Abstract of Master’s Dissertation
No.1
CourseMaster of TropicalMedicine

NameAugustin Tshibaka
KABONGO

Thesis“Purification and biochemical characterization of malate: quinone

Title

oxidoreductase from Campylobacter jejuni, a potential drug target”

Abstract of Master’s Dissertation
Background: Campylobacter jejuni is the most common cause of bacterial foodborne illness

worldwide. Infection is asymptomatic in most patients, and occasionally this disease leads toseverecomplications

suchasGuillain-Barrésyndrome.

Recently,resistanceofCampylobacter spp. to antibiotics commonly used for clinical treatment has been reported.
Therefore, the development of new drugs is an important strategy, along with othermeasures. There is a growing interest in electron transport chains (ETC) of microorganisms
as drug target. Many bacterial species express malate:quinone oxidoreductase (MQO); thisenzyme catalyses the oxidation of malate to oxaloacetate, a crucial step in the tricarboxylic
acid cycle, but also transfers electrons to ETC, contributing to cellular bioenergetics. MQO is
an interesting drug target because it is not conserved in mammalian cells and has beenfound to be essential for survival in several pathogens. In C. jejuni, the gene encoding MQO
can be identified in the genome, but no study has been conducted to characterize thisenzyme or evaluate its potential as a drug target.
Objective : This thesis aims to investigate and evaluate MQO from C. jejuni (CjMQO) as anovel drug target.
Methods: Putative CjMQO sequence was codon optimized for expression in Escherichia coli.

TOPO cloning vector (pET101/pET151) was used to prepare the expression plasmids. The
positive colonies were selected using carbenicillin as the marker with plasmid purified,sequenced and confirmed. The plasmid harbouring the correct sequence was used totransform the conventional E. coli expression system [NiCo21(DE3)]. Next, the CjMQO wasoverexpressed on the membrane fractions of NiCo21(DE3). Furthermore, I have optimized
the protocol for CjMQO purification and conducted a biochemical characterization. Finally,
the effect of two classic inhibitors of MQOs (ferulenol and embelin) were assessed on theactivity of CjMQO and growth of C. jejuni strain JCM2013 in vitro.

＊ The abstract, containing background, objectives, methods, results and conclusion should notexceed 300-500words and printed double sided on A4 paper)

Abstract of Master’s Dissertation
No.2
CourseMaster of TropicalMedicine

Name

Augustin Tshibaka
KABONGO

Results : In solution, the purified CjMQO exhibited dimeric and tetrameric states. It had

an optimal temperature of 40°C and a pH of 7. CjMQO showed higher affinity for
ubiquinones (UQ) with long side chains and a maximum velocity of 48.8, 106.7, 111.4, 9.9,
76.3 µmol/min/mg for UQ0, UQ1, UQ2, UQ4, and decyl-UQ (dUQ), respectively. Substrateinhibition of CjMQO was observed above 10 µM for UQ2 and dUQ and 2 µM for UQ4.
CjMQO showed a bisubstrate sequential Bi Bi reaction mechanism. Ferulenol and embelin
inhibited CjMQO at IC50 of 0.018 and 0.087 µM, and the growth of C. jejuni JCM2013 atIC50 of 0.322 and 20.130 µM, respectively.
Conclusion : This is the first study reporting the purification of an active MQO from C.

jejuni and its biochemical characterization. Interestingly, CjMQO displayed specificactivity higher than the one reported for Plasmodium falciparum and Toxoplasma gondii
mitochondrial MQOs, and is likely essential for C. jejuni survival and an important drugtarget, as growth of this pathogen was severely impaired in the presence of both MQO
inhibitors. Nevertheless, future studies are needed to further elucidate the physiologicalrole of this enzyme in C. jejuni, and validate it as a drug target.
Key words: malate:quinone oxidoreductase; purification; biochemical characterization;
drug target.
Key words: malate:quinone oxidoreductase; purification; biochemical characterization;

drug target

Word count : 500