I had seen parts of  this animation that beautifully shows the central dogma of molecular biology, without knowing the creator and his other pieces of work. I finally discovered Drew Berry, the artist behind this work through his TED talk. There, he shares his passion and inspirations and shows pieces of his original and recent animations. Excited by the talk, I Googled him and found that has also produced a video describing the life cycle of Plasmodium. The first and second parts of this short but entertaining and educational video can be watched here and here. This video and similar works by this animator can and should be used as powerful teaching aids.

Drew Berry has tried to be as scientifically accurate as possible by getting protein structures from the protein data bank (PDB) and reading research papers to correctly animate the mechanisms according to them. Read more about his work on his website.

“Well, in our country,” said Alice, still panting a little, “you’d generally get to somewhere else — if you run very fast for a long time, as we’ve been doing.”

“A slow sort of country!” said the Queen. “Now, here, you see, it takes all the running you can do, to keep in the same place. If you want to get somewhere else, you must run at least twice as fast as that!”

***

The Red queen hypothesis was first proposed to explain why the fitness of some species stays the same, although they are constantly evolving. Arms race between a predator and prey, or a host and a pathogen are classic examples. Constant evolution occurs when the species is in a constantly changing environment. For instance, the host is infected by more virulent species of the pathogen each generation; therefore it is under constant selection for having a better and better immune system.  Similar selection pressure holds for the pathogen that is facing stronger and stronger immune systems, so it is under selection for more virulence. In such situations, the host and the pathogen coevolve, but their fitness doesn’t change, ‘it is as the Red queen says it takes all the running you can do to keep in the same place’.

There is another important implementation of the Red Queen hypothesis and that is in explaining sexual reproduction. In species that reproduce sexually, males charge an extra cost. They contribute little or nothing to the offspring, compared to females or hermaphrodites. They consume resources that could be otherwise consumed by offspring or females that could actually produce offspring. Theoretically, an asexually reproducing population of females could grow faster and produce more offspring than a sexually reproducing one. But it doesn’t just end there. Sexual reproduction creates a new selective force called sexual selection. This selective pressure is exerted on one sex that is being chosen by the other. It can result in selection of very bizzare traits that might actually reduce the fitness of the individual (think of the Peacock’s mganificant tail). Then why are higher eukaroytes reproducing sexually? What are the males for?

The big advantage of sexual reproduction is generation of genetic diversity. Without shuffling of genes through sexual reproduction, it would take a lot longer (or never) for a new advantageous mutation to be fixed in the population. In addition, new genotypes are created because different array of alleles are put together with each round of shuffling. Without this shuffling, these potentially advantageous allele combinations might never occure. Yeasts switch from asexual reproduction to sexual reproduction when they are under stress or in an unhospitable environment. In other words, although they are well fit to live in that environment, they hope that by shuffling their genes, some of the offspring would have better chances. Obviously if they continue reproducing asexually, their offspring would be identical to them and doomed for extinction.

However, this sexual-asexual switch does not exist in animals and plants (there are always exceptions in biology). This would make you infer that they are in a constantly changing environment that requires constant evolution or it would lead to extinction. In other words, it is like running with the red queen to keep fitness at the same place. But what is it that is constantly changing and imposing this strong selective pressure for such a high costly method of reproduction? It was proposed that presence of constantly evolving pathogens and parasites puts their hosts under selective pressure for adaptation. Sexual reproduction offers the selective advantage of genetic diversity and possibility of more resistance in the offspring. Same argument stands for selfing versus outcrossing strategies. Outcrossing is more costly. It requires actively seeking (dating!) another outcrossing partner. Also, outcrossing populations may have mixed subpopulations of hermaphrodites and males. In this case again, the males are an extra cost, while selfing limits the genetic diversity of the offspring.

It is difficult to create controlled evolutionary systems in a lab environment, thus evolutionary hypotheses are difficult to test. This is what makes the paper by Morran et al. published in Science in July 2011 very exciting. They tested the validity of the Red Queen hypothesis in the coevolution of hosts and pathogens.

