Division of Molecular Biology and Human Genetics
EDCTP FUNDED STUDIES (IN PROGRESS)
This project TMA2018SF-2458 is part of the EDCTP2 programme supported by the European Union.
PROJECT: Deciphering the epidemiology and evolution of drug-resistant tuberculosis to inform policy
PRINCIPLE INVESTIGATOR: Professor Elizabeth Streicher
GRANT NUMBER: TMA2018SF-2458
Drug-resistant tuberculosis (DR-TB), specifically extreme drug-resistant tuberculosis (XDR-TB), is challenging to diagnose, complicated to treat, and consumes a disproportionate amount of TB control programs' resources. Improper treatment means that patients with DR-TB often remain infectious for prolonged periods, resulting in transmission in healthcare settings, households and communities. Successful transmission of DR-TB depends on the TB strains' fitness, local conditions, and disease control strategies.
The Western Cape Province of South Africa has a very high burden of XDR-TB (> 800 cases in the past ten years). Yet, the local XDR-TB population structure and its development over time have never been studied in detail despite the potential relevance of such investigations for targeting appropriate control measures. This shortage of information can be addressed by applying next-generation whole genome sequencing (WGS) since WGS is more discriminatory than the classical genotyping methods and allows simultaneous investigation of the entire genome.
In the proposed study, we have three main research questions: 1) compare outbreaks of DR-TB in terms of genetic polymorphisms, including drug-resistance conferring and compensatory mutations; 2) study the impact of changes in treatment strategies such as new diagnostic algorithms and TB drug regimens of XDR-TB on the population structure of M. tuberculosis strains in the region over time; and 3) assess the extent of the DR-TB epidemic in other Southern African countries.
Our results will improve the understanding of mechanisms driving the evolution and success of XDR-TB at the population level in a high TB and HIV co-endemic setting. This could pave the way for improved DR-TB prevention and control. Furthermore, the proposed study will develop significant capacity and transfer knowledge to Southern Africa. This project contributes to capacity development in Southern Africa. To establish researchers, several students will be able to receive postgraduate qualifications, and the knowledge base will be expanded in Southern Africa to inform TB policy for the possible eradication of TB disease ultimately.
This project TMA2018SF-2470 is part of the EDCTP2 programme supported by the European Union.
PROJECT: Genomic Surveillance to Control Pathogen Infections in Africa (GenPath Africa)
PRINCIPLE INVESTIGATOR: Professor Tulio de Oliveria
GRANT NUMBER: TMA2018SF-2470
PROJECT WEBSITE: https://www.genpath-africa.org
The overall goal of GenPath Africa is to control pathogen infections in Africa. Our consortium of African and European investigators proposes to build on our collective experience in responding to SARS-CoV-2 to better diagnose, monitor and clinically manage current and future epidemics in Africa. We propose expanding genomics capacity to combat drug-resistant HIV-1 and TB through precision medicine and using genomic epidemiology to guide the public health response. GenPath Africa will also apply recent wastewater and health surveillance developments to detect emerging pathogens. Capacity development activities will include degree training in South Africa and technology transfer to the National Public Health Institute of Mozambique.
GenPath Africa will advance the EDTCP3 work program by i) providing researchers and public health professionals with skills in genomic epidemiology to better understand infectious disease epidemiology and drug resistance, ii) strengthening capability in southern and East Africa to respond to current and emerging epidemics rapidly, and iii) providing researchers with training to advance their scientific careers in Africa and establish themselves as scientific and public health leaders.
The TB Genomics research group will focus on work package 2 (WP2). The overall goal of WP2 is to develop, implement, assess and build capacity for detecting and containing emerging drug-resistant TB (DR-TB) strains through precision medicine and precision public health as adjuncts to the current healthcare approaches. These will consist of the use of Mycobacterium tuberculosis next-generation sequencing (NGS) in 'real-time' to identify a strain's drug resistance profile to detect emerging resistance to the current standard of care shorter DR-TB regimens and the novel BPaLM regimen and design effective treatment regimens; the use clinical and genomic epidemiology, including 'real-time' phylogenetics to monitor the spread of DR-TB and implement targeted contact and source investigation strategies to prevent the spread of (pre)-XDR Mtb (with pre-XDR-TB defined as resistance to rifampicin and fluoroquinolones and XDR-TB defined as resistance pre-XDR-TB plus resistance to bedaquiline and/or linezolid).
