Mutation in the Two-Component System Regulator YycH Leads to Daptomycin Tolerance in Methicillin-Resistant Staphylococcus aureus upon Evolution with a Population Bottleneck

Abstract

Adaptive laboratory evolution (ALE) is a useful tool to study the evolution of antibiotic tolerance in bacterial populations under diverse environmental conditions. The role of population bottlenecks in the evolution of tolerance has been investigated in Escherichia coli, but not in a more clinically relevant pathogen, methicillin-resistant Staphylococcus aureus (MRSA). In this study, we used ALE to evolve MRSA under repetitive daptomycin treatment and incorporated population bottlenecks following antibiotic exposure. We observed that the populations finally attained a tolerance mutation in the yycH gene after 2 weeks of evolution with population bottlenecks, and additional mutations in yycI and several other genes further increased the tolerance level. The tolerant populations also became resistant to another glycopeptide antibiotic, vancomycin. Through proteomics, we showed that yycH and yycI mutations led to the loss of function of the proteins and downregulated the WalKR two-component system and the downstream players, including the autolysin Atl and amidase Sle1, which are important for cell wall metabolism. Overall, our study offers new insights into the evolution of daptomycin tolerance under population bottlenecking conditions, which are commonly faced by pathogens during infection; the study also identified new mutations conferring daptomycin tolerance and revealed the proteome alterations in the evolved tolerant populations. IMPORTANCE Although population bottlenecks are known to influence the evolutionary dynamics of microbial populations, how such bottlenecks affect the evolution of tolerance to antibiotics in a clinically relevant methicillin-resistant S. aureus (MRSA) pathogen are still unclear. Here, we performed in vitro evolution of MRSA under cyclic daptomycin treatment and applied population bottlenecks following the treatment. We showed that under these experimental conditions, MRSA populations finally attained mutations in yycH, yycI, and several other genes that led to daptomycin tolerance. The discovered yycH and yycI mutations caused early termination of the genes and loss of function of the proteins, and they subsequently downregulated the expression of proteins controlled by the WalKR two-component system, such as Atl and Sle1. In addition, we compared our proteomics data with multiple studies on distinct daptomycin-tolerant MRSA mutants to identify proteins with a consistent expression pattern that could serve as biological markers for daptomycin tolerance in MRSA.

Keywords: MRSA; WalKR; YycH; YycI; daptomycin; evolution; population bottleneck; tolerance; two-component system.

FIG 1

Laboratory evolution using population bottleneck-generated daptomycin-tolerant populations bearing a mutation in the yycH gene. (a) Experimental protocol used during the evolution experiment. Stationary-phase MRSA was repetitively treated with daptomycin (30 mg/liter) for 6 h. The survivors were subjected to a bottleneck (either a large bottleneck, 1:10 dilution, or small bottleneck, 1:100 dilution), subsequently regrown overnight, and the cycle was repeated. Three parallel populations were used for each experimental protocol. (b) Time-kill curves of the ancestral and evolved populations after 6 (left) and 12 cycles (right) of evolution upon daptomycin treatment (10 mg/liter) for 3 h (mean ± standard error of the mean [SEM], n = 3). (c) MDK₉₉ (minimum duration for killing 99% of the population) measurements of the evolved populations after 6 and 12 cycles of evolution (mean ± SEM, n = 3). The horizontal dashed line shows the mean MDK₉₉ of the ancestral population. Significance of difference with the ancestral: *, Bonferroni-adjusted P < 0.05; **, Bonferroni-adjusted P < 0.01; ns, not significant (two-tailed t test on log-transformed values, followed by Bonferroni correction). (d) Summary of the evolution experiment. Three parallel MRSA populations were evolved with large bottlenecks (B1-1, B1-2, and B1-3) and three were evolved with small bottlenecks (B2-1, B2-2, and B2-3). After 12 cycles, the populations were subjected to tolerance and resistance assays and whole-genome sequencing. B2-1 and B2-2 harbored a mutation only in the yycH gene, whereas B1-1, B1-2, B1-3, and B2-3 harbored mutations in yycH, thrC, yycI, lacE, and a hypothetical protein (hp) gene (for details of the mutation, see Table S2 and Table S3 in the supplemental material). The evolution experiment was prolonged for 6 cycles on B2-2 population using small bottlenecks, generating B2-2-1, B2-2-2, and B2-2-3, which all bore a mutation in the yycH gene. Pie charts show the proportion of specific mutations in the population based on the number of alternate reads divided by the total number of reads at the locus. Black color indicates the percentage of reference reads, whereas blue, red, yellow, purple, and orange colors indicate the percentage of alternate reads for the yycH, yycI, thrC, lacE, and hp genes.

FIG 2

Proteomics analysis of the tolerant population. (a) Venn diagram for proteome comparison of the B2-2 tolerant (YycH) to the ancestral population in the absence of antibiotics. (b) Volcano plot of the B2-2 tolerant (YycH) population compared to the ancestral in the absence of antibiotics. Differentially expressed proteins (DEPs) were those with P values of <0.05 and an absolute fold change of >1.5, corresponding to the colored dots. (c) Protein-protein interaction network of the DEPs of the B2-2 tolerant population compared to the ancestral population, as predicted by STRING v11.5. The lines represent protein interactions (thicker lines indicate higher confidence), and the dots in different colors represent different protein functions. Nodes without function enrichment are colored gray. Nodes with black outlines are proteins with higher expression in the tolerant population, and nodes with no outlines are proteins with higher expression in the ancestral population. Node corresponding to the mutated gene in the tolerant population (yycH) is shown with twice the size of the other nodes. Nodes that belong to the WalKR two-component system are marked. (d) Venn diagrams for proteome comparison of the ancestral and B2-2 tolerant population upon daptomycin treatment (WTD and YYCHD) versus those before treatment (WT and YYCH). (e) Volcano plots of the ancestral and B2-2 tolerant population upon daptomycin treatment, compared to those before treatment. (f) GO analysis and pathway enrichment study (KEGG) by use of DAVID of the DEPs of the ancestral and B2-2 tolerant population after daptomycin treatment, compared to those before treatment. Fold enrichment is defined as the ratio of the proportion of the input information to the background information. Negative values indicate downregulation, and positive values indicate upregulation.