Intervening along the spectrum of tuberculosis: meeting report from the World TB Day nanosymposium in the Institute of Infectious Disease and Molecular Medicine at the University of Cape Town

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Tuberculosis (TB) is a debilitating communicable disease affecting millions of people worldwide, with the highest incidence and mortality rates in Southern Africa. In 2017 an estimated 1.6 million people died of TB worldwide, making it a leading cause of death due to a single infectious agent¹. TB is transmitted through airborne inhalation of Mycobacterium tuberculosis (Mtb) that seeds in alveolar spaces in the lungs. Interestingly, almost one-third of the world population that is exposed to Mtb infection is able to control the infection and remain asymptomatic, otherwise known as latently infected². However, 5–10% of these latently infected individuals progress to active TB and manifest symptoms of the disease². More recently, the situation has been made more complex by the emergence of drug-resistant bacteria and co-infections with the human immunodeficiency virus (HIV). In 2017, the WHO estimated that there were 160,000 new cases of multi-drug resistant TB (MDR-TB) worldwide and approximately 5-10% of these patients progressed to extremely-drug resident TB (XDR-TB)¹. This underpins an urgent need to develop better and efficacious vaccines than the currently available Bacille Calmette-Guérin (BCG) and to develop novel drugs for tuberculosis.

Identifying new drug targets

Mtb has a sophisticated metabolic repertoire: it is able generate its own nutrients, but it can also scavenge for some nutrients from the host³. Studies have shown that Mtb is able to synthesise essential amino acids such as L-arginine and tryptophan, where deletion of these key metabolites restricts Mtb growth in culture and renders it more susceptible to host immune pressure reducing its survival^4,5. Dr Melissa Chengalroyen, a postdoctoral research fellow in the Molecular Mycobacteriology Research Unit, under the directorship/supervision of Prof Valerie Mizrahi, opened the morning session and spoke about the key elements required for Mtb survival and how Mtb sequesters these elements to evade host-recognition by non-conventional T cells. She then described a complex de novo riboflavin metabolic pathway and different tools to create Mtb mutants lacking key enzymes involved in this pathway to facilitate understanding of each step in the pathway. Her study showed that not all the enzymes involved in the riboflavin pathway are essential—some play redundant roles, while others are absolutely necessary for Mtb survival, and these may hold promise as new candidate drug targets for TB or play an essential role in alerting non-restricted T cell immune arm for faster clearance of the bacteria.

The second speaker in the morning session was Dr Kehilwe Nakedi, a postdoctoral research fellow in the laboratory of Prof Jonathan Blackburn. Her research project was aimed at identifying novel substrates for mycobacterial protein kinase G (PknG) using a mass spectrometry-based phosphoproteomics approach to elucidate the mechanisms mycobacteria interferes with the host signalling during LTBI. She identified 3164 phosphopeptides with high confidence using label-free data analysis⁶. Moreover, she identified 63 host phosphopeptides that were phosphorylated in macrophages infected with M. bovis BCG only and not those infected with the mutant lacking PknG. Further analysis of the data revealed that these substrates phosphorylated in the presence of PknG play a key role in regulating actin polymerisation and cytoskeleton integrity⁶. This work suggest that pathogenic mycobacteria survives inside the host macrophages during early tuberculosis infection through interfering with the host’s cytoskeletal dynamics mediated by PknG.

Development of host-directed drug therapies

Although the currently available TB treatment regimens are effective at killing the bacteria, the emergence of drug resistance and the long duration of treatment threaten their long-term efficacy. This underpins an urgent need to develop new anti-TB drugs and exploration of other treatment strategies to control the disease. One such strategy is to develop host-directed drug therapies (HDTs) with the aim of boosting the host’s innate ability to fight the infection and also limit the deleterious tissue pathology⁷. Although this field is still in its infancy, it holds a huge potential as adjunctive therapy for tuberculosis in clinical settings with a high disease burden.

