Phosphatases for and against: Trichuris vs. Leishmania

Posted by Kasra

Trichuris trichiura adult male

Trichuris trichiura adult male – Image taken from DPDx

Trichuris, is an intestinal roundworm, also known as whipworm, that can be transmitted through ingestion of food contaminated with its eggs. The larvae hatch inside the small intestine and complete their life cycle to adults in the cecum. After maturation, which can take about 3 months, the female worm lays thousands of eggs per day. The parasite can stay in the intetine between 1-5 years. Trichuris trichiura is a parasite of humans, while Trichuris muris is a mouse parasite, used usually as the animal model to study its infection.

In contrast to intracellular pathogens, a Th1 response is non-protective in infection with large extracellular pathogens such as intestinal helminths. For instance, during infection with Trichuris muris, a Th2 response comprising IL-4 and Ig-E production leads to resolution of infection, while a Th1 response comprising IFN-gamma, IL-12 and IL-18 is not protective.

S Hadidi et al. look at regulation of the immune response to T. muris and focus on the importance of the macrophage lipid phosphatase Ship1. Ship1 or Sh-2 containing inositol 5′ phosphatase 1 is a regulator of the PI3K pathway. Hadidi et al. show that Ship1 expression is upregulated steadily following T. muris infection. Ship1-/- mice have higher parasite burden and IFN-gamma while lower levels of IL-13. Also, Ship1-/- macrophages produce more IL-12. Blocking IL-12 or IFN-gamma by blocking antibodies rescued the phenotype by reducing worm burden and increase in IL-13. Thus, they found how activity of this phosphatase can direct the immune response against T. muris infection. It would be very interesting now to see what stimuli induce upregulation of Ship1 and also what are this enzyme’s substrates, which are so important for production of IL-12 by macrophages.

Similar to this story, a few years ago, Abu-Dayyeh et al. and Gomez et al. showed that activating phosphatases is important for Leishmania to establish its infection. Being an intracellular parasite, a Th1 response, with large amounts of IFN-gamma would be protective against Leishmania. So in this context, Leishmania-mediated activation of many phosphatases (most importantly SHP-1) leading to inhibition of IL-12 production leads to disease progression, because it skews the immune response towards Th2. In this situation, Leishmania takes advantage of the phosphatase’s function.

Hadidi S, Antignano F, Hughes MR, Wang SK, Snyder K, Sammis GM, Kerr WG, McNagny KM, & Zaph C (2012). Myeloid cell-specific expression of Ship1 regulates IL-12 production and immunity to helminth infection. Mucosal immunology, 5 (5), 535-43 PMID: 22535180

Abu-Dayyeh I, Shio MT, Sato S, Akira S, Cousineau B, & Olivier M (2008). Leishmania-induced IRAK-1 inactivation is mediated by SHP-1 interacting with an evolutionarily conserved KTIM motif. PLoS neglected tropical diseases, 2 (12) PMID: 19104650
Gomez MA, Contreras I, Hallé M, Tremblay ML, McMaster RW, & Olivier M (2009). Leishmania GP63 alters host signaling through cleavage-activated protein tyrosine phosphatases. Science signaling, 2 (90) PMID: 19797268
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The Manipulator and the Opportunist: Leishmania and HIV infection of monocytes

Posted by: Maryam Ehteshami and Kasra Hassani

It has been documented that HIV infection can render leishmaniasis harsher and reduce the chances of treatment response. On the other hand, Leishmania infection also accelerates HIV infection and disease progression. In this blog post, we summarize a recent article published in PLoS Pathogens, that explores the mechanism through which Leishmania can help HIV replication. As it turns out, human macrophages are a key part of the equation.

It is no secret that both HIV and Leishmania can infect macrophages. So when Mock et al. wanted to examine the relationship between these two microorganisms, macrophages were the first place they looked.

Macrophages are non-dividing cells with a low nucleotide pool. Nucleotide synthesis is regulated at the S phase, in other words, cell activation. So in resting macrophages the nucleotide levels are very low. Additionally, It was previously thought that human monocytes do not further proliferate once they leave the bone marrow. Recent studies however, have shown that monocytes may be far more heterogeneous than previously thought, and that a subset of them can go on to enter the cell cycle in response to certain stimulations. For example, cells stimulated with GM-CSF (Granulocyte-monocyte colony-stimulating factor) were shown to go on to proliferate.

