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Genomic and Long-Term Transcriptomic Imprints Related to the Daptomycin Mechanism of Action Occurring in Daptomycin- and Methicillin-Resistant Staphylococcus aureus Under Daptomycin Exposure.

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.

SUBMITTER: Cafiso V

PROVIDER: S-EPMC7456847 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Genomic and Long-Term Transcriptomic Imprints Related to the Daptomycin Mechanism of Action Occurring in Daptomycin- and Methicillin-Resistant Staphylococcus aureus Under Daptomycin Exposure.

Cafiso Viviana V Stracquadanio Stefano S Lo Verde Flavia F De Guidi Irene I Zega Alessandra A Pigola Giuseppe G Stefani Stefania S

Frontiers in microbiology 20200814

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 remain ...[more]

PMID: 32922373

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Project description:Elasnin is a recently reported antibiofilm agent that is effective against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). Remarkably, we observed that elasnin has a superior activity in eradicating daptomycin-resistant MRSA strain biofilm, with a lower minimum biofilm eradication concentration (MBEC) value of 0.625 μg/mL, compared to 2.5 μg/mL for the wild type. Confocal microscopy further confirmed the higher biofilm eradication on the daptomycin-resistant strain, displaying ∼53% decrease in cell density upon elasnin treatment, while the wild-type strain was only decreased by ∼15%. Quantitative proteomics revealed that the daptomycin-resistant strain has a lower expression of the membrane, cell wall, and extracellular proteins, and also proteins involved in the arginine biosynthesis, pathogenesis, and cell adhesion compared to the wild type, which may result in weaker biofilm development. This study highlights the potential clinical application of elasnin through its superior biofilm eradication activity against a daptomycin-resistant MRSA strain, and revealed the associated processes governing this superior activity through proteomics analysis. IMPORTANCE Due to the increased use of daptomycin for the treatment of MRSA infections, the emergence of daptomycin-resistant strains has become prevalent in recent years. In this study, we discovered that elasnin, a newly reported antibiofilm compound, has a superior activity in eradicating daptomycin-resistant MRSA strain biofilms compared to the wild type. Follow-up analysis revealed the reason behind this superior activity, which is the lower expression of key proteins that play a role in pathogenesis and cell adhesion in the daptomycin-resistant strain, leading to weaker biofilm development. This showcases the potential use of elasnin in clinical settings where daptomycin-resistant strains and biofilm formation are prevalent. Altogether, our study provides new insights into the mechanism of elasnin in MRSA biofilm cells and identified its superior biofilm eradicating activity in the daptomycin-resistant strain.

Project description:Methicillin-resistant Staphylococcus aureus (MRSA) threatens human health in hospital and community settings. The lipopeptide antibiotic daptomycin (DAP) is a frequently used treatment option for MRSA infection. DAP exposure can cause bacterial resistance because mutations are induced in genes implicated in cell membrane and cell wall metabolism. Adaptations aimed at surviving antimicrobial pressure can affect bacterial physiology and modify in vivo aptitude and pathogenesis. In this study, clinical DAP-susceptible (DAPs) and DAP-resistant (DAPr) MRSA isolates were used to investigate associations between DAP resistance and staphylococcal virulence. We previously found that VraSR is a critical sensor of cell membrane/wall homeostasis associated with DAP acquisition during MRSA infection. The present study found that DAPr CB1634 and CB5014 MRSA strains with vraSR upregulation were less virulent than their susceptible counterparts, CB1631 and CB5013. Differential gene-transcription profile analysis revealed that DAPr CB1634 had decreased agr two-component system expression, virulence factors, and highly suppressed hemolysis activity. Functional genetic analysis performed in DAPr CB1634 strains using vraSR inactivation followed by gene complementation found that vraSR acted as a transcriptional agrA regulator. These results indicated that VraSR has a broad range of regulatory functions. VraSR also appeared to affect DAPr adherence to epithelial cells, which would affect DAPr strain colonization and survival in the host. The correlation between DAP resistance and decreased virulence was also found in the CB5013 (DAPs) and CB5014 (DAPr) pair. Taken together, these findings are the first evidence that DAP resistance and MRSA virulence are tightly connected and involve compromised expression of regulatory and virulence determinants.IMPORTANCE Methicillin-resistant S. aureus continues to develop resistance to antimicrobials, including those in current clinical use as daptomycin (DAP). Resistance to DAP arises by mutations in cell membrane and cell wall genes and/or upregulation of the two-component VraSR system. However, less is known about the connection between the pathogen and virulence traits during DAP resistance development. We provide new insights into VraSR and its regulatory role for virulence factors during DAP resistance, highlighting coordinated interactions that favor the higher persistence of MRSA DAP-resistant strains in the infected host.

Dataset Information

Genomic and Long-Term Transcriptomic Imprints Related to the Daptomycin Mechanism of Action Occurring in Daptomycin- and Methicillin-Resistant Staphylococcus aureus Under Daptomycin Exposure.

Publications

Genomic and Long-Term Transcriptomic Imprints Related to the Daptomycin Mechanism of Action Occurring in Daptomycin- and Methicillin-Resistant <i>Staphylococcus aureus</i> Under Daptomycin Exposure.

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