Gram-negative bacteria (GNB) are major drivers of antimicrobial resistance (AMR) in humans and animals. These pathogens pose substantial economic and public health threats, emphasizing the need for a comprehensive understanding of resistance and pathogenic mechanisms in order to develop novel therapeutics and diagnostics. These mechanisms are primarily mediated by proteoforms, and therefore proteoform-level analyses of GNB offers a significant advantage in understanding bacterial pathogenesis, the mechanisms of AMR, and the identification of virulence factors.
Comprehensive proteomic analyses are dependent on efficient, robust sample preparation that extract the greatest number of proteoforms. Common extraction protocols include: (1) cell lysis and homogenization, (2) protein extraction and solubilization, and (3) the removal of unwanted analytes or contaminants (i.e. lipids, nucleic acids, salts etc.) that may interfere with downstream analyses. While GNB are relatively easy to lyse relative to other microbes (e.g Gram-positive or sporulated organisms), an efficient homogenization protocol is still required to effectively disrupt the rigid cell wall and membrane to extract proteoforms. Using Klebsiella pneumoniae and Escherichia coli, we systematically compare the extraction efficiency of bead-beating with flash frozen and lyophilized cell pellets from GNB.
Using shotgun-based proteomics, we demonstrate that lyophilization prior to protein extraction increases protein extraction yield, enriching for basic hydrophobic proteins and membrane proteins (i.e. open reading frame (ORF) products), including multidrug efflux pumps and proteins involved in lipopolysaccharide (LPS) synthesis. We detected 71 and 33 unique membrane proteins when lyophilizing prior to homogenisation compared to 10 and 7 membrane proteins detected when only flash freezing prior to homogenisation in E. coli and K. pneumoniae, respectively.
Membrane proteins play a central role in AMR resistance mechanisms, therefore improving the capacity to isolate and identify them can further aid in understanding the resistance mechanisms and molecular mechanisms associated with multidrug-resistant GNB.