Multi-strain Tn-Seq reveals common daptomycin resistance determinants in Staphylococcus aureus

Abstract

Antibiotic-resistant Staphylococcus aureus remains a leading cause of antibiotic resistance-associated mortality in the United States. Given the reality of multi-drug resistant infections, it is imperative that we establish and maintain a pipeline of new compounds to replace or supplement our current antibiotics. A first step towards this goal is to prioritize targets by identifying the genes most consistently required for survival across the S. aureus phylogeny. Here we report the first direct comparison of multiple strains of S. aureus via transposon sequencing. We show that mutant fitness varies by strain in key pathways, underscoring the importance of using more than one strain to differentiate between core and strain-dependent essential genes. We treated the libraries with daptomycin to assess whether the strain-dependent differences impact pathways important for survival. Despite baseline differences in gene importance, several pathways, including the lipoteichoic acid pathway, consistently promote survival under daptomycin exposure, suggesting core vulnerabilities that can be exploited to resensitize daptomycin-nonsusceptible isolates. We also demonstrate the merit of using transposons with outward-facing promoters capable of overexpressing nearby genes for identifying clinically-relevant gain-of-function resistance mechanisms. Together, the daptomycin vulnerabilities and resistance mechanisms support a mode of action with wide-ranging effects on the cell envelope and cell division. This work adds to a growing body of literature demonstrating the nuanced insights gained by comparing Tn-Seq results across multiple bacterial strains.

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

“…Our data, supported by previous findings on laboratory induced DAP R MRSA of specific clones HG003, USA300-TCH1516, MSSA476, MW2, and MRSA252 mutants (Coe et al, 2019), first described and defined in two clinical DAP R/S MRSA of genomic backgrounds (ST8 and ST398) strain-dependent shared and functional protein clusters as hot spots of genomic variations. In particular, membrane protein (transmembrane proteins, lipoproteins, and transporters), transcriptional regulator (Sigma-70, RpoB, RpoC, RsbU, GraX, SarR, SarU, SarX, ArlS, WalK, AgrC/A, MsrR, Msa, KdpD, SAOUHSC_02390, and SAOUHSC_00673), and cell-envelope modification (Sle1, UgtP, DltB, FmtA, LspA, Cls1, MprF, and SAOUHSC_01063) protein coding-gene clusters emerged as accumulation sites of mutational events related to the DAP-R onset, randomly occurring in the same gene.…”

Lipoprotein N -Acylation in Staphylococcus aureus Is Catalyzed by a Two-Component Acyl Transferase System

“…Construction of bacterial deletion strains and plasmids. Gene deletions were constructed using the temperature-sensitive shuttle vector pKFC in S. aureus, as previously described (84,85). Plasmids were assembled from two separate 1-kb DNA fragments flanking the gene targeted for deletion and obtained by PCR.…”

Bacillus anthracis Responds to Targocil-Induced Envelope Damage through EdsRS Activation of Cardiolipin Synthesis

“…Although the mechanism by which cardiolipin protects against daptomycin remains unclear, a follow-up study found that addition of exogenous cardiolipin to liposomes diminished the pore-forming activity of daptomycin (55). However, a transposon insertion sequencing (Tn-Seq) study in S. aureus found that mutation of a cardiolipin synthase conferred resistance to daptomycin (79). These results suggests that bacterial cells can increase the abundance of cardiolipin within their outer membranes to repair damage by antimicrobials that breach this barrier, similarly to the findings presented here in B. anthracis, but that the consequence of these modifications can be species specific.…”