- Stellenbosch University (SU), South Africa
- Centre for Epidemic Response and Innovation (CERI)
- South African National Health Laboratory Service (NHLS), South Africa
- University of Antwerp (UA), Belgium
EDCTP FUNDED STUDIES (CONCLUDED)
This project TMA2017CDF-1885 was part of the EDCTP2 programme supported by the European Union.
PROJECT: The role of Mycobacterium tuberculosis compensatory mutations in metabolic fitness via the structure and function of mycolic acids (InforMATB)
PRINCIPLE INVESTIGATOR: Dr Marisa Klopper
GRANT NUMBER: TMA2017CDF-1885
Despite early work indicating that drug-resistant Mycobacterium tuberculosis is less fit than its drug-susceptible counterparts, M. tuberculosis, resistant to increasing numbers of drugs, continues to emerge and spread. Compensatory mutations exist that may explain the ability of the resistant bacilli to retain fitness. Mutations in the inhApromoter are well-known to cause resistance to at least two drugs. These mutations have been suggested to be a gateway to extensively drug-resistant TB (XDR-TB, resistant to at least four key drugs). We propose that, in addition to causing resistance, inhA promoter mutations act as compensatory mechanisms, overcoming the negative effects of drug resistance. We tested this hypothesis by targeted mutagenesis, structural and functional investigations and fitness assays. In particular, inhA promoter mutations cause the upregulation of genes involved in mycolic acid synthesis (mabA and inhA) and haem biosynthesis (hemZ). We aimed to show that this upregulation causes an increase in the amount of mycolic acids synthesised, which in turn increases the functionality of mycolic acids. We hypothesise that this function facilitates the sequestration of ferrous iron, nitric oxide or oxygen. The overall goal of the research was to determine the role of the promoter of the mabA-inhA-hemZ operon in the physiological mechanisms involved in M. tuberculosis's compensation for fitness cost.
We have generated different mutants from a single progenitor (M. tuberculosis H37Rv) to study the role of the inhApromoter mutation. We used site-directed mutagenesis to generate an inhA promoter mutant and add a rpoB mutation in a spontaneous mutagenesis experiment. An H37Rv strain with only the rpoB mutation was also available. Whole genome sequencing (Illumina and PacBio) analysis has confirmed that the progenitor and mutant strains differ only by the mutation(s) of interest. These strains were used to compare their haem and mycolic acid productions and for additional investigations.
We conducted RNAseq experiments (funded by the Harry Crossley Foundation and Research Centre Borstel), which demonstrated that the influence of the inhA promoter mutation on the transcriptome is restricted to the inhA-mabA-hemZ operon. We attempted classical competition assays, but due to the need to avoid Tween, a surfactant used to limit clumping during culturing but which also affects the cell wall, the results were inconclusive. We are currently investigating alternative methods. Preliminary growth curve data suggest no significant difference between the growth rates of the different strains under standard culturing conditions.
We have examined the relative amounts of heme and iron in the progenitor and inhA promoter mutant (IPM) strains to determine whether upregulation of the hemZ gene resulted in perturbation of the homeostasis of these molecules. Quantification of heme by mass spectrometry was inconclusive due to difficulties with purchased standards. Iron content in the bacilli of the IPM and wild-type strains was investigated using different methods, including fluorescence measurements, imaging and ICP-MS. While most of the techniques used were not sensitive enough to detect minute differences in iron content, analysis of our images led to some surprising findings regarding bacillary length and iron distribution within the bacilli.
Our cell wall (mycolic acid) investigations revealed that an additional band or peak was present in the mutant strains on thin-layer chromatography and nuclear magnetic resonance analysis, respectively, suggesting a significant change in the cell wall lipid composition. Our transmission electron microscopy findings support these findings. Further investigations are underway.