Vegetation Formation in Staphylococcus Aureus Endocarditis Inversely Correlates With RNAIII and sarA Expression in Invasive Clonal Complex 5 Isolates

Abstract

Infective endocarditis (IE) is one of the most feared and lethal diseases caused by Staphylococcus aureus. Once established, the infection is fast-progressing and tissue destructive. S. aureus of the clonal complex 5 (CC5) commonly cause IE yet are severely understudied. IE results from bacterial colonization and formation of tissue biofilms (known as vegetations) on injured or inflamed cardiac endothelium. S. aureus IE is promoted by adhesins, coagulases, and superantigens, with the exotoxins and exoenzymes likely contributing to tissue destruction and dissemination. Expression of the large repertoire of virulence factors required for IE and sequelae is controlled by complex regulatory networks. We investigated the temporal expression of the global regulators agr (RNAIII), rot, sarS, sarA, sigB, and mgrA in 8 invasive CC5 isolates and established intrinsic expression patterns associated with IE outcomes. We show that vegetation formation, as tested in the rabbit model of IE, inversely correlates with RNAIII and sarA expression during growth in Todd-Hewitt broth (TH). Large vegetations with severe sequelae arise from strains with high-level expression of colonization factors but slower transition towards expression of the exotoxins. Overall, strains proficient in vegetation formation, a hallmark of IE, exhibit lower expression of RNAIII and sarA. Simultaneous high expression of RNAIII, sarA, sigB, and mgrA is the one phenotype assessed in this study that fails to promote IE. Thus, RNAIII and sarA expression that provides for rheostat control of colonization and virulence genes, rather than an on and off switch, promote both vegetation formation and lethal sepsis.

Keywords: AGR; CC5; RNAIII; SarA; endocarditis; staphylococcus aureus.

Figure 1

Differential development of infective endocarditis and lethality in S. aureus CC5 strains. Rabbit model of native valve IE and sepsis. Rabbits were injected intravenously with 1.3–3.6 x 10⁷ CFUs of S. aureus CC5 isolates after mechanical damage of aortic valve and monitored over 4 days. (A) Total weight of vegetations dissected from aortic valves. *, p < 0.05, **, p < 0.005, ****, p < 0.0001, one-way ANOVA, non-parametric Kruskal-Wallis with uncorrected Dunn’s multiple comparison test to BA1372 (IE-deficient). Bars represent mean value. (B–E) Percent survival. (B) BA0206 and BA0211. (C) BA1372 and BAA1791. (D) IE0560 and IE1420. (E) IE1789 and IE2295. p < 0.03, Log-rank (Mantel-Cox) of IE1789 compared to each strain.

Figure 2

Acute kidney injury and bacteremia resulting from S. aureus CC5 IE. Rabbit model of native valve IE and sepsis. Rabbits were injected intravenously with 1.3–3.6 x 10⁷ cfu of S. aureus CC5 isolates after mechanical damage of aortic valve and monitored over 4 days. (A) Kidney gross pathology grading scale (grades 0-3). 0 = no lesions, 1 = rare, small (<4mm) multifocal lesions, 2 = numerous large (>5mm) multifocal lesions, 3 = extensive to coalescing to diffuse lesions. Arrows indicate ischemic and/or hemorrhagic lesions, arrowhead indicates a necrotic lesion. (B) Scoring of kidney lesions post-mortem. One-way ANOVA with Fisher’s LSD multiple comparisons test across strains. p < 0.002, IE1789 compared to the rest of the strains. (C) Bacterial counts per milliliter of blood recovered from rabbits post-mortem. Lines represent median value. *, p < 0.02, one-way ANOVA Kruskal-Wallis test with uncorrected Dunn’s multiple comparison test across strains.

Figure 3

RNAIII and rot expression in S. aureus CC5 isolates. Quantitation of S. aureus CC5 gene expression during growth in TH broth by RT-qPCR standard curve quantitation method. (A, B) RNAIII expression and (D, E) rot expression at indicated cell densities. Error bars (standard deviation) not shown are smaller than symbol. Asterisks indicate data points significantly different than the rest at that specific cell density. (C, F) Area under the curve (mean ± SEM). Data is the result of three biological replicates. *, p < 0.05, **, p < 0.005, ***, p < 0.0005, ****, p < 0.0001, one-way ANOVA with Holm-Šídák’s multiple comparisons test across strains.

Figure 4

Hemolytic activity in S. aureus CC5 isolates. Overnight cultures of S. aureus CC5 isolates were washed and spotted onto (A) 5% rabbit blood and (B) 5% sheep blood TSA II agar plates. Zones of hemolysis were measured after overnight growth. (Top) Representative images of hemolytic activity in strains IE1789 and BA1372. (Bottom) Relative levels of hemolysin production as measured in an erythrocyte lysis assay. Data are represented as mean ± SEM. *, p < 0.05, **, p < 0.005, ***, p < 0.0005, ****, p < 0.0001, one-way ANOVA with Holm-Šídák’s multiple comparisons test to the non-hemolytic strain IE1789.

Figure 5

sarA and sarS expression in S. aureus CC5 isolates. Figure 3 . Quantitation of S. aureus CC5 gene expression during growth in TH broth by RT-qPCR standard curve quantitation method. (A, B) sarA expression and (D, E) sarS expression at indicated cell densities. Error bars (standard deviation) not shown are smaller than symbol. Asterisks indicate data points significantly different than the rest at a specific cell density. (C, F) Area under the curve (mean ± SEM). Data is the result of three biological replicates. *, p < 0.05, **, p < 0.005, ***, p < 0.0005, one-way ANOVA with Holm-Šídák’s multiple comparisons test across strains.

Figure 6

sigB and mgrA expression in S. aureus CC5 isolates. Quantitation of S. aureus CC5 gene expression during growth in TH broth by RT-qPCR standard curve quantitation method. (A, B) sigB expression and (D, E) mgrA expression at indicated cell densities. Error bars (standard deviation) not shown are smaller than symbol. Asterisks indicate data points significantly different than the rest at a specific cell density. (C, F) Area under the curve (mean ± SEM). Data is the result of three biological replicates. *, p < 0.05, **, p < 0.005, ***, p < 0.0005, one-way ANOVA with Holm-Šídák’s multiple comparisons test across strains.

Figure 7

Differential expression of egc SAgs and selx. Quantitation of egc mRNA in S. aureus CC5 during growth in TH broth by RT-qPCR standard curve quantitation method. egc and selx expression in (A) IE1789 and (B) BA1372 at indicated cell densities. Error bars (standard deviation) not shown are smaller than symbol. Data is the result of three biological replicates. Two-way ANOVA with Holm-Šídák’s multiple comparisons test across superantigen pairs in IE1789 and BA1372 during exponential growth.

Figure 8

Vegetation formation inversely correlates with RNAIII and sarA expression in S. aureus CC5 isolates. (A) Expression correlation between RNAIII and rot (top panel) or rot and sarS (bottom panel). (B) Expression correlation between sarA and sarS (top panel) or sarA and rot (bottom panel). (C) Expression correlation between sigB and sarA (top panel) or RNAIII and mgrA (bottom panel). (D) Correlation between vegetation size and RNAIII expression (top panel) or sarA expression (bottom panel). Pearson correlation coefficients (Pearson r), correlation p value, and best-fit line shown. Expression correlation between RNAIII and sigB is not shown; Pearson r = -0.34 (p = 0.5).