Finding new vaccine and diagnostic targets using Immunoproteomics

Posted by Kasra

One of the complexities in studying eukaryotic parasites is the multiplicity of their life stages. Even the simplest life cycles of eukaryotic parasites can consist of two very different stages, with different morphologies, gene expression, proteome profiles, and surface antigens. These variations often result in confusion of the immune system and disease progression instead of healing. What makes this more complicated is that very often one or more of these stages, usually the one inside the mammalian host can be difficult to culture and study in vitro. For instance, in the case of Leishmania parasites, the clinically important amastigote stage is intracellular. Methods for their axenic growth do exist; still their validity and authenticity remains controversial among researchers. Nevertheless, I believe authentic or not, axenic Leishmania amastigotes can be good tools for studying this aloof life stage of the parasite. As the famous statistician George P.E. Box says ‘Essentially, all models are wrong, but some of them are useful’.

Yet another complexity of working with these ancient species is presence of a great percentage of what genome annotators call ‘Hypothetical proteins’. These proteins appear after bioinformatic analyses of the genome sequences in search for genes. There is no other evidence rather than clues from the sequence for their existence, so they are labeled hypothetical. In addition, in many cases they have no homology to any protein with a known function, thus their function remains a big question mark, which brings me to the two papers I want to discuss!

These papers both came out last year and used immunoproteomics to hunt for new diagnostic and vaccine targets for leishmaniasis. Vinicio T. S. Coelho et al. ran 2D gels of promastigote and axenic amastigotes of Leishmania chagasi, a visceral leishmaniasis-causing species in Latin America, and blotted them against pooled sera of infected, uninfected or nonsympomatic dogs. Míriam M. Costa et al. used the colourful 2D-Difference Gel Electrophoresis (DIGE) method to look at differentially expressed proteins between promastigotes and amastigotes and also blotted them against pooled sera of uninfected, 30 day infected and chronically infected dogs to compare levels of early and late (IgM and IgG) antibodies. Both studies aimed to find immunogenic proteins as candidates for diagnosis and vaccination. For those who are not familiar with the ecology of Leishmania, I should mention that leishmaniasis is a zoonosis, and dogs are an important reservoir of the parasite that keep the cycle going, even if we prevent it in humans. Thus, vaccination of dogs against both cutaneous and visceral leishmaniasis is among the important priorities for disease control.

A combination of Difference Gel Electrophoresis and western blotting using sera allows for identification of common and stage-specific antigens. Míriam M. Costa et al. J. Proteome Research, 2011

The importance of these two studies is the application of both promastigote and amastigote proteins as sources for antigen discovery, as well as the use of sera from asymptomatic versus symptomatic dogs to characterize antibodies that arise at different stages of infection. This allows for identification of proper markers for early and advanced stages of the disease as well as knowledge about expression and antigenicity of proteins from each life stage of the parasite. Not surprisingly, in both studies, a decent number of hypothetical proteins show up. On one hand, these are not the best candidates one may look for, since we have no knowledge about their expression, function and so on. But on the other hand, I would see them as potentially interesting targets that could be worth studying. At least, we are narrowing down all the hypothetical proteins to ones for which we have data on expression and antigenicity.

In addition, the results of these studies and other studies of the similar nature should be cross-referenced in the public gene and protein databases, so that other researchers can readily access the new knowledge that has become available about these hypothetical proteins when looking them up. Once these sorts of data from various stydies start to accumulate in the databases, new patterns and insights might emerge that can lead us to an understanding of their function and possible roles in pathogenicity.

Coelho VT, Oliveira JS, Valadares DG, Chávez-Fumagalli MA, Duarte MC, Lage PS, Soto M, Santoro MM, Tavares CA, Fernandes AP, & Coelho EA (2012). Identification of Proteins in Promastigote and Amastigote-like Leishmania Using an Immunoproteomic Approach. PLoS neglected tropical diseases, 6 (1) PMID: 22272364

Costa MM, Andrade HM, Bartholomeu DC, Freitas LM, Pires SF, Chapeaurouge AD, Perales J, Ferreira AT, Giusta MS, Melo MN, & Gazzinelli RT (2011). Analysis of Leishmania chagasi by 2-D difference gel electrophoresis (2-D DIGE) and immunoproteomic: identification of novel candidate antigens for diagnostic tests and vaccine. Journal of proteome research, 10 (5), 2172-84 PMID: 21355625

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Science answers WHY questions: programmed cell death in unicellular parasites

Posted by Kasra

Famous geneticist Josh Haldane once famously said “I would lay down my life for two brothers or eight cousins”.

