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Site-directed mutagenesis of charged amino acid residues on the CpxASD and CpxP in E. coli, potentially important for signaling and direct interaction
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- Author / Creator
- Margain Quevedo, Rodrigo
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Bacterial pathogens must endure diverse environmental stresses that they encounter while colonizing and infecting a host. Two-component signal transduction systems (TCSTs) are the most widespread regulatory systems in bacteria, but importantly absent in mammals. In general, TCSTs sense and interpret stimuli, then use such information to respond by modulating the repertoire of genes expressed, allowing to ultimately mediate adaptations for survival. The CpxRA TCST envelope stress response (ESR) system in Escherichia coli and other Gram-negatives has been linked to virulence factors, antibiotic resistance and envelope stress. The CpxRA system is made up of the sensor histidine kinase (HK) CpxA, the cytosolic response regulator (RR) CpxR, and the periplasmic accessory inhibitor CpxP. In the absence of inducers, the periplasmic sensor domain of CpxA has been proposed to interact with CpxP, inhibiting CpxA autokinase activity and causing it to act as a phosphatase for CpxR~P dephosphorylation, maintaining the pathway in an off state. Intriguingly, the mechanism by which CpxA detects and transduces signals, as well as the one allowing for it to directly interact with CpxP, are still not completely understood. In the present study, I sought to determine whether some of the recently identified surface-exposed amino acid residues comprising large negatively charged patches in the CpxASD of E. coli are important for pathway activation and partner interaction (specifically with CpxP). Experimental analyses were performed by following a site-directed specific mutational approach, based on structural and electrostatic analysis of the CpxASD. The effects of cpxA lysine and alanine mutations were analyzed by transforming them into a ΔcpxA E. coli strain. Such strains were grown in either acid, neutral or alkaline pH, or in the presence of nlpE or cpxP overexpression. Cpx activity levels were then determined using a Cpx-regulated lacZ reporter gene (cpxP'-lacZ+) to indirectly measure pathway activation through β-galactosidase assays. The cpxA strains harboring the E91K, D113K or E91K+D113K mutated alleles displayed constitutive pathway hyperactivation activities and lost sensitivity toward overproduction of CpxP and NlpE. Protein levels however did not seem to be affected by the introduced mutations. I also investigated the potential for specific positively charged residues in CpxP to be direct interaction points of the previosuly characterized negatively charged residues in CpxASD, using complementary charge mutations. I determined that a specific direct-contact site could be located between the D113 negatively charged amino acid residue in the CpxASD and the R139 positively charged residue in the CpxP concave surface. This represents the first time in which a specific localized interaction site between two amino acid residues of the CpxA and CpxP components has been described. A better understanding and characterization of such interaction could open the path for targeted inhibition of the pathway in the future, leading to alternative therapeutic treatments for diseases such as infectious diarrhea.
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- Graduation date
- Spring 2019
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- Type of Item
- Thesis
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- Degree
- Master of Science
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- License
- Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.