Degradation of the intestinal mucus barrier by whipworm

Posted by: Kasra

I wrote recently about modulation of the host by intestinal worm Trichuris muris. Here is another brilliant study looking at the secreted proteins of this nematode and how they interact with the small intestine mucus.

 Hasnain et al. published in PLoS NTD that secreted proteins of T. muris contain serine proteases that are able to degrade the mucus barrier, especially Muc2. Interestingly, they observed that the components of the mucus barrier are different during acute versus chronic infection. When worm expulsion begins in acute infection of T. muris, Muc5a is also detected in the mucus, a protein which is normally not expressed in the intestinal mucus but in the lung. Muc5a is resistant to degradation by serine proteases of the parasite and probably helps in worm expulsion. This specific host response and change in mucus does not happen during chronic infection which results in continued stay of the worm in the intestine.

Secreted Proteins of T. muris degrade the mucus in chronic but not acute infection. This is due to upregulation of Muc5a in acute infection, which is resistant to the proteases. From Hasnain et al. PLoS NTD, doi:10.1371/journal.pntd.0001856.g003

Below is a schematic diagram of the structure of the mucus layer during acute and chronic infection and how Excreted Secreted Proteins (ESPs) of the parasite interact with it.

A schematic of how the mucus layer looks like during acute or chronic infection with T. muris. From Hasnain et al. PLoS NTD doi:10.1371/journal.pntd.0001856.g003

Hasnain SZ, McGuckin MA, Grencis RK, & Thornton DJ (2012). Serine Protease(s) Secreted by the Nematode Trichuris muris Degrade the Mucus Barrier. PLoS neglected tropical diseases, 6 (10) PMID: 23071854

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Communication between intestinal commensal bacteria and the host via membrane vesicles

Posted by Kasra

Releasing outer membrane vesicles or OMVs of by bacteria can be considered one of their protein secretion pathways. This pathway is especially important for carrying messages to longer distances than what other mechanisms such as type III secretion system can do.

Although the gut is largely colonized, there is not much of direct cell to cell contact between the microbiota and the host cells due to presence of a thick mucosal layer and other factors. In a recent study, Shen et al. show that bacterial OMVs can make up for this distance and allow for communication between the microbiota and host. They show that orally administered OMVs collected from Bacteroides fragilis can protect mice from chemically induced colitis. Furthermore, they show that this protection is dependent on presence of a capsular polysaccharide (PSA) on the OMV surface. Shen et al. suggest that PSA-containing OMVs are picked up by dendritic cells and induce IL-10 production, thus ameliorating colitis. Specifically, they show that production of IL-10 by DCs is dependent on recognition of PSA by TLR2. Therefore, stimulation of TLR2 by B. fragilis OMVs leads to tolerance instead of inflammation, which is necessary for homeostatic maintenance of the gut.

 

Top shows B. fragilis releasing OMVs. Bottom shows purified B. fragilis OMVs from wildtype and non-PSA producing strains. From Shen et al. Cell host & Microbe. Oct. 2012

Shen Y, Torchia ML, Lawson GW, Karp CL, Ashwell JD, & Mazmanian SK (2012). Outer membrane vesicles of a human commensal mediate immune regulation and disease protection. Cell host & microbe, 12 (4), 509-20 PMID: 22999859

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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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From Ivory Towers to Public Tribunes

Posted by Kasra

This is in continuation of a post by my old friend and classmate in his new exciting blog Genophoria. He expressed his rightful concerns about the rise of “Entertainment Science”, where he says scientists are coming out of their Ivory towers and shouting out their impressive and sometimes controversial findings to the public. It often happens that these controversial findings, or at least their conclusions in that regard are wrong. Scientists can accept that. Science is by nature self-correcting. But at the same time, for the public, they lose their credibility as truth-seekers which they claim to be.

It just happens that at this very time, PNAS has published a study on the statistics of retracted publications. Let’s not exaggerate. The percentage of retracted papers compared to number of publications is very very small. Still, their results were a bit surprising at least to me: 67% of them were retracted due to misconduct, either fraud or suspected fraud. Only 20% or so were due to error. Many questions arise: Has it always been like this? Only is it because there are more publications now and more screening? What percentage goes unnoticed? Most importantly, what were the underlying reasons for these fraudulent publications? Were they desperate Postdocs or PIs trying to win a Cell or a Nature to renew a fellowship or a grant? Or were they seeking something further, a socioeconomical, political or cultic purpose beyond science? These questions seek immediate attention and hopefully clear answers. Without any doubt, the fight for budget has become fiercer; and no, most scientists can no longer live in ivory towers, indifferent to the public and their attention – if they ever did. By the way, hadn’t you said earlier that by turning away from the public we turned from high-ranked academics into socially excluded geeks? We need to interact with the public, to rebuke false claims and promote logical thinking. I guess as you say, we are doing it wrong.

