Invited talk at the Institut Pasteur

Jumped on to the Eurostar to Paris on the 7th October and visited the https://research.pasteur.fr/en/ 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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Invited talk at the Centre for Immunology & Infection, York

28th May was spent at the University of York presenting data from our various T. brucei RNAi library screens. It was nice to get out of London and to meet up with Pegine Walrad, hear about her developing lab, and meet some of the other people doing great work on Leishmania at the Centre for Immunology & Infection.

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Why Trypanosoma brucei is sensitive to human serum

Cathepsin-L can resist lysis by human serum in Trypanosoma brucei brucei.
PLoS Pathogens 10: e1004130
Featured at: LSHTM, Wellcome Trust
See also: commentary in Microbial Cell 1:270-2

Most African trypanosomes, including the veterinary species Trypanosoma brucei brucei and T. congolense (causative agents of the wasting disease nagana in cattle), are susceptible to lysis by two trypanolytic factors in human serum – TLF1, a component of high density lipoprotein, and TLF2, an IgM/apolipoprotein-A1 complex; both complexes contain apolipoprotein-L1, which forms pores in the parasite’s lysosomal membrane (Vanhollebeke & Pays, 2010).

In contrast, the human-infective sub-species, T. b. rhodesiense and T. b. gambiense, are able to evade lysis by human serum through a variety of mechanisms. Both express truncated variant surface glycoproteins: SRA and TgsGP, respectively (Xong et al, 1998; Uzureau et al, 2013; Capewell et al, 2013). However, whereas SRA prevents APOL1 accessing the lysosome (Stephens et al, 2011), TgsGP expression only serves to hinder the membrane-APOL1 interaction (Uzureau et al, 2013). Instead, human serum resistance in T. b. gambiense also requires reduced lytic factor uptake through the expression of a mutated version of the parasite’s haptoglobin-haemoglobin receptor (Kieft et al, 2010; DeJesus et al, 2013).

Clearly, human serum serum resistance in African trypanosomes has evolved independently on more than one occasion, requiring both gain and loss-of-function mutations. But, are there other proteins that define the sensitivity of some African trypanosomes to lysis by human serum? Are there other routes to human serum resistance? Using this information, can we identify strategies to render resistant parasites sensitive to attack by human serum TLFs? In work funded by the Medical Research Council and the Wellcome Trust, we set out to define the parasite proteins that make T. b. brucei sensitive to the trypanolytic factors in human serum. Using our bloodstream-form RNAi library, we hoped to identify the full set of TLF eficacy determinants expressed by T. b. brucei.

Given the massive size (>0.5 MDa) and complexity of the lytic factors in human serum, we identified surprisingly few loss-of-function mutants that are able to confer significant human serum resistance onto T. b. brucei. As expected, TbHpHbR was the dominant hit in the RNAi library following selection in human serum. In addition, we identified the lysosomal membrane protein, p67, which is known to be important for proper lysosomal function and has previously been shown to influence sensitivity to human serum (Peck et al, 2008). Finally, we identified two proteins not previously known to influence sensitivity to human serum, ‘inhibitor of cysteine peptidase’ (or ICP) and a putative trans-membrane protein (Tb927.8.5240). ICP was subsequently, shown to be important in this pocess by Etienne Pays’ group (Uzureau et al, 2013); however, their analysis did not identify the regulated peptidase presumably responsible for lytic factor destruction.

We focussed our efforts on characterising the role of ICP in determining the human serum sensitivity of T. b. brucei. The parasite expresses two cathepsins, which constitute likely targets for regulation by ICP. Perturbations of CATB and/or CATL by chemical or genetic means had no effect on the sensitivity of wild-type cells to human serum, suggesting that ICP is able to modulate its cathepsin-suppressive effect in response to enzyme activity levels. Loss of ICP resulted in reduced sensitivity to human serum, presumably due to increased cathepsin activity against the lytic factor. Repeating the chemical and genetic perturbations of CATB and CATL in the absence of ICP, revealed that only CATL loss has a dramatic effect on the parasite’s susceptibility to lysis by human serum. Thus, we were able to conclude that ICP’s suppression of CATL activity in T. b. brucei contributes to the sensitivity of these parasites to human serum, sugesting that CATL targets the lytic factor for destruction in the lysosome.

CATL expression is essential for growth of bloodstream-form T. b. brucei, and is now recognised as an excellent target for anti-HAT drug development (Steverding et al, 2012). The above data suggests that its inhibition may render human infective sub-species susceptible to human serum lytic factors. Therefore, drugs that target this enzyme may synergise with trypanolytic factors, rendering otherwise resistant trypanosomes, sensitive to human serum. However, it should be noted that CATL may influence the production and processing of SRA and TgsGP in the endocytic networks of T. b. rhodesiense and T. b. gambiense. So, while targeting CATL may synergise with human serum, we may yet see antagonism resulting in human infective sub-species that are more resistant to human serum. Clearly, further work needs to be done to characterise the complex interactions between these factors in T. b. gambiense and T. b. rhodesiense.