The question was whether outcrossing offers selective advantage over selfing in the presence of a coevolving pathogen. They used Caenohabditis elegans and the deadly bacteria Serratia marcescenes as the host-pathogen couple. Three different strains of C. elegans were used:  strictly selfing, strictly outcrossing and wildtype (a mixture of both). ‘Coevolution’ of C. elegans and S. marcescenes was done by sequentially infecting surviving C. elegans worms with bacteria that were isolated from dead worms in the same culture. This was in comparison with ‘evolution’ of C. elegans where it was being reinfected with the ancestral strain that both cultures had started with. In the coevolution model, the bacteria were also supposedly under selection for more infectivity since the surviving worms were more resistant to infection. This experiment was carried over for 30 generations.

What would the red queen hypothesis predict for these conditions? Infection with deadly bacteria is supposed to induce more outcrossing as opposed to selfing in order to increase the chances of resistance in the offspring. However, once resistance has been acheieved, we would expect the worms to revert back to selfing as they are no longer under selective pressure. However, if resistance never occurs, outcrossing rates are expected to remain high compared to control. And this is what Morran et al. observed with the wildtype C. elegans worms. While evolving C. elegans worms that were exposed to a nonevolving strain of S. mersescenes peaked in outcrossing rate within 8 generations, by generation 30 the outcrossing rate was down back to control levels. However, the outcrossing coevolving worms showed continuously increasing outcrossing rates plateauing after 16 generations.

This clearly supports the red queen hypothesis in explaining the underlying reason for outcrossing and presence of non-offspring-bearing males in a population. However, the authors did not stop here and further backed up their argument:

They showed that the coevolving bacteria indeed have higher infectivity in strictly selfing, or non-coevolving C. elegans. Furthermore, the strictly selfing worms did not adapt to infection. Higher mortality rates were observed after multiple generations and also the coevolving bacteria were shown to be more infectious compared to the ancestral strain; this was not the case with the strictly outcrossing or wildtype strains.

So all together, this is an elegantly performed evolution experiment that highlights the importance of outcrossing in adaptation to pathogens. As it is beautifully put in Science2.0: The Red Queen is not dead. Long live the Red Queen.

Genes are not the only things that get better with sex. Matt Ridley, the author of The Red Queen , discusses how technology advances in the human society when ideas have sex in a TED talk.

Oh, and happy 2012!

Morran LT, Schmidt OG, Gelarden IA, Parrish RC 2nd, & Lively CM (2011). Running with the Red Queen: host-parasite coevolution selects for biparental sex. Science (New York, N.Y.), 333 (6039), 216-8 PMID: 21737739

Th2 immune response is highlighted by IL-4 production. But when looking in vivo, it is impossible to find the source of the IL-4 that is present in the biological fluids. So in their study published in Immunity in 2005, the authors used an intestinal infection by nematode  H. polygyrus  as their model. This worm resides solely in the intestine and induces a robust Th2 response. they engineered a GFP sequence preceded by  an internal ribosome entry site (IRES) after the final exon of the IL-4 gene. In this way, when the IL-4 mRNA gets transcribed, the ribosome can bind separately to the IRES and translate GFP. Therefore, cells that are producing IL-4 mRNA could be identified. What they surprisingly found was that, CD4+ GFP+ T cells were not always producing IL-4 unless secondarily stimulated. So, they replaced the IL-4 allele on the other chromosome with a Human CD2 gene. Presence of huCD2 on cell surface would now be indicative of IL-4 production.

They therefore showed that CD4+ Tcells could be either ”IL-4 competent” or ”IL-4 producing”. They observe a very strong post-transcriptional control and selective secretion of IL-4 . Various types of cells start producing IL-4 mRNA but only select ones in certain areas actually produce the cytokine after receiving the proper secondary signal. This idea was tried on the key Th1 cytokine IFN-gamma in another study using influenza virus and Toxoplasma as infection models and similar results were found. Interestingly, the iconic pro-inflammatory cytokine IL-1beta also takes a similar route, but in this case, both control levels are post-translational: First signal induces pro-IL-1beta production and second signal induces its maturation via the inflammasome complex. These key cytokines have receptors on plenty of cells all through the body and their aberrant release can cause serious problems, no wonder multiple controls levels are set for their release.  Studies of this kind should really alert us on interpretation of microarray or qRT-PCR data.

