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Mechanistic insight into the regulation of virulence factors in pathogenic E. coli by rhomboid protease GlpG

ABSTRACT: With the rise in antimicrobial resistance, understanding the virulence factors utilized by pathogenic E. coli is essential for the development of alternative therapeutics. While previous work has shown that disruption of the rhomboid protease gene glpG leads to defects in bacterial colonization, here we provide mechanistic insight into the loss of fitness. We show GlpG is essential for the formation of virulence factors, namely type 1 pili, required for the colonization of eukaryotic cells. As adhesion is critical for biofilm formation and bacterial persistence, the absence of GlpG proteolytic activity also reduces the production of biofilm. Working towards new potential antimicrobial targets for treating infections, we show that biofilm formation is hampered by GlpG inhibition. Our data demonstrates that GlpG plays a key role in protein quality control of virulence factors and alters the paradigm for GlpG proteolysis, previously implicated in the cleavage of only membrane embedded substrates.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Escherichia Coli K-12 (ncbitaxon:83333)

SUBMITTER: M. Joanne Lemieux

PROVIDER: MSV000097249 | MassIVE | Thu Mar 06 15:37:00 GMT 2025

REPOSITORIES: MassIVE

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Project description:Honey has been widely used against bacterial infection for centuries. Previous studies suggested that honeys in high concentrations inhibited bacterial growth due to the presence of anti-microbial compounds, such as methylglyoxal, hydrogen peroxide, and peptides. In this study, we found that three honeys (acacia, clover, and polyfloral) in a low concentration as below as 0.5% (v/v) significantly suppress virulence and biofilm formation in enterohemorrhagic E. coli O157:H7 affecting the growth of planktonic cells while these honeys do not harm commensal E. coli K-12 biofilm formation. Transcriptome analyses show that honeys (0.5%) markedly repress quorum sensing genes (e.g., AI-2 import and indole biosynthesis), virulence genes (e.g., LEE genes), and curli genes (csgBAC). We found that glucose and fructose in honeys are key compounds to reduce the biofilm formation of E. coli O157:H7 via suppressing curli production, but not that of E. coli K-12. Additionally, we observed the temperature-dependent response of honeys and glucose on commensal E. coli K-12 biofilm formation; honey and glucose increase E. coli K-12 biofilm formation at 37Â°C, while they decrease E. coli K-12 biofilm formation at 26Â°C. These results suggest that honey can be a practical tool for reducing virulence and colonization of the pathogenic E. coli O157:H7, while honeys do not harm commensal E. coli community in the human. For the microarray experiments, E. coli O157:H7 EDL933 was inoculated in 250 ml of LB in 1000 ml flasks with overnight cultures that were diluted at 1:100. Cells were shaken with 10g of glass wool at 100 rpm and 37Â°C for 7 hrs. Cells were immediately chilled with dry ice and 95% ethanol (to prevent RNA degradation) for 30 sec before centrifugation in 50 ml centrifuge tubes at 13,000 g for 2 min; cell pellets were frozen immediately with dry ice and stored -80Â°C. RNA was isolated using Qiagen RNeasy mini Kit (Valencia, CA, USA). To eliminate DNA contamination, Qiagen RNase-free DNase I was used to digest DNA. RNA quality was assessed by Agilent 2100 bioanalyser using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and quantity was determined by ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., DE, USA).

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Mechanistic insight into the regulation of virulence factors in pathogenic E. coli by rhomboid protease GlpG

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