Pneumococcal Gene Complex Involved in Resistance to Extracellular Oxidative Stress

All strains used in the present study are listed in Table 1 and were stored in 15% glycerol at −80°C. S. pneumoniae was routinely grown as standing cultures in brain heart infusion (BHI) broth (Oxoid) at 37°C, Lactococcus lactis NZ9000 was grown standing in M17 broth (Oxoid) (60) containing 0.5% glucose (GM17) at 30°C, and Escherichia coli was grown in LB broth in shaken cultures at 37°C. L. lactis and S. pneumoniae were grown, respectively, on GM17 and GM17 agar plates with 3% defibrinated sheep blood (Oxoid), whereas E. coli was cultivated on LB agar. When necessary, chloramphenicol, spectinomycin, and trimethoprim were added to the growth medium in the following concentrations: 2.5, 150, and 15 μg/ml, respectively, for S. pneumoniae; 4 μg/ml chloramphenicol for L. lactis; and 20 μg/ml kanamycin for E. coli. For in vitro complementation analyses, all strains were exposed to nisin (3 ng/ml) during early log phase and at least 2 h before the application of oxidative stress.

The sequences of the predicted proteins in the genome of S. pneumoniae R6 derived from the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/GenBank/) were sequentially compared to the predicted protein sequences in the genomes of S. pneumoniae TIGR4, Neisseria meningitidis MC58, Neisseria meningitidis Z2491, and Haemophilus influenzae Rd KW20 using BlastP. Proteins were considered homologous when the E value was <10⁻¹³. Subsequently, the list of R6 proteins with a homologue in the other genomes was compared to the sequences of the predicted proteins in the genome of L. lactis IL1403 using BlastP, and a protein was considered nonhomologous when the E value was >10⁻¹⁹.

Strain R6 nisRK was generated by the introduction of the nisRK genes into the bgaA locus of R6 as described by Kloosterman et al. (27). Deletion of spd0572 was accomplished using primers SPD571for/SPD571rev and SPD573for/SPD573rev using plasmid pOri28 as described by Kloosterman et al. (26). The spd0571-0573 genes were deleted from the D39 genome by allelic replacement mutagenesis (45). Briefly, primers SPD570for/571rev and 574for/574rev (Table 2), respectively, were used to generate 5′ and 3′ flanking fragments of tlpA and the operon spd0571-0573, respectively. These fragments were fused to a spectinomycin resistance gene amplified with primers specfor and specrev (3). Subsequently the PCR fragments were transformed into various S. pneumoniae strains. Generation of the correct deletion mutant of tlpA and the operon spd0571-0573 was verified by PCR using the primers ctrldtlpAfor/ctrltlpArev and ctrl571-573for/ctrl571-573rev, which are located outside the original deletion region; additionally the tlpA mutant was verified by Western blotting.

RNA of S. pneumoniae D39 was extracted as described before (14). Briefly, cells were harvested in mid-exponential phase, immediately frozen in liquid nitrogen, and stored at −80°C. RNA extractions were performed as described previously (13); to remove any possible DNA contamination, DNase I treatment was performed with TURBO DNA-free reagent (Ambion, Austin, TX), and the quality of the RNA was assessed using an Agilent RNA Nano Chip (Agilent Technologies, CA). A TaqMan reverse transcription-PCR (RT-PCR) kit (Life technologies) was used for the generation of cDNA from 100 ng of RNA. Control reactions were performed without reverse transcriptase to confirm the absence of contaminating genomic DNA. The resulting cDNA was used as a template for PCR.

