Hydroxamate-based compounds are potent inhibitors of Toxoplasma gondii HDAC biological activity.

Toxoplasmosis is a critical health issue for immune-deficient individuals and the offspring of newly infected mothers. It is caused by a unicellular intracellular parasite called Toxoplasma gondii that is found worldwide. Although efficient drugs are commonly used to treat toxoplasmosis, serious adverse events are common. Therefore, new compounds with potent anti-T. gondii activity are needed to provide better suited treatments. We have tested compounds designed to target specifically histone deacetylase enzymes. Among the 55 compounds tested, we identified three compounds showing a concentration of drug required for 50% inhibition (IC50) in the low 100 nM range with a selectivity index of more than 100. These compounds are not only active at inhibiting the growth of the parasite in vitro but also at preventing some of the consequences of the acute disease in vivo. Two of these hydroxamate based compound also induce a hyper-acetylation of the parasite histones while the parasitic acetylated tubulin level remains unchanged. These findings suggest that the enzymes regulating histone acetylation are potent therapeutic targets for the treatment of acute toxoplasmosis.

A potent HDAC inhibitor blocks Toxoplasma gondii tachyzoite growth and profoundly disrupts parasite gene expression.

INTRODUCTION: Toxoplasmosis is a major health issue worldwide, especially for immune-deficient individuals and the offspring of newly infected mothers. It is caused by a unicellular intracellular parasite called Toxoplasma gondii. Although the drugs commonly used to treat toxoplasmosis are efficient, they present serious side effects and adverse events are common. Therefore, there is a need for the discovery of new compounds with potent anti-Toxoplasma gondii activity. METHODS: This study tested compounds designed to target enzymes that are involved in the epigenetic regulation of gene expression. RESULTS: Among the most active compounds, an HDAC inhibitor showing an IC50 of 30 nM with a selectivity index above 100 was identified. MC1742 was active at inhibiting the growth of the parasite in vitro but also at preventing the consequences of the acute disease in vivo. This compound induced hyper-acetylation of histones, while the acetylated tubulin level remained unchanged. After MC1742 treatment, the parasite expression profile was profoundly changed with the activation of genes preferentially expressed in the sexual stages that are normally repressed in the tachyzoite stage. CONCLUSIONS: These findings suggest that this compound disturbs the Toxoplasma gondii gene expression program, inducing parasite death.

Primary brain cell infection by Toxoplasma gondii reveals the extent and dynamics of parasite differentiation and its impact on neuron biology.

Toxoplasma gondii is a eukaryotic parasite that forms latent cysts in the brain of immunocompetent individuals. The latent parasite infection of the immune-privileged central nervous system is linked to most complications. With no drug currently available to eliminate the latent cysts in the brain of infected hosts, the consequences of neurons' long-term infection are unknown. It has long been known that T. gondii specifically differentiates into a latent form (bradyzoite) in neurons, but how the infected neuron responds to the infection remains to be elucidated. We have established a new in vitro model resulting in the production of mature bradyzoite cysts in brain cells. Using dual, host and parasite RNA-seq, we characterized the dynamics of differentiation of the parasite, revealing the involvement of key pathways in this process. Moreover, we identified how the infected brain cells responded to the parasite infection revealing the drastic changes that take place. We showed that neuronal-specific pathways are strongly affected, with synapse signalling being particularly affected, especially glutamatergic synapse signalling. The establishment of this new in vitro model allows investigating both the dynamics of parasite differentiation and the specific response of neurons to long-term infection by this parasite.

TgAP2IX-5 is a key transcriptional regulator of the asexual cell cycle division in Toxoplasma gondii.

Apicomplexan parasites have evolved efficient and distinctive strategies for intracellular replication where the timing of emergence of the daughter cells (budding) is a decisive element. However, the molecular mechanisms that provide the proper timing of parasite budding remain unknown. Using Toxoplasma gondii as a model Apicomplexan, we identified a master regulator that controls the timing of the budding process. We show that an ApiAP2 transcription factor, TgAP2IX-5, controls cell cycle events downstream of centrosome duplication. TgAP2IX-5 binds to the promoter of hundreds of genes and controls the activation of the budding-specific cell cycle expression program. TgAP2IX-5 regulates the expression of specific transcription factors that are necessary for the completion of the budding cycle. Moreover, TgAP2IX-5 acts as a limiting factor that ensures that asexual proliferation continues by promoting the inhibition of the differentiation pathway. Therefore, TgAP2IX-5 is a master regulator that controls both cell cycle and developmental pathways.

