Amino acid transport and drug efficacy

Back in 2012, we published our study decoding the anti-T. brucei drug efficacy determinants – or parasite proteins critical to drug action, such as drug transporters and activators. This identified a slew of proteins that drive the action of the five anti-HAT drugs. More recently, we’ve been following up on a few of these proteins, leading to our 2015 paper on the role of two deubiquitinases in suramin action. In July, we published a paper in the FASEB Journal describing a pair of ornithine-transporting amino acid transporters (or AATs) that impact suramin and eflornithine efficacy.

AAT10.1 was identified in our original RNAi library screen. We confirmed that it’s loss led to reduced suramin efficacy, but remarkably we found AAT10.1 loss also increased eflornithine sensitivity. This led us to speculate that it might be transporting ornithine, as loss of ornithine-spermidine pathway enzymes has similar opposing effects on these drugs. Work by Juan Macedo while in Doris Rentsch’s lab confirmed this, and added to the complexity, identifying a second AAT not seen in the original screen. Intriguingly, AAT2.4’s ability to transport ornithine is dependent on exogenous histidine levels – when they’re high, ornithine can only be taken up by AAT10.1, probably explaining why we didn’t identify AAT2.4 in our original screen (histidine is very abundant in T. brucei culture media).

These results highlight how distant processes can influence drug action – neither transporter interacts with either drug, yet both can have a significant influence on drug efficacy. Finally, the impact of AAT10.1 loss on eflornithine action, highlights a possible means to potentiate the efficacy of this frontline anti-HAT drug – specific inhibition of AAT10.1 may enable the use of considerably lower doses of eflornithine. However, the high degree of conservation between AATs may preclude such a development.

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Welcoming Juan Macedo to the lab

Dr Juan Macedo joined the lab on the 7th March, supported by a Swiss National Science Foundation early mobility post-doctoral fellowship.

Juan has spent the last few years at the University of Bern working with Peter Butikofer and Doris Rentsch on T. brucei biochemistry and amino acid transport. After collaborating for the last couple of years on the amino acid transporter project, it’s a great pleasure to welcome Juan to LSHTM.

Juan’s now planning to explore the processes underlying rDNA position effect in T. brucei and its potential role in rDNA transcriptional regulation in T. brucei.

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The 34th annual SwissTryp meeting in Leysin

Thanks to Pascal Maser (SwissTPH) for inviting me to present our latest apoL1 and anti-leishmanial drug RNAi library findings at the 2017 SwissTryp meeting in Leysin, Switzerland (11th-13th January, 2017). It was a great opportunity to hear about some of the great trypanosomatid research going on in Switzerland – social motility, mitchondrial biology, drug resistance, lipid metabolism and gene expression regulation, to name just a few areas that were addressed during the two day programme.

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Old projects come to fruition in 2016

After leaving David Horn’s lab almost four years ago in August 2013, when David and his group moved to the University of Dundee, we’re still managing to publish papers together. That’s the consequence of spending ten years in the same small but very productive group that also maintains great collaborative relationships – you get to be involved in multiple projects and, the best part, see them come to fruition! 2016 saw two such papers – Lucy and Seb’s on Vex1, and the NUP2 work by Luke Maishman and colleagues in Mark Field’s group.

Vex1 is a key controller of VSG monoallelic gene expression in Trypanosoma brucei; it localises close to the site of RNA polymerase I mediated VSG transcription, the expression site body. Remarkably, depletion or overexpression of Vex1 leads to loss of monoallelic control, indicating its fundamental role in controlling this process.

NUP2 is a second component of the T. brucei nuclear lamina, which interacts with NUP1 to form a basket-like structure under the nuclear membrane, essential for nuclear integrity and apropriate gene expression regulation. These proteins, though exhibiting functional conservation, are highly divergent from the nuclear lamins of the Ophistokonta, which includes all the standard model organisms (S. cerevisiae, A. thaliana, C. elegans, X. Laevis, D. melanogaster, as well as us!). This is yet another confirmation of the benefit of exploring beyond the narrow confines of the Ophistokonta! Not only can we find many disease-causing organisms in the other groups (Excavata – T. brucei; Alveolates [SAR] – Plasmodium), but we also gain fascinating evolutionary insights through their study.

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Ubiquitination influences suramin efficacy

Suramin is one of the oldest drugs still in use today – it’s been used to treat early stage human African trypanosomiasis caused by T. b. rhodesiense since the early 1920s. Back in 2012 we showed that suramin uptake was dependent on receptor mediated endocytosis and relied on a complex network of proteins from ISG75 in the flagellar pocket to cathepsin-L and MFST in the lysosome (Alsford et al 2012).

