Understanding the molecular processes of infectious diseases for clinical drug design and discovery

The Agenda for Sustainable Development calls for the world to “ensure healthy lives and promoting well-being for all at all ages” by 2030 (SDG3). Although there has been progress in many health areas, the UN reported in 2020 that the rate of improvement has slowed and may not be sufficient to meet SDG 3 targets, particularly with the Covid-19 pandemic disrupting progress around the world.[1] This also applies to related Sustainable Development Goals (SDGs), such as ensuring availability and sustainable management of water and sanitation for all (SDG 6). Currently billions of people throughout the world still lack access to safely managed water and sanitation services and basic handwashing facilities at home. The need for solutions, the UN reports, is vast and accelerating: “Countries need comprehensive health strategies and increased spending on health systems to meet urgent needs and protect health workers, while a global coordinated effort is needed to support countries in need”.[2]

Rural communities in South Africa. Photo credit: Rebekka Stredwick. ©Diamond Light Source

Scientists are increasingly at the forefront of global and local efforts to tackle these challenges, not least in clinical drug design and discovery and vaccine development for countries where some of the need is the greatest. This includes the small but growing community of structural biologists in Africa, many of whom are just starting out on their careers.

Established in 2013, the Structural Biology Research Group at the University of Pretoria in South Africa focuses its research on molecules involved in mechanisms of infectious diseases – primarily from Mycobacterium tuberculosis (Mtb, causing tuberculosis or TB), enterotoxigenic Escherichia coli (travellers’ diarrhoea), and Listeria monocytogenes (listeriosis). The aim is to elucidate the molecular processes that allow these microorganisms to invade and/or persist within the human body or individual cells to improve our understanding of these diseases for clinical drug design and discovery. 

Collaborating with the GCRF START grant – three case studies investigating infectious diseases

In 2016, the Group moved to newly renovated laboratories in the University of Pretoria’s Agricultural Science building designed for molecular biology, protein production and structural analyses, and in 2018 commenced collaborating with the GCRF START grant programme. The benefit of being part of this international grant and research network is explained by Group leader and GCRF START Co-I, Prof. Wolf-Dieter Schubert,

“The GCRF START grant has enabled the Group to access the UK’s national synchrotron, Diamond Light Source (Diamond) for experiments using state-of-the- art synchrotron techniques not available on the African continent,” Prof. Schubert says. “It has also exposed students to international networks and workshops/training to share knowledge and build capacity in the field of structural biology for solutions to infectious diseases rife on our continent and beyond. Our Group currently includes 6 PhD students and 4 MSc students from countries as far afield as Nigeria, Cameroon, Namibia and Zimbabwe, in addition to South Africa, and we welcome two Honours students during 2021”.

The case studies below highlight the research of three emerging scientists from across Africa who are studying for their PhD’s in the Structural Biology Research Group at the University of Pretoria.  Each describes how their careers and their research benefit from the collaboration between the Group and the GCRF START grant.

Clifford Manyo Ntui: Using synchrotrons in the fight against infectious diseases

My name is Clifford Manyo Ntui and I am a PhD student in Biochemistry. I was born in Ewelle village, in South West Province of Cameroon. Growing up in the village where no clear explanation was given for the cause of countless illnesses and deaths was always a nightmare to me. To seek answers, I decided to pursue a career in the field of infectious diseases. My path towards this journey gained ground when I joined the Structural Biology Research Group at the University of the Western Cape under the supervision of Prof. Wolf-Dieter Schubert in 2012 for my Honour’s degree, with a focus on the molecular basis of infectious diseases. This was followed by an MSc working on Mtb in the same group but now at the University of Pretoria. My PhD studies on Escherichia coli (ETEC), have benefited greatly from the Group’s collaboration with the GCRF START grant.

Clifford Manyo Ntui, PhD student in the Structural Biology Research Group at the University of Pretoria, South Africa. Photo credit: Clifford Ntui. ©Diamond Light Source

The field of Biochemistry is one of great interest and importance in Africa as attested by the need for more medicinal drugs towards the fight against infectious diseases. My training and expertise in the field of structural biology of infectious diseases makes me one of only a handful of scientists working in this area on the entire continent. I have worked with more than one synchrotron light source, which has given me skills that are scarce on the African continent. Moreover, I have a strong background and interest in communicating structural biology skills to student learners and the public.

