Abstract of Master’s Dissertation
No.1
CourseHealth Innovation

Name

Tee Joseph

ThesisDevelopment of transgenic Leishmania major expressing, red-shifted

title

luciferase/mNeonGreen

Abstract of Master’s Dissertation
Background: Leishmaniasis is an infectious disease affecting at least 12 million people
globally and 350 million people are at risk of this infection. According to the World HealthOrganisation (WHO), it is one of the neglected tropical diseases caused by more than 20
Leishmania spp. and transmitted to mammalian hosts by sand fly vector. Ambisome, a drug
used for the treatment of leishmaniasis works well for visceral leishmaniasis (VL) while
Miltefosine, an expensive oral drug, treats all forms of leishmaniasis. However, the drug
toxicity and resistance are considerable. New antileishmanial drugs and effective vaccines areneeded to circumvent the limitations. The application of reporter genes has become animportant area that enhances drug and vaccine development. Transgenic parasites expressing
reporter proteins have become valuable tools for understanding the disease pathogenesis thatenhances the screening of novel compounds. For this reason, a chimeric protein, expressing
red-shifted luciferase and mNeonGreen (RE9H-mNeonGreen) Leishmania major wasengineered. This strain will allow us to monitor and visualise the parasite growth both in vitro
and in vivo studies.
Objective: This study focused on the development of transgenic Leishmania major parasites,
expressing RE9H-mNeonGreen to apply the bioluminescence and fluorescence imaging
technology for drug and vaccine screening.
Methods: The sequence of RE9H-mNeonGreen was codon-optimised and inserted into thePUC-GW-Kan vector. The codon-optimised RE9H-mNeonGreen was subcloned into thepLEXSY-pac2.1 expression system at the BglII and Not1 restriction sites and used to transformOne Shot™ TOP10 Escherichia coli by heat-shock. L. major were transfected at a logarithmic
stageofthepromastigotes

withlinear

Swa1-targetingfragment

obtained

frompLEXSY-pac-HX1-RE9H-mNeonGreen following the Amaxa short protocol. The transfectants
were characterised for their stability and viability in vitro.

No.2
CourseHealth Innovation

NameTee Joseph

Results: Results indicated, a successful generation of RE9H-mNeonGreen+L.major lines that
could serve as a basis for further research to facilitate the development of new antileishmanial
drugs or vaccines in vivo and in vitro. Using a microplate reader, a linear and robust positive
correlation (r = 0.96, p≤0.001) was observed between the number of promastigotes and the lightemitted in vitro. Fluorescence microscopy conducted on the promastigotes confirmed thesuccessful integration of RE9H-mNeonGreen into L. major, as uniform green fluorescence wasobserved

throughouttheparasite’s

population. The

limitofdetectionof

theluciferase-expressing L. major strain was 101 promastigotes for each well, indicative of thesensitivity of the bioluminescence-based approach.
Conclusion:

L.

major

promastigote

lines

weresuccessfullytransfectedwith

RE9H-mNeonGreen. The parasites were observed to have acquired both fluorescent andbioluminescent properties that will allow for easy track and visualisation. In addition, thesetransgenic L. major parasites can be used in the downstream application for various studies invitro and in vivo to facilitate the development of new drugs and vaccines.

(441 words)