MRSA lineage USA300 isolated from bloodstream infections exhibit altered virulence regulation

Abstract

The epidemic community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 lineage has recently become a leading cause of hospital-associated bloodstream infections (BSIs). Here, we leveraged this recent introduction into hospitals and the limited genetic variation across USA300 isolates to identify mutations that contribute to its success in a new environment. We found that USA300 BSI isolates exhibit altered virulence regulation. Using comparative genomics to delineate the genes involved in this phenotype, we discovered repeated and independent mutations in the transcriptional regulator sarZ. Mutations in sarZ resulted in increased virulence of USA300 BSI isolates in a murine model of BSI. The sarZ mutations derepressed the expression and production of the surface protein ClfB, which was critical for the pathogenesis of USA300 BSI isolates. Altogether, these findings highlight ongoing evolution of a major MRSA lineage and suggest USA300 strains can optimize their fitness through altered regulation of virulence.

Keywords: GWAS; MRSA; SarZ; USA300; bloodstream infections; gene regulation; pathogenesis; virulence.

Figure 1.. Phenotypic screen for cytotoxicity reveals altered virulence regulation in USA300 BSI isolates.

(A) Schematic of cytotoxicity screen. (B) Clinical S. aureus isolates grown in TSB were assessed for the ability of their supernatants to kill hPMNs. Percent death of 5% supernatants was normalized across experiments to control AH-LAC. Each point on the graph represents the mean cytotoxicity of a single isolate (n = 5–6 donors). Center line, median; box limits, upper and lower quartiles; whiskers; min and max. (C) Heat map depicting data from panel A, with the addition of 5% supernatant cytotoxicity data from bacteria grown in YCP media. Any normalized value above 100% is colored as 100%. Colored bars under heat map depict the cytotoxicity classification of each isolate: High Cytotoxicity = TSB >50%, Inducible = TSB <50% and YCP >40% increased from TSB value, Low Cytotoxicity = TSB <50% and not inducible. Strain Δluk is AH-LAC with deletion of all leukocidins. Exact values depicted in heat map can be found in Table S2. (D) Proportions of USA300 BSI isolates classified by cytotoxicity, separated into groups by the year the isolate was collected. Total number of USA300 BSIs for each year is shown under each pie chart. (E) Maximum-likelihood phylogenetic tree produced from core genome SNVs identified from Parsnp whole-genome alignments of USA300 BSIs. Strains used for further phenotypic analysis are highlighted in red.

Figure 2.. Loci associated with inducible cytotoxicity USA300 BSI isolates.

(A) Variant matrix for each of the operons that was significantly associated with the inducible phenotype (right) compared to the non-inducible isolates (left). The type of variant observed is indicated with a colored square. Between panel A and panel B, maximum-likelihood phylogenetic trees generated with parsnp are shown. (B) Same as panel A, but showing the genes in which the variants are observed. (C) Graphical view of the location and type of variants for each of the genes shown in panel B, with correspondingly colored lines. Core genome multilocus sequence typing (cgMLST) trees of (D) USA300 and (E) USA100 MRSA genomes deposited in Genbank between 2000 and 2020. Branch lengths correspond to the number of cgMLST locus differences. Genomes with ≤10 differences are collapsed into a single node. Nodes are sized proportionally to the number of genomes they represent and are colored according to which mutations or combinations of mutations were present.

Figure 3.. SarZ inducible-associated alleles lead to altered virulence regulation.

(A) Supernatants from AH-LAC and mutant strains grown to stationary phase in TSB or YCP media were assessed for their cytotoxicity to hPMNs (n = 4 donors). Data for 5% supernatant shown, full titration curves can be found in Fig. S4A/B. (B) Extracellular infection of hPMNs with AH-LAC strains or USA300 BSI isolates at MOI 50 (n = 7 donors). (C) Amino acid sequence of sarZ alleles. WT = wild type USA300 sarZ allele found in AH-LAC. The four mutant alleles found in USA300 BSI isolates are aligned below with differences highlighted in red. The major structural features of SarZ are shown above, where alpha helices are blue, beta sheets are represented by green arrows, and the critical cysteine (Cys13) is highlighted in yellow. (D) USA300 BSI isolates were grown to stationary phase and 15uL of whole cell lysates were run on a gel to detect SarZ by Western blot. (E) Supernatant cytotoxicity of AH-LAC sarZ::bursa strains complemented with the 5 different sarZ alleles, grown to stationary phase in TSB. Data from 1.25% supernatant shown, full titration curves can be found in Fig. S4C/D (n = 8 donors). SarZ Western blot shown below: AH-LAC sarZ::bursa complemented strains were grown to stationary phase and 5uL of whole cell lysates were run on a gel for Western blot. (F) Promoter pulldown (biotinylated Pssp) of WT compared to mutant SarZ purified protein (n=6). SarZ Western blot shows one representative experiment. Input Protein is 15ng of purified SarZ. Band intensity of pulldowns using 50nM and 25nM protein were normalized to Input Protein for each allele. Quantification of 25nM band intensity with WT 50nM = 100% is shown above. Statistical significance using a 2-way ANOVA with Sidak’s multiple comparisons test. Error bars indicate SEM. NS not significant.