Analysis of the interactome of Schistosoma mansoni histone deacetylase 8.

BACKGROUND: Histone deacetylase 8 from Schistosoma mansoni (SmHDAC8) is essential to parasite growth and development within the mammalian host and is under investigation as a target for the development of selective inhibitors as novel schistosomicidal drugs. Although some protein substrates and protein partners of human HDAC8 have been characterized, notably indicating a role in the function of the cohesin complex, nothing is known of the partners and biological function of SmHDAC8. METHODOLOGY/PRINCIPAL FINDINGS: We therefore employed two strategies to characterize the SmHDAC8 interactome. We first used SmHDAC8 as a bait protein in yeast two-hybrid (Y2H) screening of an S. mansoni cDNA library. This allowed the identification of 49 different sequences encoding proteins. We next performed co-immunoprecipitation (Co-IP) experiments on parasite extracts with an anti-SmHDAC8 antibody. Mass spectrometry (MS) analysis allowed the identification of 160 different proteins. CONCLUSIONS/SIGNIFICANCE: SmHDAC8 partners are involved in about 40 different processes, included expected functions such as the cohesin complex, cytoskeleton organization, transcriptional and translational regulation, metabolism, DNA repair, the cell cycle, protein dephosphorylation, proteolysis, protein transport, but also some proteasome and ribosome components were detected. Our results show that SmHDAC8 is a versatile deacetylase, potentially involved in both cytosolic and nuclear processes.

A multistrain approach to studying the mechanisms underlying compatibility in the interaction between Biomphalaria glabrata and Schistosoma mansoni.

In recent decades, numerous studies have sought to better understand the mechanisms underlying the compatibility between Biomphalaria glabrata and Schistosoma mansoni. The developments of comparative transcriptomics, comparative genomics, interactomics and more targeted approaches have enabled researchers to identify a series of candidate genes. However, no molecular comparative work has yet been performed on multiple populations displaying different levels of compatibility. Here, we seek to fill this gap in the literature. We focused on B. glabrata FREPs and S. mansoni SmPoMucs, which were previously demonstrated to be involved in snail/schistosome compatibility. We studied the expression and polymorphisms of these factors in combinations of snail and schistosome isolates that display different levels of compatibility. We found that the polymorphism and expression levels of FREPs and SmPoMucs could be linked to the compatibility level of S. mansoni. These data and our complementary results obtained by RNA-seq of samples from various snail strains indicate that the mechanism of compatibility is much more complex than previously thought, and that it is likely to be highly variable within and between populations. This complexity must be taken into account if we hope to identify the molecular pathways that are most likely to be good targets for strategies aimed at blocking transmission of the parasite through the snail intermediate host.

Reciprocal immune benefit based on complementary production of antibiotics by the leech Hirudo verbana and its gut symbiont Aeromonas veronii.

The medicinal leech has established a long-term mutualistic association with Aeromonas veronii, a versatile bacterium which can also display free-living waterborne and fish- or human-pathogenic lifestyles. Here, we investigated the role of antibiotics in the dynamics of interaction between the leech and its gut symbiont Aeromonas. By combining biochemical and molecular approaches, we isolated and identified for the first time the antimicrobial peptides (AMPs) produced by the leech digestive tract and by its symbiont Aeromonas. Immunohistochemistry data and PCR analyses evidenced that leech AMP genes are induced in the gut epithelial cells when Aeromonas load is low (starved animals), while repressed when Aeromonas abundance is the highest (post blood feeding). The asynchronous production of AMPs by both partners suggests that these antibiotic substances (i) provide them with reciprocal protection against invasive bacteria and (ii) contribute to the unusual simplicity of the gut microflora of the leech. This immune benefit substantially reinforces the evidence of an evolutionarily stable association between H. verbana and A. veronii. Altogether these data may provide insights into the processes making the association with an Aeromonas species in the digestive tract either deleterious or beneficial.