If the scientific community is willing to share the excitement of discoveries and controversies with the public, it should be more stringent in the peer-reviewing process of such claims. In retrospect, how many of the fraudulent retracted papers can be labelled as editorial or peer-review failures? Publishing in high-impact journals is getting harder and harder. But maybe during the peer-review, there should be a new focus on skepticism and a shrewd eye for biased claims, besides asking for more and more control experiments. At the same time, when presenting discoveries to the press, more transparency and accuracy about their nature and details are needed, so that a susceptibility SNP doesn’t turn into a cancer gene and an in vitro-tested compound into its ultimate cure.

Fang FC, Steen RG, & Casadevall A (2012). Misconduct accounts for the majority of retracted scientific publications. Proceedings of the National Academy of Sciences of the United States of America PMID: 23027971

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Did fungi help mammals dominate Earth?

Image

From SMBC-Comics

 

Posted by Kasra

 

The Cretaceous mass extinction is one of the most exciting topics in evolutionary biology. There are always discussions on what caused the mass extinction, what happened during the extinction, what happened to all the dinosaurs, why did the mammals and birds survive, and so on. A recent article in PLoS Pathogens by Arturo Casadevall brings forward an interesting hypothesis: Fungi might have given mammals an evolutionary advantage during this period of time.

Casadevall mentions that humans and other mammals are generally resistant to fungal infections and most pathogenic fungi are in fact only opportunistic pathogens. He suggests that this is due to the mammalian control of body temperature – higher than optimal growth temperature of fungi – and evolution of powerful adaptive immunity. On the other hand, he brings forward examples of amphibians, being ectothermic, and primitive mammals, having lower body temperature, are more susceptible to fungal infections.

He next states that the Cretaceous-Tertiary (K-T) boundary included a cooling period with plenty of dust in the atmosphere and lack of sufficient sunlight. This led to a fungal bloom on the Earth and possible growth of pathogenic fungi. The hypothesis states that this higher than normal presence of fungi selected against surviving ectothermic reptiles and in favour of endothermic mammals. Thus, fungi might have indirectly helped mammals and possibly warm-blooded birds by killing off their competitors for the limited food resources.

Like any other scientific hypothesis, this one also needs to be tested. Unfortunately fossil records cannot tell us much of how common fungal infections were at the time. However, one can first look more closely if warm-bloodedness or higher body temperature does indeed aid in protection from fungal infection, given similar immune systems. Comparing today’s amphibians, reptiles and mammals can be tricky as the host-pathogen interactions may greatly differ among the groups. This could lead to conclusions that are confounded by differences in pathogenicity of fungi or power of the host immune system. Pooling data together from larger numbers host-pathogen pairs can lead to more robust conclusions. Application of heat-resistant fungi can also be beneficial for performing more controlled experiments rather than comparing natural histories. Casadevall himself suggests that climate change can promote evolution of fungi that can better survive in elevated temperatures and be threats as emerging pathogens. In this case, we would be a step in advance knowing what to expect, should these new pathogens emerge.

Casadevall A (2012). Fungi and the rise of mammals. PLoS pathogens, 8 (8) PMID: 22916007

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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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Wanted Dead (But also Alive): The Hepatitis C Virus Polymerase

Posted by: Maryam Ehteshami

I want to dedicate today’s blog post to a little virus known as Hepatitis C virus. Having worked on HIV before, and now moving onto work with HCV, I am tempted to compare the history of these two viruses and I find a lot of parallels. Both viruses were discovered in the 80s (1983 and 1989, for HIV and HCV, respectively), they can both have a long incubation period, and they both infect a whole lot of people worldwide. Of course there are many differences as well. For one, HIV comes with a 100% mortality rate. HCV doesn’t get integrated into the human genome and about 25% of people infected can clear the virus on their own. The remaining 75% of the HCV-infected individuals remain at the mercy of the current anti-HCV treatment (20-80 % of them can expect to be cured, depending on the infecting HCV genotype). It is in fact the available treatment options that most set HIV and HCV apart.  Today, an HIV-positive person living in the first world can chose from an array of over 25 antiretroviral compounds. The development of these drugs has been the fruit of over 20 years of aggressive research, supported by grass-root activism and heavy funding from both government and industry. It has also been aided along the way with the development of appropriate animal models, cell-culture systems, and in vitro enzymatic assays that corresponded well with patient outcomes. In contrast, anti-HCV antiviral development has been very slow. For one, cell-culture growth of the virus (or the related replicon model) was not possible as recently as the mid-2000s. Developing in vitro systems for the study of HCV proteins have not always been straight forward either. Despite these many hurdles, anti-HCV drug development researchers have not given up. In fact, we are at the beginning of an exciting era. Last year saw the FDA approval of two new protease inhibitors for HCV. Gilead recently made an 11 billion dollar purchase of a small pharmaceutical company called Pharmasset which possessed an anti-HCV compound showing 100% cure rate in recent phase 2 clinical trials. Many different pharmaceuticals are also in possession of a number of very promising leads and are competing for the eventual market. Putting all recent finding together, one is tempted to speculate that this decade is the beginning of the end for hepatitis C.

