Identification of the response pathways of Escherichia coli and Enterococcus faecalis to glyphosate and it's major breakdown product Aminomethyl phosphonic acid (AMPA)

Abstract

Glyphosate is the active ingredient in non-selective herbicides, and disrupts the shikimate pathway of plants and bacteria by inhibiting the synthesis of aromatic amino acids. Application of glyphosate has increased exponentially worldwide due to the increasing adoption of genetically modified crops. Studies have shown that residues of glyphosate and its major break-down product aminomethyl phosphonic acid (AMPA) accumulate in some genetically modified (GM) food crops including soybeans, cowpea, coffee, as well as meat and dairy products from cattle and pig. Glyphosate can also disperse into the ecosystem and reach water systems. Therefore, resident bacteria of the gut will be exposed, in their respective niches, to glyphosate and AMPA at varying sub-lethal concentrations. Limited studies have been conducted on the response of gut bacteria to glyphosate and AMPA at sub-lethal concentrations. This study is especially important because of the crucial role played by gut bacteria in human and animal health. The aim of this research was to employ proteomic approaches to analyse expression profiles of Escherichia coli and Enterococcus faecalis exposed to sub-lethal concentrations of glyphosate and AMPA. Extraction of proteins was done using a commercially available kit, followed by quantification with the bicinchoninic acid assay. The TheromFisher Scientific TMT Mass Tagging Kits and Reagents were employed for labelling peptides. Data analysis was done using mass spectreophotometer and Proteome Discovere software. Pathways were analysed and mapped using KEGG PATHWAY and STRING online protein databases. Glyphosate seem to greatly affect nitrate metabolism and iron uptake in E. coli through inhibition of respiratory nitrate reductase (NarGH) and up-regulation of enterobactin biosynthesis (EntA, B, E, F, H) and iron transport proteins (TonB, FepA, ExbD), respectively. AMPA exerts a similar response on iron uptake in E. coli. In E. faecalis glyphosate and AMPA interferes with translation through up-regulated of proteins involved in aminoacyl-tRNA biosynthesis. Interestingly, glyphosate and AMPA may induce oxidative stress at sub-lethal concentrations in E. coli and E. faecalis. At sub-lethal concentrations glyphosate and AMPA negatively affect energy and growth of E. coli through nitrate metabolism pathway and stress response pathway. Additionally, glyphosate and AMPA may stimulate pathogenesis in E. coli through increasing the bacteria iron scavenging potential. Glyphosate and AMPA interrupt cell proliferation in E. faecalis through antimicrobial activity on aminoacyl-tRNA biosynthesis pathway and ribosomes. Glyphosate and AMPA also caused changes in expression of hypothetical proteins in E. coli and E. faecalis, indicating that some physiological responses remain uncharacterized. Majority of deferentially expressed proteins are involved in energy metabolism, iron uptake and transport, carbohydrate metabolism, transport and stress response. An indication of a complex set of interactions indicating glyphosate affects pathways other than the shikimate pathway. These interactions serve an important role in bacterial fitness, survival, and adaptive resistance to antibacterial agents. Thus, glyphosate and AMPA may serve as environmental cue for antibiotic resistance, virulence expression and habitat adaptation of E. coli and E. faecalis.