TriTryp – a database for genomics of Trypanosomatids

posted by: Kasra Hassani

EupathDB in collaboration with GeneDB have started up a database dedicated to genomics and functional genomics of Leishmania and Trypanosoma. The beta version of this website can be found here. It provides us with various tools and resources for genome, proteome and transcriptome browsing and sequence retreival as well as BLAST search and links to pubmed NCBI. I am sure once debugged and fully functional, this database can be very useful for those of us working on Trypanosomatids.

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.

Dawkin’s “extended phenotype”, an extension or a revolution?

Posted by: Issa Abu-Dayyeh

The extended phenotype, a relatively longer and a more difficult reading than Dawkin’s “The selfish gene”, is in my opinion a book worth the reading effort for several reasons:

1-Although a big portion of the book was dedicated to rebuttal critics that showered Dawkins with accusations of being a genetic determinist and a reductionist (Based on his views in the Selfish gene), Dawkin’s replies to those criticisms are pretty logical and organised. In fact, Dawkins almost did not have to retract any of the claims he made 6 years before “the extended phenotype” was written.

2-The rest of the book sets to establish a new vision on the extent to which a gene can act.

Many of us would agree that an organisms’ behaviour is selected to maximize the success of the replication of the genes residing inside this organism. As tempting as this statement might be, this vision definitely pictures the body as the gene’s prison. It is the boundary, the wall,the farthest limit upon which a gene can act.

Dawkins suggests in “the extended phenotype” that the action of genes goes way beyond their ability to produce proteins for the bodies they reside in. In fact, genes can have effects on inaminate objects (such as the type of house an animal would build) or on other living beings. An example given by Dawkins is a trematode that lives in snails. This trematode codes for proteins that drive the snail to produce thicker shells than ususal. This provides greater protection for the trematode while diverting the snail’s energy from practices that could benefit the snail but not the trematode such as: reproduction. The author goes on and on giving examples of how genes can act at a distance!

But how influential is this extended phenotype argument? After reading the book, my initial thought was that it is really no revolution! This is simply an extension of our vision of how far genes should be seen to go. On a deeper thought, I believe this book is revolutionary from a different perspective. First, it places more emphasis on the interactions of genes (regardless of the organism that carries them) on the overall evolution of complex traits and the natural selection they undergo. The principle also explains how a parasite can alter the host’s behaviour to its advantage (therefore suggesting what was formerly thought as mal-adaptation of a host gene as good adaptation of the parasite gene), and how some parasites can end up as symbionts and ultimately interested in increasing the reproductive success of the host and, soon, very difficult to even be seen as  parasites (ex: the mitochondria and chloroplast endosymbiont theory).

This book simply modifies a vision: from behaviour maximizing the success of the genes inside the organism to behaviour maximizing the success of genes that code for that specific behaviour, no matter in whose body those genes are found. This definition reorganizes the genetic vision in a way highly compatible with dawkins’ selfish gene view of evolution and natural selection. Is he right about the extended phenotype or is he wrong? I think most of us would agree it is a logical extension of what we perceive as a direct effect of a gene, but what really matters is that it is different… and a different view is sometimes what we need to reevaluate our current vision and devise new experiments to expand our knowledge. Not to mention the importance of such a vision on the mathematics of genetic contributions to phenotypes. In a nutshell, this is a book worth reading!

MHC I and MHC II, simple and clear, once and for all!

posted by: Issa Abu-Dayyeh

Every couple of months I ultimately get into a discussion about the role of MHC class I and II in the activation of immune functions. What drives their transcription/translation, what cells produce them, how are the peptides found and loaded on them; these are many questions that do not seem to go in the long-term memory section of my brain as well as many others.

Here I decided -once and for all- to summarize the dogmas in the aspects mentioned previously.