In 2014, four years before the GCRF START collaboration, I started on my MSc studies at the University of Pretoria working on the crystals structure determination of novel drug targets of Mycobacterium tuberculosis, the bacterium that elicits tuberculosis. I was quite successful in my endeavour, solving the structure of one potential new drug target, thiamine phosphate kinase. As part of my MSc training, I twice travelled to the European Synchrotron Radiation Facility (ESRF) in Grenoble, France, to undertake diffraction experiments using highly brilliant and monochrome X‐rays. South Africa is a member of the ESRF, providing access to world class, cutting‐edge equipment. The data collected on these trips allowed me to solve the crystal structure of the protein mentioned above. In addition, I was able to collect diffraction data for a number of colleagues, again leading to the successful determination of a range of crystal structures in the field of Infection Biology as well as Microbial Ecology and Genomics. This formed the foundation of my training in synchrotron techniques which I could use in my PhD research. 

For my PhD, I am working on EtpA protein from enterotoxigenic Escherichia coli (ETEC), a bacterium that causes severe diarrhoea in many African countries. This disease leads to the death of hundreds of thousands of children under the age of five all over the developing world.[3] EtpA is an adhesin which has been found to associate with flagellin from flagella allowing for bacterial adherence and toxin delivery. Currently, there is no crystal structure of EtpA from ETEC. This research aims to structurally characterise EtpA alone, as well as in complex with flagellin from ETEC as a possible vaccine target against ETEC diarrhoeal disease.

This project has produced the first crystal structure of the secretion domain of EtpA protein from ETEC using X-ray diffraction on the beamline I04 at the Diamond Light Source synchrotron, the experiments conducted at Diamond remotely from our labs at the University of Pretoria. The project is the product of a successful research programme thanks to funding provided by the GCRF START grant which has ensured the smooth running of our molecular biology laboratory. START has also contributed to organising workshops which have been invaluable for enhancing my protein structural biology knowledge, such as the CCP4 workshop of which GCRF START is a sponsor.

Vukosi Edwin Munyai: Characterising the protein structures of Listerial adhesion protein (LAP)

My name is Vukosi Edwin Munyai. I was born and raised at Kurhuleni village, located in Limpopo Province, South Africa. Collaborating with the GCRF START grant, provides me with the opportunity to contribute to Global challenges in line with the UN’s Sustainable Development Goals for creating a better future for all. As one of the first students to enrol for a PhD in science in my village, I am so grateful to do research that impacts human lives.

The GCRF START grant has given us a platform for learning in structural biology aspects, conducting experiments, associating with other scientists, and access to the Diamond synchrotron. I have the opportunity to learn more professionally and to learn how to handle different tasks as an independent researcher. In the future, I would like to spend time in academia, teaching and influencing young people, using my own story and research to inspire them.

Vukosi Edwin Munyai, PhD student in the Structural Biology Research Group at the University of Pretoria, South Africa.
Photo credit: Vukosi Edwin Munyai ©Diamond Light Source

In 2011, I completed my BSc degree in Biochemistry and Microbiology at the University of Venda, South Africa. From 2012 to 2014, I was working at United National Breweries in South Africa as a laboratory assistant when I decided to go back to study. In 2015, I enrolled for an Honours degree in Microbiology and a year later, for a Master’s degree in Biotechnology at the University of Western Cape, which I completed in 2018. After completing my Master’s degree, I relocated to Pretoria in 2019, where I enrolled for PhD in Biochemistry and structural biology.

My main objective is to characterise the protein structures of Listerial adhesion protein (LAP) and also its complex structure with human HSP60 (‘heat shock protein’). It has been proposed recently[4] that L. monocytogenes uses alcohol and acetaldehyde dehydrogenase/LAP interaction with human intestinal HSP60 to enable its para-cellular translocation to the sites of its pathogenicity/cause of disease. Characterising the complex structure of LAP and HSP60 will help to explain how L. monocytogenes infects its host alongside the established modes of invasion through the Internalin surface proteins. Understanding the LAP/HSP60 interaction may help in developing clinical drugs to prevent listerial infections. Dehydrogenase enzymes for industrial applications are presently in short supply. The structure of LAP will thus enhance knowledge about its roles in both disease and industrial applications.

Maria Hamuyela: Studying the functions and the structure of the ETEC EatA passenger domain

My name is Maria Hamuyela and I study the secreted EatA protein of enterotoxigenic Escherichia coli (ETEC) bacteria. I am currently employed as a technologist for the University of Namibia in Windhoek. After more than a year of searching for opportunities, I came across Prof Wolf-Dieter Schubert and he accepted me to join the Structural Biology Research Group at the University of Pretoria to carry out my PhD studies whilst retaining my job as a technologist in Namibia.

