mice each) were infected intranasally with either serotype 2 (strain D39) or serotype 4 (strain TIGR4) or with their ΔhtrA mutants. We chose to examine the effect of HtrA in two genetic backgrounds as it was recently shown that the genetic background can have a major influence on the role of virulence factors (6). After administering the infectious dose (10⁶ CFU/mouse in the case of D39 and either 10⁵ or 10⁶ CFU/mouse in the case of TIGR4), we observed the mice and recorded the development of symptoms over a period of 8 days. Mice infected with the D39 wild type started to show symptoms of illness at 24 h postinfection, and all mice were moribund by 72 h. However, none of the D39 ΔhtrA-infected mice showed any signs of illness throughout the experiment (Fig. 3A). In the case of the TIGR4 strain, which is more virulent than D39, all mice infected with a dose of 10⁵ CFU of TIGR4 ΔhtrA survived the experiment, while 80% of those infected with wild-type TIGR4 became moribund (Fig. 3B). With a dose of 10⁶ CFU/mouse, mice infected with wild-type TIGR4 became moribund very rapidly (all were sacrificed by 29 h postinfection), while the first moribund case of TIGR4 ΔhtrA-infected mice was not recorded until 120 h postinfection and 60% of the animals were healthy at the end point of the experiment, at 8 days (Fig. 3C).

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FIG. 3.

Survival of MF1 mice infected intranasally with 10⁶ CFU of D39 or D39 ΔhtrA/mouse (A) or with 10⁵ or 10⁶ CFU of TIGR4 or TIGR4 ΔhtrA/mouse (B and C, respectively). *, P < 0.05 for longer survival times for the ΔhtrA mutant than for the wild type.

Growth of D39 wild type and ΔhtrA mutant in lungs and blood after intranasal infection.The behavior of the ΔhtrA mutant compared to that of the D39 wild type in the lung airways, lung tissues, and blood after intranasal infection was studied 0, 6, 12, 18, 24, and 48 h after infection. In the lung airways, both the D39 wild type and the ΔhtrA mutant were cleared over time. However, the ΔhtrA mutant was cleared more rapidly. The number of ΔhtrA cells recovered was significantly lower than that of D39 wild-type cells at 6 and 12 h postinfection (P < 0.05) (Fig. 4A). In lung tissues, however, while D39 grew very well, the ΔhtrA mutant was unable to survive (Fig. 4B). Furthermore, D39 appeared in the blood at 12 h postinfection and grew dramatically, whereas the ΔhtrA mutant was never detected in the blood (Fig. 4C).

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FIG. 4.

Time course of mean (± SEM) bacterial growth in lung lavage fluid (A), lung tissue (B), and blood (C) after intranasal infection with 10⁶ CFU of D39 wild type or its ΔhtrA mutant/mouse. Horizontal dashed lines represent the limits of detection of the assay. *, P < 0.05 for lower bacterial loads for the ΔhtrA mutant than for D39.

TIGR4 ΔhtrA mutant does not cause bacteremia after intranasal infection.The ability of the TIGR4 ΔhtrA mutant to cause bacteremia in mice after intranasal infection was also tested. Mice were bled 24 and 48 h after a challenge with 10⁵ CFU of either the TIGR4 wild type or the ΔhtrA mutant/mouse, and the numbers of pneumococci in the blood were determined by plating of the cells onto BAB. The number of TIGR4 wild-type cells was between log 5 and log 6.8 in 60% of the mice after 24 h of infection, while no bacterial count was detected in the rest of the group. Mice with detectable bacterial counts died, and those with no count survived at least until 48 h postinfection. In contrast, the TIGR4 ΔhtrA mutant was not found in the blood of mice at any time point (Table 3).

Growth of D39 wild type and ΔhtrA mutant in blood after intravenous infection.To determine if the role played by HtrA was specific to the lungs, we injected organisms directly into the bloodstream to bypass the respiratory tract (Fig. 5). Wild-type D39 grew in the blood and resulted in all injected animals becoming moribund, while the ΔhtrA mutant caused no disease and was cleared from the blood. HtrA therefore plays a critical role in the survival and growth of the pneumococcus in the vascular compartment.

