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STUDIES ON THE FUNCTION OF THE LEISHMANIA MAJOR 

TELOMERASE TERT COMPONENT IN TELOMERES 

MAINTENANCE, CELL PROLIFERATION, AND INFECTIVITY 

MARK EWUSI SHIBURAH 

BOTUCATU, SP 

2024 



                

 
 

 

UNIVERSIDADE ESTATUAL PAULISTA 

 “Júlio de Mesquista Filho” 

INSTITUTO DE BIOCIÊNCIAS DE BOTUCATU 

 

 

ESTUDOS SOBRE A FUNÇÃO DO COMPONENTE TERT NA 

MANUTENÇÃO DOS TELÔMEROS E NA PROLIFERAÇÃO 

CELULAR DE L. MAJOR 

 

 

 

CANDIDATO: MARK EWUSI SHIBURAH 

ORIENTADORA: PROFA. DRA. MARIA ISABEL NOGUEIRA CANO 

 

 

 

Tese apresentada ao Instituto de Biociências, Campus 

de Botucatu, UNESP, como parte dos pré-requisitos 

necessários para obtenção do título de Doutor no 

Programa de Pós-Graduação em Ciências Biológicas 

(Genética) 

 

 

 

 

 

 

BOTUCATU, SP 

2024 



UNIVERSIDADE ESTATUAL PAULISTA 

 “Júlio de Mesquista Filho” 

INSTITUTO DE BIOCIÊNCIAS DE BOTUCATU 

STUDIES ON THE FUNCTION OF THE LEISHMANIA MAJOR 

TELOMERASE TERT COMPONENT IN TELOMERES 

MAINTENANCE, CELL PROLIFERATION, AND INFECTIVITY 

CANDIDATE: MARK EWUSI SHIBURAH 

SUPERVISOR: PROF. DR. MARIA ISABEL NOGUEIRA CANO 

Thesis presented to the Institute of Biosciences, 

Botucatu Campus, UNESP, as part of the prerequisites 

necessary to obtain the title of Doctor in the 

Postgraduate Program in Biological Sciences 

(Genetics)

BOTUCATU, SP 

2024 



Catalog page/ Ficha catalográfica 



Minutes from the defense/ Ata da defesa 



ii | P a g e

Dedication 

This work is dedicated to the God of my fathers and the memory of my beloved grandmother, Aba 

Ewusiwaa.  



iii | P a g e

Acknowledgments 

Sincerest gratitude to everyone who has been a part of my journey and the people of Sao Paulo 

whose taxes support FAPESP to push the frontiers of science! 



 

iv | P a g e  
 

Resumo 

 

Mais de 98 países e territórios notificam casos de leishmaniose, uma doença que afeta quase um 

milhão de pessoas anualmente, e contra a qual não há tratamento e controle eficazes. Nosso objetivo 

foi estudar a função do componente transcriptase reversa da telomerase (TERT) em Leishmania 

major (LmTERT) e verificar se a LmTERT poderá ser considerada alvo para o desenvolvimento 

de novos medicamentos para tratar a leishmaniose. O componente TERT contém o núcleo catalítico 

da telomerase, a enzima responsável pelo alongamento dos telômeros e pela manutenção da 

estabilidade do genoma. Neste estudo utilizamos um sistema CRISPR-Cas9 já padronizado, para 

induzir o nocaute total e por substituição do gene TERT e inibição da telomerase em L. major. 

Southern blot usando sondas específicas, PCR e sequenciamento Sanger do tipo “primer-walking” 

confirmaram que as duas metodologias induziram a deleção eficiente dos dois alelos do gene TERT 

em L. major. O nocaute do gene TERT teve efeitos no crescimento do parasito, induzindo parada 

do ciclo celular e problemas na proliferação. O encurtamento progressivo dos telômeros, a marca 

registrada da ausência de TERT, foi observado tanto de forma qualitativa por Southern TRF como 

quantitativa por Flow-FISH. Além disso, as linhagens mutantes apresentavam aumento de danos 