We recently contributed to a study led by Prof Mark Field’s group at the University of Dundee, which revealed that two ubiquitin hydrolases (TbUsp7 and TbVdu1) modulate the surface expression and turnover of ISG75 (as well as several other proteins), thereby affecting parasite sensitivity to suramin.

Modulation of the surface proteome through multiple ubiquitylation pathways in African trypanosomes.
PLoS Pathogens 11: e1005236

SILAC analyses revealed that both hydrolases have a high degree of specificity. The effect of TbVdu1 depletion decreases ISG75 and ISG65 protein levels in T. brucei, while loss of TbUsp1 impacts ISG75 and a small cohort of other endosomal proteins, including an orthologue of VAMP7b and MBAP1. These changes in ISG75 protein levels elicited by the loss of either of these regulatory proteins impacts drug efficacy, rendering the parasite significantly less sensitive to suramin.

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DELTAS programme at WACCBIP, University of Ghana

On the 12th/13th November the WACCBIP DELTAS programme inception meeting took place in the Biochemistry, Cell & Molecular Biology department at the University of Ghana, Accra. This five year programme led by Dr Gordon A. Awandare is funded by the Wellcome Trust to provide PhD and post-doctoral opportunities to African scientists in Africa in partnership with several international institutions. The programme is currently accepting applications for post-doctoral fellowships (closing date 30th November 2015) and the call for applications to the PhD stream will be released soon.

The DELTAS programme is based at the West African Centre for Cell Biology of Infectious Pathogens (WACCBIP) set up by Gordon with support from the World Bank. However, it is a pan-African programme with projects available at several other African institutions – PhD students and post-doctoral scientists will be able to work with groups at WACCBIP, MRTC (Mali), MRC ( The Gambia), KEMRI (Kenya) and the University of Cape Town (South Africa). Faculty members of the programme’s international partners (LSHTM and the Universities of Oxford, Cambridge, Pittsburgh and Cape Town) will provide co-supervision, mentoring, and six month secondments in their laboratories.

The fundamental aim of the programme is to expand disease research capacity in Africa, supporting African scientists to carry out high quality research on the diseases (both communicable and non-communicable) that assail the continent.

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Royal Society Pfizer Awards, 19th October 2015

For ten years, the Royal Society-Pfizer Awards have been recognising outstanding African Scientists. For the first time this year, on the 19th October, there were two awardees. The advanced award was presented to Prof Jean-Jacques Muyembe-Tamfum, Institut National de Recherche Biomedicale (Democratic Republic of Congo), in recognition of a lifetime working on Ebola. A mid-career award was presented to Dr Gordon A. Awandare of the University of Ghana.

With funding from the World Bank, Gordon recently set up the West African Centre for Cell Biology of infectious Pathogens (WACCBIP) for the delivery of high quality African-led pathogen research and postgraduate teaching. More recently, and in partnership with a several international institutions, including LSHTM, Gordon won prestigious Wellcome Trust DELTAS funding to establish a pan-African PhD and post-doctoral programme based at the University of Ghana (more on this in an upcoming post!).

A few of us from LSHTM were lucky enough to be invited to the award ceremony. This included a day of talks highlighting the achievements of the 2015 awardees, as well as those of past winners. There were also fascinating supporting talks from Prof Peter Piot (LSHTM) on Ebola and Prof Mark Carrington (University of Cambridge) on human serum sensitivity in African trypanosomes.

Listening to the talks of past recipients and the acceptance speeches of the 2015 winners, it was clear just how important recognition from august institutions such as the Royal Society is. These awards make a real difference to the research careers of African scientists, acknowledging the high quality of their work, and boosting their ability to secure the funding necessary to build on their successes and to expand their research programmes. Ultimately, such recognition goes some way to support the continued development of a dynamic and productive scientific research community in Africa, something the whole continent (and the international community) can benefit from.

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Invited talk at the Institut Pasteur

Jumped on to the Eurostar to Paris on the 7th October and visited the Institut Pasteur on the 8th October to give a seminar (‘Decoding toxin-parasite interactions in Trypanosomatids‘). Thanks to Philippe Bastin and Gerald Späth for hosting (and for treating me to some a fine French dining the evening before!), and to all their colleagues for making me welcome on the 8th.

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Invited talk at UCL

20th October was spent at University College London (just up the road!) presenting data from our various T. brucei RNAi library screens – something a bit diferent for all the virologists who work in the Division of Infection & Immunity at UCL! Dr Richard Milne and Prof Greg Towers hosted, and made sure I got to meet a lot of the PIs in the Division – it was nice to hear about some of the great virology research that’s going on there.

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