My (Our) project - Cell division in Plasmodium Berghei

What am I doing?

I am investigating the role of an Aurora-like protein kinase, PbARK2, in cell division in Plasmodium berghei, the rodent model for Plasmodium falciparum.

Why am I doing this?

Plasmodium undergoes cell division in a very atypical way – while most eukaryotes undergo open mitosis, in which the nuclear membrane breaks down before sister chromatids are pulled apart, Plasmodium cells maintain their nuclear envelope at all times. This is called closed mitosis. In addition to closed mitosis, their nuclear division is asynchronous – in a multinucleate cell, each nucleus divides independently of the ones around it. This suggests that there is no global control of mitosis within a cell, and a recent study highlighted that CDC20, a protein essential for mitotic regulation in all eukaryotes is in fact dispensable in the blood stages of parasite development.

Given that Plasmodium uses a drastically different system for cell division, many of the proteins involved may be very different to those in humans, making them good drug targets as off-target effects will be limited. By understanding the underlying biology of the system, we can better make predictions and investments into drug development.

How am I doing this?

ARK2 has already been shown to be essential for blood stage development in P. berghei. The plan was therefore as follows: place ARK2 under the control of an alternative promoter that is active in the blood stage to escape the lethal phenotype, allowing phenotypic analysis at later stages of development. This was to be achieved using a PTD construct (promoter trap using double homologous recombination), generated using molecular cloning techniques in the lab, before transfecting it into wild-type parasites.

Schematic representation of the PTD construct for placing Ark2 under the control of ama1

To provide additional insight into the function of ARK2, a previously generated parasite line expressing an ARK2-GFP tag was to be used to visualise the cellular distribution of the protein and determine expression patterns throughout the life cycle. This would be complimented by qPCR data produced for each life cycle stage.

What’s the story so far?

Last semester started pretty well – there are four of us MSci students in the lab doing very similar projects with different proteins so we felt like we weren’t all on our own. The initial molecular cloning went well, with the direct supervision of Declan, our resident lab-God, knower-of-all-things, provider-of-reagents and all-round-science-boss (the lab technician). With the 3’ fragment successfully inserted into the vector, we were left to do the 5’ section ourselves – this is where things started to go wrong. I managed to get lucky and only need to do one step twice, but Jack had to repeat his bacterial transformation four or five times. However, after a slightly shaky semester we had our constructs completed, sequenced to check accuracy, and ready for transfection. Two weeks before the end of the semester we were poised to begin our transfections and get on with the meat of the project. December 5^th came around, everything went well, nothing could have gone wrong, we felt confident and excited until a week later, when nothing survived the drug selection step, meaning our transfections hadn't worked.

Merry Christmas!

Four weeks later we were back and ready to get back into it. We knew the protocol better this time, we had read more about it, we wanted it more this time. And then it didn’t work. Neither did the third attempt, but the positive control worked just fine. Slightly disheartened, we decided to move on with getting our Western blots to show our protein was correct in the GFP-tagged lines. Carrie has a fantastic post telling you just how well that went… While a few weeks went by desperately hunting for meaning in the Rorschach tests we were creating, we started to learn how to use the fluorescence microscope to visualise our proteins. Jack has a detailed post talking about some of the trials and tribulations we experienced with the microscope, particularly the bit where more than four people need to use one microscope to do time sensitive investigations. Somewhere along the line we went from “just taking practice images” to being expected to produce a full collage of every life cycle stage – I’m still not sure exactly when that was, but I found that this was something I could confidently do. In addition to regular life cycle stages, we discovered that my protein was only transiently expressed during ookinete development, meaning we needed to image timepoints in ookinete development. This is where it gets exciting, because I got my first actual set of data usable in my write-up!

Collage of ookinete developmental stages showing punctate distribution of ARK2 dependent on stage.

Having finally got a decent set of images, I hoped that shortly after everything else would click into place – my Western would come out beautifully, my transfection would be successful and I’d have a clear picture of everything-ARK. However, the next transfection also failed and I still, 3 months and 14 Westerns later, don’t have a band for my protein. We’ve got a few plans for how to fix this but time is running out and it’s getting a bit worrying.

That being said, having spoken to my friends who are doing PhDs, and the post-docs in the lab, in research you have to get used to failure. The trick to being a good scientist is to persevere despite things not going right and work out how to fix it. Use the scientific method and think logically about what you can change, and maybe, just maybe, you’ll get the result you want.

If you’ve had a project come right down to the wire, or want to share any of your science woes please let me know in the comments so I can feel like it’s not just me that’s had this experience.

How bacteria might win the fight against malaria, dengue and Zika

Have you heard of the most prevalent parasitic microbe in the world? 