Mohrs K, Wakil AE, Killeen N, Locksley RM, & Mohrs M (2005). A two-step process for cytokine production revealed by IL-4 dual-reporter mice. Immunity, 23 (4), 419-29 PMID: 16226507

Research on parasites is important, even if most of them are not direct health concerns to the developed world! Millions of years of coevolution of parasites along with their hosts have made them masters in manipulation of the immune system and in coexistence with it. Many parasitic infections such as those with Toxoplasma, Trypanosoma and some Leishmania sp. elicit innate and adaptive immune responses that can result in life-long immunity to reinfection. However, the parasite might be carried chronically for life, keeping the antibody titers up.

The research team of Ricardo Gazzinelli have taken advantage of the stealth yet stimulatory property of parasites to target cancer. For their purpose, they expressed a cancer antigen in a strongly attenuated strain of Trypanosoma cruzi, the causative agent of Chagas disease in the Americas. This live vaccine showed great protection against melanoma in both prophylactic and therapeutic models.

T. cruzi was chosen as the vector for many reasons: it intrinsically possesses TLR ligands and thus induces a strong proinflammatory response; like many other parasites, it has ways of staying inside the body for a very long time; and it propagates inside the cytoplasm, therefore it can induce a Th1 type immune response and activation of cytotoxic CD8⁺ T cells, which are very important against cancer. It is interesting to me that although they did not observe any disease or parasitemia, such strong immune response and protection was observed. I think it is important to see if and how many residual parasites are sticking around and where and how long do they stay in the body. This might also teach us more about how this model actually works.

The study shows that if vaccinated before tumor induction, mice are completely protected from cancer. It is also shown that this antigen-carrying vector can delay tumor growth and lethality if given after. One might ask: what advantages does this vector provide over vaccination with traditional recombinant protein, or other vectors such as attenuated viruses? The authors compared the protection and immune response resulted from recombinant T. cruzi to the canonical recombinant antigen (NY-ESO1) with Alum or CpG adjuvants. Recombinant T. cruzi showed better protection and stronger immune response compared to canonical vaccination strategies. Doing a quick search through pubmed, I didn’t find other strategies such live or viral vectors rather than variations of recombinant protein being used so far. I might be wrong, but still this is a novel idea and this recombinant parasite deserves a chance of being further studied, especially for prophylactic uses. There are plenty of other cancer or maybe even infectious antigens that can be targeted by this method. But of course, there are plenty of biohazard issues that also need to be addressed with injecting an attenuated form a dangerous pathogenic parasite.

Here is the link to the article abstract on Pubmed.

Junqueira C, Santos LI, Galvão-Filho B, Teixeira SM, Rodrigues FG, Darocha WD, Chiari E, Jungbluth AA, Ritter G, Gnjatic S, Old LJ, & Gazzinelli RT (2011). Trypanosoma cruzi as an effective cancer antigen delivery vector. Proceedings of the National Academy of Sciences of the United States of America, 108 (49), 19695-700 PMID: 22114198

In silico prediction of protein-protein interactions within a species is an advancing field. Especially now that relatively large amounts of empirical data are available for training and validation, more and more in silico methods are being presented. However, as a host-parasite interactions enthusiast, I always had the question if the interactions between the host and pathogen proteins can be predicted. Although already done a few times for viruses such as HSV, HCV, HIV and Influenza, creation of an empirical inter-species interactome is not an easy and always affordable task. Still having an interactome database provides very valuable data in host-pathogen research. It not only reveals systemic overviews about the nature of the interaction occuring, but it can also open doors for more accessible and feasible research by suggesting a shorter list of proteins, pathways or interactions to focus on.