ao DNA e problemas de replicação.  As estruturas subcelulares das células nocaute LmTERT 

comparadas ao tipo selvagem por microscopia eletrônica e de varredura, evidenciaram 

modificações incomuns no citoplasma e uma abundância de autofagossomos, sugerindo um 

mecanismo autofágico pró-sobrevivência. Um mecanismo altruísta utilizado pelos parasitas foi 

abolido, e a análise proteômica demonstrou a existência de alterações na constituição do 

lipofosfoglicano (LPG) de superfície com expressões significativamente alteradas de 

leishmanolisina Gp63. Alterações nas proteínas do domínio META (expressas principalmente nas 

formas metacíclicas) e um número superior ao normal de parasitos de fase estacionária que 

aglutinam lectina de amendoim, foram igualmente observadas nas linhagens nocaute. Os resultados 

cumulativos que sugerem um comprometimento do potencial infeccioso do parasita foram 

confirmados pelo estudo in vivo de infecção em camundongos BALB/c e infecção in vitro usando 

macrófagos derivados da medula óssea de camundongos BALB/c. Foi observado desenvolvimento 

significativo de lesões nos camundongos infectados com parasitos controle contra aqueles 

infectados com as linhagens nocaute. Um índice de infectividade consistentemente mais alto foi 

observado para linhagens controle versus nocaute 48 horas pós-inoculação. Testes preliminares 

usando um inibidor não-nucleosídico da telomerase, o BIBR1532, resultou em telômeros mais 

curtos e diminuição do crescimento do parasito. Juntos, esses efeitos pleiotrópicos causados pela 

ausência ou inibição da LmTERT sugerem fortemente que ela apresenta grande potencial para ser 

explorada como alvo para o desenvolvimento de medicamentos contra a leishmaniose. 

 

Palavras-chave: TERT, leishmaniose, encurtamento de telômeros, CRISPR-Cas9, autofagia, 

parada do ciclo celular, alterações de crescimento e ultraestruturais, proliferação celular 

comprometida, danos ao DNA, perda de infectividade, inibição da telomerase por BIBR1532  



 

v | P a g e  
 

Abstract 

 

Over 98 countries and territories have reported cases of leishmaniasis, a disease affecting nearly 

a million individuals annually but with ineffective remedies. Our goal was to study the function of 

the telomerase reverse transcriptase (TERT) in Leishmania major (LmTERT) and leverage this 

knowledge in developing new drugs. The TERT component contains the catalytic core of 

telomerase, the enzyme responsible for elongating telomeres and maintaining genome stability. A 

loss of function study using CRISPR-Cas9 and inhibition of the telomerase in L. major was 

conducted. Probe-specific Southern blot, PCR, and primer walk Sanger sequencing confirmed the 

efficient deletion of the TERT gene in L. major. The knockout of the TERT gene resulted in parasite 

growth defects, DNA fragmentation, cell cycle arrest, and problematic replication measured by 

flow cytometry. Progressive telomere shortening, the hallmark of TERT absence, was observed by 

Southern TRF and Flow-FISH assessments. We also assessed the subcellular structures of the 

LmTERT knockout cells against a wild type using scanning and electron microscopy and found 

unusual modifications in the cytoplasm, and an abundance of autophagosomes, suggesting a pro-

survival autophagic mechanism. Changes in the metacyclic domain proteins were seen equally in 

the knockout lines. An altruistic mechanism used by the parasites was abolished. The cumulative 

results suggesting a compromise on parasite infective potential was confirmed by in vivo BALB/c 

mice infection study and in vitro bone-marrow derived macrophage infection. Significant lesion 

development was observed in the mice infected with control parasites against those infected with 

the knockout lineage. A consistently higher infectivity index was observed for control versus 

knockout lineages at 48 h post-inoculation. Consistent with the growth challenges and telomere 

shortening, preliminary tests of the inhibition of the telomerase using BIBR1532, a non-nucleoside 

small molecule, resulted in shorter telomeres and poor parasite growth. These results together 

suggest the usefulness of LmTERT to the parasite, putting the protein in a space to be explored for 

drug development against leishmaniasis.  