For complementation analysis, a pNZ8048 (11) derivative was constructed that contained the tlpA gene or the spd0571-0573 operon under the control of nisA, a nisin-inducible promoter. Primers tlpAF and tlpaR or primers operonF and operonR, which contained NcoI and XbaI restriction sites, were used to amplify the tlpA gene and the spd0571-0573 operon from the chromosome of strain D39. The resulting PCR products were introduced into the pNZ8048 plasmid using the restriction sites mentioned above, resulting in pNZ8048::tlpA and pNZ8048::spd0571-0573, respectively. Subcloning of the latter plasmid using AvrII/XbaI, NcoI/BseYI, and NcoI/AvrII unique restriction sites in the operon and the plasmid resulted in pNZ8048::spd0571-0572, pNZ8048::spd0572-0573, and pNZ8048::spd0573, respectively. The plasmids were transferred to S. pneumoniae and L. lactis for complementation studies.

Bacteria were grown until mid-log phase, and 200 μl of the cultures was added to 200 μl of medium with 60 mM paraquat (Supleco) or without paraquat (61). Both samples were incubated at 37°C for 2 h. The percent survival was calculated by dividing the number of CFU of the cultures after exposure to paraquat by the number of CFU of the control without paraquat.

The hydrogen peroxide sensitivity assay was performed essentially as described by Pericone et al. (48). Briefly, bacteria were grown in BHI broth until mid-log phase (optical density at 600 nm [OD₆₀₀] of 0.2 to 0.3), and 100-μl aliquots of each culture were added to 100 μl of medium or 100 μl of medium containing 40 mM H₂O₂ (Merck), resulting in an exposure to 20 mM H₂O_2. The bacterial cultures were incubated at 37°C for 30 min. The reaction was stopped by the addition of 200 U of bovine liver catalase (Sigma); after serial dilutions from each well were prepared, aliquots were spotted onto blood agar plates. The percentage of survival of hydrogen stress was determined and calculated as described above.

TIGR4 and the operon mutant, which contained a spectinomycin resistance cassette, were diluted 1:100 in a 1:1 ratio; as a control, the mutant was also diluted separately to 1:100 in BHI broth. Directly after cultures were mixed, CFU counts were determined at the start of and during exponential (OD₆₀₀ of ∼0.2) and stationary (OD₆₀₀ of ∼ 0.5) phases and 24 h later. All serial dilutions were plated on blood agar plates; in addition, the mixed cultures were also plated on plates with spectinomycin to determine the number of mutant bacteria. CFU counts were determined after overnight incubation. To calculate the ratio wild type to mutant, the number of spectinomycin-resistant colonies was subtracted from the total number of colonies. The number of CFU of the mutant derived from the mixed culture plated on spectinomycin plates corresponded with the number of mutant CFU derived from the individual culture plated on blood agar. This indicated that there were no negative effects of the spectinomycin and the coculturing with the wild type. Furthermore, the ratio of the wild type to mutant did not change more than 10-fold over the course of 24 h.

Experiments with female BALB/c mice 6 weeks of age were performed at the University of Texas Health Science Center at San Antonio, Texas, under Institutional Animal Care and Use Committee protocol 09022x-34. To minimize distress, mice were anesthetized with 2.5% vaporized isoflurane during all experimental procedures including inoculation and collection of nasal lavage and prior to euthanasia. Mice were infected with either TIGR4 and TIGR4 Δspd0571-0573 (Sp^r) at a ratio of 1:1 or TIGR4 and TIGR4 Δspd0571-0573 nisRK complemented with pNZ8048E or pNZ8048::spd0571-0573 at a ratio of 1:1. In the latter experiments mice received drinking water supplemented with nisin (2 μg/liter) to drive the expression of the operon from the nisA promoter on the plasmid. For intranasal challenge, mice were held up manually, and the left nostril was inoculated with 2.0 × 10⁶ CFU in 25 μl of phosphate-buffered saline (PBS) in a dropwise fashion using a pipette. Immediately afterwards, mice were hung over their cage by their incisors on a wire until they awoke and removed themselves, typically within 20 to 30 s. For determination of bacterial titers in the nasopharynx, total numbers of pneumococci in nasal lavage fluids were quantified. Briefly, using the same protocol as challenge, 10 μl of PBS was inoculated into the left nostril, followed by its aspiration after 5 to 10 s. Typically, 2 to 4 μl was recovered per mouse. For determination of bacterial titers in the blood, 2 to 5 μl of blood was collected from the tail vein. Bacterial titers in nasal lavage fluid and blood samples were determined by serial dilution, plating on blood agar plates, and extrapolation from bacterial counts following overnight incubation. For determination of bacterial titers in the lungs, mice were sacrificed, and the lungs were excised, weighed, and homogenized. Bacterial titers were determined per gram of homogenized tissue. All dilutions were plated in replicate using plates with the appropriate antimicrobial to discriminate between strains. Statistical analysis of bacterial counts was done using a nonparametric Mann-Whitney rank sum test using SigmaStat statistical analysis software (Aspire Software, Ashburn, VA).