Schistosoma mansoni mucin gene (SmPoMuc) expression: epigenetic control to shape adaptation to a new host.

The digenetic trematode Schistosoma mansoni is a human parasite that uses the mollusc Biomphalaria glabrata as intermediate host. Specific S. mansoni strains can infect efficiently only certain B. glabrata strains (compatible strain) while others are incompatible. Strain-specific differences in transcription of a conserved family of polymorphic mucins (SmPoMucs) in S. mansoni are the principle determinants for this compatibility. In the present study, we investigated the bases of the control of SmPoMuc expression that evolved to evade B. glabrata diversified antigen recognition molecules. We compared the DNA sequences and chromatin structure of SmPoMuc promoters of two S. mansoni strains that are either compatible (C) or incompatible (IC) with a reference snail host. We reveal that although sequence differences are observed between active promoter regions of SmPoMuc genes, the sequences of the promoters are not diverse and are conserved between IC and C strains, suggesting that genetics alone cannot explain the evolution of compatibility polymorphism. In contrast, promoters carry epigenetic marks that are significantly different between the C and IC strains. Moreover, we show that modifications of the structure of the chromatin of the parasite modify transcription of SmPoMuc in the IC strain compared to the C strain and correlate with the presence of additional combinations of SmPoMuc transcripts only observed in the IC phenotype. Our results indicate that transcription polymorphism of a gene family that is responsible for an important adaptive trait of the parasite is epigenetically encoded. These strain-specific epigenetic marks are heritable, but can change while the underlying genetic information remains stable. This suggests that epigenetic changes may be important for the early steps in the adaptation of pathogens to new hosts, and might be an initial step in adaptive evolution in general.

Chromatin regulation in schistosomes and histone modifying enzymes as drug targets.

Only one drug is currently available for the treatment and control of schistosomiasis and the increasing risk of selecting strains of schistosome that are resistant to praziquantel means that the development of new drugs is urgent. With this objective we have chosen to target the enzymes modifying histones and in particular the histone acetyltransferases and histone deacetylases (HDAC). Inhibitors of HDACs (HDACi) are under intense study as potential anti-cancer drugs and act via the induction of cell cycle arrest and/or apoptosis. Schistosomes like other parasites can be considered as similar to tumours in that they maintain an intense metabolic activity and rate of cell division that is outside the control of the host. We have shown that HDACi can induce apoptosis and death of schistosomes maintained in culture and have set up a consortium (Schistosome Epigenetics: Targets, Regulation, New Drugs) funded by the European Commission with the aim of developing inhibitors specific for schistosome histone modifying enzymes as novel lead compounds for drug development.

Chromatin regulation in schistosomes and histone modifying enzymes as drug targets

Only one drug is currently available for the treatment and control of schistosomiasis and the increasing risk of selecting strains of schistosome that are resistant to praziquantel means that the development of new drugs is urgent. With this objective we have chosen to target the enzymes modifying histones and in particular the histone acetyltransferases and histone deacetylases (HDAC). Inhibitors of HDACs (HDACi) are under intense study as potential anti-cancer drugs and act via the induction of cell cycle arrest and/or apoptosis. Schistosomes like other parasites can be considered as similar to tumours in that they maintain an intense metabolic activity and rate of cell division that is outside the control of the host. We have shown that HDACi can induce apoptosis and death of schistosomes maintained in culture and have set up a consortium (Schistosome Epigenetics: Targets, Regulation, New Drugs) funded by the European Commission with the aim of developing inhibitors specific for schistosome histone modifying enzymes as novel lead compounds for drug development.

Coral bleaching under thermal stress: putative involvement of host/symbiont recognition mechanisms.