 

 

Keywords: TERT, Leishmaniasis, telomere shortening, CRISPR-Cas9, Autophagy, Cell cycle 

arrest, growth, and ultrastructural changes, compromised cell proliferation, DNA damage, loss of 

infectivity, BIBR1532 inhibition. 

  



 

vi | P a g e  
 

THESIS ARRANGEMENT:  

This thesis begins with a general introduction, followed by a section on the objectives, followed 

by a section entitled Introduction to the Chapters where the materials, methods, and results for the 

project are presented as two separate Chapters. Each chapter is presented as an article and begins 

with an introduction followed by a conclusion and references. A brief general conclusion section 

ends the chapters followed by a reference section referring specifically to citations from the general 

introduction.  

To aid clarity, the lists of figures and tables are divided into various sections: e.g., a list of figures 

for the general introduction, a list of figures for Chapter 1, and a list of figures for Chapter 2. 

 

Table of Contents 

Catalog page/ Ficha catalográfica ................................................................................................ i 

Minutes from the defense/ Ata da defesa ..................................................................................... i 

Dedication .................................................................................................................................. ii 

Acknowledgments .................................................................................................................... iii 

Resumo ...................................................................................................................................... iv 

Abstract ....................................................................................................................................... v 

Table of Contents ....................................................................................................................... vi 

List of Figures ......................................................................................................................... viii 

List of tables................................................................................................................................ x 

List of Abbreviations ................................................................................................................. xi 



vii | P a g e

1. Introduction .......................................................................................................................... 1 

1.1 Leishmaniasis, a Neglected Tropical Disease and a quest for new remedies ................ 4 

1.2 Biology of Leishmania parasites .................................................................................... 5 

1.3 Telomeres, End replication problem and Hayflick limit ................................................ 9 

1.4 The telomerase enzyme ................................................................................................ 16 

1.5 Alternative mechanisms in telomere maintenance: ALT pathways ............................. 19 

1.6 Justification: Telomerase as a drug target in Leishmania ............................................ 20 

2. Objectives ........................................................................................................................... 22 

2.1 Main objective .............................................................................................................. 22 

2.2 Specific Objectives ....................................................................................................... 22 

3. Materials, Methods, Results and Discussion: Introduction to the chapters ........................ 23 

3.1 Chapter 1 ...................................................................................................................... 24 

3.2 Chapter 2 ...................................................................................................................... 93 

4. General conclusion and perspectives ................................................................................ 107 

5. References ........................................................................................................................ 108 

6. Appendix ............................................................................................................................. 116 



 

viii | P a g e  
 

List of Figures in Introduction 

Figure 1. Distribution of leishmaniasis co-infection with HIV across the globe.. ........................... 2 

Figure 2. Visceral leishmaniasis distribution. .................................................................................. 3 

Figure 3. Cutaneous leishmaniasis global assessment...................................................................... 4 

Figure 4. Life cycle of the Leishmania parasite. .............................................................................. 7 

Figure 5. A cartoon of the end replication problem. ...................................................................... 11 

Figure 6. Schematic representation of the 3'-G overhang structural conformations. ..................... 12 

Figure 7. The association of telomeres with the shelterin complex. .............................................. 14 

Figure 8.  L. major and L. amazonensis chromosome termini. ...................................................... 15 

Figure 9. Graphic representation of TERT domains and motifs. ................................................... 18 

Figure 10. Secondary Structure of Telomerase RNA (TER) in different organisms.. ................... 18 

List of figures in Chapter 1 (Page 24 - 92) 

Fig 1. Defective growth, DNA synthesis, and accumulation of DNA damage in TERT-depleted 

parasites. .................................................................................................................................. 33 

Fig 2. Absence of cell death and increased PNA-negative parasites among the TERT-depleted L. 

major promastigotes ................................................................................................................ 36 

Fig 3. TERT-depleted parasites show a senescent-like phenotype. ............................................... 39 

Fig 4. TERT depletion induces telomere attrition in L. major promastigotes. The deletion of 

