S. aureus USA500 (Diep et al., 2006) was used for construction of gene knockout and complementation strains. E. coli DC10B (Monk et al., 2012) was used for shuttle plasmid construction. Luria Broth medium was composed of 1% tryptone (Oxoid), 0.5% yeast extract (Oxoid) and 0.5% NaCl; BM (B-Medium) was composed of 1% tryptone, 0.5% yeast extract, 0.5% glucose, 0.1% K₂HPO₄ and 0.5% NaCl; BM and TSB (Tryptic soy broth, Oxoid) were used for S. aureus cultivation. Bacterial strains were inoculated in BM, and their growth rate at 37°C was monitored by measuring the OD values at 600 nm. Anhydrotetracycline (ATc) was used for induction of secY antisense RNA during gene knockout. Antibiotics were added to medium at the following concentrations: chloramphenicol, 10 μg/ml; ampicillin, 100 μg/ml, levofloxacin, 50 μg/ml.

We constructed plasmid pMX10 by replacing ccdB element with multiple cloning sites in pKOR1 (Bae and Schneewind, 2006) and used it for construction of gene knock out strains. Primers pMX10-f and pMX10-r were mixed equally to a final concentration of 100 uM, incubated at 72°C for 20 min and slowly cooled to 4°C. The resulting dimers were digested with BamHI and KpnI and ligated to pKOR1 backbone digested with the same restriction enzymes. To construct ΔctaB in USA500, the upstream (us) fragment (about 1000 bp) at the upstream of ctaB gene of USA500 strain was amplified with primer ctaB-uf and ctaB-ur, while the downstream (ds) fragment with primers ctaB-df and ctaB-dr. The two fragments were then used as templates for fusion PCR with primer ctaB-uf and ctaB-dr. The final PCR product was digested with KpnI and MluI and then ligated into pMX10. The recombinant plasmids was transformed into USA500 by electroporation and mutants were selected according to the method reported by Bae et al. (Bae and Schneewind, 2006). To construct the complementation strain ΔctaB::pRBctaB, a fragment containing the promoter region and coding sequence of ctaB gene was amplified with primers cp-ctaB-f and cp-ctaB-r. The PCR product was digested with EcoRI and BamHI and then ligated into plasmid PRB473. The resulting plasmid was transformed into the ΔctaB mutant via electroporation. The sequences of primers are listed in Table 1.

To compare pigment production, USA500 and USA500ΔctaB were dropped onto TSA plates and USA500ΔctaB with pRB473 or pRBctaB on TSA plates with 10 μg/ml chloramphenicol. The plates were incubated at 37°C for 24 h and pictured. For quantitate assay of pigment production, the same strains were cultured in TSB at 37°C for 24 h. For each sample, pigment was extracted with methanol and detected with a parameter (GeneSpec III, Hitachi, Japan), following a previously reported protocol (Morikawa et al., 2001). For hemolytic activity determination, the strains were analyzed by growing the strains on 5% sheep blood agar at 37°C for 48 h. The result represents three independent experiments.

The mouse virulence test was performed on Balb/C mice. USA500 and the ΔctaB mutant strains were cultured for 18 h and 1 ml of the each culture was mixed with 2% Cytodex-1 beads by 1:1. The mice were randomized into two groups (5 mice/group). Each mouse was challenged with 200 μl bacterial mixture (each containing approximately 2 × 10⁵ bacterial cells) via injection under skins on the back. After 48 h, the mice were sacrificed and the abscess under skin was homogenized in 2 ml PBS. The samples were diluted and plated on TSA plates at 37°C for 18 h. CFU counting was performed and a Student's t-test was used for statistical analysis using Microsoft Excel.

Animal studies on mice were performed according to relevant national and international guidelines (the Regulations for the Administration of Affairs Concerning Experimental Animals, China) and were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Medical College, Fudan University (IACUC Animal Project Number: 20110630). Standard operation procedures were followed to carry out animal experiments in bio-safety level 2 labs.

The MIC of each antimicrobial compound was determined in triplicate by a conventional broth microdilution technique in TSB medium, following the protocol previously published (Andrews, 2001) and the CLSI guidelines. The MIC was defined as the lowest antibiotic concentration that inhibited visible bacterial growth (also according to OD600 measurements) after 24 h of incubation at 37°C.

To determine the number of persister cells in exponential phase, cells were grown overnight in 4 ml and were inoculated to 10 ml of fresh medium to an initial OD600 of 0.05. Cultures were shaken for 1.5–2 h (for normally growing cells), until an OD600 of approximately 0.5 was reached. To determine the number of persister cells in stationary phase, overnight cultures (16 or 24 h) were used without dilution.

For heat stress assay, stationary phase cultures were incubated at 57°C for up to 3 h. For oxidative stress assay, stationary phase cultures were diluted by 1:100 in TSB that contained 50 mM hydrogen peroxide (H₂O₂) for 4 h. For starvation stress assay, stationary phase cultures were centrifuged, washed and resuspended in 3% NaCl. The survival of bacteria was determined by CFU counting at each hour. All stress assays were conducted using at least three biological replicates.

