Alsford Lab

After a couple of months on BioRxiv our first pre-print has been accepted for publication in Antimicrobial Agents & Chemotherapy!

‘Chemogenomic profiling of anti-leishmanial efficacy and resistance in the related kinetoplastid parasite Trypanosoma brucei‘

Selecting our T. brucei RNAi library in the anti-HAT drugs revolutionised our understanding of their mode of action and the ways the parasite can develop resistance. Unfortunately, an equivalent approach doesn’t yet exist in the related kinetoplastid parasite, Leishmania, which causes leishmaniasis, a devastating disease that’s endemic to a wide swathe of the tropics and sub-tropics.

However, the T. brucei and Leishmania genomes exhibit remarkable synteny, and these parasites share much in their biochemistry and cell biology (though their life styles are different – extra and intracellular, respectively). Therefore, we decided to select the T. brucei RNAi library in the anti-leishmanial drugs to see what insights we could gain into anti-leishmanial drug action and potential resistance mechanisms.

We identified 44 proteins, including T. brucei orthologues of the two known Leishmania drug transporters. The remaining 42 hits hadn’t previously been associated with anti-leishmanial drug action. Several of these were validated in T. brucei, highlighting the importance of a lysosomal transporter to paromomycin, and membrane trafficking and structure to amphotericin-B, as well as identifying a potential contributor to amphotericin-B/miltefosine cross-resistance.

This cohort of proteins represents a valuable resource for future study in Leishmania and has revealed some intriguing biology in T. brucei.

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.

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 several of the standard model organisms (S. cerevisiae, 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.

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.