To avoid problems associated with the purification of proteins covalently attached to the membrane such as lipoproteins, tlpA without its signal sequence and its stop codon was amplified from the D39 genome using primers 572SQ and 572SC. The PCR product was cloned into the pET-26b(+) expression vector (Novagen, Inc.) using the NdeI and XhoI restriction sites. The resulting plasmid pET-26b::tlpA was subsequently transformed into E. coli strain BL21(DE3) for high-level TlpA production. Expression of tlpA including a C-terminal His₆ tag, which is provided by the pET26b(+) vector, was induced by 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG), and its production was confirmed by Western blot analysis using an anti-His tag antibody (Zymed-Invitrogen). To purify the protein, a 1-liter culture of E. coli strain BL21(DE3) was induced with 1 mM IPTG; after 3 h the cells were harvested by centrifugation and resuspended in binding buffer (20 mM sodium phosphate buffer, 300 mM NaCl, 10% [vol/vol] glycerol, 5 mM imidazole, 3 mM dithiothreitol [DTT], pH 7.4). Cells were disrupted with a bead beater (Bertin) using three cycles of 30 s and after centrifugation at 14,000 rpm for 5 min; the clarified supernatant fraction was applied to a 5-ml His-Trap HP column (GE Healthcare). After the unbound proteins were washed off the column with binding buffer, the His-tagged TlpA protein was eluted from the column using elution buffer (20 mM sodium phosphate buffer, 300 mM NaCl, 10% [vol/vol] glycerol, 3 mM DTT, pH 7.4) with a gradient of increasing imidazole concentrations (up to 500 mM imidazole). The purity of the eluted protein fraction was determined using SDS-PAGE and Coomassie staining, and 1.14 mg of purified protein was used for the generation of a polyclonal antibody (Eurogentec, Seraing, Belgium).

Fractionation of S. pneumoniae cells was performed essentially as described previously (38). S. pneumoniae was grown to the desired OD in BHI broth, after which the bacteria were spun down for 10 min at 7,000 rpm. The supernatant was precipitated using 50% trichloroacetic acid (TCA), and the cell pellet was taken up in 1/20 volume of lysis buffer (100 mM Tris-HCl, pH 8.0, 20% sucrose, 20 mM MgCl₂) to which fresh lysozyme (5 mg/ml), mutanolysin (200 U/ml), and EDTA-free protease inhibitor (Roche) were added, and the mixture was incubated for 30 min at 37°C. The protoplasts were spun down at 3,000 × g for 10 min at 4°C, resuspended in 0.5 ml of sucrose buffer (20% sucrose, 10 mM Tris-HCl, pH 8.0), and disrupted by the addition of 9.5 ml of 100 mM Tris-HCl with EDTA-free protease inhibitor and 1 mM EDTA; undisrupted protoplasts were removed by centrifugation (4,000 × g for 10 min at 4°C). The membrane and cytoplasm fractions were separated by centrifugation at 100,000 × g for 30 min at 4°C. Samples were taken up in loading buffer, incubated for 5 min at 95°C, and used for SDS-PAGE and Western blot analysis.

