Overexpression of <scp>l</scp>,<scp>d</scp>-Transpeptidase A Induces Dispensability of Rod Complex in <i>Escherichia coli</i>

Antimicrobial resistance (AMR) is a significant global threat, and the presence of resistance-determinant genes is one of the major driving forces behind it. The bacterial rod complex is an essential set of proteins that is crucial for cell survival due to its role in cell wall biogenesis and shape maintenance. Therefore, these proteins offer excellent potential as drug targets; however, compensatory mutations in nontarget genes render this complex nonessential. The MreB protein of this complex is an actin homologue that rotates along the longitudinal axis of the cell to provide rod shape to the bacteria. In this study, using chemical-chemical interaction profiling and FtsZ suppression assay, we identified the MreB targeting activity of IITR07865, a previously discovered small molecule in our lab. <i>Escherichia coli</i> suppressors against IITR07865 revealed mutations in two cell division-associated genes, <i>min C</i> and <i>pal</i>, that have not been previously implicated in rod complex essentiality. IITR07865 resistant mutants were found to inactivate and render the rod complex nonessential, making the rod complex inhibitors ineffective. Further, through transcriptome analysis, we reveal the primary cause of resistance in suppressor strains to be the overexpression of an l, d-transpeptidase A enzyme, which is involved in peptidoglycan and Braun's lipoprotein cross-linking. Our results demonstrate a novel mechanism of resistance development in rod-shaped Gram-negative bacterial pathogen <i>E. coli</i> involved in UTIs where mecillinam, a clinically used antibiotic that targets rod complex, is a drug of choice.