Role of Staphylococcus aureus Catalase in Niche Competition against Streptococcus pneumoniae

Abstract

Nasal colonization by Staphylococcus aureus is a major predisposing factor for subsequent infection. Recent reports of increased S. aureus colonization among children receiving pneumococcal vaccine implicate Streptococcus pneumoniae as an important competitor for the same niche. Since S. pneumoniae uses H₂O₂ to kill competing bacteria, we hypothesized that oxidant defense could play a significant role in promoting S. aureus colonization of the nasal mucosa. Using targeted mutagenesis, we showed that S. aureus expression of catalase contributes significantly to the survival of this pathogen in the presence of S. pneumoniae both in vitro and in a murine model of nasal cocolonization.

Staphylococcus aureus causes a wide range of infections ranging from minor skin infections to life-threatening invasive diseases. The emergence of methicillin-resistant strains with high virulence potential in both hospital and community settings is contributing to a current public health crisis (9, 12, 13).

A major risk factor for S. aureus infection is antecedent colonization of the nasal mucosa (19). Successful colonization depends not only on the ability of S. aureus to survive host factors (4, 6) but also on coexistence with other bacteria (16, 21).

The latter concept has been underscored by two recent reports that implicate Streptococcus pneumoniae as a primary competitor for niche colonization (3, 15).

Specifically, one surveillance study performed in an area where pneumococcal vaccination was not practiced showed that the S. pneumoniae carriage rate in children was negatively associated with S. aureus nasal carriage (15). The other study showed that children with recurrent otitis media vaccinated with the 7-valent pneumococcal vaccine had an increased incidence of S. aureus-related acute otitis media and S. aureus colonization after vaccination (3), suggesting that there is a natural competition for colonization between S. aureus and S. pneumoniae.

S. pneumoniae produces H₂O₂ as an antimicrobial factor to reduce competition by other upper respiratory pathogens, such as Haemophilus influenzae, Neisseria meningitides, Moraxella catarrhalis, and S. aureus (14, 16). Since S. aureus is a natural colonizer of the human nares, we hypothesized that its success derives in part from a relative resistance to H₂O₂ killing by other microflora. Here we tested this hypothesis by generating a catalase knockout mutant strain of S. aureus and examining the role of enzymatic H₂O₂ inactivation in niche competition with S. pneumoniae.

MATERIALS AND METHODS

Bacterial strains, media, and mice.

S. aureus strains were cultured at 37°C in Todd-Hewitt broth (THB) or on Todd-Hewitt agar (THA) (Difco). S. pneumoniae TIGR4 was cultured in THB with 0.5% yeast extract (THY) at 37°C in a 5% CO₂ incubator. Eight- to 10-week old female CD1 mice were purchased from Charles River Laboratories, Wilmington, MA. When included, antibiotics were added at the following concentrations: 100 μg ampicillin/ml, 50 μg erythromycin/ml, and 100 μg spectinomycin/ml.

Generation of catalase-deficient S. aureus ΔKatA mutant.

In-frame allelic replacement of the S. aureus katA gene with a spectinomycin adenyltransferase (spec) cassette was performed using PCR-based methods as described previously (11), with minor modifications. Primers were designed based on the previously published S. aureus katA sequence cross-referenced to the genome of S. aureus strain N315 (10). PCR was used to amplify 500 bp upstream of katA with primers katAupF (5′-ATGGTCGACTATGACATCAACACTTGTAAC-3′) and katAupR (5′-TCAAATATATCCTCCTCATCCCTCCACAATTTATAATAAT-3′) along with 500 bp of sequence immediately downstream of katA with primers katAdownF (5′-AATAACAGATTAAAAAAATTATAAATTTGATATGTAGTTTCTATA-3′) and katAdownR (5′-ATCGGATCCTACCCAGAATTACTTCGTACT-3′). The katAupR and katAdownF primers were constructed with 25-bp 5′ extensions corresponding to the 5′ and 3′ ends of the spec gene, respectively. The upstream and downstream PCR products were then combined with a 650-bp amplicon of the complete spec gene for use as templates in a second round of PCR using primers katAupF and katAdownR. The resultant PCR amplicon, containing an in-frame substitution of katA with spec, was subcloned into temperature-sensitive vector pMAD (1) to create the knockout plasmid. This vector was transformed initially into permissive S. aureus strain RN4220 and then into S. aureus strain Newman by electroporation. Transformants were grown at 30°C and shifted to the nonpermissive temperature for plasmid replication (40°C), and differential antibiotic selection and blue-white color selection with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) were used to identify candidate mutants. Allelic replacement of the katA allele was confirmed unambiguously by PCRs that documented targeted insertion of spec and the absence of katA in chromosomal DNA isolated from the final mutant, which was designated the ΔKatA mutant.