MHC class I are molecules produced by all nucleated cells, their production is augmented by IFN-alpha, IFN-beta, IFN-gamma, and TNF-alpha. They are loaded with peptides that result from proteasome-mediated degradation of proteins found in the cytosol, they are transported to be expressed on the surface of cells, and MHC I molecules bound to foreign peptides activate CD8+ T cells by binding to their TCR.

In other words, these MHC molecules keep the internal contents of molecules in check. This is consistent with the fact that such a mechanism is very useful against cancer cells and cells infected with viruses that are actively producing their proteins inside the cells.

On the other hand, MHC class II molecules are produced by antigen presenting cells of the immune system: mainly dendritic cells, macrophages, and B cells. Their production is primarily driven by IFN-gamma (and not the other interferons), they are loaded with peptides that are generated by peptidases found inside phagolysosomes, and upon their translocation to the cell surface, they activate CD4+ T cells.

Two main comments come to my mind upon writing those “facts”:

1- It is impressive how those two MHC types seem to complement each other’s function. MHC I screens for internal “problems”: viruses, cancer, and other antigens that are hiding inside cells away from immune detection, while MHC class II are directed against obvious intruders that go inside the cells by phagocytosis (or any surface detection mechanism ex: TLR) such as:  bacteria and parasites. Together, those two molecules work hand in hand in keeping the organism as alert as possible to all sorts of invaders.

2-Although it is interesting per se that interferons and other pro-inflammatory cytokines upregulate the production of those molecules, it is even more interesting to see that molecules such as IFN-gamma which is not typically involved in counter-acting viral infections can upregulate MHC type I molecules. To me, this suggests that an invasion of the immune system by viruses must somehow lead to the production of danger signals recognized by IFN-gamma producing cells (or their activators) and ultimately leads to a response against those viruses through MHC I upregulation (Future work in the field will be the judge!)

In the end, it is as if the immune system is on “code red” and asks each cell to rapidly disply its ID card to the immune police….Normal cells will show the good ID and pass, suspicious cells will display a “wanted” ID and are destined to be eliminated.

Isn’t it amaizing???

Put the parasite on scale

Posted by Hamed Shateri Najafabadi

For the hardcore molecular biologists and those of us who used to look for protein names in articles, this is going to be a boring post; but to those who like surprises I should say this will be thrilling.

How much do you think the parasites weigh in an ecosystem? A team of researchers from 10 institutes, eight from USA, one from Mexico and one from Republic of Panama have spent five years as a part of a two million-dollar project to find out that parasites can even outweigh the top predators including birds and fish. The biomass of the transmission stage of trematodes all by itself exceeds the biomass of all birds. Their paper which was published a week ago in Nature accentuates the energetic implications of this finding. It is definitely a worthy paper to take a look at. Next time I’m thinking of something huge, I will think of the pile that all parasites from California would make!

What kills YOU may make THEM stronger – cross-resistance between an antimalarial and an antibiotic

Posted by Marie-Luise Winz

Ross J. Davidson et al. recently published their article on the occurrence of resistance against ciprofloxacin, a fluoroquinolone antibiotic among E.coli strains isolated from rectal swabs of patients from six remote villages in Guyana between 2002 and 2005. Ciprofloxacin is a fluoroquinolone broad-spectrum antibiotic that inhibits the bacterial gyrase and topoisomerase IV. Both type II topoisomerases are responsible for relaxing positive supercoils prior to DNA replication. Their inhibition ultimately inhibits cell replication.