Interestingly, Mock et al. showed that Leishmania infection can promote monocyte viability and proliferation similar to GM-CSF. It has long been seen that intracellular protozoan parasites such as Leishmania, Trypanosoma and Toxoplasma can inhibit apoptosis of their host cell and thereby increase their lifespan (Heussler et al. 2001). Mock et al. take this to the next step showing that Leishmania­ might further induce proliferation of the infected monocytes. This could help spreading of the parasitic infection, and indeed Mock et al. show that the cells remain infected after proliferation (Figure 1, PKH dye shows that monocytes are infected with Leishmania). However, if this is good or bad for Leishmania, still needs to be determined, especially that an activated macrophage could be able to kill internalized parasites.

Another theory as to why Leishmania promotes cell cycle progression in macrophages is the following: Leishmania parasites lack the machinery necessary for synthesizing purine nucleotides. Resting cells have low levels of nucleotides. Therefore, by activating the cell cycle, Leishmania ensures that sufficient levels of nucleotides become available for its replication. In fact Mock et al. examined the effect of macrophage infection by Leishmania on expression levels of ribonucleotide reductase (RNR) enzyme. This enzyme is responsible for converting ribonucleotides to nucleotides. Western blotting revealed that RNR levels increased in the presence of Leishmania infection, similar to those elevated levels seen in the presence of GM-CSF. This elevation also corresponded with nucleotide level increases. Overall, this presents one theory for why Leishmania would want to induce macrophage stimulation.

As mentioned earlier, macrophages are also a target for HIV infection. But HIV replicates at a very high rate. And high rate of replication requires high levels of intracellular nucleotides. In fact, the Kim group have previously shown that HIV reverse transcription is severely reduced under conditions which mimic macrophage intracellular levels of nucleotides and that HIV replication is lower in macrophages as compared to T cells (Kennedy et al. 2010) .  Based on their observations with Leishmania and HIV, they showed that HIV replication in macrophages may increase when the cells are co-infected with the parasite (Figure 1, Green GFP-HIV proliferation only occurs in GM-CSF-treated or Leishmania-infected monocytes). They also showed that this is the result of Leishmania-induced cell proliferation and increased nucleotide levels (figure not shown). In other words, Leishmania manipulates the macrophage to create a friendlier environment for its own survival and HIV ceases this opportunity and uses these changes in the macrophage for its own gain.

Figure 1. Monocytes were transduced with a GFP-HIV vector. Increased fluorescence signifies increased viral replication. PKH indicates presence of Leishmania. (From Mock et al. 2012, PLoS Pathogens)

It is likely that there are many different mechanisms involved in Leishmania/HIV co-infection that were not discussed here. Almost certainly many of them involve immune modulation. Here, Mock et al. have shed light on a unique biochemical mechanism for the observed increased infection by either microorganism. As suggested by the authors, it would be interesting to examine other macrophage-infecting microorganisms such as Mycobacterium tuberculosis in this context.

This topic raises many exciting questions. Most fundamentally, what is the underlying mechanism for induction of proliferation in Leishmania-infected monocytes? And what is the implication of monocyte proliferation to spread or control of Leishmania infection? But also, what happens to HIV in macrophages in the absence of a parasitic infection? These could perhaps be the topics of next research projects and next blog posts.

References:

Mock DJ, Hollenbaugh JA, Daddacha W, Overstreet MG, Lazarski CA, Fowell DJ, & Kim B (2012). Leishmania induces survival, proliferation and elevated cellular dNTP levels in human monocytes promoting acceleration of HIV co-infection. PLoS pathogens, 8 (4) PMID: 22496656

Kennedy EM, Gavegnano C, Nguyen L, Slater R, Lucas A, Fromentin E, Schinazi RF, & Kim B (2010). Ribonucleoside triphosphates as substrate of human immunodeficiency virus type 1 reverse transcriptase in human macrophages. The Journal of biological chemistry, 285 (50), 39380-91 PMID: 20924117

Heussler VT, Küenzi P, & Rottenberg S (2001). Inhibition of apoptosis by intracellular protozoan parasites. International journal for parasitology, 31 (11), 1166-76 PMID: 11563357

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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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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.