BACKGROUND: Coral bleaching can be defined as the loss of symbiotic zooxanthellae and/or their photosynthetic pigments from their cnidarian host. This major disturbance of reef ecosystems is principally induced by increases in water temperature. Since the beginning of the 1980s and the onset of global climate change, this phenomenon has been occurring at increasing rates and scales, and with increasing severity. Several studies have been undertaken in the last few years to better understand the cellular and molecular mechanisms of coral bleaching but the jigsaw puzzle is far from being complete, especially concerning the early events leading to symbiosis breakdown. The aim of the present study was to find molecular actors involved early in the mechanism leading to symbiosis collapse. RESULTS: In our experimental procedure, one set of Pocillopora damicornis nubbins was subjected to a gradual increase of water temperature from 28 degrees C to 32 degrees C over 15 days. A second control set kept at constant temperature (28 degrees C). The differentially expressed mRNA between the stressed states (sampled just before the onset of bleaching) and the non stressed states (control) were isolated by Suppression Subtractive Hybridization. Transcription rates of the most interesting genes (considering their putative function) were quantified by Q-RT-PCR, which revealed a significant decrease in transcription of two candidates six days before bleaching. RACE-PCR experiments showed that one of them (PdC-Lectin) contained a C-Type-Lectin domain specific for mannose. Immunolocalisation demonstrated that this host gene mediates molecular interactions between the host and the symbionts suggesting a putative role in zooxanthellae acquisition and/or sequestration. The second gene corresponds to a gene putatively involved in calcification processes (Pdcyst-rich). Its down-regulation could reflect a trade-off mechanism leading to the arrest of the mineralization process under stress. CONCLUSION: Under thermal stress zooxanthellae photosynthesis leads to intense oxidative stress in the two partners. This endogenous stress can lead to the perception of the symbiont as a toxic partner for the host. Consequently, we propose that the bleaching process is due in part to a decrease in zooxanthellae acquisition and/or sequestration. In addition to a new hypothesis in coral bleaching mechanisms, this study provides promising biomarkers for monitoring coral health.

Halocyntin and papillosin, two new antimicrobial peptides isolated from hemocytes of the solitary tunicate, Halocynthia papillosa.

We report here the screening of five marine invertebrate species from two taxa (tunicates and echinoderms) for the presence of cationic antimicrobial peptides (AMP) in defence cells (hemocytes). Antimicrobial activities were detected only in the two tunicates Microcosmus sabatieri and Halocynthia papillosa. In addition, we report the isolation and characterization of two novel peptides from H. papillosa hemocytes. These molecules display antibacterial activity against Gram-positive and Gram-negative bacteria. Complete peptide characterization was obtained by a combination of Edman degradation and mass spectrometry. The mature molecules, named halocyntin and papillosin, comprise 26 and 34 amino acid residues, respectively. Their primary structure display no significant similarities with previously described AMP.

Controlled chaos of polymorphic mucins in a metazoan parasite (Schistosoma mansoni) interacting with its invertebrate host (Biomphalaria glabrata).

Invertebrates were long thought to possess only a simple, effective and hence non-adaptive defence system against microbial and parasitic attacks. However, recent studies have shown that invertebrate immunity also relies on immune receptors that diversify (e.g. in echinoderms, insects and mollusks (Biomphalaria glabrata)). Apparently, individual or population-based polymorphism-generating mechanisms exists that permit the survival of invertebrate species exposed to parasites. Consequently, the generally accepted arms race hypothesis predicts that molecular diversity and polymorphism also exist in parasites of invertebrates. We investigated the diversity and polymorphism of parasite molecules (Schistosoma mansoni Polymorphic Mucins, SmPoMucs) that are key factors for the compatibility of schistosomes interacting with their host, the mollusc Biomphalaria glabrata. We have elucidated the complex cascade of mechanisms acting both at the genomic level and during expression that confer polymorphism to SmPoMuc. We show that SmPoMuc is coded by a multi-gene family whose members frequently recombine. We show that these genes are transcribed in an individual-specific manner, and that for each gene, multiple splice variants exist. Finally, we reveal the impact of this polymorphism on the SmPoMuc glycosylation status. Our data support the view that S. mansoni has evolved a complex hierarchical system that efficiently generates a high degree of polymorphism-a "controlled chaos"-based on a relatively low number of genes. This contrasts with protozoan parasites that generate antigenic variation from large sets of genes such as Trypanosoma cruzi, Trypanosoma brucei and Plasmodium falciparum. Our data support the view that the interaction between parasites and their invertebrate hosts are far more complex than previously thought. While most studies in this matter have focused on invertebrate host diversification, we clearly show that diversifying mechanisms also exist on the parasite side of the interaction. Our findings shed new light on how and why invertebrate immunity develops.