Figure 4.. Increased virulence in a murine BSI model due to mutations in sarZ.

(A) Survival of mice infected i.v. (5×10⁷ CFU) with USA300 BSI isolates. Inducible USA300 (I1-I5) that have mutations in sarZ are blue. Closely related high cytotoxicity USA300 (H1–5) that have a WT sarZ locus are red. Dark lines represent pooled data for the 5 inducible isolates and for the 5 high cytotoxicity isolates (n = 65). Faded lines represent survival data for each of the 10 isolates (n = 10–15). Data shown in faded lines is also depicted in Fig. S7, separated to show differences within pairs. (B) CFU burden of mice infected i.v. (1×10⁷ CFU) with USA300 BSI isolates. Pairs 1 and 2 were used to infect mice and organs were harvested 1-day post-infection to determine CFU burden (n = 10). Statistical analysis using unpaired two-tailed t tests with Welch’s correction. (C) Survival of mice infected i.v. (7.5×10⁷ CFU) with H2 and H2 sarZ::tet (n = 10–15). (D) Survival of mice infected i.v. (8×10⁷ CFU) with H2 sarZ::tet complemented with WT and FS1 sarZ alleles (n = 10). Statistical analysis for survival curves done with the Log-rank (Mantel-Cox).

Figure 5.. Characterization of the SarZ regulon in USA300.

(A) Hierarchical clustering of 696 differentially expressed genes between the AH-LAC sarZ::bursa + vector transposon mutant (ΔsarZ), or the AH-LAC sarZ::bursa + sarZ^WT overexpression mutant (OE), compared to wild-type AH-LAC + vector (WT) during exponential (3h) and/or or stationary (5h) growth. Columns correspond to comparisons with labels indicated at the bottom. Log2 ratio color shades and intensity represent the difference in normalized log2 counts per million (CPM), with a color key shown on the far-right. Significant expression changes (FDR q<0.05) between three replicate experiments are highlighted in yellow in the matched panel on the left. (B) Same as panel A, but focusing on the 49 genes with opposite changes between the AH-LAC sarZ deletion and overexpression experiments, and including results for the sarZ loss-of-function mutants (H2 ΔsarZ and I1) compared to their (complemented) wild-type strains for two USA300 BSI isolates. Gene names and descriptions are shown on the right, and significant changes are marked by dots. (C) Same as B, after further restricting to the 16 genes with opposite regulation between sarZ deletion and overexpression experiments in AH-LAC and USA300 BSI isolate backgrounds.

Figure 6.. Increased ClfB in sarZ mutants contributes to lethality.

(A) Promoter pulldown of 50nM WT SarZ purified protein to biotinylated promoter DNA (n=5). SarZ Western blot shows one representative experiment. 15X unlabeled promoter DNA was used as a competitor. Quantification of band intensity normalized to Pssp = 100% is shown above. (B) ClfB Western blot and quantification of whole cell lysates grown 3h in TSB. Band intensity normalized to AH-LAC = 1 (n=2–3) (C) Survival of mice infected i.v. (1–2×10⁷ CFU) with I2 and I2 clfB::tet (n = 10). (D) Survival of mice infected i.v. (3×10⁷ CFU) with I2 clfB::tet and I2 clfB::clfB (n = 10). Statistical significance using a 2-way ANOVA with Sidak’s multiple comparisons test. Error bars indicate SEM. Statistical analysis for survival curves done with the Log-rank (Mantel-Cox).