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Cryptosporidium parvum is a highly prevalent zoonotic and anthroponotic protozoan parasite that causes a diarrheal syndrome in children and neonatal livestock, culminating in growth retardation and mortalities. Despite the high prevalence of C. parvum, there are no fully effective and safe drugs for treating infections, and there is no vaccine. We have previously reported that the bacterial-like C. parvum lactate dehydrogenase (CpLDH) enzyme is essential for survival, virulence and growth of C. parvum in vitro and in vivo. In the present study, we screened compound libraries and identified inhibitors against the enzymatic activity of recombinant CpLDH protein in vitro. We tested the inhibitors for anti-Cryptosporidium effect using in vitro infection assays of HCT-8 cells monolayers and identified compounds NSC158011 and NSC10447 that inhibited the proliferation of intracellular C. parvum in vitro, with IC50 values of 14.88 and 72.65 μM, respectively. At doses tolerable in mice, we found that both NSC158011 and NSC10447 consistently significantly reduced the shedding of C. parvum oocysts in infected immunocompromised mice’s feces, and prevented intestinal villous atrophy as well as mucosal erosion due to C. parvum. Together, our findings have unveiled promising anti-Cryptosporidium drug candidates that can be explored further for the development of the much needed novel therapeutic agents against C. parvum infections. [ABSTRACT FROM AUTHOR]

The essential and distinct functions of Protein Phosphatase type 1 (PP1) catalytic subunit in eukaryotes are exclusively achieved through its interaction with a myriad of regulatory partners. In this work, we report the molecular and functional characterization of Gametocyte EXported Protein 15 (GEXP15), a Plasmodium specific protein, as a regulator of PP1. In vitro interaction studies demonstrated that GEXP15 physically interacts with PP1 through the RVxF binding motif in P. berghei. Functional assays showed that GEXP15 was able to increase PP1 activity and the mutation of the RVxF motif completely abolished this regulation. Immunoprecipitation assays of tagged GEXP15 or PP1 in P. berghei followed by immunoblot or mass spectrometry analyses confirmed their interaction and showed that they are present both in schizont and gametocyte stages in shared protein complexes involved in the spliceosome and proteasome pathways and known to play essential role in parasite development. Phenotypic analysis of viable GEXP15 deficient P. berghei blood parasites showed that they were unable to develop lethal infection in BALB/c mice or to establish experimental cerebral malaria in C57BL/6 mice. Further, although deficient parasites produced gametocytes they did not produce any oocysts/sporozoites indicating a high fitness cost in the mosquito. Global proteomic and phosphoproteomic analyses of GEXP15 deficient schizonts revealed a profound defect with a significant decrease in the abundance and an impact on phosphorylation status of proteins involved in regulation of gene expression or invasion. Moreover, depletion of GEXP15 seemed to impact mainly the abundance of some specific proteins of female gametocytes. Our study provides the first insight into the contribution of a PP1 regulator to Plasmodium virulence and suggests that GEXP15 affects both the asexual and sexual life cycle. [ABSTRACT FROM AUTHOR]