Capable of infecting up to 70% of all insects? 

With a preference for murdering males in cold blood? 

Known as “Wolbachia”, this genus of bacteria is found in many arthropods, including those responsible for the transmission of many human pathogens; such as malaria, dengue fever, and the Zika virus. It gets transmitted vertically, meaning that infected females pass it directly to their offspring. Different strains of Wolbachia infect different hosts and can have many different effects. In filiarial nematodes Wolbachia plays a mutualistic role, with elimination of the symbiotic bacteria causing host sterility and sometimes death. In mosquitos however, Wolbachia has a more sinister role - causing something known as “cytoplasmic incompatibility”. I’ll explain what exactly that means in a minute, but the end result is that infected females will always produce infected offspring, and uninfected females have half the chance of producing offspring. This is a case where it’s easier to show, than tell.

This picture, taken from the Werren lab Wolbachia biology page, shows how cytoplasmic incompatibility leads to an increase in the proportion of infected hosts.

The exact mechanism for this cytoplasmic incompatibility (CI) isn’t yet clear. What is known is that some kind of modification must take place in the infected male during spermatogenesis, as mature sperm cells do not contain Wolbachia. We also know that rescue must occur at some point in the fertilised infected egg, as the presence of Wolbachia prevents CI from occurring, but uninfected eggs don’t produce offspring. The main consequence of CI is that the male pronucleus enters mitosis later than the female pronucleus, which means the genetic information from the male does not segregate properly during the first mitosis. This leads to the production of haploid daughter cells which is embryonically lethal, preventing any offspring from being formed.

In May last year, a paper by Daniel LePage et al., was published which found that just two genes in the Wolbachia genome were needed to cause CI. The sequences for these genes were found to originate from viral DNA, integrated from the bacteriophage WO genome at some distant point in Wolbachia’s evolutionary history. These two genes, named cifA and cifB (cytoplasmic incompatibility factors A and B), were initially identified as part of a pool of 113 genes shared between CI-causing strains of Wolbachia. This pool was narrowed down by removing proteins known to be very different or absent in a non-CI-causing strain, wAu, and by removing proteins known to be expressed by infected ovarian tissue as these might be generally expressed and not play a direct role in CI. This left just two genes, WD0631 and WD0632.

In section a, a Venn diagram shows 113 genes are shared between four common CI-causing strains of Wolbachia. In section b, a Venn diagram shows how these 113 genes were narrowed down to just two candidates for CI-causing genes – WD0631 and WD0632.

To investigate if these two were indeed the genes responsible for CI, transgenic lines of Drosophila melanogaster were created with both genes under the control of a promoter that is active in germ line cells. Males of this line were crossed with infected and uninfected females and the relative hatch rate calculated. It can be seen in the graph below that having both genes caused a significant change in the embryo hatch rate when crossed with uninfected females, and that this was rescued when crossed with infected females – as would be expected if these were the culprits for CI.

A comparison of relative embryo hatch rate in D. melanogaster. White symbols indicate uninfected males and females, black symbols indicate those infected with a CI-causing strain of Wolbachia.

Further to this the team investigated whether the cytological appearance was the same between Wolbachia infected embryos and transgenic WD0631⁺/WD0632⁺ lines, further strengthening their case that these were the responsible genes.

Panels a-f show different classifications of cytological appearance in recently fertilised ovaries of D. melanogaster. a) indicates unfertilised eggs, b) shows normal nucleated cells at 1h of development, c) shows normal embryos at 2h of development, d) shows failure of nuclear division after two to three mitoses, e) shows chromatin bridging and f) shows regional mitotic failure. In section g) the relative abundance of each cytological appearance is shown in different crossing scenarios.

The paper doesn’t go on to speculate on or provide any investigation into the mechanism of action of these two genes, but sets the stage for further work into this system and tantalisingly closes with the statement 

“Finally, cifA and cifB are important for arthropod pest and vector control strategies, as they could be an alternative or adjunct to current Wolbachia-based efforts aimed at controlling agricultural pests or curbing arthropod-borne transmission of infectious diseases”

This brings me on to the conclusion of this post and refers all the way back to the title – how can bacteria be used to fight insect-vector based disease?

There are already programs in place that are attempting to use Wolbachia-based methods of eradicating the insect vector of the dengue fever virus, the Aedes aegypti mosquito. By introducing large numbers of infected males into an uninfected population, the population can be reduced without the use of pesticides. The work done by LePage could provide the means to supplement Wolbachia in insect systems or possibly induce CI in other systems. An alternative to herbicides, or maybe maintenance-free pest control – can you think of any other applications? Let me know in the comments.