Expression analysis of highly polymorphic mucin proteins (Sm PoMuc) from the parasite Schistosoma mansoni.

The co-evolutionary dynamics that exist in many host-parasite interactions sometimes lead to a compatibility polymorphism, of which the molecular bases are unknown. To identify key molecules involved in this phenomenon in the S. mansoni/B. glabrata model, we developed a comparative proteomics approach for the larval stages that interact with the invertebrate host. The comparison of the proteomes of compatible and incompatible parasite strains led to the identification of a new family of schistosome antigens that share molecular characteristics with the molecules of the mucin family. In particular, they possess a domain containing a variable number of tandem repeats (VNTR). The pronounced polymorphism of these proteins, that distinguishes compatible and incompatible parasite strains, led us to further investigate the role that this protein family plays in the compatibility polymorphism in our model. In the present study, we examine precursor structure, report analysis of mucin-like expression and describe their polymorphism. Our data show that these proteins share structural characteristics with highly glycosylated secreted mucins. The proteins are (i) only expressed in larval stages that interact with the mollusc, (ii) located in the apical gland of miracidia and sporocysts and (iii) secreted and released in excretion-secretion products. Finally, we show that these mucins display a high degree of polymorphism and that extensive differences are observed between S. mansoni strains. These different characteristics led us to name this novel gene family "S. mansoni polymorphic mucins" (Sm PoMuc).

Molecular determinants of compatibility polymorphism in the Biomphalaria glabrata/Schistosoma mansoni model: new candidates identified by a global comparative proteomics approach.

The co-evolutionary dynamics that exist in host-parasite interactions sometimes lead to compatibility polymorphisms, the molecular bases of which are rarely investigated. To identify key molecules that are involved in this phenomenon in the Schistosoma mansoni/Biomphalaria glabrata model, we developed a comparative proteomics approach using the larval stages that interact with the invertebrate host. We used qualitative and quantitative analyses to compare the total proteomes of primary sporocysts from compatible and incompatible parasite strains. The differentially expressed proteins thus detected belong to three main functional groups: (i) scavengers of reactive oxygen species, (ii) components of primary metabolism, and (iii) mucin-like proteins. We discuss the putative roles played by these protein families as determinants of compatibility polymorphism. Since mucins are known to play key roles in the host-parasite interplay, we consider the newly discovered S. mansoni mucin-like proteins (SmMucin-like) as the most promising candidates for influencing the fate of host-parasite interactions. An analysis of their expression is presented in a paper published in the same journal issue.

Excretory-secretory proteome of larval Schistosoma mansoni and Echinostoma caproni, two parasites of Biomphalaria glabrata.

Schistosoma mansoni and Echinostoma caproni are two trematode species that use different strategies (mimicry and immunosuppression, respectively) to interfere with the snail innate immune system. Parasites excretory-secretory (ES) products have been shown to play a key role in these host-parasite immune interactions. However, they remain largely uncharacterized in larval trematodes. We developed a global proteomic approach to characterize the ES proteome of S. mansoni and E. caproni primary sporocysts. In ES products of both parasites, we found proteins involved in reactive oxygen species scavenging, glycolysis, signalling or calcium binding (superoxide dismutase Cu/Zn; glutathione S-transferase; aldo-keto-reductase; triose-phosphate isomerase; glyceraldehyde-3-phosphate dehydrogenase; aldolase, enolase, MICAL-like, calreticulin). According to their predicted functions, we propose a model in which these proteins (i) are involved in antioxidant activity, (ii) prevent hemocyte encapsulation process or (iii) favor invasion and migration of sporocysts in host tissues. These results suggest that S. mansoni and E. caproni sporocysts develope a strong immune protection during the first hours of infection giving them enough time to build up a long lasting immune evasion strategy relying on molecular mimicry or immunosuppression, respectively.