The pathogenesis of Plasmodium falciparum malaria is linked to the variant surface antigen PfEMP1, which mediates tethering of infected erythrocytes to the host endothelium and is encoded by approximately 60 var genes per parasite genome. Repeated episodes of malaria infection result in the gradual acquisition of protective antibodies against PfEMP1 variants. The antibody repertoire is believed to provide a selective pressure driving the clonal expansion of parasites expressing unrecognized PfEMP1 variants, however, due to the lack of experimental in vivo models there is only limited experimental evidence in support of this concept. To get insight into the impact of naturally acquired immunity on the expressed var gene repertoire early during infection we performed controlled human malaria infections of 20 adult African volunteers with life-long malaria exposure using aseptic, purified, cryopreserved P. falciparum sporozoites (Sanaria PfSPZ Challenge) and correlated serological data with var gene expression patterns from ex vivo parasites. Among the 10 African volunteers who developed patent infections, individuals with low antibody levels showed a steep rise in parasitemia accompanied by broad activation of multiple, predominantly subtelomeric var genes, similar to what we previously observed in naïve volunteers. In contrast, individuals with intermediate antibody levels developed asymptomatic infections and the ex vivo parasite populations expressed only few var gene variants, indicative of clonal selection. Importantly, in contrast to parasites from naïve volunteers, expression of var genes coding for endothelial protein C receptor (EPCR)-binding PfEMP1 that are associated with severe childhood malaria was rarely detected in semi-immune adult African volunteers. Moreover, we followed var gene expression for up to six parasite replication cycles and demonstrated for the first time in vivo a shift in the dominant var gene variant. In conclusion, our data suggest that P. falciparum activates multiple subtelomeric var genes at the onset of blood stage infection facilitating rapid expansion of parasite clones which express PfEMP1 variants unrecognized by the host’s immune system, thus promoting overall parasite survival in the face of host immunity. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is a prevalent protozoan parasite that can infect any nucleated cell but cannot replicate outside of its host cell. Toxoplasma is auxotrophic for several nutrients including arginine, tryptophan, and purines, which it must acquire from its host cell. The demands of parasite replication rapidly deplete the host cell of these essential nutrients, yet Toxoplasma successfully manages to proliferate until it lyses the host cell. In eukaryotic cells, nutrient starvation can induce the integrated stress response (ISR) through phosphorylation of an essential translation factor eIF2. Phosphorylation of eIF2 lowers global protein synthesis coincident with preferential translation of gene transcripts involved in stress adaptation, such as that encoding the transcription factor ATF4 (CREB2), which activates genes that modulate amino acid metabolism and uptake. Here, we discovered that the ISR is induced in host cells infected with Toxoplasma. Our results show that as Toxoplasma depletes host cell arginine, the host cell phosphorylates eIF2 via protein kinase GCN2 (EIF2AK4), leading to induced ATF4. Increased ATF4 then enhances expression of the cationic amino acid transporter CAT1 (SLC7A1), resulting in increased uptake of arginine in Toxoplasma-infected cells. Deletion of host GCN2, or its downstream effectors ATF4 and CAT1, lowers arginine levels in the host, impairing proliferation of the parasite. Our findings establish that Toxoplasma usurps the host cell ISR to help secure nutrients that it needs for parasite replication. [ABSTRACT FROM AUTHOR]

The article highlights the connection between Plastid–endomembrane in apicomplexan parasites. Topics discussed apicoplast is a nonphotosynthetic plastid found in most apicomplexan parasites, including Toxoplasma gondii and Plasmodium spp.; mentions the relation between apicoplast and endomembrane system; and highlights the role of Autophagy machinery and phosphoinositides in apicoplast segregation.

Therapeutics with novel modes of action and a low risk of generating resistance are urgently needed to combat drug-resistant Plasmodium falciparum malaria. Here, we report that the peptide vinyl sulfones WLL-vs (WLL) and WLW-vs (WLW), highly selective covalent inhibitors of the P. falciparum proteasome, potently eliminate genetically diverse parasites, including K13-mutant, artemisinin-resistant lines, and are particularly active against ring-stage parasites. Selection studies reveal that parasites do not readily acquire resistance to WLL or WLW and that mutations in the β2, β5 or β6 subunits of the 20S proteasome core particle or in components of the 19S proteasome regulatory particle yield only hundred-fold decreases in susceptibility. We observed no cross-resistance between WLL and WLW. Moreover, most mutations that conferred a modest loss of parasite susceptibility to one inhibitor significantly increased sensitivity to the other. These inhibitors potently synergized multiple chemically diverse classes of antimalarial agents, implicating a shared disruption of proteostasis in their modes of action. These results underscore the potential of targeting the Plasmodium proteasome with covalent small molecule inhibitors as a means of combating multidrug-resistant malaria. [ABSTRACT FROM AUTHOR]

