Role of Staphylococcus aureus catalase in niche competition against Streptococcus pneumoniae (original) (raw)

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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 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% CO 2 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 g ampicillin/ml, 50 g erythromycin/ml, and 100 g spectinomycin/ml. Generation of catalase-deficient S. aureus ⌬KatA 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 (5Ј-ATGGTCGACTATGACATCAACACTTGTAAC-3Ј) and katAupR (5Ј-TCA AATATATCCTCCTCATCCCTCCACAATTTATAATAAT-3Ј) along with 500 bp of sequence immediately downstream of katA with primers katAdownF (5Ј-AA TAACAGATTAAAAAAATTATAAATTTGATATGTAGTTTCTATA-3Ј) and katAdownR (5Ј-ATCGGATCCTACCCAGAATTACTTCGTACT-3Ј). The katAupR and katAdownF primers were constructed with 25-bp 5Ј extensions corresponding to the 5Ј and 3Ј 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-␤-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 ⌬KatA mutant. Complementation studies. Primers katAF_KpnI (5Ј-ATAGGTACCTCCCAT GGTAAAGCCAAGAG-3Ј) and katAR_BamHI (5Ј-ATAGGATCCTTTACGC GCACGTTAAACAC-3Ј) 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 ⌬KatA mutant by electroporation. For the complementation studies, the isogenic WT and ⌬KatA 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. H 2 O 2 susceptibility assay. H 2 O 2 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 concentra