Programmed cell death, otherwise known as apoptosis is a well justified procedure in multicellular organisms. All cells within a multicellular organism are originated from a single zygote and are genetically identical (except for some sets of immune cells that go through somatic mutation and genomic rearrangements, but that is different story). The purpose of the organism is to pass on the genetic material to the next generation and to maximize this genetic transfer. Therefore, if programmed death of some cells within the organism would help this purpose (i.e. increase the organism’s fitness), its evolution is logical. After all, often only a handful of cells (the gametes) get to pass their genetic material to the next generation and rest die eventually anyway.

This said, occurrence programmed cell death in unicellular organisms brings up an evolutionary dilemma. Scientists have found markers of apoptosis in unicellular organisms as wide as Plasmodium, Trypanosoma, Giardia and Saccharomyceses. Unicellular organisms within an ecosystem are usually competing with one another for resources, the same way animals and plants do in a larger scale. Therefore, evolution of a trait as strong and costly as altruistic death is worth a closer look, both mechanistically and rationally.

Reece et al. have discussed this matter in a recent review article published in PLoS Pathogens. Mainly focusing on Plasmodium, this review addresses hard questions such as why would altruism evolve in unicellular organisms? What could be the benefits of it? What are the factors that control its occurrence?

Discussing the ideas and hypotheses presented in the review here would be spoilers for those who are eager to read it. In that case you may stop right now and download the open access article right here. If not, you can continue reading as I bring up a few highlights of the paper that I found most interesting.

Hamilton’s rule in evolutionary biology states that cooperation can evolve under these circumstances:

rb – c > 0

Where <r> is relatedness, (for instance the ratio of relatedness between siblings is 0.5 because they share 50% of their genome), <b> is the benefit the receiver gets, and <c> is the cost of the giver. So if benefit x relatedness is larger than cost, then cooperation or sacrifice can be actually be worth it. It is with reference to this rule that Josh Haldane was willing to give away his life for two brothers or eight cousins.

Within multicellular organisms, r = 1 because all cells have been deriven from a single clone. Therefore, wherever, b > c, cell death can evolve. But in unicellular organisms, the story is different. For instance, the population of Plasmodium or Leishmania parasites in the mosquito can be genetically very close or very diverse. Now the evolutionary theory would predict that if occurrence of apoptosis is for altruism and cooperation, it is favourable if the parasites within the population are genetically close to one another. This is a theory that can be tested in a lab: both genetic diversity of different parasite populations and occurence of apoptosis within those populations are measurable with today’s techniques.

Going further from the evolutionary strategy behind evolution of programmed cell death in unicellular organisms, we should also think about what could be possibly the benefits of the receivers that would favor death of the others. One of the theories is that overgrowth of parasites can result in limitation of resources and/or harm to the vector (in this case the Phlebotomine mosquito). If the vector gets overwhelmed by the parasites, it cannot transfer them to the next stage in the life cycle. Thus as Reece et al. suggest, death can be density dependent to control the parasite popultion, another hypothesis which is also readily testable.

And finally, we get to (in my opinion) the difficult part, which is to discover the mechanism underneath these strategies. How can parasites detect relatedness or density? It is possible that sophisticated strategies and mechanisms simply do not exist in certain populations because infecting populations have always been clonal and measurement of relatedness has not been needed. But it cannot always be case. Reece et al. point out to some studies which show evidence of detection of genetic diversity by parasites and existence of mechanisms similar to bacterial quorum sensing.

There is still a lot more to learn and be amazed with.

Reece SE, Pollitt LC, Colegrave N, & Gardner A (2011). The meaning of death: evolution and ecology of apoptosis in protozoan parasites. PLoS pathogens, 7 (12) PMID: 22174671

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