The highly conserved proteins of the 14-3-3 family are universal adaptors known to regulate an enormous range of cellular processes in eukaryotes. However, their biological functions remain largely uncharacterized in pathogenic protists comprising of several 14-3-3 protein isoforms. In this study, we report the role of 14-3-3 in coordinating cytoskeletal dynamics during phagocytosis in a professional phagocytic protist Entamoeba histolytica, the etiological agent of human amebiasis. There are three isoforms of 14-3-3 protein in amoeba and here we have investigated Eh14-3-3 Protein 3 (EhP3). Live and fixed cell imaging studies revealed the presence of this protein throughout the parasite phagocytosis process, with high rate of accumulation at the phagocytic cups and closed phagosomes. Conditional suppression of EhP3 expression caused significant defects in phagocytosis accompanied by extensive diminution of F-actin at the site of cup formation. Downregulated cells also exhibited defective recruitment of an F-actin stabilizing protein, EhCoactosin at the phagocytic cups. In addition, mass spectrometry based analysis further revealed a large group of EhP3-associated proteins, many of these proteins are known to regulate cytoskeletal architecture in E histolytica. The dynamics of these proteins may also be controlled by EhP3. Taken together, our findings strongly suggest that EhP3 is a novel and a key regulatory element of actin dynamics and phagocytosis in E. histolytica. [ABSTRACT FROM AUTHOR]

The single mitochondrion of apicomplexan protozoa is thought to be critical for all stages of the life cycle, and is a validated drug target against these important human and veterinary parasites. In contrast to other eukaryotes, replication of the mitochondrion is tightly linked to the cell cycle. A key step in mitochondrial segregation is the fission event, which in many eukaryotes occurs by the action of dynamins constricting the outer membrane of the mitochondria from the cytosolic face. To date, none of the components of the apicomplexan fission machinery have been identified and validated. We identify here a highly divergent, dynamin-related protein (TgDrpC), conserved in apicomplexans as essential for mitochondrial biogenesis and potentially for fission in Toxoplasma gondii. We show that TgDrpC is found adjacent to the mitochondrion, and is localised both at its periphery and at its basal part, where fission is expected to occur. We demonstrate that depletion or dominant negative expression of TgDrpC results in interconnected mitochondria and ultimately in drastic changes in mitochondrial morphology, as well as in parasite death. Intriguingly, we find that the canonical adaptor TgFis1 is not required for mitochondrial fission. The identification of an Apicomplexa-specific enzyme required for mitochondrial biogenesis and essential for parasite growth highlights parasite adaptation. This work paves the way for future drug development targeting TgDrpC, and for the analysis of additional partners involved in this crucial step of apicomplexan multiplication. [ABSTRACT FROM AUTHOR]

Plasmodium parasites invade and multiply inside red blood cells (RBC). Through a cycle of maturation, asexual replication, rupture and release of multiple infective merozoites, parasitised RBC (pRBC) can reach very high numbers in vivo, a process that correlates with disease severity in humans and experimental animals. Thus, controlling pRBC numbers can prevent or ameliorate malaria. In endemic regions, circulating parasite-specific antibodies associate with immunity to high parasitemia. Although in vitro assays reveal that protective antibodies could control pRBC via multiple mechanisms, in vivo assessment of antibody function remains challenging. Here, we employed two mouse models of antibody-mediated immunity to malaria, P. yoelii 17XNL and P. chabaudi chabaudi AS infection, to study infection-induced, parasite-specific antibody function in vivo. By tracking a single generation of pRBC, we tested the hypothesis that parasite-specific antibodies accelerate pRBC clearance. Though strongly protective against homologous re-challenge, parasite-specific IgG did not alter the rate of pRBC clearance, even in the presence of ongoing, systemic inflammation. Instead, antibodies prevented parasites progressing from one generation of RBC to the next. In vivo depletion studies using clodronate liposomes or cobra venom factor, suggested that optimal antibody function required splenic macrophages and dendritic cells, but not complement C3/C5-mediated killing. Finally, parasite-specific IgG bound poorly to the surface of pRBC, yet strongly to structures likely exposed by the rupture of mature schizonts. Thus, in our models of humoral immunity to malaria, infection-induced antibodies did not accelerate pRBC clearance, and instead co-operated with splenic phagocytes to block subsequent generations of pRBC. [ABSTRACT FROM AUTHOR]

