Wall teichoic acid structure governs horizontal gene transfer between major bacterial pathogens

Abstract

Mobile genetic elements (MGEs) encoding virulence and resistance genes are widespread in bacterial pathogens, but it has remained unclear how they occasionally jump to new host species. Staphylococcus aureus clones exchange MGEs such as S. aureus pathogenicity islands (SaPIs) with high frequency via helper phages. Here we report that the S. aureus ST395 lineage is refractory to horizontal gene transfer (HGT) with typical S. aureus but exchanges SaPIs with other species and genera including Staphylococcus epidermidis and Listeria monocytogenes. ST395 produces an unusual wall teichoic acid (WTA) resembling that of its HGT partner species. Notably, distantly related bacterial species and genera undergo efficient HGT with typical S. aureus upon ectopic expression of S. aureus WTA. Combined with genomic analyses, these results indicate that a 'glycocode' of WTA structures and WTA-binding helper phages permits HGT even across long phylogenetic distances thereby shaping the evolution of Gram-positive pathogens.

Figure 1. ST395 is resistant to Φ11 and Φ80α-mediated HGT of SaPIs.

(a) Various S. aureus sequence types and ST395 mutants lacking restriction modifications systems SauUSI or SauUSI plus HsdR were analysed for capacities to acquire SaPIbov1 or SaPI1 via helper phages Φ11 or Φ80α, respectively. SaPI donor strains were JP1794 (SaPIbov1) and JP3602 (SaPI1). Values represent the ratio of transduction units (TRU; transductants per ml phage lysate) to plaque-forming units (PFU; plaques per ml phage lysate on S. aureus RN4220 w.t.) given as means (n=3)±s.d. No TRU were observed in controls lacking phages or SaPI particles. (b) S. aureus PS187 w.t. and mutants lacking restriction modification systems were analysed for capacities to acquire the tetracycline resistance plasmid pT181 via electroporation. Values represent the CFU per microgram DNA and given as means (n=3)±s.d. CFU, colony forming units. No CFU were observed in controls lacking donor DNA. ΔhsdR, no type I restriction modification system. ΔsauUSI, no type IV restriction modification system, lacks SAOUHSC_02790 homologue of NCTC8325. Statistically significant differences compared with wild type (w.t.) calculated by the unpaired two-tailed Student’s t-test are indicated: NS, not significant, P>0.05; *P<0.01 to<0.05; **P<0.001 to 0.01; ***P<0.001.

Figure 2. Φ187 mediates HGT of SaPIs among ST395 and to other species and genera.

S. aureus ST395 strains with or without WTA or other Gram-positive bacteria were analysed for capacities to acquire SaPIbov1 or SaPI187β via helper phage Φ187. SaPI donor strains were VW1 (SaPIbov1) and VW7 (SaPI187β). Values represent the ratio of transduction units (TRU; transductants per ml phage lysate) to plaque-forming units (PFU; plaques per ml phage lysate on S. aureus PS187 w.t.) given as means (n=3)±s.d. No TRU were observed in controls lacking phages or SaPI particles. ΔtagO, no WTA. c-tagO, ΔtagO complemented with tagO. ND, not determined due to marker resistance or spontaneous occurring clones.

Figure 3. ST395 branches deeply from other S. aureus lineages and bears a novel WTA type and WTA gene cluster.

(a) Phylogenetic relationships of S. aureus sequence types based on DNA sequences from 1,147 orthologous genes. The genome of S. epidermidis RP62A was used for rooting the tree. Asterisks indicate 100% branch support in both, the maximum-likelihood tree and the Bayesian maximum clade credibility tree. (b) ST395 bears a novel WTA-biosynthetic gene cluster. Genetic organization of the RboP-GlcNAc WTA-biosynthetic tar cluster found in all S. aureus genomes (upper cluster) that is replaced by a new gene cluster containing putative WTA-biosynthetic genes (green) in ST395 strain PS187 (lower cluster). Gene locus numbers are indicated. Protein sequence alignments of the characteristic ST395 WTA proteins with homologues from CoNS can be found in Supplementary Fig. S6. (c) The chemical structure of the WTA repeating unit of S. aureus strain PS187 WTA (GroP-α-D-GalpNAc).

Figure 4. Characteristic MGEs of S. aureus PS187.

(a) Genetic organization of the two genomic islands found in PS187. The organization of PS187 vSAα is similar to that of other S. aureus genomes. The vSAβ of PS187 lacks typical enterotoxin or lantibiotic clusters found in all other vSAβ types sequenced so far. (b) Comparison of SaPI187α from PS187 and SaPI3 from typical S. aureus genomes. SaPI187α shares the att site with SaPI1, SaPI3 and SaPI5 and was found to be similarly organized as SaPI3 from S. aureus Col. Note the presence of a novel enterotoxin in SaPI187α designated as enterotoxin SeW. (c) Comparison of SaPI187β from PS187 and SaPI122 from S. aureus RF122. Although SaPI187β shares the att site with SaPI122 the SaPI122-characteristic gene encoding a multidrug resistance protein is absent in SaPI187β. Instead SaPI187β encodes a new serine protease. (d) Comparative map of mercury and cadmium resistance operons on a large plasmid from PS187 and corresponding genes from the S. epidermidis ATCC12228 SCC composite island (right) and the S. epidermidis RP62A integrated plasmid vSE1 (left). Dotted lines indicate sequence identities above >92% (DNA level) and 93% (protein level). Gene locus numbers are listed and ORFs are coloured according to functional categories as indicated.

Figure 5. WTA structure determines the capacity of SaPIs to traverse even long phylogenetic distances.

S. aureus RN4220 (ST8) strains with, with altered or without WTA (a) or S. aureus PS187 (ST395) or other Gram-positive bacterial species expressing genes for biosynthesis of RboP-GlcNAc WTA (b) were analysed for capacities to acquire SaPIbov1 or SaPI1 via helper phages Φ11 or Φ80α, respectively. SaPI donor strains were JP1794 (SaPIbov1) and JP3602 (SaPI1). Values represent the ratio of transduction units (TRU; transductants per ml phage lysate) to plaque-forming units (PFU; plaques per ml phage lysate on S. aureus RN4220 w.t.) given as means (n=3)±s.d. No TRU were observed in strains expressing WTA other than RboP-GlcNAc. ΔtagO, no WTA; ΔltaS (4S5), no lipoteichoic acid; c-tagO, ΔtagO complemented with tagO; ΔtarM ΔtarS, no WTA glycosylation; c-tarM and c-tarS, ΔtarM ΔtarS complemented either with tarM or tarS. WTA hybrid strains expressing additional RboP-GlcNAc WTA are indicated with H.