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FIG. 5.

Survival of MF1 mice (A) and time course of mean (± SEM) bacterial growth in blood (B) after intravenous challenge with 10⁵ CFU of D39 wild type or its ΔhtrA mutant/mouse. *, P < 0.05 for shorter survival times for D39 than for the ΔhtrA mutant (A) and P < 0.05 for lower bacterial loads for the ΔhtrA mutant than for D39 (B).

The ΔhtrA mutant induces less IL-6 and TNF-α than does the D39 wild type.To study the inflammatory response toward the ΔhtrA mutant compared to that toward the D39 wild type, we measured IL-6 and TNF-α levels in the lung lavage fluids and lung tissues of MF1 mice (Fig. 6). D39 induced high levels of IL-6 at 24 h postinfection which were reduced after 36 h in the lung lavage fluids and increased with time in the lung tissues. No or very low IL-6 levels were detected in both the lung lavage fluids and the lung tissues in the case of the ΔhtrA mutant. D39 caused peak TNF-α levels in the lung lavage fluids after 24 h which then declined sharply, while TNF-α concentrations increased over time in the lung tissues. In contrast, the ΔhtrA mutant induced very low TNF-α levels in the lung lavage fluids, and the levels in the lung tissues were less than those induced by D39. Although these results are complicated by the fact that the lungs contained different numbers of organisms, it is clear that ΔhtrA induces much less inflammation in the lung.

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FIG. 6.

Median (plus median absolute deviation) levels of IL-6 and TNF-α induced in lung lavage fluids and lung tissues of MF1 mice infected intranasally with 10⁶ CFU of D39 wild type or its ΔhtrA mutant/mouse. (A) Levels of IL-6 in lung lavage fluid; (B) levels of IL-6 in lung tissue; (C) levels of TNF-α in lung lavage fluid; (D) levels of TNF-α in lung tissue. *, P < 0.05 for lower cytokine levels for the ΔhtrA mutant than for D39.

Histology.A histological examination revealed that the influx of inflammatory cells into the lungs of mice infected with wild-type bacteria was larger than that in the lungs of mice infected with the ΔhtrA mutant. At 48 h postinfection, the lungs of mice infected with D39 contained more lesions, especially at the pleural interface (Fig. 7). These areas were swollen and congested with recruited cells (the nuclear morphology indicates that the majority of cells were neutrophils). In contrast, the lungs from mice infected with the ΔhtrA mutant had few inflammatory lesions.

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FIG. 7.

Lung tissue sections from MF1 mice infected with 10⁶ CFU of D39 wild type (A) or its ΔhtrA mutant (B)/mouse. Mice were sacrificed at 48 h postinfection, and their lungs were removed, fixed, and stained with hematoxylin and eosin. The sections show a blood vessel (Bv) in close proximity to the pleural interface (Pl). The pleural interface is narrow and healthy in the lung infected with the ΔhtrA mutant but is swollen with inflammatory cells in the lung infected with D39. Inflammatory cells can also be seen moving out of the blood vessel towards the pleural interface between the two large arrowheads in panel A.

Complementation with HtrA reverts the D39 ΔhtrA mutant to full virulence.To confirm that the attenuation of virulence in the ΔhtrA mutant was entirely due to the lack of HtrA, we complemented this mutant with the pAL2HtrA plasmid, which expresses HtrA from the gram-positive ami promoter. When the HtrA level was restored in the ΔhtrA mutant, the strain was again fully virulent in a pneumonia model of infection (Fig. 8). The virulence phenotype of the ΔhtrA mutant was therefore confirmed to be solely due to the deletion of htrA.

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FIG. 8.

Effect of complementation with HtrA on virulence. The ΔhtrA strain had reduced virulence, as judged by the survival times of mice after intranasal infection with 10⁶ CFU/mouse. Complementation with HtrA (from plasmid pAL2HtrA) reverted this strain to full virulence.