Abstract
Background
Persistent MRSA bacteremia is common with high morbidity and mortality despite appropriate antibiotics. Persistent infections are associated with antibiotic tolerance and can arise from perturbations in cellular pathways. We performed whole genome sequencing of clinical isolates to identify the genetic bases of antibiotic tolerance.
Methods
Whole genomes of MRSA from patients with persistent bacteremia were sequenced, which identified 8 isolates harboring different citZ mutations. To assess the effect of the mutations directly on citrate synthase activity, purified recombinant enzymes were assayed using a commercially available kit. The same kit was used to measure activity in whole cell lysates of MRSA isolates harboring wild-type and mutant citZ alleles. Enzyme kinetic parameters were determined by fitting initial rate data to the Michaelis-Menten equation using nonlinear regression.
Figure 1. Whole Genome Sequencing of Persistent MRSA Bacteremia
Whole genome sequencing of 206 blood cultures from 20 patients with persistent MRSA bacteremia (defined as >7 days) under the hypothesis that continuous antibiotic exposure will give rise to - and enrich for - mutations that convey antibiotic tolerance through in-host evolution.
Results
Analysis of whole genomes from 206 blood cultures from 20 patients with persistent MRSA bacteremia identified citZ, which encodes for citrate synthase, the first step of the tricarboxylic acid cycle, as the most repeatedly mutated gene. The data revealed parallelism in its evolution, as citZ was mutated 8 independent times, a rate far greater than from chance alone. To characterize the impact of the mutations on enzyme activity, recombinant proteins were expressed in E. coli and purified for enzymatic assays. As compared to wildtype enzyme, two mutants had reduced activity, and four mutants had near-absent activity; two mutants were unable to be expressed due to destabilizing mutations. Michaelis-Menten analysis of the two mutants with residual activity show that, as compared to wildtype, both had lower affinity for its substrates, and lower maximum activity. Furthermore, we found the cell was unable to compensate for the loss of citrate synthase activity, as lysates harboring these mutations also displayed analogous reductions in activity.
Figure 2. Purified citrate synthase mutant proteins have reduced enzyme activity
Citrate synthase catalyzes the reaction between acetyl-CoA and oxaloacetic acid to form citric acid with CoA-SH as a byproduct. CoA-SH can interact with DTNB to form TNB, which can be measured spectrophotometrically at A412nm to indirectly measure the activity of citrate synthase. Wildtype citrate synthase and the D141N mutant have comparable activity whereas the remaining study mutants have either reduced (mutants A313P and A313V) or near-absent (mutants G7D, G7D + D141N, S201P, P354S) activity. The three active site mutants (H248G, D309G, H219G) also have near-absent activity and serve as negative controls. Positive control is citrate synthase provided in the Sigma Citrate Synthase Assay Kit. No enzyme control replaces enzyme with assay buffer. All error bars are ± 1 SD, calculated from at least three replicate experiments.
Figure 3. A313P and A313V mutants have decreased substrate affinity and decreased maximal activity
Panels A and B show plots of the initial rate of citrate synthase activity (Y-axis, in units of µmole/mL/sec) versus OAA/AcCoA substrate concentration (X-axis, in millimolar units), for wildtype citrate synthase and the A313P and A313V mutants. Panel (A) shows enzymatic parameters for wildtype, A313P mutant, and A313V mutant with variable OAA (0-0.625 mM) and fixed AcCoA (0.3 mM). Panel (B) shows enzymatic parameters for wildtype, A313P mutant, and A313V mutant with fixed OAA (0.5 mM) and variable AcCoA (0-2.5 mM). Three replicates were performed for each condition, with error bars showing ± 1 SD.
Figure 4. Citrate synthase mutants disrupt functional dimerization and destabilize alpha-helix packing
Identified citrate synthase mutants mapped onto the crystal structure of T. thermophilus citrate synthase (PDB 1IOM) show that the mutations are located either at dimerization interfaces or within the interior of hydrophobic packing interfaces.
Conclusion
Cellular metabolism and virulence regulation are interconnected in S. aureus, as alterations in TCA cycle activity lead to increased persister formation and host macrophage inactivation. Our findings that inactivating citZ mutations are enriched can provide a potential explanation for the mechanism of persistent bacteremia.
Disclosures
All Authors: No reported disclosures
DNA extraction from primary liquid blood cultures for bloodstream infection diagnosis using whole genome sequencing