LmTERT leads to a significant reduction in telomeres. .......................................................... 41 

Fig 5. TERT-depleted parasites showed reduced or no capacity to infect BALB/c mouse models 

and bone marrow macrophages ............................................................................................... 47 

 



ix | P a g e

S1 Fig. Expression of pTB007 in L. major (Lm007) does not affect the growth and cell cycle of the 

parasite. ................................................................................................................................... 83 

S2 Fig. Strategies for the deletion of LmTERT and the methods of confirmation.. ...................... 84 

S3 Fig. Representative gating paths used in analyzing flow cytometry data.. ............................... 85 

S4 Fig. Fluorescent antibody test of Leishmania major infection in experimentally infected mice.

 ................................................................................................................................................. 86 

S5 Fig. SCG gene amplifications. .................................................................................................. 87 

S6 Fig. Plasmid engineering for LmTERT reintroduction in knockout lineages (complementation).

 ................................................................................................................................................. 88 

S7 Fig. Differential protein expression........................................................................................... 89 

List of figures in Chapter 2 (Page 92 – 106)

FIG 1. Growth comparisons of BIBR1532 treated and non-treated parasites. (A) Growth 

comparisons between untreated parasites and parasites treated with three different 

concentrations of BIBR1532. (B), (C), (D) comparative proliferation in percentages of 

parasites at 24, 48, and 72 h respectively. ............................................................................... 97 

FIG 2. Telomere length attrition due to BIBR1532 treatment. (A) Southern blot TRF assessment of 

the sizes of telomeres in the parasites 72 h after treatment of BIBR1532. (B) WALTER 

intensity profiles of the TRF sizes obtained from the southern blot. ...................................... 99 



 

x | P a g e  
 

List of tables 

Tables in Chapter 1 (Pages 24-92) 

Table 1 Comparative changes in telomere size .............................................................................. 42 

 

S1 Table. List of oligonucleotides used in this study ..................................................................... 91 

  



 

xi | P a g e  
 

List of Abbreviations 

NTDs- Neglected Tropical Diseases 

L. major- Leishmania major 

LmTERT- Leishmania major TERT 

ORF- Open Reading Frame 

TERT- Telomerase reverse transcriptase 

TEN- telomerase essential N-terminal  

TRBD- telomerase RNA binding domain 

RT- reverse transcriptase  

CTE- C-terminal extension  

TER- Telomerase RNA 

TBE- template boundary element 

STE- stem terminus element 

CRISPR- Clustered Regularly Interspaced Short Palindromic Repeats 

Cas9- CRISPR-Associated protein 9 

T7RNAP- T7 RNA Polymerase 

pTB007- Expression plasmid carrying Cas9, T7RNAP and Hygromycin genes used in transfection  

Lm007- Leishmania major parasite carrying plasmid pTB007 

sgRNA- single guide RNA 

tracrRNA- trans-activating CRISPR RNA 

DT- donor template 

EdU- 5-ethynyl-2'-deoxyuridine 

LPG- Lipophosphoglycan 

PBS- phosphate-buffered saline 

BIBR1532-  2-[[(E)-3-naphthalen-2-ylbut-2-enoyl]amino]benzoic acid 

SCG- scβ-Galactosyltransferases 

 

 



1. Introduction

The search for an alternative therapeutic strategy in treating leishmaniasis caused by protozoan 

parasites from the Leishmania genus  is a matter of global public health interest [1]. Leishmania 

parasites have a digenetic lifecycle that involves living inside a mammalian host and a 

phlebotomine insect vector. This lifecycle imposes on the parasite a need to develop strategies to 

survive two different extreme environments using two major developmental phases: intracellular 

amastigotes and extracellular promastigotes, with observable differences but shared basic 

molecular and cellular characteristics [1–4].  

Over 20 species of Leishmania can cause the disease and may manifest in different clinical 

forms depending on the parasite species and host immune system. The three main clinical forms of 

leishmaniasis are Cutaneous, Mucocutaneous, and Visceral [5,6]. Most of the cutaneous forms are 

self-healed in contrast to the dilacerating and mutilating mucocutaneous and the lethal visceral 

forms [6–9].  

