Essential trypanosome transporters hint at new therapeutic targets

By Rebecca Hall

Twitter: @RebeccaJHall13

Blog: http://www.ananxiousscientist.wordpress.com

 

Human African trypanosomiasis (HAT), more commonly known as sleeping sickness, is a disease caused by the protozoan parasite Trypanosoma. Endemic in 36 sub-Saharan African countries, HAT causes fever, headaches, joint pain and, once the parasite has crossed the blood-brain barrier, the characteristic sleep cycle disturbances that give the condition its colloquial name. Trypanosomes have a complex life cycle, residing partly inside its tsetse fly host and infecting mammals in a separate stage. The adaptations that the parasite has undergone in order to thrive inside humans enable it to evade the immune system; by ‘putting on’ a unique ‘coat’ of glycoproteins, trypanosomes ensure that the immune cells cannot keep up with its disguises. As such, developing drugs to combat sleeping sickness and nagana, its equivalent in cattle, is a complex and frequently unsuccessful process.

The drugs that are available currently to treat HAT are limited by a risk of toxicity and are not always effective. There is also increasing concern that resistance may arise and so there is a lot of interest in teasing out the biology of trypanosomes, with transport and metabolism being one key area. The hope is that they may be able to find new therapeutic targets by identifying essential components of the parasite.

Amino acid uptake is hugely important for trypanosomes. When they transition from mammalian to insect host they are required to adapt to very different environments. Blood is the sole diet of the tsetse and therefore the parasite must be able to survive on amino acids as their energy source when they are in this stage of their life cycle. They are also auxotrophic for a number of amino acids, meaning they cannot produce them themselves and instead rely on importing them to survive. The transporters for these therefore provide a potential drug target; block the ability to uptake essential metabolites and the parasite will die.

A paper published in early January describes two transporters that could become potential therapeutic targets. Mathieu et al. looked at two amino acids, arginine and lysine, that are essential for trypanosome survival. They identified candidate transporters by constructing a phylogenetic tree and transformed them into Saccharomyces cerevisiae mutants. These mutants were unable to uptake different amino acids and so the group were able to establish what these proteins transported by assessing the ability of the mutants to grow on various substrates. They identified transporters that enabled growth on lysine and arginine in strains of S. cerevisiae that would otherwise have been unable to grow.

The team then used transport assays to reveal that these transporters have both high affinity and selectivity for their substrates. Transcriptomics suggested that they are highly expressed and analysis of cMyc-tagged trypanosomes indicated that these transporters localise in the plasma membrane. They finally assessed the essentiality of these proteins by down-regulating their expression through RNA interference and found that growth of these parasites was significantly reduced.

These transporters are therefore interesting therapeutic candidates because of the reliance of the trypanosome on them for survival. Importantly, these are not related to uptake systems in humans and so any drug that worked against them would not run the risk of off-target effects.

 

Source: Arginine and lysine transporters are essential for Trypanosoma brucei, Mathieu et al. (2017), PLOS ONE

 

 

ABC transporter implicated in parasite drug resistance

An ABC transporter in Leishmania potentially confers resistance to the antimony used in leishmanicidal drugs by sequestering the compound in vesicles and exporting them via the parasite’s flagellar pocket.

Leishmaniasis is a neglected tropical disease (NTD) caused by the protozoan parasite Leishmania. It is responsible for 20 000 – 30 000 deaths every year in countries including India, Bangladesh and South Sudan. The World Health Organisation (WHO) estimates that 310 million are at risk of developing visceral leishmaniasis.

Leishmania has two distinct life cycles, one in its mammalian host and one in its sandfly vector.  The sandfly injects promastigotes into the skin during a blood meal. These promastigotes are then taken up by macrophages where they transform into amastigotes and multiply. They are eventually released from the infected cell into the bloodstream from where they may be taken up by another sandfly during its next blood meal.

Slide1
Leishmania have two distinct life cycle stages, one within their mammalian hosts and one within the sand fly vector. The parasites are taken up during an infected blood meal and replicate within mammalian cells before being transferred to the sand fly during the next meal. Adapted from CDC (http://www.cdc.gov/parasites/leishmaniasis/biology.html).

Current treatments for leishmaniasis, including amphotericin B, miltefosine and pentavalent antimonials, can be both toxic and expensive. This coupled with the ever-increasing issue of drug resistance means that the disease is in danger of reaching crisis point. Scientists have been attempting to elucidate the various ways in which resistance could arise in the hope of curtailing some of the problems facing Leishmania control.

A team from Spain have done just that, identifying an ATP-binding cassette (ABC) transporter in Leishmania which they believe might be involved in resistance to antimony. Leishmania has 42 ABC genes yet few have been characterised. The team led by Ana Perea looked at SbV, an antimony-based drug which is taken up by the amastigote (intracellular) form of the parasite. It becomes reduced to SbIII and activated once inside the macrophages. Leishmania encodes enzymes that are capable of reducing SbV to SbIII, which then combines with thiols that are effluxed from the parasite.

The transporter in question is LABCG2. It was chosen as related transporters LABCG4 and LABCG6 had previously been implicated in resistance to the drug miltefosine. LABCG2 is involved in phosphatidylserine (PS) externalisation during infection of the host macrophages. They found that overexpressing LABCG2 resulting in the promastigotes becoming 7-fold more resistant to the antimony-based compound. This resistance was however not seen in other leishmanicidal drugs such as miltefosine.

The team then delved into exactly what was behind the resistance to SbIII. The parasites were incubated in antimony and after 60 minutes the accumulation of the compound was measured. The mutants which overexpressed LABCG2 were found to have accumulated 76% of the total amount of SbIII that the controls had. They interpreted this as an indication that the LABCG2 transporter mediates the elimination of antimony from the parasite.

Finally, they looked to establish whether thiols, which bind to and export heavy metals, could play a role in Leishmania antimony resistance. They found that thiol efflux from the parasites was greater in the presence of antimony and, following tagging by green fluorescent protein (GFP) discovered that the transporter does localise at the plasma membrane.

Overexpressing the LABCG2 ABC transporter might therefore protect Leishmania against otherwise toxic antimonic drugs by effluxing them as a complex bound to thiols. They believe that this could be a mechanism by which Leishmania may become drug resistant, although emphasise the need for LABCG2 knockout mutants to really establish what role the transporter plays in the parasite.

 

Source: Perea et al. (2016). The LABCG2 transporter from the protozoan parasite Leishmania is involved in antimony resistance. Antimicrobial Agents and Chemotherapy, 60, 3489 – 3496.

Rebecca Hall

Twitter: @RebeccaJHall13

Blog https://ananxiousscientist.wordpress.com/