Wall Teichoic Acid Glycosylation Governs Staphylococcus aureus Nasal Colonization

Abstract

Nasal colonization by the human pathogen Staphylococcus aureus is a major risk factor for hospital- and community-acquired infections. A key factor required for nasal colonization is a cell surface-exposed zwitterionic glycopolymer, termed wall teichoic acid (WTA). However, the precise mechanisms that govern WTA-mediated nasal colonization have remained elusive. Here, we report that WTA GlcNAcylation is a pivotal requirement for WTA-dependent attachment of community-acquired methicillin-resistant S. aureus (MRSA) and emerging livestock-associated MRSA to human nasal epithelial cells, even under conditions simulating the nutrient composition and dynamic flow of nasal secretions. Depending on the S. aureus strain, WTA O-GlcNAcylation occurs in either α or β configuration, which have similar capacities to mediate attachment to human nasal epithelial cells, suggesting that many S. aureus strains maintain redundant pathways to ensure appropriate WTA glycosylation. Strikingly, a lack of WTA glycosylation significantly abrogated the ability of MRSA to colonize cotton rat nares in vivo. These results indicate that WTA glycosylation modulates S. aureus nasal colonization and may help to develop new strategies for eradicating S. aureus nasal colonization in the future.

Importance: Nasal colonization by the major human pathogen Staphylococcus aureus is a risk factor for severe endogenous infections and contributes to the spread of this microbe in hospitals and the community. Here, we show that wall teichoic acid (WTA) O-GlcNAcylation is a key factor required for S. aureus nasal colonization. These data provide a mechanistic explanation for the capacity of WTA to modulate S. aureus nasal colonization and may stimulate research activities to establish valuable strategies to eradicate S. aureus nasal colonization in high-risk hospitalized patients and in the general community.

FIG 1

Impact of nasal growth conditions on WTA composition and WTA glycosylation. (A and B) S. aureus susceptibility to the WTA GlcNAc-dependent bacteriophage Φ11 (A) along with Φ11 adsorption levels (B). (C) qRT-PCR analysis of WTA glycosyltransferase gene expression levels in S. aureus USA300. mRNA was isolated from lag-, log-, or stationary (stat.)-phase-grown bacteria. For bacteriophage susceptibility and adsorption analysis or analysis of gene expression levels, strains were grown under rich conditions (B-medium [BM]; white columns) or nasal growth conditions (SNM3 [containing 0.2 mM bipyridine]; black columns). Values are given as means and standard deviations (SD; n = 3 to 6). Statistically significant differences calculated using an unpaired two-tailed Student’s t test are indicated as follows: ns (not significant), P > 0.05; *, P < 0.05, **, P < 0.01; ***, P < 0.001.

FIG 2

WTA glycosylation affects interaction of MRSA with epithelial cells. Data represent binding capacities of S. aureus variants to A549 lung epithelial cells (MOI30) (A), cotton rat nasal epithelial primary cells (CRNEC) (MOI20) (B), or human nasal epithelial primary cells (HNEpC) (MOI30) (C) under static conditions. The binding capacities of various strains encompassing the CA-MRSA USA300 strain and a LA-MRSA CC398 strain (82086) to confluent epithelial cell monolayers were analyzed. The S. aureus wild-type (w.t.) strain and strains lacking WTA (ΔtagO) or WTA glycosylation (ΔtarM ΔtarS [ΔMΔS] or ΔtarS [ΔS], LA-MRSA CC398 strain 82086) and the complemented mutants (ΔMΔS c-tarM, ΔMΔS c-tarS, or ΔS c-tarS, LA-MRSA CC398 strain 82086) are indicated. Values are given as means and standard deviations (SD; n = 3 to 4). Statistically significant differences calculated using one-way analysis of variance (ANOVA) with Bonferroni’s multiple-comparison test are indicated as follows: ns (not significant), P > 0.05; *, P < 0.05, **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

WTA glycosylation mediates adherence to primary epithelial cells under dynamic flow conditions. (A) Principle of flow-chamber-based attachment assays mimicking dynamic nasal conditions. (B and C) The binding capacities of S. aureus variants to monolayers of confluent cotton rat nasal epithelial primary cells (CRNEC) (MOI60) (B) or human nasal epithelial primary cells (HNEpC) (MOI60) (C) under conditions of mild shear stress were analyzed. The S. aureus USA300 wild-type (w.t.) strain, strains lacking WTA (ΔtagO) or WTA glycosylation (ΔtarM ΔtarS [ΔMΔS]), and the complemented mutants (ΔMΔS c-tarM and ΔMΔS c-tarS) are indicated. Values are given as means and standard deviations (SD; n = 3 to 4). Statistically significant differences calculated using one-way ANOVA with Bonferroni’s multiple-comparison test are indicated as follows: ns (not significant), P > 0.05; *, P < 0.05, **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 4

WTA glycosylation affects S. aureus nasal colonization in vivo. Cotton rats were intranasally challenged with the S. aureus USA300 wild-type (w.t.) strain and the USA300 ΔtarM ΔtarS (ΔMΔS) mutant lacking WTA glycosylation. Colonization status was analyzed 3 days after bacterial instillation. Statistically significant differences calculated using a D’Agostino and Pearson omnibus normality test and a subsequent Mann-Whitney test are indicated as follows: *, P < 0.05.