USA300 and USA500 clonal lineages of Staphylococcus aureus do not produce a capsular polysaccharide due to conserved mutations in the cap5 locus

Abstract

The surface capsular polysaccharide (CP) is a virulence factor that has been used as an antigen in several successful vaccines against bacterial pathogens. A vaccine has not yet been licensed against Staphylococcus aureus, although two multicomponent vaccines that contain CP antigens are in clinical trials. In this study, we evaluated CP production in USA300 methicillin-resistant S. aureus (MRSA) isolates that have become the predominant community-associated MRSA clones in the United States. We found that all 167 USA300 MRSA and 50 USA300 methicillin-susceptible S. aureus (MSSA) isolates were CP negative (CP(-)). Moreover, all 16 USA500 isolates, which have been postulated to be the progenitor lineage of USA300, were also CP(-). Whole-genome sequence analysis of 146 CP(-) USA300 MRSA isolates revealed they all carry a cap5 locus with 4 conserved mutations compared with strain Newman. Genetic complementation experiments revealed that three of these mutations (in the cap5 promoter, cap5D nucleotide 994, and cap5E nucleotide 223) ablated CP production in USA300 and that Cap5E75 Asp, located in the coenzyme-binding domain, is essential for capsule production. All but three USA300 MSSA isolates had the same four cap5 mutations found in USA300 MRSA isolates. Most isolates with a USA500 pulsotype carried three of these four USA300-specific mutations, suggesting the fourth mutation occurred in the USA300 lineage. Phylogenetic analysis of the cap loci of our USA300 isolates as well as publicly available genomes from 41 other sequence types revealed that the USA300-specific cap5 mutations arose sequentially in S. aureus in a common ancestor of USA300 and USA500 isolates.

Importance: The USA300 MRSA clone emerged as a community-associated pathogen in the United States nearly 20 years ago. Since then, it has rapidly disseminated and now causes health care-associated infections. This study shows that the CP-negative (CP(-)) phenotype has persisted among USA300 isolates and is a universal and characteristic trait of this highly successful MRSA lineage. It is important to note that a vaccine consisting solely of CP antigens would not likely demonstrate high efficacy in the U.S. population, where about half of MRSA isolates comprise USA300. Moreover, conversion of a USA300 strain to a CP-positive (CP(+)) phenotype is unlikely in vivo or in vitro since it would require the reversion of 3 mutations. We have also established that USA300 MSSA isolates and USA500 isolates are CP(-) and provide new insight into the evolution of the USA300 and USA500 lineages.

FIG 1

CP immunoblots of USA300 and USA500 clinical isolates. Capsule serotyping was performed by a colony immunoblot method with the use of CP5-specific antibodies as described previously (43). The first row shows the results from the CP5-positive (CP5⁺) control strains Reynolds and Newman, the CP5- and CP8-negative (CP⁻) control strains LAC and 8325, and the CP8⁺ control strains ST80 and MN8. The second and third rows include six representative USA300 and USA500 clinical isolates, respectively.

FIG 2

Pulsed-field gel electrophoresis (PFGE) documenting the SmaI digestion patterns of USA500 isolates relative to the USA500 reference strain NRS385 (USA500ref) and relevant typing characteristics. The USA300 reference strain (USA300ref) has PFGE pattern USA300-0114 and was obtained from the Network for Antimicrobial Resistance in Staphylococcus aureus (NARSA). “Cp5/8” refers to capsule polysaccharide types 5 and 8, “CapE 223” refers to the nucleotide at position 223 in cap5E, and “CapG 478” refers to the nucleotide at position 478 in cap5G.

FIG 3

USA300 cap5 locus showing universal mutations in USA300 relative to the cap5 reference strain Newman. Orange arrows depict open reading frames (ORFs). “P_cap5” refers to the cap5 promoter region. The inverted repeat sequence, as reported by Ouyang et al. (32), is shown as opposing arrows beneath P_cap5 indicating the T→C mutation in strain USA300 relative to strain Newman. The yellow line indicates a disruption of the cap5D ORF due to insertion of an A at nt 994 in USA300 (994AA). The cap5E G→T mutation at nt 223 (G223T) and the cap5G T→C mutation at nt 478 (T478C) are indicated beneath each ORF. (B) Lines beneath the map indicate the cap5 regions from strain Newman that are cloned in the complementation plasmids pKOR19 (cap5ABC plus truncated cap5D), pKOR18 (cap5ABCD plus a portion of cap5E), pCap16 (cap5ABCDE), and pCap17 (which contains cap5ABCDEF). (C) CP5 serotyping results from USA300 MRSA strain 923 transformants harboring the cap5 complementation plasmids depicted in panel B.

FIG 4

A maximum likelihood phylogeny based on whole-genome alignments of publicly available S. aureus genomes. (A) Phylogenetic analysis of 104 S. aureus strains from 41 STs showing the distribution of conserved mutations in cap5 in USA300 (Table 2). There are 14 USA300 strains in the tree that are representative of 146 strains from our study and 319 publicly available USA300 genomes. The remaining 90 genomes are from non-USA300 isolates. Strain names are shown as either the common name or the accession number followed by the sequence type (ST) in parentheses. Strains with red and blue text, respectively, are previously confirmed USA300 and USA500 isolates. The clade in orange is clonal complex 8 (CC8). Other common CCs are also highlighted. For simplicity, only a representative strain from each ST with the same pattern of cap5 mutations was included. Carriage of PVL and ACME is indicated with black and red diamonds, respectively, due to their association with the USA300 subclone. cap5 mutations are shown as solid or open circles as indicated. (B) High-resolution phylogeny of CC8 strains (a subset from panel A). USA300 and USA500 strains are annotated as in panel A.