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Invited talk at the Institute for Molecular Infection Biology, Wurzburg

Just got back from a couple of days in Wurzburg, Germany. Nicolai Siegel invited me to talk on the Microbiology Colloquium (at the Institute for Molecular Infection Biology) – on 29th April I presented our latest data on the characterisation of trypanosome factors responsible for drug and human serum efficacy.

The Institute has a great strength in bacteriology, so it was mostly an audience of bacteriologists with only a smattering kinetoplastid researchers; I may have catered more to the latter, but the questions suggested that a few of the former also found it interesting! (Some judicious editing will be required before I present this again at the end of May at the University of York!)

Apart from the talk, it was a great opportunity to catch up with Nicolai Siegel, who invited me, and Chris Janzen and Susanne Kramer at the Biozentrum, and to hear about the great work they and their teams are doing.

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Accession Numbers – Anything to Declare?

Some people get very exercised by accession numbers – the unique identifiers associated with genes and proteins.

When publishing results in a paper it’s accepted, and indeed rightly mandated by scientific publishers, that all associated accession numbers should be declared, so enabling follow-up analyses by other groups.

Should the same apply to conference presentations? In this case, results are often incomplete, representing work in progress, which may not enter the published record for months or even years. In contrast, peer review and publication gives a scientific paper public recognition, and also endorses its quality and contribution to the field. Papers also tend to represent complete or near complete pieces of work. Not being peer-reviewed, and rarely being complete, conference presentations have nothing like the cachet of a scientific paper.

In spite of this, many groups declare the accession numbers of the proteins they’re working on, even in conference presentations. However, others don’t. Instead, giving their proteins catchy names, so protecting their identities, while still sharing the results that make them worthy of study.

Some people find this irritating, believing that such behaviour contradicts the collegiate and collaborative foundation of science.

They believe that if you want to present your work you should present all the details, including the full identity of the protein(s) that are its subject. The assumption being that if you’re willing to present your data in a public forum, you must be so far ahead of your competitors that it won’t be worth their while jumping on the band wagon that you’ve just wheeled out! And anyway, if they did, it would be so obvious to the rest of the community that their reputation would be hammered; though what the real world consequences of this would be are anyone’s guess.

Maybe, whether or not you’re a ‘declarer’ depends on the nature of the work, the competitiveness of your field, or the characters within it – or at least your preception of any or all of these elements. After all, some results have the potential to have a huge impact on a field, but many more are incremental; though all are worthy, the former have the potential to cement reputations and make careers. Some fields are fiercely competitive. Others are populated by individuals of whom, rightly or wrongly, the moral majority are wary. Or, maybe we’d simply rather not put temptation in others’ way, and have to spend the next months (or years) worrying about what might or might not happen.

In my view, an idealistic one-size-fits-all rule can’t apply, and, ultimately, the decision to declare (or not) should be down to the individual. As long as the data are honestly presented, I’ll happily look forward to reading the published paper, if and when it comes out. When it comes to my own research, I’ll continue to deal with this issue on a case-by-case basis, depending on just how close I think I am to a complete story of any significance.

Probably, the idealists shouldn’t rush to judge, and the rest of us should be a little less paranoid. But, perhaps, as scientists we’re all just a little conflicted – wanting to work for the wider community, and often doing so, but at the same time knowing that we have to look after ourselves. After all, it’s inherent to a system where funding is short-term and hard to get, and for the most part insecurity is the only guarantee, that we sometimes feel the need to guard our most important discoveries, in the hope that we’ll be able to keep doing the science we love for the long term.

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BSP Meeting Cambridge 2014

6-9th April was spent at the University of Cambridge attending the British Society for Parasitology annual meeting, including the Tryp-Leish symposium. Thanks to Mark Field (and colleagues) for organising a great meeting – his final act in Cambridge before packing up his lab and moving to the University of Dundee!

There were many great talks, but highlights included the latest findings from Federico Rojas (Keith Matthews, University of Edinburgh) on bloodstream-form T. brucei differentiation (SIF receptor, anybody?), and Lucy Glover’s talk on VEX1, a significant player in the regulation of antigenic variation in T. brucei (David Horn, University of Dundee). Mike Barrett’s CA Wright medal talk (drug resistance and modes of action) and the plenary talks by Keith Gull (trypanosome cell biology) and David Roos (apicomplexan cell biology) were also fascinating.

Finally, the meeting provided a great opportunity to talk science and catch up with friends over a beer or two in a great setting.

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