In this blog post, I would like to focus on one of the major antiviral targets, namely NS5B, the viral RNA-dependent RNA-polymerase, which is essential for replication viral genomic RNA.  I would like to focus on some of the issues that the field has faced when it comes to assessing NS5B activity in vitro and some new findings that will impact anti-NS5B drug development.

As the viral polymerase, the NS5B protein is a major target for therapy. Both nucleoside analogues and non-nucleoside analogues are in development for inhibiting this protein (namely, the 11 billion dollar investment by Gilead was for a nucleoside analogue called GS-7977). But working with the NS5B enzyme has proven to be a challenge and may in part account for some of the delay that the field has seen in drug development. Almost all publications to date (dating back to mid 1990s) report an in vitro activity of below 1% to 5% for this enzyme.  This low activity is what makes working with this enzyme so difficult and this is why I was very excited to read a February 2012 publication in the Journal of Biological Chemistry (Jin et al. 2012) that claimed that with some minor protocol changes, the in vitro activity of the enzyme can be increased to 65%!!

But lets take a step back for a moment and examine some of the rationales that were previously proposed to explain this low level of activity. There are almost 100 crystal structures available for NS5B and a recent April 2012 publication in Journal of Virology even shows the protein bound to the RNA substrate (Mosley et al 2012). A close inspection of these structures revealed an intriguing property for this enzyme. It appears that near the C-terminus of NS5B, there exists a beta-hairpin loop that folds right into the active site (see Figure 1, the loop is highlighted in red). It is suggested that the presence of this loop in the active site, prevents the double stranded RNA/RNA substrate from binding, and if no RNA is bound, no activity can be had! Some have gone on to call this loop the “auto-inhibitory” beta-hairpin loop. This was supported by the recent crystal structure, where the beta-hairpin loop was taken out and replaced by two glycine residues and lo and behold! A 100-fold increase in activity was observed! The absence of the loop also allowed for the co-crystalization of NS5B with its RNA substrate (Figure 2).

Figure 1.Crystal structure of NS5B genotype 1b. The enzyme is shown in pink. The beta-hairpin loop is highlighted in red (Bressanelli et al 1999).

Figure 2. Crystal structure of NS5B Genotype 2a. The enzyme is shown in pink. The glycine residues replacing the beta-hairpin loop are shown in red. The co-crystalized RNA/RNA substrate is shown bound to the active site.

Of course, all of this begs the question why would a naturally occurring part of the enzyme, prevent the very activity that it is supposed to do? No one really knows the answer yet, but it is speculated that this loop serves some sort of a regulatory function, especially relevant in vivo where other host and viral proteins are present and impact viral replication.  The issue is that we really have very little information on how NS5B interacts with itself and other proteins in the context of replication. But I digress!

Of course synthetically taking out a structural loop of the enzyme can be an immediate solution to the problem of low enzyme activity. But this also raises questions about the in vivo relevance of such a model. And this is what makes the Jin et al 2012 publication exciting. Their findings indirectly corroborate previous findings arguing that the rate-limiting step in in vitro activity is the slow binding of the enzyme to the RNA substrate. Based on this, they show that it is not necessary to further modify the enzyme, but rather just give the enzyme more time to achieve what it needs to achieve. They show that 24 hour pre-incubation of the enzyme, with the RNA substrate, with the addition of a few starting nucleotides, gives the enzyme enough time to form active elongation complexes. Under these modified conditions, they found that 65% of the enzyme was capable of forming elongation complexes, which in turn could incorporate nucleotides as good as the next polymerase!

If the findings in this publication are repeated and confirmed, I am sure that many NS5B enzymologists will take a breath of relief. With the projection that the GS-7977 compound will be the next compound to get FDA approval, we are looking at many years of nucleoside analogue therapy for individuals infected with HCV. As treatment usage increases, we expect to see more drug resistance, which will bring us back to the drawing board for the development of next-generation nucleoside analogues. Therefore, any knowledge we gain now about the functions of NS5B in vitro will surely help us better prepare for the future. As the development of GS-7977 will likely represent a turning point in HCV therapy history, I am hopeful that the two publications highlighted here will come to represent a turning point for in vitro studies of the HCV polymerase.

Jin Z, Leveque V, Ma H, Johnson KA, & Klumpp K (2012). Assembly, purification, and pre-steady-state kinetic analysis of active RNA-dependent RNA polymerase elongation complex. The Journal of biological chemistry, 287 (13), 10674-83 PMID: 22303022
Mosley RT, Edwards TE, Murakami E, Lam AM, Grice RL, Du J, Sofia MJ, Furman PA, & Otto MJ (2012). Structure of HCV Polymerase in Complex with Primer-Template RNA. Journal of virology PMID: 22496223
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