“Malaria drugs fail for first time on patients in UK”

A recent news article by the BBC reported that four cases of malaria in the UK failed to respond to artemether-lumefantrine treatment, the current front-line drug combination recommended by the WHO. Drug resistance to older antimalarial drugs, like chloroquine, is already widespread in many countries with endemic malaria. It spreads faster in areas where treatments have been dispensed inappropriately, because this fosters selection for resistant strains, similarly to antibiotic resistance. WWARN (worldwide antimalarial resistance network) has an excellent series of interactive maps that you can use to explore the available data on antimalarial resistance.

Of particular concern is resistance to artemisinins – a group of drugs which are the key component in the artemisinin-combined therapies recommended by the WHO. These are well tolerated therapies with high effectiveness, assuming the full course of treatment is taken. However, in certain areas where healthcare has not been well managed, or access to drugs is available without appropriate medical advice, artemisinin resistance has spread rapidly and pervasively. This is shown well in the map produced by Ashley et al., which demonstrates the prevalence of a certain cause of resistance in South East Asia.

A map produced by Ashley et al., showing prevalence of PfKelch13-associated artemisinin resistance in S.E. Asia, and the limited spread of PfKelch13-independent resistance in Africa.

The exact mechanism of resistance to artemisinin is still contested, with several ideas proposed so far but no consensus has yet been achieved. Part of the reason for this is that we still don’t really know how artemisinin kills the parasite in the first place… What we do know however, by using Genome Wide Association Studies of parasite genomes, is that mutations in the “Kelch-propeller domain” of Plasmodium falciparum Kelch13 are strongly associated with cases of artemisinin resistance. Kelch-propeller domains are used for protein:protein interactions, and can be very specific. K13 belongs to a superfamily of proteins known to mediate ubiquitin-regulated protein degradation and oxidative stress-responses, which plays into some theories suggesting that artemisinin acts by generating reactive oxygen species, thereby causing oxidative stress.

While mutations in PfK13 are certainly common in resistant cases (again, particularly in S.E. Asia), they aren’t constant. As seen in the map above, African cases of artemisinin-resistance are mostly not found to have any mutations in PfK13. Instead, variable artemisinin efficacy appears to be linked to multi-locus genotypes involving other resistance-associated proteins such as PfMDR1 (a multi-drug resistance pump) and PfCRT (a chloroquine resistance-associated transporter protein), along with less well understood proteins such as PfUBP1 and PfAP2MU. Worryingly, the diverse causes of artemisinin resistance suggest multiple, independent evolutions of resistance in isolated Plasmodium populations.

Going back to the original point of this post, the BBC article “Malaria drugs fail for the first time on patients in the UK”, we should look at the source of this information. This is a brief report of four case histories of patients with imported malaria that were initially treated with artemether-lumefantrine but presented with recurrent parasitaemia within 6 weeks of treatment with no intervening travel to malaria-endemic areas. In English, this means patients that caught malaria outside of the UK were treated to the point where they seemed healthy, but were then readmitted at a later date with the same symptoms without an opportunity to catch it again. Two patients had been to Uganda, one to Liberia and one to Angola – all in Africa. As you might be able to guess, when profiled for resistance markers, none of the parasites these patients were infected with had any PfKelch13 mutations. Instead, they had a variety of mutations in other loci known to be associated with resistance, shown in table 1.

Ultimately all patients were successfully treated with alternative drug combinations such as atovaquone/proguanil, quinine/doxycycline or artemether/lumefantrine with doxycycline so the initial headline may be slightly exaggerated when you take the full picture into account. However, this is still an important news story as it is the first time artemisinin resistance has been seen in the UK. As resistance spreads throughout Africa these cases will only become increasingly common, so finding appropriate alternatives is a necessary task. 

The BBC article includes an interview with Dr Sutherland, the lead researcher, who stresses that while this is not yet a national health crisis it is important for doctors to be aware that drugs may not work and says drug guidelines should be reviewed. He also suggested that large scale studies of drug efficacy need to be urgently undertaken in Africa to determine the severity and scale of the problem we could be facing very soon.

Personally, I feel that while this is a very important news story, the fact that just four UK cases made headlines while thousands occur every year abroad is telling of people’s attitudes in the UK. The spread of drug resistant malaria is very real and a very serious concern to people living in malaria endemic areas, causing death on a huge scale, but is not on the radar for most people until it directly concerns them. I think awareness needs to be raised about this as an issue that faces other countries, not just ours. I’d like to know if you agree or disagree, and if you have any ideas about ways we could achieve this goal – let me know in the comments.