A whole-genome assay identifies four principal gene functions that confer tolerance of meropenem stress upon Escherichia coli

Abstract

We report here the identification of four gene functions of principal importance for the tolerance of meropenem stress in Escherichia coli: cell division, cell envelope synthesis and maintenance, ATP metabolism, and transcription regulation. The primary mechanism of β-lactam antibiotics such as meropenem is inhibition of penicillin binding proteins, thus interfering with peptidoglycan crosslinking, weakening the cell envelope, and promoting cell lysis. However, recent systems biology approaches have revealed numerous downstream effects that are triggered by cell envelope damage and involve diverse cell processes. Subpopulations of persister cells can also arise, which can survive elevated concentrations of meropenem despite the absence of a specific resistance factor. We used Transposon-Directed Insertion Sequencing with inducible gene expression to simultaneously assay the effects of upregulation, downregulation, and disruption of every gene in a model E. coli strain on survival of exposure to four concentrations of meropenem. Automated Gene Functional Classification and manual categorization highlighted the importance at all meropenem concentrations of genes involved in peptidoglycan remodeling during cell division, suggesting that cell division is the primary function affected by meropenem. Genes involved in cell envelope synthesis and maintenance, ATP metabolism, and transcriptional regulation were generally important at higher meropenem concentrations, suggesting that these three functions are therefore secondary or downstream targets. Our analysis revealed the importance of multiple two-component signal transduction mechanisms, suggesting an as-yet unexplored coordinated transcriptional response to meropenem stress. The inclusion of an inducible, transposon-encoded promoter allowed sensitive detection of genes involved in proton transport, ATP production and tRNA synthesis, for which modulation of expression affects survival in the presence of meropenem: a finding that would not be possible with other technologies. We were also able to suggest new targets for future antibiotic development or for synergistic effects between gene or protein inhibitors and existing antibiotics. Overall, in a single massively parallel assay we were able to recapitulate many of the findings from decades of research into β-lactam antibiotics, add to the list of genes known to be important for meropenem tolerance, and categorize the four principal gene functions involved.