An accessory wall teichoic acid glycosyltransferase protects Staphylococcus aureus from the lytic activity of Podoviridae

Abstract

Many Staphylococcus aureus have lost a major genetic barrier against phage infection, termed clustered regularly interspaced palindromic repeats (CRISPR/cas). Hence, S. aureus strains frequently exchange genetic material via phage-mediated horizontal gene transfer events, but, in turn, are vulnerable in particular to lytic phages. Here, a novel strategy of S. aureus is described, which protects S. aureus against the lytic activity of Podoviridae, a unique family of staphylococcal lytic phages with short, non-contractile tails. Unlike most staphylococcal phages, Podoviridae require a precise wall teichoic acid (WTA) glycosylation pattern for infection. Notably, TarM-mediated WTA α-O-GlcNAcylation prevents infection of Podoviridae while TarS-mediated WTA β-O-GlcNAcylation is required for S. aureus susceptibility to podoviruses. Tracking the evolution of TarM revealed an ancient origin in other staphylococci and vertical inheritance during S. aureus evolution. However, certain phylogenetic branches have lost tarM during evolution, which rendered them podovirus-susceptible. Accordingly, lack of tarM correlates with podovirus susceptibility and can be converted into a podovirus-resistant phenotype upon ectopic expression of tarM indicating that a "glyco-switch" of WTA O-GlcNAcylation can prevent the infection by certain staphylococcal phages. Since lytic staphylococcal phages are considered as anti-S. aureus agents, these data may help to establish valuable strategies for treatment of infections.

Figure 1. The α-O-GlcNAc WTA glycosyltransferase TarM protects S. aureus from the lytic activity of Podoviridae.

(a) S. aureus RN4220 and USA300 susceptibility to the broad-host-range lytic phage ΦK (Myoviridae), and to the lytic phages Φ44AHJD, Φ66 and ΦP68 (Podoviridae) was analyzed using a soft-agar overlay approach. A representative experiment is shown. (b) Podovirus ΦP68 adsorption rates (%) to S. aureus RN4220 and USA300 variants. S. aureus wild type and strains lacking WTA (ΔtagO), WTA α-O-GlcNAcylation (ΔtarM), WTA β-O-GlcNAcylation (ΔtarS), WTA glycosylation (ΔtarM ΔtarS), and the complemented mutants (ΔtarM ΔtarS pRB474-tarM, ΔtarM ΔtarS pRB474-tarS) are indicated. Values are given as means and standard deviations (SD, n = 3). Statistical significant differences calculated by one-way ANOVA with Bonferroni’s multiple comparison test are indicated: not significant (ns), P > 0.05; *P < 0.05, **P < 0.01.

Figure 2. Point mutations in TarM render Φ66 propagation strain PS66 susceptible to Podoviridae.

(a) A sequence alignment of wild-type TarM and PS66 TarM is shown. Position of mutations (Gln-453 with Lys; Ala-464 with Glu) and the end of the open reading frame (493) are indicated. (b) S. aureus RN4220 susceptibility to the broad host-range lytic phage ΦK (Myoviridae), and to the lytic phages Φ44AHJD, Φ66, and ΦP68 (Podoviridae) was analyzed using a soft-agar overlay approach. S. aureus RN4220 wild type and strains lacking WTA α-O-GlcNAcylation (ΔtarM), and the complemented mutants (ΔtarM pRB474-tarM, ΔtarM pRB474-tarM (Q453K; A464E) are indicated. A representative experiment is shown.

Figure 3. Ectopic expression of TarM protects podovirus-susceptible S. aureus against Podoviridae.

The α-O-GlcNAc WTA glycosyltransferase TarM was ectopically expressed in various tarM-lacking and podovirus-susceptible S. aureus strains, and the phage susceptibility using a phage panel encompassing the lytic phages Φ44AHJD, Φ66 and ΦP68 (Podoviridae) was analyzed using a soft-agar overlay approach. Various dilutions of phage lysates, S. aureus wild type strains (tarS positive, but tarM negative (or encoding a mutated tarM, strain PS66)), and engineered strains expressing tarM (pRB474-tarM), or empty plasmid control (pRB474) are indicated. A representative experiment is shown.

Figure 4. Phylogenetic distribution of tarM reveals an ancient origin in other staphylococci and vertical inheritance during S. aureus evolution.

(a,b) Phylogenetic network representing the inferred relationship of 98 S. aureus strains and two closely related species, S. argenteus and S. schweitzeri. Strains are indicated by their multilocus sequence types (STs). ST* and ST** are single-locus variants of ST30 and ST1148, respectively. Strains encoding tarM are indicated in black, while strains lacking tarM are indicated in red, purple, and blue. (c) Genetic organization of the tarM region in S. aureus. The intact tarM region is shown in the upper cluster. Gene locus numbers refer to S. aureus strain COL (GenBank accession no. CP000046). Lower clusters indicate distinct deletion events involving tarM.