For antibiotic exposure, 2 ml of the overnight or the exponential phase cultures was transferred to 14 ml culture tubes (Greiner), antimicrobials were added at 100-fold MIC as indicated and the cultures were shaken for 12 h, or for 7 days during long-term experiments. For CFU determination, 100 μl was taken before and during antimicrobial challenge on an hourly basis during the first 8 h and after 24 h, or after 1, 2, 3, 5, 6 days during long-term experiments. Cells were washed in PBS and spotted as 10 μl aliquots of serial dilutions onto TSA plates. Colonies were counted after incubation for 24 h at 37°C. The lower limit of quantification was 100 CFU/ ml. All time-kill experiments were conducted using at least three biological replicates.

S. aureus USA500 parent strain and ΔctaB mutant were cultured for 6 or 24 h as log phase and stationary phase cultures. The cultures were divided into 3 aliquots and treated with RNAprotect (Qiagen) and frozen at −80°C. Total RNA was extracted from bacterial cells using the RNeasy Mini kit (Qiagen) as described (Atshan et al., 2012). The quality of RNA samples was examined with Bioanalyzer 2100 RNA-6000 Nano Kit. To remove 16S and 23S rRNAs, 10 μg of high-quality total RNA was processed using the Ribo-Zero™ Gold Kit before precipitating with ethanol and resuspending into 25 μL of nuclease-free water. The cDNA libraries with 150- to 250-bp multiplexed cDNA were generated from the enriched mRNA samples using the TruSeq Illumina kit (Illumina, San Diego, CA), following instructions from the manufacturer.

Sequencing was performed with HiSeq2500 (Illumina). The Cufflinks suite of tools were used to assess and quantify the total number of reads. With the program Cuffdiff as part of the suite, transcripts were quantified by assessing the total number of reads for the entire transcript. Briefly, reads were mapped to annotated coding sequences (CDSs) from genome of S. aureus USA300 TCH1516 strain since USA500 is the progenitor of USA300. The samples to be compared were evaluated for variance and tested for differential expression. Reads' P-values were determined, and significance was assessed by conducting Benjamini-Hochberg correction for multiple testing. The transcript sequencing data were submitted to the NCBI Sequence Read Archive, available for access under a RUN number RSS3919726.

For quantitative Real-time PCR, the same RNA samples were taken from that used for RNA-seq. After reverse transcription with cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA), qRT-PCR was performed using SYBR Green PCR reagents (Takara Biotechnology) to determine the relative expression levels of the target genes with gene-specific primers listed in Table 1. The housekeeping gene rrs1 (16s RNA) was used as an endogenous control. All qRT-PCR experiments were carried out in triplicate with independent RNA samples and the 2^−ΔΔCT method was performed for analysis of relative gene expression data (Livak and Schmittgen, 2001).

The significance of experimental differences in pigment production, hemolytic activity, survival in vivo and persister assay was evaluated by unpaired Student's t-test.

To investigate the functions of CtaB, we constructed a ctaB deletion mutant, ΔctaB, via homologous recombination, as well as made a complemented strain ΔctaB::pRBctaB by inserting ctaB with its own promoter into plasmid pRB473. When grown in TSB, the ΔctaB mutant showed a slight growth defect, compared with the parent strain USA500 (Figure 1A). The ΔctaB mutant displayed enhanced golden pigment production when grown on TSA for 24 h compared with the control strain (Figure 1B), and complementation of the ΔctaB mutant reduced pigment production to normal levels (Figure 1B). Quantification of pigment production by extracting carotenoid products confirmed that CtaB depletion afforded enhanced pigmentation than USA500 strain (Figure 1C).

Hemolytic ability is an important aspect of S. aureus virulence (Wang and Muir, 2016). We analyzed the level of bacterial growth and hemolysis of the ΔctaB mutant on sheep blood agar plates. While all strains showed similar sized colonies, deletion of ctaB generated a strain with reduced hemolytic activity, which could be restored by complementation of the ΔctaB mutant with the wild type ctaB gene (Figure 2A).

Having observed that the ΔctaB mutant enhanced pigment production but reduced hemolytic activity, we wondered whether ΔctaB mutant would affect virulence in vivo. Skin is one of the most frequently targeted sites for S. aureus infection (Liu, 2009). To determine whether CtaB is associated with virulence during S. aureus infection, we compared the ΔctaB mutant and the parent strain in a mouse model of skin abscess. Colony counting of bacteria from mouse skin lesions showed that inactivation of ctaB attenuated bacterial survival in vivo. After 24 h of infection, the CFU counting of USA500 increased from 6.18 ± 0.46E + 6 to 6.79 ± 1.02E + 6. Within contrast, the ΔctaB mutant survived less well with a decrease of CFU, from 4.74 ± 0.57E + 6 to 2.96 ± 1.3E + 6. (Figure 2B).