Complementation studies.

Primers katAF_KpnI (5′-ATAGGTACCTCCCATGGTAAAGCCAAGAG-3′) and katAR_BamHI (5′-ATAGGATCCTTTACGCGCACGTTAAACAC-3′) were used to amplify the katA gene from the chromosome of wild-type (WT) S. aureus strain Newman. The fragment was directionally cloned into the shuttle expression vector pDCerm (8), and the recombinant plasmid (pKatA) was used to transform the S. aureus ΔKatA mutant by electroporation. For the complementation studies, the isogenic WT and ΔKatA S. aureus strains were transformed with the control pDCerm plasmid. Strains containing the pDCerm or pKatA plasmid were maintained in THB or on THA containing erythromycin.

H₂O₂ susceptibility assay.

H₂O₂ susceptibility assays were performed using overnight S. aureus cultures grown at 37°C with shaking. Bacteria were harvested by centrifugation, suspended in phosphate-buffered saline (PBS) at a concentration of 5 × 10⁷ CFU/ml, and mixed with various concentrations of H₂O₂. The killing assay was terminated after 2 h of incubation at 37°C by addition of 5,000 U/ml of catalase (Sigma), which was followed by enumeration of surviving bacterial CFU on THA.

Susceptibility of S. aureus to S. pneumoniae killing in vitro. (i) Plate assay.

Overnight S. aureus cultures were centrifuged, washed, and suspended in PBS at a concentration of 5 × 10⁸ CFU/ml. Two hundred microliters was plated on THY plates, and a paper disk impregnated with 1.5 × 10⁹ log-phase S. pneumoniae cells was placed in the center of each plate. The zone of S. aureus growth inhibition was measured after 24 h of incubation at 37°C in the presence of 5% CO₂.

(ii) Liquid culture-based assay.

Overnight S. aureus cultures were centrifuged, washed in PBS, diluted to obtain a concentration of 1 × 10⁹ CFU/ml, and mixed with log-phase S. pneumoniae at a ratio of 1:1, 1:5, or 1:10 in THY. After 4 h of incubation at 37°C in the presence of 5% CO₂, the remaining H₂O₂ was quenched with 50 μl of a 5,000-U/ml exogenous catalase solution, and the surviving S. aureus cells were diluted in PBS and plated on THA plates. As a control, parallel experiments were performed in an identical fashion in the presence of 1,000 U/ml catalase.

Murine nasal cocolonization studies.

Mice were inoculated intranasally with a 10 μl of a mixture containing 10⁸ WT cells and 10⁸ S. aureus ΔKatA cells. After 30 min, the mice were divided into two groups and given either 10 μl of THY or 3 × 10⁸ early-stationary-phase S. pneumoniae cells in THY. After 3 days, the mice were sacrificed, the nasal tissue was homogenized and vortexed for 5 min in PBS, and the CFU were enumerated on THA with or without spectinomycin after appropriate dilution. Occasional contaminants were excluded during counting of the CFU by the morphology or color of the bacterial colonies. Animal experimentation guidelines were followed in the animal studies.

Statistical analysis.

The significance of experimental differences in H₂O₂ sensitivity and S. pneumoniae killing in vitro was evaluated by using the unpaired Student t test. The results of the mouse in vivo challenge studies were evaluated by using the nonparametric two-tailed Wilcoxon and Mann-Whitney tests.

RESULTS

To address the role of catalase in niche competition, a KatA deletion mutant of S. aureus strain Newman was generated by allelic replacement of the katA gene with a spectinomycin acetyltransferase cassette. Deletion of the katA gene was confirmed by PCR and by the absence of effervescence upon exposure of the ΔKatA mutant to H₂O₂ (data not shown).

To assess the effect of katA deletion on S. aureus susceptibility to H₂O₂, the WT and ΔKatA strains were exposed to a range of H₂O₂ concentrations in PBS. In the absence of catalase, S. aureus was highly susceptible to H₂O₂ killing (Fig. 1A). Complementation with pKatA restored the ability of the ΔKatA mutant to resist H₂O₂ killing (Fig. 1B). The pDCerm vector used for complementation was also placed into the ΔKatA mutant, and it had no impact on H₂O₂ susceptibility.

FIG. 1.

S. aureus catalase confers resistance to H₂O₂ killing. (A) Susceptibility of WT and ΔKatA S. aureus strains to different concentrations of H₂O₂. (B) Restoration of resistance to H₂O₂ killing upon complementation of the ΔKatA mutant with pKatA. All experiments were performed at least three times, and similar results were obtained.