With this in mind, I had great pleasure in reading this single author paper by Stefan Wuchty published recently in PloS ONE that provides a computational interactome of Plasmodium falciparum and Homo sapiens. This paper actually led me to a whole body of research that has been done on experimental and computational determination of host-pathogen interactomes, albeit mostly on viruses.

In order to map P. falciparum – H. sapiens interactome, Wuchty used experimentally determined host-parasite interactions plus orthologous protein groups between the two species as starting point. The false-positives were removed with various filtering methods that are beyond the reach of my knowledge of mathematics and informatics. Biological criteria such as co-expression of the interacting proteins in the host cell and specific parasite phase were also required.  Interstingly, the pattern of interactions he found, showed similarity with what was already observed with viral pathogens. In order to take control of the cell, intracellular pathogens appear to attack the host both at the protein as well as the pathway level. Hub proteins – proteins that interact with many other proteins and are envolved in multiple metabolic/siganling pathways – have been found to be an attack target not only by P. falciparum but also other pathogens. In addition, Wutchy saw that a relatively small number of human proteins interact with a big number of parasite proteins, suggesting that the parasite utilizes all it has got to take over the key proteins of the host.

This study and other works of the same style certainly provide precious knowledge about host-parasite interactions both in terms of systems biology and also hints for hands on research. I look forward to seeing these interactomes created and expanded for other pathogens and also their experimental validation and usage. Furthermore, an online database of these host-parasite interactomes would definitely make them more accessible.

Wuchty S (2011). Computational Prediction of Host-Parasite Protein Interactions between P. falciparum and H. sapiens. PloS one, 6 (11) PMID: 22114664

Plasmodium falciparum parasites invade different groups of cells during their life cycle. Upon injection into humans, sporozoites pass through the skin and travel in the blood to be picked up by hepatocytes. After completion of the liver phase, merozoites come back to the blood and invade red blood cells. Finally, there is another sort of invasion happening inside the midgut of the mosquito, different from the vertebrate host. The invasion of red blood cells by merozoites is the most accessible of the three for studying. Of great scientific and theraputic interest are the proteins that allow for this binding and internalization of the parasites to occur. Blocking the blood stage of malaria would essentially abrogate the majority of its pathological complications.

Many surface proteins of Plasmodium and red blood cells had been previously proposed to be involved in this host-parasite interaction and binding. However, in almost all cases, great redundancy was shown in the protein-protein interactions; meaning that knocking out one surface protein or blocking one interaction would only replace it by another one. In some cases this would result in a change in the tendency of the Plasmodium parasites to bind mature vs. Immature red blood cells, but invasion would still happen. But now, recent work has found a definitive receptor for invasion of red blood cells by P. falciparum merozoites. The beauty of this work is not only in finally finding a ligand and receptor for this stage of P. falciparum life-cycle, but also in my opinion in making great use scientific knowledge already available for making this discovery. So here is the brief story for those of you who do not feel like reading this short yet elegant letter to Nature:

In search for a definitive receptor, Crosnier and colleagues decided to study a surface protein of P. falciparum that was found by another group to be essential for parasite growth: PfRh5. In order to find its binding protein, they went through the already published proteome of the red blood cell and picked up the secreted and surface expressed proteins. Using an ‘Avidity-based extracellular interaction screen’ (AVEXIS) they screened for binding of PfRh5 to recombinantly produced secreted or surface proteins of the red blood cell and luckily they got a single hit: Basigin or BSG. They then validated this direct interaction using Surface Plasmon Resonance and showed that the interaction occurs independently from glycosylations. Next, they showed that adding soluble BSG, blocking it by a specific antibody or shRNA knockdown inhibits invasion of red blood cells by all clinical and lab strains of P. falciparum. As I previously mentioned, inhibition of invasion was never seen before for any of the receptor-ligand pairs suggested.

