Insights on virulence from the complete genome of Staphylococcus capitis

Abstract

Staphylococcus capitis is an opportunistic pathogen of the coagulase negative staphylococci (CoNS). Functional genomic studies of S. capitis have thus far been limited by a lack of available complete genome sequences. Here, we determined the closed S. capitis genome and methylome using Single Molecule Real Time (SMRT) sequencing. The strain, AYP1020, harbors a single circular chromosome of 2.44 Mb encoding 2304 predicted proteins, which is the smallest of all complete staphylococcal genomes sequenced to date. AYP1020 harbors two large mobile genetic elements; a plasmid designated pAYP1020 (59.6 Kb) and a prophage, ΦAYP1020 (48.5 Kb). Methylome analysis identified significant adenine methylation across the genome involving two distinct methylation motifs (1972 putative 6-methyladenine (m6A) residues identified). Putative adenine methyltransferases were also identified. Comparative analysis of AYP1020 and the closely related CoNS, S. epidermidis RP62a, revealed a host of virulence factors that likely contribute to S. capitis pathogenicity, most notably genes important for biofilm formation and a suite of phenol soluble modulins (PSMs); the expression/production of these factors were corroborated by functional assays. The complete S. capitis genome will aid future studies on the evolution and pathogenesis of the coagulase negative staphylococci.

Keywords: CoNS; SMRT sequencing; coagulase-negative staphylococci; genomics; methylation.

Figure 1

Neighbor-joining tree showing the relationship between S. capitis AYP1020 and other sequenced CoNS strains. Phylogeny was inferred using the MLST scheme developed for S. epidermidis. S. capitis AYP1020 is represented in bold.

Figure 2

Circular representation of the S. capitis AYP1020 chromosome and its plasmid, pAYP1020. For the chromosome, the first (innermost) ring indicates the GC skew, followed by the GC content (second ring). The third ring indicates the degree of amino acid identity across all CDS of S. capitis AYP1020 compared to S. epidermidis RP62a, as determined by BLASTp and scaled according to percent identity as indicated in the key (Altschul et al., 1997). Colored arrows on the fourth and seventh rings represent the following COG categories (Tatusov et al., 2000); A, B, J, K, L, information and storage and processing; D, O, M, N, P, T, U, V, W, Y, Z, cellular processes and signaling; C, G, E, F, H, I, Q, metabolism; R and S, poorly characterized, on the reverse and forward strands, respectively. The fifth and sixth rings represent the CDS (blue), tRNA (maroon), and rRNA (purple) on the reverse and forward strands, respectively. For the genome, the scale displayed in the center of the graphic represents 500 kb. For the plasmid, the scale represents 5 kb. The images were generated using CGview (Grant et al., 2012).

Figure 3

Orthologous classification of CDS of S. capitis AYP1020 compared to S. epidermidis RP62a. Orthologs were defined by bidirectional BLASTp with an e-value cut off of 10e-30 (A). Non-orthologous CDS were grouped based on COG functional categories (B).

Figure 4

Biofilm formation of S. capitis AYP1020. Scanning electron micrograph of biofilm formed by S. capitis AYP1020 on polyurethane at 10,000X magnification (A). Biofilm was quantified on polystyrene microtitre plates. Data are expressed as mean ± SEM (^*P < 0.05) (B). Schematic ClustalW alignment revealed high similarity (71–83% identity) between the IcaRADBC proteins of S. capitis AYP1020 and those of S. epidermidis. White regions represent the same amino acid, gray regions represent similar amino acids and black regions represent non-similar amino acids (C).

Figure 5

Predicted phenol-soluble modulin (PSM) genes and amino acid sequences in S. capitis AYP1020. The genetic arrangement of phenol soluble modulin genes in AYP1020 (A) is highly similar when compared to S. epidermidis RP62a (B). In each case, PSMs are found on 4 distinct genetic loci. Predicted PSMs of S. capitis have high amino acid sequence identity when compared to S. epidermidis RP62a (C). AYP1020 has both α- and β-type PSMs, which are classed based on length. Each PSM contains an amphipathic α-helix, which is highlighted in yellow. AYP1020 has three distinct PSM β1 genes (a, c, and d), whereas RP62a has two identical copies of PSM β1 (a and b).