Since S. pneumoniae produces H₂O₂ in quantities sufficient to kill other bacterial species (14), we tested whether catalase has an important survival function for S. aureus when it is cultured in the presence of S. pneumoniae. As shown in Fig. 2A, a disk impregnated with log-phase S. pneumoniae cells partially inhibited growth of the WT S. aureus strain on a THY plate but had a much more profound effect on the growth of the isogenic ΔKatA mutant.

FIG. 2.

Catalase protects S. aureus against S. pneumoniae killing in vitro. (A) Effect of a disk impregnated with S. pneumoniae on growth of the WT or ΔKatA S. aureus strain. (B) Survival of the WT or ΔKatA S. aureus strain upon coculture with S. pneumoniae at ratios of 1:1, 1:5, and 1:10. (C) Restoration of resistance to S. pneumoniae killing upon complementation of the ΔKatA mutant with pKatA. All experiments were repeated at least three times, and similar results were obtained.

In a more quantitative liquid culture-based assay, at a ratio of S. aureus to S. pneumoniae of 1:1, minimal killing of WT or mutant S. aureus was noted (Fig. 2B). However, at a ratio of 1:5 or 1:10, the survival of the ΔKatA mutant in the presence of S. pneumoniae was reduced by as much as 8 logs compared to the survival of the parent strain (Fig. 2B). The differential killing was most likely a result of H₂O₂ production by S. pneumoniae, since no killing of S. aureus was observed if an exogenous source of catalase was added to the culture at the start of the assay (Fig. 2B). Complementation with pKatA restored the ability of the ΔKatA mutant to resist S. pneumoniae killing (Fig. 2C).

Next, to extend the biological relevance of these findings, the role of S. aureus catalase was assessed using a murine model of nasal colonization. In this study, mice were inoculated intranasally with equal numbers of WT and ΔKatA S. aureus cells with or without S. pneumoniae. After 3 days, the surviving WT and ΔKatA cells were harvested from the noses of the mice. As shown in Fig. 3, the survival of the WT strain and the survival of the ΔKatA strain in noses of mice did not differ significantly at day 3 when they were inoculated alone, but a notable difference in the levels of survival in favor of WT S. aureus was apparent in mice given S. pneumoniae as a competitor for the same niche.

FIG. 3.

Catalase protects S. aureus against S. pneumoniae killing in a murine model of nasal colonization. Mice were inoculated intranasally with a 1:1 mixture of the WT and ΔKatA S. aureus strains. After 30 min, the mice were inoculated in the same nostrils with either buffer or S. pneumoniae at a ratio of S. pneumoniae to S. aureus of 3:1. Surviving bacteria from the nostrils were quantitated after 3 days. The graph on the left shows the ratios of the surviving WT S. aureus strain to the surviving the ΔKatA S. aureus mutant for individual mice. The numbers of surviving WT and ΔKatA S. aureus CFU recovered from each mouse are plotted on the right. Mice that were poorly colonized (≤5 WT CFU and ≤5 ΔKatA CFU as enumerated on THA plates) were excluded from the surviving ratio plot (left) but were included in the survival graphs on the right. The data were compiled from three experiments performed in the same way. The minimum detection level of the assay is 20 CFU. S.p, S. pneumoniae.

DISCUSSION

Multiple studies have shown that colonization of the upper airway with S. pneumoniae is negatively correlated with S. aureus colonization, and introduction of the S. pneumoniae vaccine has increased the rate of S. aureus nasal colonization (3, 15). By eradicating carriage of S. pneumoniae vaccine strains, immunization removes an important niche competitor that utilizes H₂O₂ to restrict colonization by other bacteria. In this study, we showed that the S. aureus catalase is a major factor in S. aureus defense against S. pneumoniae killing due to neutralization of secreted H₂O₂. H₂O₂ is used as an antimicrobial factor by many other microbes (2, 18), including Streptococcus sanguinis in the oral cavity and lactobacilli in the vagina, two sites frequently cocolonized by S. aureus. Thus, it could be speculated that in S. aureus catalase is an important tool for securing a niche on multiple mucosal surfaces in the human host. The presence of catalase may also explain the preferential survival of WT S. aureus compared to the ΔKatA mutant in the cotton rat model of nasal colonization previously reported by Cosgrove and coworkers (5).

S. aureus encodes a number antioxidants, including, alkyl hydroperoxide reductase, and staphyloxanthin, which may supplement catalase in defense against H₂O₂-producing organisms, such as S. pneumoniae.

Although catalase is a factor produced by many bacteria, several studies have failed to establish a function for catalase in systemic virulence (7, 17, 20). Our finding that catalase plays an important role in S. aureus in mucosal niche competition points to an alternative role that catalase could play in the most proximal steps of disease pathogenesis. The S. aureus catalase could thus be a novel pharmacologic target for decolonization strategies, a desirable therapeutic endpoint in many clinical scenarios.