The presence of various proteins was detected by Western blot analysis. Protein fractions were separated by SDS-PAGE using precast NuPAGE gels from Invitrogen and then semidry blotted (1.25 h at 100 mA per gel) onto a nitrocellulose membrane (Protran; Schleicher and Schuell). Detection of antibodies was carried out with fluorescent IgG secondary antibodies (IRDye 800 CW goat anti-rabbit; LiCor Biosciences) in combination with the Odyssey Infrared Imaging System (LiCor Biosciences). For antibody detection, fluorescence at 800 nm was recorded using the linear range. Signals on the Western blots were quantified as follows: non-gamma-transformed images with nonsaturated signals, as determined with the plot profile function of ImageJ (rsb.info.nih.gov/ij), were analyzed using the gel function of the same program. Comparisons of the amount of signal for each type of sample were performed on the same Western blot using the “gel lane” analysis function of the same program. The only digital manipulation performed on the scans was the switching of lanes to obtain the desired order.

Thioredoxin activity was determined as described by Holmgren (18). Briefly, 2 μg of purified TlpA or 2 μg of thioredoxin from E. coli (Sigma) and 100 mM DTT were mixed, and the reaction was started by the addition of 1 mg of insulin from bovine pancreas (Sigma) in 1 ml of 20 mM potassium phosphate buffer, pH 8.0; reactions were monitored for 1 h by measuring the reduction of disulfide bonds in insulin as determined by the precipitation of the protein, which is measured at 650 nm.

Statistical significance of the hydrogen peroxide stress survival was determined using the independent sample t test using SPSS, version 16.0. For the analysis of the animal model a nonparametric Mann-Whitney rank sum test using SigmaStat statistical analysis software (Aspire Software, Ashburn, VA) comparing the ratios to a value of 1 was performed.

Three important human pathogens colonize the nasopharynx, cause similar diseases, and are naturally competent. To identify genes that they have in common and that might contribute to virulence and oxidative stress resistance, all predicted open reading frames (ORF) in the S. pneumoniae R6 and TIGR4 genomes were compared to those in the N. meningitidis MC58 and H. influenzae Rd KW20 genomes using BLASTP (http://blast.ncbi.nlm.nih.gov). We then selected those ORFs that did not display significant homology to any predicted protein in the genome of Lactococcus lactis IL1403, which is related to S. pneumoniae but is not a pathogen and does not colonize the nasopharynx. This resulted in the identification of several ORFs (Table 3). One ORF is involved in folate synthesis and resistance to sulfonamides (spd0269, sulA) (15), and another (spd1029) belongs to the multiantimicrobial extrusion (MATE) family and might also be involved in drug resistance. Four ORFS are or seem to be involved in amino acid metabolism and uptake (spd0372, spd0377, spd1158, and spd1328) (27). Two ORFs (spd0429 and spd0430) encode homologues of proteins that form part of the Trk potassium transport system, which has also been implicated in resistance to antimicrobial peptides (43). Another ORF (spd0864) is a homologue of tehB and is most likely involved in tellurite resistance (30), and one (spd0953) is involved in the pyruvate metabolism of S. pneumoniae. One ORF, spd0572, is predicted to encode a protein belonging to the thioredoxin-like protein family, of which TlpA, ResA, and DsbE are members (32; http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi? uid = cd02966); thus, we named this protein TlpA. Proteins belonging to this family have been shown to play an important role in the response against oxidative stress (7, 9, 19, 32). Interestingly, the ORF was identified in two previous signature-tagged mutagenesis (STM) studies as being required for successful animal passage (10, 28). Therefore, we investigated the role of TlpA during intracellular and extracellular oxidative stress in S. pneumoniae.