Finding such a well-fit receptor for invasion of P. falciparum brings up an evident question: Are people with mutations in the bsg gene resistant to malaria? The authors found very few nonsynomymous single nucleotide polymorphisms (meaning SNPs that lead to a different protein sequence) in some populations in the databases. Blood donations from some of those SNP carriers showed actually resistance to P. falciparum invasion. Unfortunately, population genetics data is seriously lacking in areas afflicted with malaria, so whether this gene has been through positive selection or not cannot be determined at the moment. The authors are hopefull to be able to answer this question after some genome projects currently in progress in Africa are complete.

This ligand-receptor interaction appears to be specific to P. falciparum and other Plasmodium species have not been mentioned in the paper (I am pretty sure they have been checked). This makes in vivo drug testing difficult as P. falciparum is not used in the murine malaria infection model. Nontheless, P. falciparum is the most prevalent and lethal malaria-causing species. The discovery of BSG as a receptor for invasion opens many doors towards a generation of theraputics and prophylaxis, bringing us hopefully one step closer to its elimination.

Crosnier C, Bustamante LY, Bartholdson SJ, Bei AK, Theron M, Uchikawa M, Mboup S, Ndir O, Kwiatkowski DP, Duraisingh MT, Rayner JC, & Wright GJ (2011). Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum. Nature, 480 (7378), 534-7 PMID: 22080952

It has been more than a year since my last post. Apparently doing science has taken over writing about science. I am going to try to put more frequent updates which means that I get to explore research being done on parasites again! I would also like to state again that parasite diary would gladly accept your diaries as well. May it be your own research, or work by somebody else that you find fascinating. It doesn’t need to be recently published either. I am pretty sure nobody has read everything about everything. Therefore, all stories about parasites (whether eukaryotic or not) can be exciting and informative to read. If you are interested in participating, send us your diaries to  parasitediary AT gmail DOT com and we will publish them under your name.

As a start to the new era of the parasite diary, I would like to introduce a podcast  that I think anyone with the slightest interest in parasites should not miss. This Week in Parasitism (TWIP)  is narrated by Dr. Vincent Racaniello and Dr. Dickson Despommier from Columbia University. This podcast teaches you  about ecology, physiology and behaviour of eukaryotic parasites and tells you stories that you have never heard before about their history and impact on human life.  Their enthusiasm  for research and for parasites pumps up your energy to continue doing your boring benchwork while listening! I need say no more. Check out TWIP and its sisters (or brothers?) TWIV (Virology) and TWIM (Microbiology).

P.S. The picture in the logo of TWIP is of the nematode Trichinella spiralsis sitting comfortably inside its nurse cell in the muscle tissue.

I am appointed to do a review paper for a ‘Reading and Conference’ course on Fungi. I chose the opportunistic pathogen Aspergillus fumigatus as the focus of my review. Having studied only on Trypanosomes and innate immune cells so far, my background in mycology is close to zero. So I decided to start from scratch. I stepped upon a surprisingly neatly written review that gave me exactly as much background I needed on Aspergillus before I would go more in depth on the subject. “Aspergillus: A primer for the novice” by Dr. Joan Bennett includes scientifically exciting and still critical detail about various species of Aspergillus, their commercial, historical and culinary(yes!) importance. For once after a long time I read the whole review paper and not only the section that interested my research focus. If interested to learn more about the ubiquitous fungus Aspergillus, you can also enjoy reading this paper alongside your favorite afternoon beverage here.

Many pathogens are unable to live outside the host. Therefore, before killing or completely using up their host, they should ensure that they will be successfully transfered to another one, or one may say, those who did not never made it through evolution. Depending on their life-cycle and type, strategies to ensure transmission diffes among pathogens. In a comment for Nature Reviews in Immunology Sacks and Yazdanbakhsh comparatively discuss these strategies among air-borne pathogens, protozoan vector borne pathogens and also multicellular pathogens. Air-borne bacteria and viruses can easily spread after an acute infection and do not necessarily need to modulate immune response to avoid the up-coming sterilizing immunity. On the other hand, vector-borne parasites such as Plasmodium or the Trypanosomes require more time for efficient transmission. Therefore, parasites have developed strategies to delay life-long immunity. For instance, in African Trypanosomes (T. brucei) continuous variation of the surface glycoprotein (correctly named the variable surface glycoprotein or VSG) hiders development of a protective immune response and allows the parasite to reside in the blood for a long time. Alternatively, Leishmania infections co-inside with presence of regulatory T cells and considerable amounts of IL-10 which down-regulates the protective Th1 response. In larger parasites such as helminths rapid movement from immune-sensitive areas such as the skin or acquiring and presentation of host antigens are among the strategies that are used for delaying the immune response and buying time for transmission.

