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

Leishmania-induced IRAK-1 inactivation via host SHP-1 activation

Posted by Issa Abu-Dayyeh

I will hereby post the author summary of my most recent publication regarding modulation of macrophage signaling by the Leishmania parasite. The paper has been published on the 23rd of December 2008 in the Public Library of Science (PLoS) Neglected Tropical Diseases, and demonstrates for the first time the ability of Leishmania to inactivate IRAK-1 through its ability to activate host PTP-SHP-1.

Citation: Abu-Dayyeh I, Shio MT, Sato S, Akira S, Cousineau B, et al. (2008) Leishmania-Induced IRAK-1 Inactivation Is Mediated by SHP-1 Interacting with an Evolutionarily Conserved KTIM Motif. PLoS Negl Trop Dis 2(12): e305. doi:10.1371/journal.pntd.0000305

Here is the author summary of the paper:

Leishmania developed several methods to seize control of macrophage signalling pathways in an effort to inactivate their killing abilities. One effective method utilized by the parasite is the activation of host protein tyrosine phosphatases, specifically SHP-1. This increased phosphatase activity contributes to the inactivation of signalling molecules involved in critical macrophage functions such as NO and cytokine production. Interestingly, the absence of SHP-1 results in stronger macrophage inflammatory responses to a bacterial cell wall component known as LPS, a molecule detected by macrophages through Toll-like receptors (TLRs). This observation suggested a role for SHP-1 in the regulation of TLR signalling. Our study reveals that upon Leishmania infection, SHP-1 is able to rapidly bind to and inactivate a critical kinase (IRAK-1) in this pathway. This regulatory binding was shown to be mediated by an evolutionarily conserved motif identified in the kinase. This motif was also present in other kinases involved in Toll signalling and therefore could represent a regulatory mechanism of relevance to many kinases. This work not only reports a unique mechanism by which Leishmania can avoid harmful TLR signalling, but also provides a platform on which extensive investigation on host evasion mechanisms and regulation of cellular kinases can be gained.

For further information please check the citation below to read the whole paper. PLoS is an open-access journal and therefore offers free access to its contents to anybody anywhere in the world!

P.S.: Abstract translations are also available in the following languages: Arabic, French, Spanish, and Farsi.

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?

Model for parasitism: invasive/evase vs. pathoantigenic molecules

Posted by Kasra Hassani

A very interesting conceptual model for parasitic virulence was proposed by Chang et al. (2003) a few years ago and has been recently discussed in Leishmania and Leishmaniasis by Banuls et al. (2007). It has long been established that parasitic organisms, bacteria, protozoa, helminths etc. benefit from certain molecules that enable them establish infection within the host and cause pathogenicity. It can be said that the parasites are at the same time ‘visible’ and ‘invisible’ to the host.

Here I briefly introduce the model with Leishmania as the model organism. The idea is to define twp groups of effective molecules named invasive/evasive and pathoantigenic. The invasive/evasive molecules help the parasite to evade from the innate immune system and its microbicidal mechanisms to establish its infection. These molecules usually stay ‘invisible’ to the immune system and their expression might end after the establishment of infection. Good examples in the case for Leishmania are gp63 surface protease and the surface molecule lypophosphoglycan (LPG). Both of these molecules have crucial roles in evasion from the complement system, facilitation of phagocytosis and subversion of macrophage signalling to the parasite’s benefit. Their expression is slowed down and LPG is almost totally absent in Leishmania amastigotes. As expected, they also do not elicit an immune response.

On the other hand, another rank of molecules, which are generally intracellular in Leishmania are pathoantigenic and cause immunopathologic responses. Interestingly, the majority of Leishmania’s immunogenic proteins are intracellular rather surface proteins and are being produced as a result of parasite’s multiplication within the host. They are believed to be exposed to the immune system during cytolysis and cause the virulence phenotype. A proper example is the amastigote-specific protein A2 which is an intracellular protein and is highly immunogenic. This protein being expressed in high levels in visceral species induces visceralization of Leishmaniasis.

Chang et al. have discussed their model extensively with Leishmania infection but have described how it could beautifully fit with other types of acute and chronic diseases. For instance in schistosomiasis, the adult worm stays in the blood vessel ‘invisible’ to the immune system while releaseing highly antigenic eggs that cause immunopathology. This model can present convergent evolution of parasitic strategies in very divergent parasites.

Perhaps each parasitologist could at least conceptually fit and expand this model for their own parasite of interest to have a better overall understanding of its parasitic strategies.