Assistant Prof Reto Guler discussed in detail their published pre-clinical data on the use of statins as a potential host-directed therapy for TB in mice⁸. He then spoke about the translation of this work in a proof-of-concept phase IIB, double-blind, randomized, placebo-controlled trial launched in Khayelitsha township and funded by the European & Developing Countries Clinical Trials Partnership ((EDCTP), RIA2017T-2004). The coordinator of this consortium is Reto Guler (University of Cape Town, Division of Immunology). Chief principal investigator of the clinical trial is Friedrich Thienemann (University of Zürich), local PI in Cape Town is Sandra Mukasa (University of Cape Town). Other project partners include Robert J. Wilkinson (Imperial College London), Claudia Schacht (LINQ Management GmbH, Germany), Gunar Günther and Emmanuel Nepolo (University of Namibia). The aim of the clinical trial is to investigate the use of statins to prevent chronic lung inflammation and potentially TB relapse in patients post completion of a standard TB treatment regimen. He also talked about other potential candidates targets for HDTs such as the transcriptional factor BATF2⁹ and microRNA-143 and microRNA-365¹⁰.

Another speaker on this topic was Dr Suraj Parihar, a Senior Research Officer and contributing investigator at CIDRI-Africa in the Institute of Infectious Diseases and Molecular Medicine (IDM). His talk focused mainly on preclinical studies that investigated the efficacy of repurposed drug generally used to reduce blood glucose and cholesterol as HDTs for TB. He found that this drug was able to reduce lung inflammation in pre-clinical in vitro and in vivo models of TB. He then went on to speak about how growth factors can also be repurposed to enhance the killing ability of human and mouse macrophages. Although some of these studies are still at the pre-clinical stage, they hold a great promise and may pave way for alternative therapies and new clinical trials supported by strong pre-clinical data.

Transmission of Mtb

Mtb is transmitted through the inhalation of Mtb-containing aerosols from person to person. Transmission of the bacteria is high in places such as schools, churches, mines, hospitals and heavily congested neighbourhoods of Cape Town such as informal settlements and townships^11,12. In places with high prevalence of human immunodeficiency virus (HIV), such as Khayelitsha, the rate of new Mtb infections accounts for more than half of TB cases. In 2006, it was reported that as high as 1500/100,000 incidence rates were observed in some of the townships, exceeding that of national average¹³. The rate of transmission and subsequent disease development has been linked with high aerosol bacillary loads. There are many challenges when it comes to measuring Mtb transmission via aerosol such as the low numbers of bacilli that can be captured, contamination by other bacterial or fungal particles in patients and other airborne particulate matter¹⁴. New aerosol methods for capturing Mtb such as respiratory aerosol sampling chamber (RASC) hold promise in quantifying rate of transmission especially in high endemic areas. The capacity to measure the rate of transmission and the type of bacilli strain circulating becomes even more critical in the era of high antimicrobial resistance¹⁵.

Dr Anastasia Koch, a Carnegie Developing the Next African Leaders (DEAL) early career fellow, mentored by Prof Helen Cox and Prof Digby Warner, started off her talk by mentioning the potential of whole genome sequencing technology in identifying Mtb genotypes resistant to the first line tuberculosis drugs¹⁶. She discussed the major differences in genetic diversity observed in broth cultured Mtb populations and those derived directly from sputum, and moreover how simple culturing could result in loss of some of key genotypes. Anastasia then moved on to discussing the importance of getting a sample as close to that being transmitted by an infected person as possible in order to accurately study the strains that are being transmitted and driving disease in a community. She gave an example of how colleagues from the MMRU and the Desmond Tutu HIV Centre, have been able to capture and isolate Mtb strains from RASC bio-aerosols. She was able to culture these samples and compare whole genome of those strains with sputum induced strains. The ability to isolate Mtb from bio-aerosols and combining that with whole genome sequencing could greatly inform our understanding of TB transmission and treatment, particularly of emerging drug or multi-drug resistant strains.