What I find more interesting among all of this is the evolution of the host in the same direction. In many parasitic infections, the immune response does not lead to complete parasite clearance, rather to a residual infection with minimum or no pathology yet still transmissibility.  Read et al. have argued in a Primer in PLoS Biology that this ‘tolerance’ is a type of immunity that can arise in the host-parasite co-evolution as an alternative to ‘resistance’ where complete of the pathogen clearance occurs. Firstly, complete clearance of the pathogen can be too costly compared to its control. Secondly, In the dynamic co-evolution of the host and the parasite, genes who confer tolerance against a pathogen could be favored to those who confer resistance. Evolution of tolerance does not harm or might even favor parasite existence since tolerant host are reservoirs of the parasites within the population. Therefore, they do not prompt counter-adaptation by the parasites.

Sacks and Yazdanbakhsh conclude their comment by mentioning that these immune strategies should be taken into consideration when designing vaccines for parasitic diseases. They suggest that instead of trying to override this desire of the immune system for tolerance rather than resistance, vaccines could induce tolerance where minimal pathology is caused by a controlled persistence of the parasites. A classic example of a vaccination strategy in this line is Leishmanization wherein live Leishmania parasites used to be inoculated in soldiers or children in risk of infection and would confer immunity to further infections. With regard to development of immunological tolerance to leishmaniasis, not resistance, these types of vaccines need reconsideration.

I remember some years ago when I used to read about ecology, behavior and evolution, how fascinated I was by the classic book of G. Evelyn Hutchinson, The ecological theater and the evolutionary play (1965) and by the way that book was depicting niches in ecology and evolution. Separation of niches and consequent speciation occurs constantly and could look simple in the first look. On the other hand, the resulting diversity could be also beyond imagination. Different depths in the cork of an old tree in a tropic jungle can provide different micro-environments to the species living in them, both micro and macroscopic. Various tiny species of insects and other invertebrates could live in different depths of the cork of the tree; different species of birds could feed from them using specialized beaks that penetrate different depths of the cork, so that they would not enter the other species’ niche. It does not just end there. Species distribution varies by height of the tree as so do the mico-environments. And yet even more, in some cases speciation can be temporal with different species of birds coming to the tree at different times during the day. Thus, a tree could act as countless niches for countless species and thus countless biologic diversity.

Years later, I stumbled upon the same phenomenon in the world of parasites. As I was looking through the second chapter of “Foundations of Parasitology” by Schmidt and Roberts (2005), I realized that the vertebrate intestinal tract could be looked at the same way as a tree. Along the intestinal tract, different environments exist, in terms of physical conditions, nutrients, pH and enzymes. Therefore, we should not be surprised to see the same trend and observe different parasites adapting to different micro-environments along the tract and staying away from each other. Below is a graph of distribution of different intestinal worms along the intestine tract of a bird. The bar shows distance from the stomach. Specialization and separation of niches among different parasites can be readily seen (Stock and Holmes 1988).

Another study that the book chapter mentions is about 8 different species of nematodes that were found to be distributed in the intestine of a turtle, not only longitudinally but also radially with preferences towards either lumen or the ring of the intestine (Schad, 1965).

Sometimes we go in so deep into our molecules that we miss the big picture of biology of parasites. How they interact with each other and with their hosts in an ecological and evolutionary point of view. I guess the beauty of biology is that no matter how diverse and complicated the organisms and their relationships can get, still the same principles and patterns apply.