To determine whether TlpA is involved in the protection against intracellular oxidative stress, the gene was deleted; no obvious differences in growth compared to the wild type were observed under normal conditions. Subsequently, bacteria were exposed to 30 mM paraquat for 1 h. Paraquat is a redox compound which induces the formation of superoxide in the cytoplasm in the presence of oxygen (8). However, the parent and mutant strains were equally sensitive to paraquat (Fig. 1). Use of higher concentrations of paraquat (60 mM and 125 mM) and a longer incubation time (2 h) did not alter this (data not shown). These results suggest that TlpA is not involved in the protection of S. pneumoniae against internal oxidative stress.

The effect of deleting tlpA on extracellular oxidative stress survival was investigated by exposing the bacteria to 20 mM H₂O₂ for 30 min. Survival of the mutant was reduced 10-fold compared to the wild type in strain D39 and its unencapsulated derivative R6 (Fig. 2); D39 nisRK and R6 nisRK strains were selected to be able to complement any observed phenotypes (26). Insertion of the nisRK genes into the bgaA locus did not seem to have a negative impact on resistance to oxidative stress as survival of the D39 nisRK and D39 strains were similar (data not shown). The unencapsulated R6 derivative had a survival percentage similar to that of the encapsulated D39, indicating that the capsule may not play an important role in protection against extracellular oxidative stress (Fig. 2). Strains G54 and 670-6B were more resistant to oxidative stress than R6 and D39, as they had an approximately 2-log higher survival percentage (Fig. 2). Interestingly, in all strains, except for TIGR4 which showed a 5-fold reduction (Fig. 2), deletion of the tlpA gene led to at least a 1-log reduction in the survival of 20 mM H₂O₂, which was statistically significant (Fig. 2). Thus, TlpA plays an important role in the protection against oxidative stress in S. pneumoniae.

To verify whether sensitivity to oxidative stress was caused only by deletion of the tlpA gene, it was cloned into pNZ8048, and the resulting plasmid was transformed into D39 ΔtlpA nisRK and used for complementation analysis. For complementation experiments, all strains including controls were exposed to nisin (3 ng/ml) during early logarithmic growth and approximately 2 h before the stress condition was applied_. This was done to exclude the possibility that any observed effects in complemented strains were caused by the addition of nisin and to ensure sufficient production of the protein(s). However, expression of the gene failed to increase the survival of the tlpA mutant (Fig. 3A). Due to experimental variation, all survival percentages were 10-fold higher in these experiments, but still the difference between the mutant and wild type was 10-fold and statistically significant (Fig. 3A).

Analysis of the genomic region of tlpA showed that it is located in the middle of a putative operon (Fig. 4) with ORFS spd0571, annotated as ccdA with homology to a cytochrome c-type biogenesis protein, and msrAB (spd0573), encoding the methionine sulfoxide reductase A/B protein of S. pneumoniae (25). Although there are no orthologs of these genes in L. lactis, they were not identified in the bioinformatic analysis for following reasons. In Neisseria meningitidis, there is no ortholog of Spd0571; although it has similarity with the N-terminal part of the multidomain protein DsbD, this was not enough to pass the similarity requirements of the BLAST search. For Spd0573, the situation is different. In S. pneumoniae, this protein is a polypeptide fusion of the MsrA and the MsrB proteins that occur in many other organisms. Both an MsrA and MsrB protein ortholog are present in L. lactis, and their similarity with either the MsrA part or the MsrB part of Spd0573 was high enough to make it seem that an ortholog is also present in L. lactis.

S. pneumoniae does not contain cytochromes, strongly suggesting that Spd0571 has another function. MsrAB enzymes catalyze the reduction of oxidized methionines to methionines upon the occurrence of oxidative stress (20). The three genes belong to the core genome of S. pneumoniae (40) and are highly conserved (98% identity on the nucleotide level with all S. pneumoniae genomes in the public database). Interestingly, despite this high homology, there are some differences in the annotation of the msrAB open reading frame in the various strains; in most genomes the msrAB gene is larger than that annotated in TIGR4 and D39 (Fig. 4A). Therefore, we analyzed all intergenic regions by hand and with the BPROM software (Softberry, Mt. Kisco, NY); both methods indicated only one possible promoter, which was located in front of spd0571 in all sequenced strains. Further analysis by RT-PCR showed that the three genes, indeed, form an operon (Fig. 4B and C). Thus, deletion of tlpA probably also has a polar effect on msrAB expression, which explains why complementation of the mutant was unsuccessful.