The GCRF START programme has afforded me an opportunity to study towards my PhD studies allowing me access to world class equipment and techniques like X-ray diffraction at the UK’s Diamond synchrotron and provides me with access to the reagents that I need to do my research. I am also getting scientific training, not only in the laboratory but through workshops.

Maria Hamunyela is a technologist at the University of Namibia and a PhD student in the structural biology research group at the University of Pretoria, South Africa.

ETEC commonly causes watery diarrhoea in children younger than five years old – a cause of death due to diarrhoeal diseases in this age group, as well as malnutrition and stunting in children. Lack of access to clean water and sanitation means infectious diseases such as ETEC and Shigella spread through water and food. Studying ETEC has a potential to provide knowledge needed to develop vaccines and drugs against ETEC and other related diseases in line with the WHO/UNICEF Integrated Global Action Plan for the Prevention and Control of Pneumonia and Diarrhoea (GAPPD). The GAPPD has, as a key goal, the reduction of deaths from diarrhoea in children younger than five to less than 1 per 1000 live births by 2025 (WHO/UNICEF, 2013)[5]. Hopefully designing an inhibitor for EatA will be a fundamental step in achieving this goal.

The EatA passenger domain protein is required for the virulence of ETEC. It degrades Mucin 2, a major mucin secreted by the intestinal epithelium. Previous reports show that EatA passenger domain shows potential as a candidate vaccine for ETEC and other enteric pathogens such as Shigella flexneri. The EatA passenger domain has however not been extensively studied with regards to functional and structural properties. Therefore, the main aim of my project is to characterise both the structure and functional properties of the EatA passenger domain to design an inhibitor for EatA.

Impact of the GCRF START grant – Structural Biology students trained from seven African countries

Prof Wolf-Dieter Schubert from the University of Pretoria acknowledges the dramatic impact the GCRF START programme has had from its inception in 2018:

“Overall, GCRF START has helped support the research of six PhD students, five MSc students and six BSc Honours students in our research group. In addition to supporting individual research projects, the programme also allowed them to meet and interact with international experts, provided access to internationally competitive infrastructure such as the Diamond Light Source, and supported their participation in a number of workshops and conferences.

The impact is thus clearly multidimensional. Coupled to the fact that supported students came from seven African countries with a balanced gender distribution throughout, the long-term impact will be considerable. This will apply particularly if students are able to continue on their path towards independent research careers and possibly return to their home countries. The impact of the grant so far bodes well for structural biology and health related, and as well as industrial research throughout the African continent.”

GCRF START Co-I, Prof. Wolf-Dieter Schubert, head of the Structural Biology Research Group, University of Pretoria, South Africa. ©University of Pretoria

Read more about the UN’s Sustainable Development Goals here

[1] https://sdgs.un.org/goals/goal3 accessed 22.3.2021

[2] The Covid-19 pandemic is threatening health systems and in particular, ones with poor resources, insufficient health facilities, medical supplies and health-care workers to meet the surge in demand. For example, an estimated 10 million persons fell ill with Tuberculosis (TB) globally in 2018, and drug-resistant Tuberculosis is also a continuing threat with the goal to end TB by 2030 no longer a possibility (UN, 2020).

[3] Globally, ETEC and Shigella are estimated to cause ~400 million episodes of diarrhoea annually in young children (WHO 2009). and ETEC and Shigella episodes resulted in an estimated 2.6 million additional children with moderate stunting and 2 million additional children with severe stunting. https://www.who.int/immunization/research/meetings_workshops/6.Enterotoxigeni_Escherichia_coli_ETEC_A.Louis_Bourgeois_PDVAC_2016.pdf?ua=1 accessed 16.03.2021

[4] Kim K-P, Jagadeesan B, Burkholder KM, Jaradat ZW, Wampler JL, Lathrop AA, Morgan  MT, Bhunia AK. (2006) Adhesion characteristics of Listeria adhesion protein(LAP)-expressing Escherichia coli to Caco-2 cells and of recombinant LAP to eukaryotic receptorHsp60 as examined in a surface plasmon resonance sensor., FEMS Microbiol Lett 256, 324-332, doi:10.1111/j.1574-6968.2006.00140.x

[5] https://www.who.int/publications/i/item/the-integrated-global-action-plan-for-prevention-and-control-of-pneumonia-and-diarrhoea-(gappd) accessed 16.3.2021