Basal levels of (p)ppGpp differentially affect the pathogenesis of infective endocarditis in Enterococcus faecalis

Abstract

The alarmone (p)ppGpp mediates the stringent response and has a recognized role in bacterial virulence. We previously reported a stringent response-like state in Enterococcus faecalis isolated from a rabbit foreign body abscess model and showed that E. faecalis mutants with varying levels of cellular (p)ppGpp [Δrel, ΔrelQ and the (p)ppGpp⁰ ΔrelΔrelQ] had differential abilities to persist within abscesses. In this study, we investigated whether (p)ppGpp contributes to the pathogenesis of E. faecalis infective endocarditis (IE), a biofilm infection of the heart valves. While the stringent response was not activated in heart valve-associated E. faecalis, deletion of the gene encoding the bifunctional (p)ppGpp synthetase/hydrolase Rel significantly impaired valve colonization. These results indicate that the presence of (p)ppGpp is dispensable for E. faecalis to cause IE, whereas the ability to regulate (p)ppGpp levels is critical for valve colonization. Next, we characterized how basal (p)ppGpp levels affect processes associated with IE pathogenesis. Despite being defective in binding to BSA-coated polystyrene surfaces, the Δrel strain bound to collagen- and fibronectin-coated surfaces and ex vivo porcine heart valves as well as the parent and ΔrelΔrelQ strains, ruling out the possibility that the impaired IE phenotype was due to an attachment defect. Moreover, differences in cellular (p)ppGpp levels did not affect extracellular gelatinase activity but significantly impaired enterococcal invasion of human coronary artery endothelial cells. Taken together, this study uncovers for the first time the fact that differences in basal (p)ppGpp levels, rather than the stringent response, differentially affect processes that contribute to the pathogenesis of IE.

Keywords: Gram positive bacterial infection; biofilm; heart valve; stringent response; vegetation.

Fig. 2.

The stringent response is not activated in vivo in heart valve-associated E. faecalis cells. (a) Each symbol represents the aortic valve bacterial load in an animal from which a heart was collected at 2 or 4 days post-infection. The mean valve bacterial load (horizontal bar) increased from 5.6 log₁₀ c.f.u./valve at 2 days to 6.7 log₁₀ c.f.u./valve at 4 days, suggesting active bacterial replication at the aortic valve. (b) RNA was extracted from aortic valve homogenates collected from rabbits during early infection (2 days) or late infection (3 or 4 days; see the Methods section). RT-qPCR was used to measure the abundance of 16S rRNA transcripts in DNase-treated RNA from each valve homogenate. 16S rRNA transcript abundance, as represented by the C _t value, correlates with valve bacterial load (r ²=0.78), further suggesting that E. faecalis cells at the aortic valve are in an active growth state. Circles, early infection (n=5). Squares, late infection (n=8). (c) RT-qPCR was used to measure the relative expression of the genes encoding the E. faecalis vegetative sigma factor RpoD (sigA), the translation elongation factor Ts (tsf), the (p)pGpp synthesis proteins (rel and relQ) and several known IE virulence factors (ebpA, ace, efbA and gelE) in early and late infection aortic valve homogenates relative to IE inoculum growth conditions. EF0886 was used as the reference gene. The horizontal bars and error bars represent the geometric means and 95% confidence intervals, respectively. Confidence intervals that do not cross y=10⁰ (which represents no fold change in expression) are significantly different from the inoculum control (*, P<0.05). Transcription of sigA and tsf would be expected to be down-regulated if E. faecalis were in a stringent response state.

Fig. 3.

The transcript levels of the infective endocarditis virulence genes ace and efbA are dysregulated in strains with altered (p)ppGpp levels during in vitro biofilm growth. RNA was harvested from planktonic and biofilm cells of the strains OG1RF, ∆rel and ∆rel∆relQ that were grown under conditions known to induce in vitro expression of ace and efbA. The transcript copy number detected per microlitre of cDNA was divided by the corresponding number of c.f.u. from which RNA was extracted to obtain the absolute transcript level. Each symbol represents the absolute transcript level of the indicated gene in one biological replicate collected on separate days. The horizontal bars and error bars represent the geometric mean±geometric standard deviation. **, P=0.0094 by Tukey’s multiple comparison post hoc test after ordinary one-way ANOVA testing for differences in ace expression between OG1RF, ∆rel and ∆rel∆relQ biofilm cells. The differences in the means for the other conditions were not significant by one-way ANOVA.

Fig. 4.

Deletion of rel impairs cellular attachment to BSA-coated polystyrene but not collagen, fibronectin, or ex vivo porcine heart valve tissue sections. For the experiments shown in panels (a–c), E. faecalis OG1RF, ∆rel and ∆rel∆relQ were grown in conditions known to induce in vitro expression of ace and efbA. Bacterial attachment to polystyrene wells that were (a) coated with BSA, (b) coated with collagen, or (c) coated with fibronectin was quantified by crystal violet staining and normalized by c.f.u. Each symbol represents one biological replicate. The horizontal bars show the mean. (a) Ordinary one-way ANOVA was used to determine whether the difference between the means of ∆rel or ∆rel∆relQ and OG1RF was statistically significant (P=0.0023). **, P=0.0011 by Dunnett’s multiple comparison post hoc test. (b, c) The differences in the means were not significant by one-way ANOVA. (d) The percentages of E. faecalis OG1RF, ∆rel and ∆rel∆relQ cells that attached to ex vivo porcine heart valve sections relative to the number of planktonic bacteria in the same well after 2 hours of incubation. Each symbol represents one biological replicate collected on a separate day. The horizontal bars show the mean. The means were not significantly different by statistical analysis (one-way ANOVA).

Fig. 1.

The ∆rel strain is impaired in valve colonization in an experimental model of E. faecalis infective endocarditis. E. faecalis OG1RF and the isogenic (p)ppGpp mutant strains ∆rel, ∆relQ and ∆rel∆relQ were tested in a rabbit model of experimental infective endocarditis. Each symbol represents the aortic valve bacterial load in each animal at the experiment endpoint. The horizontal bars show the arithmetic mean of the log₁₀-transformed values. Ordinary one-way ANOVA was used to determine whether the difference among the means was statistically significant (P=0.0180). *, P=0.0151 by Tukey’s multiple comparison post hoc test.

Fig. 5.

Disruption of cellular (p)ppGpp levels does not impact on gelatinase production but is associated with diminished invasion of human coronary artery endothelial cells (HCAECs). (a) Supernatants of OG1RF, ∆rel, or ∆rel∆relQ cultures grown overnight in medium supplemented with 40% horse serum were tested for gelatinase activity in a zone-of-clearance assay. The area of the zone of clearance for OG1RF was set to 100% in each biological replicate, and this was used to calculate the relative gelatinase activity based on the zone areas for the other strains tested. The symbols represent biological replicates measured on separate days; the bars show the means. The means were not significantly different by one-way ANOVA. (b) HCAECs were infected with OG1RF, ∆rel or ∆rel∆relQ at an m.o.i. of 100 : 1 for 2 hours. The ability of each E. faecalis strain to invade is reported as the percentage of intracellular bacteria recovered after 2 hours relative to the total c.f.u. of the initial inoculum. Each symbol represents one biological replicate. The horizontal bars show the mean. Ordinary one-way ANOVA was used to determine whether the difference between the means of ∆rel or ∆rel∆relQ and OG1RF was statistically significant (P<0.0001). ****, P=0.0001 by Dunnett’s multiple comparison post hoc test.