To determine if CtaB is involved in persister formation or survival, we subjected stationary cultures of USA500, ΔctaB and ΔctaB::pRBctaB under stress conditions including heat, oxidative stress, and starvation. The CtaB mutation attenuated the ability of S. aureus to survive starvation in 3% NaCl, which provides similar osmotic pressure as TSB, and the impact was reversed by gene complementation (Figure 3A). However, CtaB knockout did not affect survival of S. aureus under treatment with heat or H₂O₂ (data not shown).

Before persister assay, we measured the antibiotic sensitivity of ctaB mutant and found no difference in MIC tests for multiple antibiotics (data not shown). Challenging the stationary phase cultures of USA500, ΔctaB and ΔctaB::pRBctaB with 100 X MIC ciprofloxacin or levofloxacin yielded disparate killing curves. As shown in Figures (3B,C), the surviving ratios of ΔctaB were similar with that of USA500 in the first 3 days, but became higher than the control strain in day four and day five. Meanwhile, complementation with plasmid pRBctaB but not pRB473 partially reversed the augmentation of persister formation caused by deletion of ctaB in the last 2 days of treatment. We also tested other antibiotics such as vancomycin, rifampicin, streptomycin, tobramycin, and gentamycin at 100X MIC concentration but found no significant difference in persister formation between the ΔctaB mutant and the parent strain from either exponential phase or stationary phase (data not shown).

The above results indicate an intriguing and paradoxical role of CtaB in persistence and virulence of S. aureus, as its deletion attenuated virulence and survival in 3% NaCl while increasing persister numbers for quinolone antibiotics. To gain insights into the role of CtaB in altered S. aureus virulence and persistence, we performed RNA-seq analysis of USA500 and ΔctaB mutant grown for 6 h (log phase) or 24 h (stationary phase) in TSB medium. Based on the results of read counts of all annotated genes, a total of 4 RNA-seq samples were clustered without supervision (Figure 4). The results indicated that the effect of CtaB knockout on the bacterial transcriptome at 24 h were more apparent than that at 6 h (Figures 5A,B).

In log phase cultures, we found 18 genes with significant changes in transcription (cutoff > 2-fold) and p-values less than 0.05 between ΔctaB mutant and the parent strain (Table 2). Most strikingly, the virulence gene hld was significantly down regulated (0.35, p = 8.47E-05) in the ΔctaB mutant. In S. aureus, gene hld is located inside the coding sequence for small regulatory RNA RNAIII which regulates the expression of many S. aureus genes encoding exoproteins and cell-wall-associated proteins. The data indicated that deletion of CtaB could attenuate expression of various virulence genes regulated by RNAIII in log phase. Interestingly, CtaB deletion did not affect expression of the Agr system (encoded by agrB, agrD, agrC, and agrA), which is the well-known upstream regulator of RNAIII and a virulence factor. The virulence gene esaB (Burts et al., 2008; Anderson et al., 2011)was also down regulated. Meanwhile, the virulence genes set18 and set19 were up regulated in the ΔctaB mutant. Two hemin ABC transporter super family genes htrB and htrA were significantly up regulated, as a consequence of lack of heme caused by deletion of CtaB (Table 2).

For the transcripts at 24 h, 119 genes showed significant changes between ΔctaB mutant and the parent strain (Table 3), indicating that CtaB has a major impact on stationary phase S. aureus. The proposed pathways that these genes participate in were analyzed (Figure 6). Nineteen genes encoding ribosome proteins were strongly down regulated, as well as nine genes involved in biosynthesis of Aminoacyl-tRNA. Genes involved in arginine, proline, cysteine, methionine, glycine, serine, threonine, lysine, phenylalanine, tyrosine, tryptophan, valine, leucine, and isoleucine were down regulated in the CtaB mutant. Expression of several ABC transporters was also up regulated, but other transporters such as OppA1, OppB3, OppC1, OppD1, and OppF1, were down regulated. Twenty two genes that encode factors for amino acid metabolism showed difference in expression. Expression of two genes (arcB2 and arcC1) involved in arginine metabolism was up regulated while the others were down regulated. The deletion of CtaB also down regulated genes from pathways involved in purine metabolism, pyrimidine metabolism, and fatty acid biosynthesis. Though out of 17 genes associated with S. aureus infection 12 were up regulated, genes in the dlt operon (dltA, dltB, dltC, and dltD) were significantly down regulated (Collins et al., 2002). The CtaB deletion also induced expression of genes of five two-component systems, including PhoPR, LgrAB, SaeRS, and LytSR, indicating that these systems might play a role when S. aureus is confronted with lack of heme biosynthesis (Table 3).

Quantitative Real-time PCR was performed to validate the RNA-seq results. Genes were chosen from the list of genes with significant changes of transcription, favoring those associated with virulence and protein production but had a p-value < 0.05. All showed similar fold change with those from the RNA-seq results (Figure 7).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.