TB/HIV co-infection

More than 36.7 million people live with HIV/IDS globally and most of these people live in sub-Saharan Africa. In 2017, it was estimated that more than 350 000 people died due to HIV/TB co-infection, making TB a highest contributor to death in people living with HIV¹⁷. HIV targets and depletes CD4 T cells at later stages of disease, including protective TB specific CD4 T cells^18,19. Early antiretroviral (ARV) drug treatment is associated with improved outcomes and helps restore CD4 T cell count, including protective TB-specific CD4 T cells. However, there are complications associated with early ARV treatment in people who also are starting TB treatment. Some of these people develop TB-associated immune reconstitution syndrome (TB-IRIS), which can be fatal, unless controlled by host-directed immune suppressants such as corticosteroids²⁰.

To set the scene, Mohau Makatsa, a PhD candidate in the laboratory of Prof Wendy Burgers in the Division of Medical Virology, talked about a particular subset of CD4 T helper cells expressing IL-22, or “Th22 cells”, which are targeted by HIV. He showed how these cells can be stimulated ex-vivo by Mtb antigens and express different surface molecules compared to Th1 cells. There is a similar magnitude of these cells compared to Th1 cells in people with latent TB, but they are depleted in the peripheral blood in active TB disease and HIV co-infection. IL-22 has been shown to be important for the control of Mtb in mice²¹. It is unclear how these cells play a role in the pathogenesis of TB in humans.

Dr Muki Shey, a Senior Research Officer & Wellcome Intermediate Fellow at CIDRI-Africa in the IDM followed and talked about identification of predictive immunological biomarkers associated with mortality in people who died of severe HIV-associated TB (HIV-TB). He detailed a set of immunological markers that were investigated that could be linked to people that died after presenting to hospital with advanced HIV with first diagnosis of TB and starting TB treatment. Identification of markers that can predict mortality in these patients could lead to better management, development of host-directed therapies and improved survival.

Engineering tools for TB

The international guest speaker at the Nanosymposium was Assistant Prof Bryan Bryson from the Massachusetts Institute of Technology. Bryan spoke about using cutting-edge single cell RNA sequencing technology to dissect activation states of macrophages infected with Mtb. He identified key regulatory proteins that are differentially expressed in granulocyte-macrophage colony-stimulating factor (GM-CSF) versus M-CSF differentiated macrophages. He showed that GM-CSF turns off IL-10 in Mtb infected macrophages. Using the same macrophages stimulated in vitro, he showed how he could use parametric stitching of single cell RNA sequencing and align in a multidimensional way these macrophage profiles with non-human primate macrophages. This method allows a greater understanding of macrophage heterogeneity and spatiotemporal localisation within granulomas. He also talked about phagosomics, a new way of measuring phagosome maturation and identifying new genes/proteins associated with phagosome formation during Mtb infection. He also talked about how to build a phagosome de novo, which allows large scale testing of Mtb host stresses such as drugs. This uses tagged Mtb strains in combination with gene expression data to allow for better understanding of phagosome transcriptional changes in the presence of multiple Mtb stresses.

Way forward

Prof Valerie Mizrahi, the director of the IDM gave the closing speech at the end of the symposium. In her concluding remarks she said, “It’s with incredible passion that people are progressing TB research, and that is because we’re living with it. It’s incumbent on all of us to think about why we’re doing what we’re doing and to remember that at the end of the day, it’s about the TB patients. Ultimately, one of the things we want to do is put ourselves out of business.”

Conclusion

It is evident from the talks given by the various speakers that a lot of research is being done to combat TB at the IDM in the Faculty of Health Sciences at the University of Cape Town. The research ranges from the identification of new candidate drug targets, investigating the utility of repurposed drugs as host-directed drug therapies, understanding the transmission of the bacteria in high burden communities, investigating the immunology of TB/HIV co-infection and identification of biomarkers for TB disease progression. An important highlight of this year’s World TB Day NanoSymposium is that a bulk of this research is being undertaken and led by early career research; thus, demonstrating the depth and breadth of talented TB researchers at the IDM.

Data availability

No data are associated with this article.