A cell-free strategy for profiling intracellular antibiotic sensitivity and resistance – characterising the proteomes of K. pneumoniae ATCC 13882, ST258-T1b and NJST258-1 and E. coli K-12 MG1655.
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ABSTRACT: Antimicrobial resistance (AMR) is a pandemic spread across multiple infectious disease microbes. To provide a new tool to study AMR, here we develop a Klebsiella pneumoniae cell-free gene expression (CFE) system. To characterise the system, we use proteomics to compare this to a Escherichia coli MG1655 CFE model, to identify relative differences and unique proteins. Then we use this native CFE system to profile antimicrobial activity in comparison to whole cell inhibition, to reveal host differences in IC50/MIC50 values. Finally, we use the CFE tool to study AMR variants, at a proof-of-concept level. As an exemplar, we show that RpoB H526L confers a 58-fold increase in CFE resistance to rifampicin – a common genotype frequently observed in rifampicin-resistant Mycobacterium tuberculosis clinical isolates. In summary, we provide a cell-free synthetic biology strategy for the profiling of antibiotic sensitivity and resistance from K. pneumoniae. While initial processing requires Biosafety Level 2, the final extracts are non-living and suitable for long-term storage, and potentially transfer to a Biosafety Level 1 lab. This bioassay has potential uses for early-stage host-specific antimicrobial development and the testing of AMR variants for structure-activity relationship studies. The data reposited is label-free high-resolution LC-MS proteomics data performed to characterise the proteins in cell-free extract of K. pneumoniae ATCC 13882 and compare to that of E. coli MG1655 to identify common and unique proteins. We also characterised the proteins of K. pneumoniae clinically resistant isolates ST258-T1b and NJST258-1, and compared them to K. pneumoniae ATCC 13882 laboratory strain.
INSTRUMENT(S): Synapt MS, MALDI Synapt MS
ORGANISM(S): Bacteria Escherichia Coli Str. K-12 Substr. Mg1655
TISSUE(S): Cell Culture
SUBMITTER: KAMESHWARI CHENGAN
LAB HEAD: Dr Simon Moore
PROVIDER: PXD046864 | Pride | 2023-11-29
REPOSITORIES: Pride
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