Posted by: Kasra Hassani

Recently antimicrobial peptides (AMPs) have received a lot of attention due to their ubiquitous presence in defence systems along with diversity of structure and function and of course putative commercial and therapeutics usages. Organisms as diverse as bacteria, fungi, insects and vertebrates possess a ‘personalized’ set of AMPs that fight invaders with low or no effect on hosts; interestingly, AMPs seems to be highly tolerant to emergence of resistance.

A study in Science by Haine et al. has suggested that insects use a two step mechanism in fighting infections. Firstly, up to 99.5% of the bacteria are killed by the phagocytic haematocytes and other immune mechanisms of the insects within the first few hours. Secondly and interestingly, the remaining low percentage which have been selected due resistance to the first immune response are ‘mopped up’ by a load of AMPs secreted from the host for the following days (up to two weeks). Because different AMPs with different properties and functions (pore forming, modulatory, inhibitory…) are secreted at the same time, very low chances for emergence of resistance remains for the surviving bacteria. The authors have highlighted that when thinking of AMPs for therapeutic purposes, their exact ecological role in nature has to be kept in mind.

Picture from Schneider and Chambers, Science 2008

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.

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.

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!

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???

Posted by: Issa Abu-Dayyeh

The exact role played by the protein tyrosine phosphatase (SHP-1) in the negative regulation of macrophage functions has been an active area of research for many years. In fact, SHP-1 deficient mice are hyper-inflammatory. They lose their hair “for God’s sake” due to exaggerated inflammatory responses in the skin area! (hence their name motheaten). But what does this tell us? It tells us a lot of pathways are simply “on fire”. To dissect every single pathway controlled by this PTP is a humongous job, and the best approach in my opinion is to try to focus, and dissect a pathway at a time and a function at a time. So, what did we attempt to do in our most recent publication (Blanchette, J. et al.) in Immunology (2008)?

The paper explores the signaling pathways that seem to be major contributors to NO production in SHP-1 deficient macrophages. NO production is driven by a gene known as iNOS whose expression is driven by several transcription factors, most importantly: Nf-kB, STAT, and AP-1. One of those transcription factors “AP-1″ is activated by a MAP kinase called JNK.

This work utilizes inhibitors of many of these members to see which of them will be able to suppress that excess NO production observed in SHP-1 deficient macrophages in an effort to understand how SHP-1 causes this increased NO production.

To save you the dull experimental details…Results showed that the exaggerated NO production in SHP-1-/- macrophages seems to be due to an increased JNK/AP-1 and not NF-kB activity.

And so what? some people might ask!

Well…I agree a finding like this might not find a cure to leishmaniasis. Nevertheless, bearing in mind that NF-kB translocation is increased in the absence of SHP-1, this paper then suggests something rather important. This increased NF-kB activity is not contributing to iNOS transcription. What is it doing then? and how can iNOS be differentially regulated? These are questions that await answers. (If somebody has answers, I will be glad to hear from them).

This work simply broadens our knowledge about where SHP-1 exerts its effects, and by knowing how, we can probably try to eventually revert some of those actions during the course of a Leishmania infection and help find an effective drug against leishmaniasis that is not as toxic as the ones available nowadays…

A block added to the wall. that is how I see it.

If you are interested in viewing the paper, please visit it here

Enjoy,

Issa

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?

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.

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!

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.