Sefkhet (sefkhet) wrote in biomed,
Sefkhet
sefkhet
biomed

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Dissertationing

I'll be graduating at the end of June, finals pending and all that, and a big part of my grade for third year (25%) comes from my dissertation. I started working on this at Christmas, before I went into the lab to do the actual investigation early this year, and I finally finished it late last night. My title is: Interaction of the Periplasmic Proteins of Escherichia coli with the Membrane Fusion Protein EmrA, and the whole project clocked in at 11,708 words. Just wanted to share the joy!

Clinical resistance to antimicrobial agents is a phenomenon that is seen with increasing frequence in the developed world, with many common human pathogens exhibiting resistance to antibiotics which have in the past been used as successful treatment strategies. One such pathogen is the bacterium Escherichia coli, which manifests itself in a number of diseases, including urinary tract infections, nosocomial bacteriaemia, neonatal meningitis, and paediatric enteritis. Resistance by various mechanisms is observed in E. coli bacteria, including active efflux from the cells mediated by a number of different efflux pumps that exist both as single entities and as tripartite complexes, with the latter being composed of an inner membrane protein, an outer membrane protein, and an outer membrane protein. One such tripartite complex is the antibiotic efflux pump EmrAB-TolC, a membrane of the proton-driven major facilitator superfamily.

The purpose of this investigation was to explore the hypothesis that other proteins from the periplasmic space of E. coli bind to the membrane fusion protein EmrA and that such interaction plays a significant role in the ability of this protein to mediate antibiotic efflux as a component of EmrAB-TolC. The investigation was carried out by overexpressing and purifying EmrA originating from transformant colonies that had been stored as glycerol stocks at -80oC, and performing a pull-down assay to demonstrate interaction between the EmrA and E. coli periplasmic proteins obtained by osmotic shock treatment. SDS-PAGE analysis of the results of this pull-down assay revealed what appeared to be two periplasmic proteins that had interacted with EmrA, but further analysis with MALDI-TOF showed one of these proteins to be an unknown transcriptional regulator from Vibrio cholerae, while it was not possible to identify the other.

It was concluded that the banding pattern observed on SDS-PAGE analysis was a false positive result, and that the results of this investigation as a whole were therefore inconclusive. In order to definitively determine whether or not the membrane fusion EmrA does bind to E. coli periplasmic proteins and therefore whether this is a factor in the efflux function of the tripartite complex EmrAB-TolC, further research is necessary. Elucidation of the mechanism by which these tripartite efflux pumps operate is imperative if a mechanism for inhibition is to be developed and the growing trend in clinical resistance halted.

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