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Abstract
Daptomycin (DAP) is one of the last-resort treatments for heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) and vancomycin-intermediate S. aureus (VISA) infections. DAP resistance (DAP-R) is multifactorial and mainly related to cell-envelope modifications caused by single-nucleotide polymorphisms and/or modulation mechanisms of transcription emerging as result of a self-defense process in response to DAP exposure. Nevertheless, the role of these adaptations remains unclear. We aim to investigate the comparative genomics and late post-exponential growth-phase transcriptomics of two DAP-resistant/DAP-susceptible (DAPR/S) methicillin-resistant S. aureus (MRSA) clinical strain pairs to focalize the genomic and long-term transcriptomic fingerprinting and adaptations related to the DAP mechanism of action acquired in vivo under DAP pressure using Illumina whole-genome sequencing (WGS), RNA-seq, bioinformatics, and real-time qPCR validation. Comparative genomics revealed that membrane protein and transcriptional regulator coding genes emerged as shared functional coding-gene clusters harboring mutational events related to the DAP-R onset in a strain-dependent manner. Pairwise transcriptomic enrichment analysis highlighted common and strain pair-dependent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, whereas DAPR/S double-pair cross-filtering returned 53 differentially expressed genes (DEGs). A multifactorial long-term transcriptomic-network characterized DAPR MRSA includes alterations in (i) peptidoglycan biosynthesis, cell division, and cell-membrane (CM) organization genes, as well as a cidB/lytS autolysin genes; (ii) ldh2 involved in fermentative metabolism; (iii) CM-potential perturbation genes; and (iv) oxidative and heat/cold stress response-related genes. Moreover, a D-alanyl–D-alanine decrease in cell-wall muropeptide characterized DAP/glycopeptide cross-reduced susceptibility mechanisms in DAPR MRSA. Our data provide a snapshot of DAPR MRSA genomic and long-term transcriptome signatures related to the DAP mechanism of action (MOA) evidencing that a complex network of genomic changes and transcriptomic adaptations is required to acquire DAP-R.
Highlights
Methicillin-resistant Staphylococcus aureus (MRSA) remains one of the major multidrug-resistant pathogens responsible for severe infections with high mortality rates (Stefani et al, 2015; Yoon et al, 2016; Britt et al, 2017)
Previous investigations on the same DAPR/S MRSA strain pairs characterized some traits related to DAP resistance (DAP-R), including a dysregulation in the two key determinants of net positive surface charge, dltA and mprF, both during exponential and stationary growth phases; a significant increase in the D-alanylated wall teichoic acids amount correlating with DltA gain-in-function; a heightened elaboration of lysyl-phosphatidylglycerol reflecting MprF gain-in-function; an increased cell membrane (CM) fluidity; a straindependent CM fatty acid perturbation due to an increase in the anteiso-branch chain species corresponding to a reduction in the major iso-branched chain and saturated fatty acids (SFAs); and a reduced susceptibility to prototypic cationic host defense peptides of platelet and leukocyte origins (Cafiso et al, 2014; Mishra et al, 2014; Boudjemaa et al, 2018)
Our data define the complex genomic and long-term transcriptomic fingerprinting and adaptations of DAPR MRSA, providing new insights into their distinctive traits focusing on targets related to DAP-mechanism of action (MOA)
Summary
Methicillin-resistant Staphylococcus aureus (MRSA) remains one of the major multidrug-resistant pathogens responsible for severe infections with high mortality rates (Stefani et al, 2015; Yoon et al, 2016; Britt et al, 2017). The model of DAP-MOA proposes that DAP binds the cytoplasmic membrane leading to its permeabilization and depolarization caused by a loss of cytoplasm potassium ions (Allen et al, 1987). This mechanism, which can account for DAP’s bactericidal effect, would correlate with several changes in the membrane components, for example, with the level of phosphatidylglycerol. In DAPR strains, yycFG can accumulate mutations (Friedman et al, 2006; Howden et al, 2011), impairing the YycFG function and altering CW homeostasis to survive the DAP action, that is, lowering peptidoglycan turnover and increasing cross-linking. A621E mutation in RpoB was associated with an increased expression of the dlt-operon and correlated with an increase in positive cell-surface charge, whereas A621E and A477D substitutions were linked to an increased CW biosynthesis and thickness (Cui et al, 2010; Bæk et al, 2015)
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