Over a million new leishmaniasis cases are reported annually [10]. According to the World 

Health Organization, 94% of leishmaniasis cases reported in 2017 were recorded in 7 countries, 

including Brazil (www.who.int/leishmaniasis/burden/en).  In the last two decades, a great number 

of HIV/leishmaniasis coinfections have also been reported (Figure 1) [11,12]. Due to the challenges 

associated with existing treatment and control methods against the disease, an alternative 

therapeutic target has become necessary [13].  

The WHO outlines a road map for tackling leishmaniasis by looking at the data from different 

perspectives, including mortalities from vector-disease transmission from 2021 to 2030. Efforts are 

still being made to combat the spread of leishmaniasis through several health interventions, 

http://www.who.int/leishmaniasis/burden/en


2 

including the Elimination Programme of Kala-azar in Southeast Asia 

(https://www.who.int/publications-detail-redirect/who-wer9635-401-419). The 200 territories and 

countries that reported cases of leishmaniasis to the WHO show nearly 50% of these regions still 

have endemic cases of leishmaniasis (Figure 2 and Figure 3) [11].  

Figure 1. Distribution of leishmaniasis co-infection with HIV across the globe.  The 2020 report on the global 

distribution of countries reporting cases of leishmaniasis co-infection with HIV. WHO classification of countries within 

the boundaries of Middle East and the Americas seems to have an increased incidence of co-infection cases. Source of 

image: WHO website. 



3 

Figure 2. Visceral leishmaniasis distribution. The burden of visceral leishmaniasis across the globe. Some regions 

in Africa including Ghana, report no cases of Visceral leishmaniasis. Source: World Health Organization. 



4 

Figure 3. Cutaneous leishmaniasis global assessment. The global burden of cutaneous leishmaniasis in different 

countries described according to their impact in the various regions. Source: World Health Organization. 

1.1 Leishmaniasis, a Neglected Tropical Disease and a quest for new 

remedies 

The WHO classifies 20 conditions mainly identified in tropical regions under the umbrella name 

‘Neglected Tropical Diseases’ (NTDs). Leishmaniasis is one such condition. A disease under the 

NTD classification like leishmaniasis affects largely impoverished communities where women and 

children are unduly affected (https://www.who.int/health-topics/neglected-tropical-

diseases#tab=tab_1).  

Even though there has been some progress in getting countries to fight these diseases, there is 

still little attention being paid to the development of new and effective remedies. Also, there is little 

https://www.who.int/health-topics/neglected-tropical-diseases#tab=tab_1
https://www.who.int/health-topics/neglected-tropical-diseases#tab=tab_1


 

 
103 

 

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107 

 

4. General conclusion and perspectives 

 

Our data on the study of the TERT as a potential drug target against leishmaniasis has provided 

useful insights on the possible effects on the biology of the parasite. Particularly, the druggability 

of the target was successfully confirmed by the use of BIBR1532. However, there are still lingering 

questions on the mechanism of action and whether the BIBR1532 acts in similar ways as in other 

tested models. Additionally, the proposed model of action after TERT ablation will need to be 

experimentally proven to aid in therapeutic modulation of the target. Future studies may look at the 

processivity of the telomerase during the inhibition at different concentrations. Toxicity levels of 

the various concentrations can also be tested to avert potential threats to normal cells with 

telomerase activity. 

Lastly, studying further the infectivity in vitro and in vivo will be an additional benefit to 

developing potential therapeutics.  

  



 

 
108 

 

5. References 

References in this section refer to the citations made before the beginning of the objectives 

section. 

 

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implications for parasite biology. Mol Microbiol 2011;79:647–62. https://doi.org/10.1111/j.1365-

2958.2010.07479.x. 

[2] Da Silva R, Sacks DL. Metacyclogenesis is a major determinant of Leishmania promastigote 

virulence and attenuation. Infect Immun 1987;55:2802–6. https://doi.org/10.1128/iai.55.11.2802-

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