Evolutionary blueprint for host- and niche-adaptation in Staphylococcus aureus clonal complex CC30

Abstract

Staphylococcus aureus clonal complex CC30 has caused infectious epidemics for more than 60 years, and, therefore, provides a model system to evaluate how evolution has influenced the disease potential of closely related strains. In previous multiple genome comparisons, phylogenetic analyses established three major branches that evolved from a common ancestor. Clade 1, comprised of historic pandemic phage type 80/81 methicillin susceptible S. aureus (MSSA), and Clade 2 comprised of contemporary community acquired methicillin resistant S. aureus (CA-MRSA) were hyper-virulent in murine infection models. Conversely, Clade 3 strains comprised of contemporary hospital associated MRSA (HA-MRSA) and clinical MSSA exhibited attenuated virulence, due to common single nucleotide polymorphisms (SNP's) that abrogate production of α-hemolysin Hla, and interfere with signaling of the accessory gene regulator agr. We have now completed additional in silico genome comparisons of 15 additional CC30 genomes in the public domain, to assess the hypothesis that Clade 3 has evolved to favor niche adaptation. In addition to SNP's that influence agr and hla, other common traits of Clade 3 include tryptophan auxotrophy due to a di-nucleotide deletion within trpD, a premature stop codon within isdH encoding an immunogenic cell surface protein involved in iron acquisition, loss of a genomic toxin-antitoxin (TA) addiction module, acquisition of S. aureus pathogenicity islands SaPI4, and SaPI2 encoding toxic shock syndrome toxin tst, and increased copy number of insertion sequence ISSau2, which appears to target transcription terminators. Compared to other Clade 3 MSSA, S. aureus MN8, which is associated with Staphylococcal toxic shock syndrome, exhibited a unique ISSau2 insertion, and enhanced production of toxic shock syndrome toxin encoded by SaPI2. Cumulatively, our data support the notion that Clade 3 strains are following an evolutionary blueprint toward niche-adaptation.

Keywords: Staphylococcus aureus; evolution; insertion sequence; pathoadaptation; pathogenicity island; pseudogene; toxin-antitoxin addiction module; virulence.

Figure 1

SaPI and ISSau2 distribution in CC30. The location of each element is mapped on the reference genome of MRSA 252 [Holden et al. (2004)]. SaPIs are indicated by colored bars, on the circular blue genome, and are labeled on the interior of the circular genome. SaPI2 and SaPI4 (yellow) are unique to Clade 3. SaPI1 (light green) is present in PT80/81 (Clade 1) and ST30spa19 CA-MRSA (Clade 2). SaPI3 and SaPIbov (red) are not in CC30, but their location is shown for reference, based on location of attS sites. ISSau2 insertions, indicated by colored lollipops above the circular genome, are named after the gene that is adjacent to each insertion. Three different 16S-23S-5S-rRNA loci are abbreviated as 5S-rRNA-1, 5S-rRNA-2, and 5S-rRNA-3.

Figure 2

SaPI structures and insertion sites. (A) SaPI4, with an illustration of the rpsF-ssb-rpsR operon, and nucleotide sequence extending from the 3′-end of rpsR. The attS of SaPI4 shaded gray, overlaps the 3′-end of the rpsR open reading frame, and letters above the sequence correspond to the C-terminus of RpsR protein. Arrows above the nucleotide sequence indicate inverted repeats, likely comprising a transcriptional terminator. Illustrations below the sequence compare SaPI4 of S. aureus MRSA 252 with that of ovine adapted strain 011. The duplicated left and right attS sequences are shaded gray, int genes are colored orange, while SAR0385 and orthologous genes, encoding a putative secreted protein, are magenta. A central outlined and shaded segment of SaPI4 is highly conserved in S. aureus phage ϕ1028, and SaPIbov of bovine adapted S. aureus ET3-1. Genes in SaPIbov encoding superantigen toxins are shaded red. The int of SaPIbov is shaded orange to emphasize function, although each SaPI family has a distinct int and attS. Structures of SaPI2 (B), SaPI3 (C), SaPI1 (D), and their genomic insertion sites. Features are labeled as in A. SaPI2 of ST30spa33 strain MN8 (B), is compared to SaPI2 of a non-related MRSA strain N315. SaPI3 (C) is not present in CC30. Structures shown are from strain USA400 [Baba et al. (2002)], which is a CA-MRSA, and strain 68111, which is a triple locus MLST variant of ST30 [Li et al. (2011)]. A clonal complex is comprised of isolates that differ from the ancestral sequence type (ST) at no more than two of seven MLST alleles. Therefore, S. aureus 68111 is distantly related to ST30, but is not within CC30. (D) shows SaPI1 structures from different strains, in comparison to SaPI1 in Clade 1 PT80/81 strains, and Clade 2 ST30spa19 CA-MRSA.

