Detecting picograms of protein in the secretome

Posted by Kasra

When designing experiments in the lab, we usually say we cannot check for everything. Well, what if we could?! Meissner et al. used only 150,000 macrophages per sample to analyze their secretome. They have been able to detect and quantify protein abundances at the picogram level in a label-free system. Picogram detection limit means that cytokines are readily quantifiable, and Meisner et al. claim it matches with the detection limit of ELISA. I could imagine that in not so long this technology can become more readily accessible, allowing researchers to acquire more pertinent data per sample. We can know the statuses of a multitude of proteins (from cytokines to nonconventionally secreted proteins) simultaneously, not through multiple ELISAs and western blots, and therefore draw much more sensible conclusions from experiments. It will actually be more affordable than separate quantification of each protein, not mentioning dramatically time-saving.

Also, this data can be integrated with other high throughput quantitative analyses of the cell. For instance, Meissner et al. have compared the changes in protein abundance in the secretome to changes in their transcript levels, and roles of different adaptor molecules (in this case MyD88 versus TRIF) and tried to explain how they all relate.

Meissner F, Scheltema RA, Mollenkopf HJ, & Mann M (2013). Direct proteomic quantification of the secretome of activated immune cells. Science (New York, N.Y.), 340 (6131), 475-8 PMID: 23620052

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

What happens during differentiation of Leishmania? From a metabolism point of view

Posted by Kasra Hassani

The differentiation of the promastigote Leishmania to the amastigote form is one of the most interesting and promising areas of study in Leishmania research. Researchers are interested to know what are the differences between these two life-stages, how do the parasites shift from one to the other and what happens during this transformation. Rosenzweig et al. have addressed these questions by proteomic comparison of Leishmania donovani’s proteome during its transformation from the promastigote to the amastigote form. Using a new labelling method called the iTRAQ, they have looked at the proteome of L. donovani after stimulation with low pH and high temperature within 2.5, 5, 10, 15, 24h and after 6 days. With their method they believe that they have been able to detect half of the parasite’s proteome. This study has shown key changes in the proteome content of L. donovani which corresponds with its metabolic needs in the phagolysosome.

The mid-gut of sandfly is a sugar-rich environment because of its nectar-diet supplemented with occasional bloodmeals. However, sugars are scarce in the phagolysosome and the parasite has to switch to fatty acids and amino acids as energy sources. According to Rosenzweig et al., starting from 10h after stimulation, glycolysis enzymes are down-regulated while enzymes required for beta-oxidation, gluconeogenesis, amino acid catabolism, TCA cycle and mitochondrial respiration are up-regulated. Furthermore, protein translation and thus metabolism slows down which corresponds to parasite’s smaller size and slower growth. In this way the parasite retools its metabolism to be able to live and multiply inside the phagolysosome. Another interesting and rather surprising finding of Rosenzweig et al. is that most of these alterations in metabolism start rather late (after 10-15h) and they take hours to maturate. This finding, raises the question that how do incompletely differentiated parasites reside in the same host environment as the mature amastigotes?

Studying the secretome of Leishmania donovani

Posted by Kasra Hassani

In this paper, Silverman et al. have pointed to two interesting subjects: first, what proteins are generally secreted from Leishmania, and second, how are these proteins secreted. In an extensive proteomic analysis, they have pointed out 151 proteins that they believe are being actively secreted out of stationary promastigotes of Leishmania donovani. These proteins belong to a wide variety of groups, such as proteases, antioxidants, nucleases etc. and each might play roles in survival of the parasite within its hosts and modulation of the immune response. Identification of these proteins opens up many opportunities for further studies that promote understanding their function and possible therapeutic targets in continuing studies.

Another interesting finding of Silverman et al. was that among these secreted proteins only 2 contain a classical amino-terminal secretion signal, which means that Leishmania largely might benefit from non-classical secretion pathways such as exosomes. Exosomes have been studied previously in human B cells and dendritic cells and it is actually interesting to point out that there is striking correspondence between the proteome content of these exosomes and Leishmania’s secretome (except for the proteins for which Leishmania does not have an ortholog). The authors have proposed the release of exosomes from the surface of the cell and especially from the flagellar pocket to be an important pathway of protein secretion by Leishmania and they have observed vesicular budding from the parasite surface by STM.