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Quantitative proteome analysis of an Escherichia coli exposed to tetracycline reveals multiple affected cytoplasmic metabolic processes
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Advisor(s)
Abstract(s)
Background: Many studies have already focused on the evaluation of variations in protein expression
caused by antibiotic exposure in tetracycline resistant microorganisms. However, little is known about
the metabolic response of genetically resistant bacterial populations to antibiotic exposure. In this
study we used a liquid chromatography-mass spectrometry-based (LC-MS/MS) proteomics to evaluate
the global cytoplasmic metabolic changes of a resistant Escherichia coli isolate, when challenged with
tetracycline.
Material/methods: IsolateEcAamb278 was recovered from a soil sample, used for the intensive
farming of tomato plants and showed nonsusceptibility to all categories of β-lactams except
carbapanems and cefoxitin, and tetracycline. Previous genomic characterization revealed the
presence of TEM-1, CTX-M-1, Sul2,TetA and TetB resistance determinants. Single colonies of E.
coliEcAamb278 were used to extract crude cytoplasmic protein fraction using non detergent cell lysis
buffer. The proteomic profile of E. coli EcAmb278 was determined by label-free LC-MS/MS upon
tetracycline stress in a comparative study, using as referencethe proteome of the same strain nonexposed
to antibiotics. The samples were analyzed by nano LC-MS/MS, using a Q-Exactive mass
spectrometer. All data was searched with VEMS. Proteins were quantified by spectral counting and
mziXIC, followed by iBAQ estimation.
Results: The complete proteome yielded about 1484 proteins, using a 1% FDR as cut off. The
comparison of the proteome profiles of the two E. coli EcAamb278 samples pointed to several proteins
with altered expression under tetracycline stress conditions. The twelve most significant (FDRcorrected
p<0.05) proteins differentially regulated by more than two-fold were involved in DNA
metabolism, transcription, virulence, intracellular trafficking and secretion, biosynthesis of vitamins,
other processes, and unknown functions. For instance, (1) acyl-CoA thioester hydrolase, involved in
biosynthesis of coenzyme A; (2) bacterial NAD+-dependent DNA ligase, plays a critical role in DNA
replication, recombination and repair in all living organisms; and (3) adhesion protein FimH, which
confirmed the pathogenicity of this environmental-borne E. coli. Overall, several other detected
proteins were associated with the process of antibiotic resistance, virulence and acquisition and
transfer of foreign DNA (AcrA, TolC, MdtE, Omps, TnsE, Colicin peptides, among others).
Conclusions: These results indicate that E. coli responses to tetracycline are related to protein
translation as well as metabolic regulation. We observed differentially regulation of several metabolic
proteins though not belonging to the canonical antibiotic resistance pathway, suggesting an integrated metabolic bacteria response to antibiotic exposure, which can potentially be explored for drug
development.
Description
Keywords
Antibacterial Resistance Mechanisms Proteomics Escherichia coli Tetracycline Stress Resistência aos Antimicrobianos
Pedagogical Context
Citation
Jones-Dias, D., Carvalho, A. S., Moura, I. B., Dill, B. D., Molina, H., Manageiro, V., Caniça, M., Matthiesen, R. Quantitative proteome analysis of an Escherichia coli exposed to tetracycline reveals multiple affected cytoplasmic metabolic processes. 2016. 26th European Congress of Clinical Microbiology and Infectious Diseases, Amsterdam, Netherlands. P0770.