Genome-wide gene expression profiling analysis of Leishmania major and Leishmania infantum developmental stages reveals substantial differences between the two species

Posted by Hamed Shateri Najafabadi

The title of this post is in fact the title of a recent paper published by Annie Rochette and her colleagues in BMC Genomics (2008, 9:255). This work, which has been done in Barbara Papadopoulou‘s lab at Laval University, reveals unexpected differences between developmental regulation of genes at mRNA level between the two closely related trypanosomatids Leishmania major and Leishmania infantum. I asked Annie to write a summary of her paper in her own point of view. I hope you agree with me that the author’s point of view should be well reflected in the abstract of the paper, so was the case for this article. Here is the abstract as Annie sent to me:

“Leishmania parasites cause a diverse spectrum of diseases in humans ranging from spontaneously healing skin lesions (e.g., L. major) to life-threatening visceral diseases (e.g., L. infantum). The high conservation in gene content and genome organization between Leishmania major and Leishmania infantum contrasts their distinct pathophysiologies, suggesting that highly regulated hierarchical and temporal changes in gene expression may be involved. We used a multispecies DNA oligonucleotide microarray to compare whole-genome expression patterns of promastigote (sandfly vector) and amastigote (mammalian macrophages) developmental stages between L. major and L. infantum. Seven percent of the total L. infantum genome and 9.3% of the L. major genome were differentially expressed at the RNA level throughout development. The main variations were found in genes involved in metabolism, cellular organization and biogenesis, transport and genes encoding unknown function. Remarkably, this comparative global interspecies analysis demonstrated that only 10-12% of the differentially expressed genes were common to L. major and L. infantum. Differentially expressed genes are randomly distributed across chromosomes further supporting a posttranscriptional control, which is likely to involve a variety of 3′UTR elements. This study highlighted substantial differences in gene expression patterns between L. major and L. infantum. These important species-specific differences in stage-regulated gene expression may contribute to the disease tropism that distinguishes L. major from L. infantum.”

Thanks to Annie and her colleagues for this beautiful paper.

I would also like to highlight another paper by Nagalakshmi and colleagues which was published in Science about a month ago. In this work, the transcriptome of yeast is analyzed, but not using microarrays. They used massive high-throughput Illumina sequencing to sequence the whole transcriptome of yeast. This approach, in addition to providing precise estimates for the extent at which each part of the genome is transcribed, gives a plethora of other information that is extremely difficult to gain by routine microarray analysis. First of all, it does not need any a priori assumption regarding the regions that are being transcribed, similar to tiling arrays with the difference that the resolution is several folds higher than any affordable tiling array. It also provides information regarding post-transcriptional modifications of RNAs, such as splicing, alternative splicing, poly-adenylation, etc (see Hani’s blog). Trypanosomatids have surprised us several times, by showing us that a mature RNA can look nothing like its precursor due to the high extent of editing and trans-splicing. They have shown us that it is possible to transcribe almost half of a complete chromosome in just one huge RNA, or that a chromosome can be extensively transcribed from both strands. I am sure these surprises will be nothing once we have the data from sequencing the whole transcriptome of a trypanosomatid species; two will be better!

Altruism in Leishmania: apoptotic parasites are required for infectivity of metacyclic promastigotes

Posted by Kasra Hassani

Suppression of the innate immune response and inhibition of activation of phagocytes that would otherwise kill the parasites has long been established as mechanisms of immune evasion and persistence among Leishmania parasites.

In their paper, van Zandbergen et al. have indicated presence of a high ratio (more than 40%) of apoptotic cells in the metacyclic/stationary phage parasites. They have characterized these cells by occurrence of phosphatidyl serine (PS) in the outer leaflet of plasma membrane as well as PS-binding protein Anexin A5(AnxA5). The majority of AnxA5+ cells have been shown to be apoptotic and different in morphology to infective parasites and they have shown that depletion of these apoptotic cells from the infective population substantially abrogates infectivity.

Apoptotic cells induce production of TGF-beta and IL-10 which are anti-inflammatory cytokines; these cytokines are produced as well by neutrophils when they phagocyte apoptotic Leishmania. Apoptotic parasites also hamper secretion of TNF-alpha, all of which results in inactivation of neutrophils and later macrophages and their inability to kill the phagocytosed parasites.

This is an interesting example of altruism among single-cell populations; the authors have suggested that apoptosis is probably triggered in late log phase and stationary phase promastigotes in the sandfly midgut due to nutrient depletion prior to their entry into the mammalian host.

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.