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Evolution of multidrug resistance during Staphylococcus aureus infection involves mutation of the essential two component regulator WalKR.

ABSTRACT: Antimicrobial resistance in Staphylococcus aureus is a major public health threat, compounded by emergence of strains with resistance to vancomycin and daptomycin, both last line antimicrobials. Here we have performed high throughput DNA sequencing and comparative genomics for five clinical pairs of vancomycin-susceptible (VSSA) and vancomycin-intermediate ST239 S. aureus (VISA); each pair isolated before and after vancomycin treatment failure. These comparisons revealed a frequent pattern of mutation among the VISA strains within the essential walKR two-component regulatory locus involved in control of cell wall metabolism. We then conducted bi-directional allelic exchange experiments in our clinical VSSA and VISA strains and showed that single nucleotide substitutions within either walK or walR lead to co-resistance to vancomycin and daptomycin, and caused the typical cell wall thickening observed in resistant clinical isolates. Ion Torrent genome sequencing confirmed no additional regulatory mutations had been introduced into either the walR or walK VISA mutants during the allelic exchange process. However, two potential compensatory mutations were detected within putative transport genes for the walK mutant. The minimal genetic changes in either walK or walR also attenuated virulence, reduced biofilm formation, and led to consistent transcriptional changes that suggest an important role for this regulator in control of central metabolism. This study highlights the dramatic impacts of single mutations that arise during persistent S. aureus infections and demonstrates the role played by walKR to increase drug resistance, control metabolism and alter the virulence potential of this pathogen.

SUBMITTER: Howden BP

PROVIDER: S-EPMC3213104 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Evolution of multidrug resistance during Staphylococcus aureus infection involves mutation of the essential two component regulator WalKR.

Howden Benjamin P BP McEvoy Christopher R E CR Allen David L DL Chua Kyra K Gao Wei W Harrison Paul F PF Bell Jan J Coombs Geoffrey G Bennett-Wood Vicki V Porter Jessica L JL Robins-Browne Roy R Davies John K JK Seemann Torsten T Stinear Timothy P TP

PLoS pathogens 20111110 11

Antimicrobial resistance in Staphylococcus aureus is a major public health threat, compounded by emergence of strains with resistance to vancomycin and daptomycin, both last line antimicrobials. Here we have performed high throughput DNA sequencing and comparative genomics for five clinical pairs of vancomycin-susceptible (VSSA) and vancomycin-intermediate ST239 S. aureus (VISA); each pair isolated before and after vancomycin treatment failure. These comparisons revealed a frequent pattern of mu ...[more]

PMID: 22102812

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Project description:Among the 16 two-component systems in the opportunistic human pathogen Staphylococcus aureus, only WalKR is essential. Like the orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodeling and is therefore intimately involved in cell division. However, despite the importance of WalKR in S. aureus, the basis for its essentiality is not understood and the regulon is poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including chromatin immunoprecipitation sequencing, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis (ltaS): translation (rplK), DNA compaction (hup), initiation of DNA replication (dnaA, hup) and purine nucleotide metabolism (prs). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by coordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Our findings further address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics. IMPORTANCE The opportunistic human pathogen Staphylococcus aureus uses an array of protein sensing systems called two-component systems (TCS) to sense environmental signals and adapt its physiology in response by regulating different genes. This sensory network is key to S. aureus versatility and success as a pathogen. Here, we reveal for the first time the full extent of the regulatory network of WalKR, the only staphylococcal TCS that is indispensable for survival under laboratory conditions. We found that WalKR is a master regulator of cell growth, coordinating the expression of genes from multiple, fundamental S. aureus cellular processes, including those involved in maintaining cell wall metabolism, protein biosynthesis, nucleotide metabolism, and the initiation of DNA replication.

Project description:The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene cluster, walRKJ.

Project description: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.

Project description:Vancomycin (VAN)-intermediate-resistant Staphylococcus aureus (VISA) is continually isolated globally, with a systematic review suggesting a prevalence of 2% in all blood culture samples. Most VISA strains exhibit common characteristics, such as a thickened cell wall, reduced autolysis, and attenuated virulence. Here, based on multi-omics approaches, we have characterized clinical VISA isolates obtained through prolonged antimicrobial treatment in a single patient. All VISA isolates were isogenic, based on multi-locus sequence typing (MLST) ST5, SCCmec type II (2A), and spa type t17639. Core-genome single nucleotide variations (SNVs) found among thirteen isolates during the patient's hospitalization, indicated clonality, but not notable genetic features of the VISA phenotype. We determined the complete genome sequence of VAN-susceptible strain KG-03 (minimum inhibitory concentration [MIC] 0.5 ?g/mL) and two VISA strains, KG-18 and KG-22 (MIC 8.0 and 4.0 ?g/mL, respectively). Comparative genome analysis showed remarkable strain-specific IS256 insertions. RNA-Seq transcriptome analysis revealed IS256-mediated overexpression of the walKR two-component system in VISA KG-18, possibly leading to modulation of cell wall integrity (lytM and sceD) and surface charge (mprF and dltABCD). In addition, secretome analysis indicated that cell wall-anchored proteins (Protein A, SasG, and SdrD) were significantly decreased. KG-18 and KG-22 exhibit thickened cell wall, and are relatively resistant to lysostaphin, which cleaves a staphylococcus-unique pentaglycine chain in the peptidoglycan. We conclude that KG-18 achieved reduced susceptibility to VAN by IS256-mediated WalKR overexpression, leading to a markedly thickened cell wall for trapping free VAN molecules with redundant D-Ala-D-Ala targets. In addition, a positively charged surface with lysyl-phosphatidylglycerol and depolarization of wall teichoic acid could contribute to inhibiting cationic daptomycin and VAN antimicrobial activity. Comparative omics approaches in this study strongly suggest that fully complete and annotated genome sequences will be indispensable for characterizing overall VISA phenotype.

Dataset Information

Evolution of multidrug resistance during Staphylococcus aureus infection involves mutation of the essential two component regulator WalKR.

Publications

Evolution of multidrug resistance during Staphylococcus aureus infection involves mutation of the essential two component regulator WalKR.

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