The (predicted) function of the proteins encoded by the genes in the operon and their similarity to parts of the N. meningitidis PilB and DsbD protein that act together to combat oxidative stress (7) strongly suggested that all three proteins are involved in resistance against H₂O₂. Thus, we deleted the whole operon (spd0571-0573) from the genome of D39 nisRK (D39 Δspd0571-0573 nisRK) and in parallel cloned this region into pNZ8048 (pNZ8048::spd0571-0573) and introduced it into the D39Δspd0571-0573 nisRK strain. Deletion of the operon resulted in a similar sensitivity to oxidative stress as the tlpA single mutants (Fig. 3A). Furthermore, induction of expression of the spd0571-0573 operon resulted in restoration of the resistance to H₂O₂ to wild-type levels in the operon mutant (Fig. 3A). Additionally, overexpression of the three genes in the wild type increased the survival of hydrogen peroxide stress (Fig. 3B). To further confirm that all three genes are necessary for hydrogen peroxide survival, we used unique restriction sites in the genes to subclone the plasmid and generate three derivatives: two lacking either spd0571 or spd0573 and one containing only spd0573. Western blot analysis of these plasmids in L. lactis using an anti-TlpA antiserum that we generated confirmed that TlpA was produced, showing that no mutations interfering with the reading frame had occurred during the procedure. The production of TlpA from these plasmids was also significantly higher than in the wild type (data not shown). Neither pNZ8048::spd0571-0572, pNZ8048::spd0572-0573, nor pNZ8048::spd0573 was able to restore survival of the operon mutant to wild-type levels upon induction (Fig. 3C). These experiments show that for the protection against oxidative stress, it is not sufficient to (i) express only msrAB, (ii) express tlpA in combination with msrAB, or (iii) express tlpA in combination with spd0571. Only expression of all three genes is able to protect S. pneumoniae against hydrogen peroxide. Therefore, we concluded that all three genes are necessary for resistance to oxidative stress in S. pneumoniae.

To determine whether spd0571-0573 are sufficient to protect against extracellular oxidative stress, the genes were heterologously expressed using the nisin-induced controlled expression (NICE) system in the related lactic acid bacterium L. lactis NZ9000. This strain is highly sensitive to oxidative stress (Fig. 5); however, induction of expression of the operon in the mid-log growth phase resulted in a dramatic, 1,000-fold higher survival of exposure to 20 mM H₂O₂ which was solely dependent on the presence of the spd0571-0573 genes on the plasmid (Fig. 5). Even in the absence of the inducer nisin, which results in low expression in L. lactis due to the leakiness of the nisA promoter (11), survival was still increased 10-fold, strongly suggesting that the level of resistance is correlated with the amounts of protein produced (Fig. 5). Furthermore, the derivatives of the plasmid containing only two genes were also unable to increase survival (data not shown). Thus, the operon is necessary and sufficient to protect bacteria against extracellular oxidative stress.

As spd0571-0573 seem specifically involved in the resistance to external oxidative stress and as TlpA is reported to be a lipoprotein (50), we wondered whether the protein is present only in the membrane or whether it is also released into the environment. To detect the presence of the protein, polyclonal antibodies were generated against the purified protein. To this end, the tlpA gene without signal sequence and including a C-terminal His₆ tag was cloned and overexpressed in E. coli. The resulting purified protein was used to generate polyclonal antibodies. Western blot analysis of D39 nisRK and its isogenic tlpA mutant showed the presence of specific anti-TlpA antibodies in addition to a nonspecific band around 7 kDa (Fig. 6A). Investigation of various fractions of S. pneumoniae with this antibody showed that the protein was exclusively present in the membrane (Fig. 6B). To control for accurate separation of the various fractions, cross-reacting antibodies against S. aureus TrxA (37) and antibodies against S. pneumoniae MgtA were used as controls for cytoplasmic and membrane proteins, respectively (Fig. 6B) (38). Thus, TlpA is a lipoprotein exclusively located in the pneumococcal membrane.