Figure 3

Illustration of ISSau2 (A) and its insertion sites (B–G) in CC30. ISSau2 is comprised of orfA and orfB (A), flanked by 39 nt inverted repeats IR-L and IR-R, with terminal 5′-TG and 3′-CA dinucleotides (A). The sequence above the illustration spans the 3′-end of orfA and 5′-end of orfB. The +1 translation of orfA, terminating at a stop codon, is shown below the sequence. Above the sequence is a translation that would result from a −1 frame-shift within AAAAAAG. The −1 translation from this point onwards continues throug to the end of orfB, and would produce a single 1569 nt trans-frame protein. (B) Illustration of oppAFBDC and trpS genome segment of non-CC30 strain USA300. Beneath this is shown the oppC-trpS intergenic sequence of USA300, aligned to that MRSA 252. Asterisks above the USA300 sequence indicate stop codons of oppC and trpS. Convergent arrows indicate inverted repeats, likely comprising a rho-independent transcription terminator stem-loop structure. In all CC30 strains, the left arm of the stem is disrupted by a flanking repeat of IS1272 (shaded gray), which in turn is disrupted by ISSau2. In MRSA 252, oppB (cross-hatched) has an in-frame internal deletion that is unique to the EMRSA-16 lineage. (C) ISSau2 insertion in the genome of S. aureus TCH60 (top), which is ST30spa19 CA-MRSA (Clade 2), and corresponding segment of MRSA 252 (bottom). PCR with primers spanning the 3′-end of the 5S rRNA and flanking ilvA (right panel) reveal that this insertion is in Clade 1 strains (M809, M1015) and another Clade 2 CA-MRSA (WBG10049), but not other CC30, including strains that pre-date the Clade 1 pandemic (ATCC12598, NRS204, ATCC25923). (D) ISSau2 insertion adjacent to a 16S-23S-5S rRNA operon, and flanking okrD gene, which is unique to Clade 3 HA-MRSA. The sequence below the illustration shows the end of the 5S rRNA transcript, and adjacent rho-independent transcriptional terminator, comprised of tandem stem-loops followed by a poly-T segment. ISSau2 disrupts the right arm of the first stem-loop, with duplication of the CAT target site. (E and F) show similar disruption of a putative stem-loop structure downstream of rplQ, and a likely transcription terminator of the sbcDC operon. (G) Unique ISSau2 insertion in ST30spa33 strain MN8 disrupts stem-loop structure adjacent to saeRS regulator. The saeRS genome segment of MRSA 252 is shown for reference, and the nucleotide sequence downstream of saeS is shown above the illustration. In strain MN8, ISSau2 inserts into the left arm of a putative stem-loop structure, with duplication of the CTC target site. This is confirmed by PCR of a genomic segment spanning saeS and adjacent SAR0757, producing a 2.8 kb amplicon in MN8 (Lane 6), and a 1.2 kb product in all other strains including PT80/81 strain M1015 (Lane 1), ST30spa19 CA-MRSA strain WBG10049 (Lane 2), ST36spa16 HA-MRSA strains PM7 and PM64 (Lanes 3 and 4), and additional ST30spa33 strains UAMS-1 (Lane 5), L516 (Lane 7), and L528 (Lane 8).

Figure 4

(A) Illustration of yefM-yoeB and flanking DNA in CC30. yefM-yoeB are flanked by repeats LDR-1 and LDR-2 (black bars) in PT80/81 and ST30spa19 CA-MRSA, whereas HA-MRSA and contemporary MSSA have only one LDR and lack yefM-yoeB. (B) Nucleotide sequence of LDR-1 and LDR-2 from strain M1015, and the single LDR of MRSA 252. The lower-case gray shaded nucleotides differentiate LDR-1 and LDR-2. The single LDR of MRSA 252 is a hybrid of LDR-1 and LDR-2, which is suggestive of a recombination event. (C) Growth of S. aureus RN4220 harboring P_cad::yefM-yoeB or P_cad::yoeB, on BHI agar, or BHI supplemented with 5 μM cadmium, to induce P_cad.

Figure 5

Assessment of CC30 secreted proteins by SDS-PAGE (A) and Western blot for detection of Hla (B). The individual strains are the same as defined in Figure 3G. For visualization of secreted proteins by Coomassie Blue staining (A), a total of 3.0 OD₆₀₀ units of cell-free culture supernatant was applied to each lane, while 0.02 OD units was applied in Figure 3B. Zones I, II, and III as outlined in the SDS-PAGE gel (A) are enlarged in Figure 3C. The numbered protein bands were excised from the gel, followed by trypsin digestion and mass spectrometry. The identity of proteins in each band is provided in Table 3.