Fig. 1: Proteomic analyses of GlpG identified substrates associated with bacterial virulence.

a Cytoscape network of periplasmic and inner membrane proteins identified after affinity pulldown of whole cell extract with glpG-3XFLAG followed by Triton ×-100 solubilization and LC-MS/MS reveal an enrichment of proteins involved in proteostasis. n = 3. b Experimental procedure for LC-MS/MS proteomics of E. coli periplasmic proteins. Created with BioRender.com. https://BioRender.com/85mschr. c The volcano plot showing decreased (blue) and increased (orange) protein abundance levels in extracts after incubation with active relative to inactive GlpG. We quantified 895 proteins with high reproducibility (~95%) between biological replicates n = 4. Protein quantification was performed using Proteome Discoverer(v 2.4.1.15). Protein abundance changes between periplasmic proteins incubated with active and inactive GlpG with a significant p-value below 0.05 (−log10 p-value > 1.30) and a fold change (FC) greater than two (log2 FC > 1), are presented. Lines represent the boundaries for a twofold change and a significant p-value of <0.05. Proteins were identified with the UniProt ID. The number of proteins quantified in one sample but not the other is identified in curly brackets. d In-gel digestion and mass spectrometric characterization of periplasmic proteins of K12 substr. MG1655 ΔglpG separated by SDS-PAGE reveal accumulation of aggregated FimA (>135 kDa MW range) when incubated with inactive GlpG, but no accumulation when incubated with active GlpG, n = 2. e Western blot of K12 WT or mutant periplasmic proteins separated by SDS-PAGE, detecting FimA aggregates. Periplasmic proteins were isolated from K12 wild-type, K12 mutant strains harboring single gene deletions: ΔglpG, ΔdegQ, ΔdegP, and rescues of the mutant strains overexpressing GlpG. K12 ΔfimA was used as a negative control. Biological replicates for western blot samples were repeated n = 3 for confirmation.