Overexpression of the operon in D39 and L. lactis increased the survival of oxidative stress. Thus, we hypothesized that the observed differences in oxidative stress survival between strains might be due to a difference in the amount and/or induction levels of TlpA. The same antiserum was used to compare protein levels under oxidative stress using the linear range of the Odyssey Infrared Imaging System, which can be used to quantify protein levels (64). Samples were equalized based on CFU numbers determined after the oxidative stress procedure and before being loaded on the gel. In addition, the nonspecific signal in the antiserum was also used to control that similar amounts of protein were used and to determine whether observed effects were specific for TlpA. Under normal growth conditions, there was no difference in TlpA levels between D39 and G54 (Fig. 6C). Next, we determined whether extracellular oxidative stress increased the protein levels of TlpA. In strain G54, TlpA levels were increased 2-fold under oxidative stress conditions, whereas there was no increase in strain D39 (Fig. 6C). Furthermore, in the presence of H₂O_2, the levels of TlpA were ∼1.5-fold higher in strain G54 than in D39 (Fig. 6D). Thus, extracellular oxidative stress increased the amount of TlpA in G54 but not in D39, which might explain the observed differences in oxidative stress survival in these two strains (Fig. 2).

To investigate the role of these genes in pneumococcal infection at various stages of disease over time, the virulence of a TIGR4 operon mutant was assessed in a competitive index pneumonia model starting from nasopharyngeal colonization. To ensure that the results were reproducible, the experiment was repeated, and the results were combined. When strains were grown individually under nonstressed cultures, no differences were observed in growth rate and yield over 24 h in vitro (data not shown). Furthermore, during in vitro competitive growth the mutant was not outcompeted by the wild type over 24 h, indicating that the function of the operon is not essential and that deletion has no negative influence on fitness under nonstressed conditions. Interestingly, the mutant was initially able to establish infection in the nasopharynx and the lungs and to progress from the lungs to the blood. However, by day 3 the mutant was severely outcompeted in the nasopharynx (P = 0.008), with two of the four surviving mice clearing the mutant by day 5 (Fig. 7A). Strikingly, after day 2 the mutant was also outcompeted in the lungs (P < 0.01) and blood (P = 0.01) (Fig. 7B and C). Thus, these in vivo experiments showed that these genes play an important role in the later stages of disease. It is of note that mice that died had high-grade bacteremia with symptoms of bacterial sepsis.

In order to validate the above results, a second set of competition index experiments was performed using mice infected intranasally with TIGR4 Δsp0658-0660 nisRK complemented with pNZ8048E or pNZ8048::spd0571-0573. In vitro experiments confirmed that the expression of the operon also restored survival to wild-type levels in a TIGR4 Δsp0658-0660 nisRK background (Fig. 3D). These mice received nisin in their drinking water to drive gene expression from the nisA promoter of the pNZ8048 plasmid; the strains contain the nisRK genes instead of the bgaA locus (26) and have the additional burden of plasmid maintenance; thus, the results from these experiments are not directly comparable to those with the wild type and the isogenic mutant. Pneumococci complemented with spd0571-0573 outcompeted those harboring the empty vector in the nasopharynx at all days tested (Fig. 8A). In the blood of these same mice the complemented mutant was present in greater numbers from day 3 onward (Fig. 8B). Thus, complementation studies confirmed that the operon was required for full virulence. Consequently, these in vivo experiments showed that this operon plays an important role in the later stages of disease, presumably because of the ROS released by cells of the immune system recruited to the site of infection at these time points.