Surprisingly, more than 5% of the samples showed evidence of ciprofloxacin resistant E.coli, although all the patients denied previous use of this antibiotic. This is a dramatically high percentage, even superior to the 4% of resistant strains found in an intensive care unit survey in the United States, where ciprofloxacin is actually being used. In order to determine which resistance mechanisms were used by the bacteria, the quinolone-resistance determining regions (QRDR) of the genes gyrA, gyrB (subunits of gyrase), parC, and parE (subunits of topoisomerase IV) were amplified by PCR and analysed, and the presence of quinolone-resistance (qnrA/qnrB) genes that protect gyrase and topoisomerase IV from inhibition by quionolones (Jacoby et al., 2006), was examined. Furthermore, the presence of energy-consuming export systems was analysed.
Ciprofloxacin resistance was found to be caused mostly by mutations of gyrA and parC, whereas mutations of gyrB, parE, or presence of qnrA, qnrB, and export systems could not be demonstrated.
Exposure to ciprofloxacin or other fluoroquinolones could be ruled out, due to the remoteness of the area. In contrast, a correlation between a significantly higher proportion of resistant E.coli strains and a P.vivax epidemic in 5 of the 6 villages included in this study, in late 2002 was observed – 10.2% resistant strains in February 2003 versus 3.8% in February 2002 (before the epidemic) and 3.5% in February 2005 (after the epidemic).
Chloroquine is one of the most common and most affordable antimalarial drugs used in many tropical countries, despite a high proportion of resistant P.falciparum strains, especially in Africa. The drug is still commonly used, and effective against infection with P.vivax. Chloroquine has been demonstrated to have weak antibiotic effects on some bacteria, including E.coli (Jain et al., 2003). Fluoroquinolones were actually derived from the chemically related quinolines, the family of compounds, to which chloroquine belongs.

ciprofloxacin (a) and chloroquine (b)

By an in vitro assay, the authors were able to demonstrate that serial treatment with chloroquine was able to confer resistance to ciprofloxacin, leading to mutations of gyrA and parC, the genes that were also mutated in the resistant strains from Guyana.
However, the occurrence of bacterial resistance to ciprofloxacin and the intake of chloroquine as antimalarial treatment in the individual patient did not correlate. Nevertheless, it is possible, that either chloroquine was taken up unintentionally e.g., through contaminated drinking water or that resistant bacteria were passed on to other patients who hadn’t undergone antimalarial treatment, through faecal contamination.
The levels of chloroquine that were observed in the drinking water (not during, but after the epidemic) do not seem to be likely to cause bacterial resistance, and only one single resistant E.coli strain was found in a water sample in 2004. Yet, it is very improbable, that the use of chloroquine and the high incidence of E.coli resistance to ciprofloxacin are unrelated events.
The authors point out that the use of chloroquine as an antimalarial drug should not be discontinued, despite their findings, owing to the good efficiency of this drug against P.vivax and to the fact that it is one of the most cost-effective antimalarial drugs at hand. However, they are planning to carry out further experiments in the future, in which they want to test other quinoline antimalarials for their ability to cause bacterial fluoroquinolone resistance, and may be able to suggest safer antimalarials for future use.
They also emphasize that the use of fluoroquionolone antibiotics in regions with high use of quinoline antimalarials might have to be reconsidered.
I found this article very interesting, having already heard about cross-resistance to different antibiotics among bacteria, but not between different drugs that are used against pathogens from two different domains – eukaryotes and (eu-)bacteria. This demonstrates, once more, how tightly seemingly different things are related in our world. For us as (future) scientists, it is thus important to keep our eyes and minds open to findings that don’t seem related to our field of interest at first sight, but might be, after all, of high relevance.

Parasites lead to evolution of robustness against gene loss in host signaling networks

Posted by Hamed Shateri Najafabadi

A new study by Marcel Salathé and Orkun Soyer reveals exciting evolutionary consequences of host-parasite interactions on the architecture of biological networks of the host. Their paper, which was published a few days ago in Molecular Systems Biology, is one of those that you read and wonder why no one had thought of it before! The approach that they use is elegant and the findings are very significant.

