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EFFECT OF METABOLIC INHIBITION ON THE GROWTH AND BIOFILM PRODUCTION OF VIBRIO CHOLERAE AND PSEUDOMONAS AERUGINOSA
Title
EFFECT OF METABOLIC INHIBITION ON THE GROWTH AND BIOFILM PRODUCTION OF VIBRIO CHOLERAE AND PSEUDOMONAS AERUGINOSA
Creator
Bunn, Dakota C.
Date
2017, 2017-05
Description
V. cholerae is a gastrointestinal pathogen which causes extreme watery diarrhea and results in over 120,000 deaths per year worldwide. It is...
Show moreV. cholerae is a gastrointestinal pathogen which causes extreme watery diarrhea and results in over 120,000 deaths per year worldwide. It is especially prevalent in developing countries that lack proper water treatment and in areas struck by natural disasters such as hurricanes. P. aeruginosa is an opportunistic pathogen that is ubiquitous in nature, and increasingly found in hospitals burn wards, sinks, catheters and other surgical equipment. Both bacteria are developing increased antibiotic resistance through several mechanisms, with one of the most common ones being the formation of a complex exopolysaccharide matrix known as a biofilm. In this study, using metabolic inhibition, we determined that Na+-NQR is essential for the growth of V. cholerae and P. aeruginosa in both nutrient rich and physiological conditions. We were also able to confirm that inhibition of this enzyme, in both growth conditions, resulted in decreased biofilm production, subsequently eliminating one of the main mechanisms for antibiotic resistance of these bacteria.
M.S. in Biology, May 2017
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Characterization of the role of His257 of vibrio cholerae ApbE in the flavin transfer reaction
Title
Characterization of the role of His257 of vibrio cholerae ApbE in the flavin transfer reaction
Creator
Yuan, Ming
Date
2019
Description
ApbE is a novel enzyme that transfers flavin cofactors into subunits NqrB and NqrC of the sodium-dependent NADH dehydrogenase (Na+-NQR). As...
Show moreApbE is a novel enzyme that transfers flavin cofactors into subunits NqrB and NqrC of the sodium-dependent NADH dehydrogenase (Na+-NQR). As the first enzyme of the bacterial respiratory chain, the function of Na+-NQR affects the survival and development of pathogenicity in many disease-causing bacteria, including Vibrio cholerae. Our preliminary studies indicate that His257 plays a key role in the catalytic activity of ApbE, and that it is an essential component in the transfer of FMN to NqrC. In order to further study how His257 is specifically involved in the catalytic reaction of ApbE, we produced and characterized four mutants: H257G, H257E, H257K, and H257T; in the presence of the activator, K+. Our data showed that mutants H257E and H257K present minimal flavin transfer activity. Interestingly, the mutants H257G and H257T showed activity several times higher compared to the other mutants, however, their activities were still smaller when compared to wild-type. The data suggests that His257 has a very important role for ApbE activity, but that it is not essential. Furthermore, steady-state kinetics showed that the mutants have similar substrate KM values with the wild-type. In addition, double reciprocal plots from bi-substrate titrations showed that ApbE follows a sequential kinetic mechanism where a ternary complex is formed during the reaction.
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Kinetic and Structural Characterization of the Vibrio cholerae Flavin Transferase ApbE
Title
Kinetic and Structural Characterization of the Vibrio cholerae Flavin Transferase ApbE
Creator
Fang, Xuan
Date
2019
Description
Cholera has long been a global concern and in the past decades traditional antibiotic treatments have failed due to the emergence of the...
Show moreCholera has long been a global concern and in the past decades traditional antibiotic treatments have failed due to the emergence of the antibiotic-resistance of its causative agent, V. cholerae. The resistance is mainly supported by a transmembrane electrochemical gradient of Na+ produced by the respiratory complex Na+-NQR coupled with an internal electron transfer pathway. The assembly and function of Na+-NQR is fulfilled by ApbE, the only known flavin transferase which covalently attaches two FMN molecules to the complex as part of its electron transport chain. Hence, ApbE is closely associated with the cause of antibiotic resistance. Because it does not have any human homologues, ApbE becomes an excellent drug target. In this work, we have investigated the physical properties of the enzyme and clarified its substrate specificity and pH dependence. For instance, our experiments indicate that divalent cations are essential for ApbE function, and that the selectivity depends largely on the size and the coordination sphere of the cation. Our data also show that ApbE regulation by pH, ADP and potassium is an important mechanism that enhances the adaptation, survival and colonization of V. cholerae in the small intestine. Moreover, pH dependence, mutagenesis, and steady-state kinetic studies have led us to identify the conserved His257 as a residue with dual roles: substrate binding and catalysis. Furthermore, bi-substrate kinetic studies have also revealed that ApbE follows a random Bi Bi mechanism. Together with structural studies, we propose a reaction mechanism where His257 functions as a base, shedding light into the understanding of the ApbE family.
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IDENTIFICATION OF THE RIBOFLAVIN BINDING SITE IN VIBRIO CHOLERAE ION PUMPING NQR COMPLEX
Title
IDENTIFICATION OF THE RIBOFLAVIN BINDING SITE IN VIBRIO CHOLERAE ION PUMPING NQR COMPLEX
Creator
Lee, Chia-Hsing
Date
2021
Description
NQR is a six-subunit complex that transfers electrons from NADH to ubiquinone, one of the essential enzymes in the bacterial respiratory chain...
Show moreNQR is a six-subunit complex that transfers electrons from NADH to ubiquinone, one of the essential enzymes in the bacterial respiratory chain of many pathogens such as Vibrio cholerae, Pseudomonas aeruginosa, Chlamydia trachomatis. Its electron transfer path requires three different flavin cofactors to facilitate: FAD, FMN, and riboflavin. The FMN in subunit B (FMNB) brings electrons to riboflavin and then transfers it to the final electron receptor UQ in subunit B, coupled with the Na+ pumping mechanism. NQR has a unique evolutionary history, and one of the pieces of evidence is that NQR has been reported as the only one flavoenzyme that uses riboflavin as its redox cofactor. However, the binding site of riboflavin has not been well understood. To gain insight into the electron transfer at this site in V.cholerae NQR, we generated mutants at the interface of subunits B, D, and E where the possible location of riboflavin is. To characterize these mutants, we assessed NQR properties with different approaches including enzyme kinetics and flavin radical profiling. We found that the mutagenesis surrounding the hydrophobic pocket disrupted the NQR activity, and cause the loss of neutral radical, but did not interfere with the binding affinity between the substrates and NQR. This study will help to understand electron transfer better in NQR and develop the drugs targeting the riboflavin binding site in the future.
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