Marcel is now a postdoc fellow at Stanford and very soon is going to start working on “questions about the non-genetic (e.g. cultural) effects on disease dynamics” (I got it from his web page). I asked him to write a synopsis of his paper for The Parasite Diary, and here it is:

“Many molecular pathways are robust against removal of parts, but why such robustness is evolutionary maintained is a question that has not been answered yet. Another, seemingly unrelated finding in recent years is the process by which parasites attack their hosts and evade an immune response from the host. Evidence is accumulating that the most frequent evasion strategy of parasites is to interfere with the protein machinery of hosts, for example by suppressing important genes that are necessary to recognize a parasite and/or mount an immune response – we cite various key papers in the study.
Our idea was to bring these two observations together: if parasites interfere with host pathways, they create selective pressure on the host to avoid such interference. One obvious solution to this problem is that hosts would evolve pathways that are robust to the suppression of a protein – if a parasite suppresses the protein, the host would still be able to respond in the appropriate fashion. We believe that part of what we see in knockout studies – which are usually performed in the lab in the absence of parasites – could be explained by this phenomenon.
To see whether our idea made sense we used a mathematical model of pathway dynamics and ran evolutionary simulations in the computer. Our findings confirmed that our proposal is plausible, and in principle it is also testable. The evolved robustness resulted either from redundancy or from specific network architecture, and was more stable when it resulted from the latter; robustness based on redundancy alone was quickly lost under subsequent stabilizing evolution (without parasite interference).
Altogether, we hope that this type of research invites biologists to look closer at the ecological aspects of systems biology properties.
Parasites are an extremely strong and continuous source of selection on any species (with maybe the notable exception of viruses), and such strong selection pressures should not be ignored when we try to understand evolutionary processes.”

Thank you Marcel for your enjoyable paper. We are looking forward to your future works.

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!

Revealing the inner beauty by 3D structured illumination microscopy

Posted by Hamed Shateri Najafabadi

John W. Sedat and his team at the University of California, San Francisco, have developed a revolutionary method for visualizing the cells. Their new “3D-structured illumination microscopy”, or 3D-SIM, analyzes the changes that happen in the light interference pattern when a fine cellular structure reflects the light, and interprets the image with a resolution of about 100nm, almost twice as good as the resolution of the state-of-the-art confocal laser scanning microscopes – shamefully, that was all I could understand from the physics of this microscope! This technique is fascinating in that, in contrast to the electrone microscopy techniques, it can be used for specific labeling of molecules using the very conventional methods such as labeling with fluorescent antibodies. There is no need to change the protocols that you currently use for preparing the specimens; just the micropscope is different. In a report in Science, Sedat and his team demonstrate the ability of this technique in resolving multicolor images of the nuclear periphery with an unprecedented precision, revealing exciting features such as presence of chromatin-deprived spaces just below the foci of nuclear pore complexes (see figure below).

Some images are so stunning:

P.S. Also see the first comment; Marie-Luise has kindly written a description on the physics behind 3D-SIM.

microRNA processing by splicing

Posted by Hamed Shateri Najafabadi

My colleage, Yuan, sent me a paper a while ago that had interesting points on possible mechanisms of microRNA processing. Ruby, Jan and Bartel from MIT in their paper, “Intronic microRNA precursors that bypass Drosha processing” (Nature 2007, 448:83-86), report a new mechanism for maturation of microRNAs from introns. This Drosha-independent mechanism recruits the splicing machinery to produce pre-miRNAs/introns that the authors, tastefully, call ‘mirtrons’ (the conventional pathway requires the enzyme Drosha to cleave pri-miRNA transcripts into pre-miRNAs, see Figure below). This paper is a beautiful example of how high-throughput sequencing, spiced by open minds and sharp eyes can lead into spectacular findings. I would like to quote the last sentences of this paper: “This mechanism, together with that of mirtron processing, would enable miRNAs to emerge in any organism with both splicing and post-transcriptional RNA silencing, even those lacking the specialized RNase III enzyme Drosha or its plant counterpart, DICER-LIKE1. In this scenario, miRNAs might have emerged in ancient eukaryotes before the advent of modern miRNA biogenesis pathways.”