With relatively few known specific transcription factors to control the abundance of specific mRNAs, Plasmodium parasites may rely more on the regulation of transcript stability and turnover to provide sufficient gene regulation. Plasmodium transmission stages impose translational repression on specific transcripts in part to accomplish this. However, few proteins are known to participate in this process, and those that are characterized primarily affect female gametocytes. We have identified and characterized Plasmodium yoelii (Py) CCR4-1, a putative deadenylase, which plays a role in the development and activation of male gametocytes, regulates the abundance of specific mRNAs in gametocytes, and ultimately increases the efficiency of host-to-vector transmission. We find that when pyccr4-1 is deleted or its protein made catalytically inactive, there is a loss in the initial coordination of male gametocyte maturation and a reduction of parasite infectivity of the mosquito. Expression of only the N-terminal CAF1 domain of the essential CAF1 deadenylase leads to a similar phenotype. Comparative RNA-seq revealed that PyCCR4-1 affects transcripts important for transmission-related functions that are associated with male or female gametocytes, some of which directly associate with the immunoprecipitated complex. Finally, circular RT-PCR of one of the bound, dysregulated transcripts showed that deletion of the pyccr4-1 gene does not result in gross changes to its UTR or poly(A) tail length. We conclude that the two putative deadenylases of the CAF1/CCR4/NOT complex play critical and intertwined roles in gametocyte maturation and transmission. [ABSTRACT FROM AUTHOR]

Malaria is a global health concern caused by infection with Plasmodium parasites. With rising insecticide and drug resistance, there is a critical need to develop novel control strategies, including strategies to block parasite sporogony in key mosquito vector species. MAPK signaling pathways regulated by extracellular signal-regulated kinases (ERKs) and the stress-activated protein kinases (SAPKs) c-Jun N-terminal kinases (JNKs) and p38 MAPKs are highly conserved across eukaryotes, including mosquito vectors of the human malaria parasite Plasmodium falciparum. Some of these pathways in mosquitoes have been investigated in detail, but the mechanisms of integration of parasite development and mosquito fitness by JNK signaling have not been elucidated. To this end, we engineered midgut-specific overexpression of MAPK phosphatase 4 (MKP4), which targets the SAPKs, and used two potent and specific JNK small molecule inhibitors (SMIs) to assess the effects of JNK signaling manipulations on Anopheles stephensi fecundity, lifespan, intermediary metabolism, and P. falciparum development. MKP4 overexpression and SMI treatment reduced the proportion of P. falciparum-infected mosquitoes and decreased oocyst loads relative to controls. SMI-treated mosquitoes exhibited no difference in lifespan compared to controls, whereas genetically manipulated mosquitoes exhibited extended longevity. Metabolomics analyses of SMI-treated mosquitoes revealed insights into putative resistance mechanisms and the physiology behind lifespan extension, suggesting for the first time that P. falciparum-induced JNK signaling reduces mosquito longevity and increases susceptibility to infection, in contrast to previously published reports, likely via a critical interplay between the invertebrate host and parasite for nutrients that play essential roles during sporogonic development. [ABSTRACT FROM AUTHOR]

Metalloproteases (MPs) have demonstrated roles in immune modulation. In some cases, these enzymes are produced by parasites to influence host immune responses such that parasite infection is facilitated. One of the best examples of parasite-mediated immune modulation is the matrix metalloprotease (MMP) leishmanolysin (Gp63), which is produced by species of the genus Leishmania to evade killing by host macrophages. Leishmanolysin-like proteins appear to be quite common in many invertebrates, however our understanding of the functions of these non-leishmania enzymes is limited. Numerous proteomic and transcriptomic screens of schistosomes, at all life cycle stages of the parasite, have identified leishmanolysin-like MPs as being present in abundance; with the highest levels being found during the intramolluscan larval stages and being produced by cercaria. This study aims to functionally characterize a Schistosoma mansoni variant of leishmanolysin that most resembles the enzyme produced by Leishmania, termed SmLeish. We demonstrate that SmLeish is an important component of S. mansoni excretory/secretory (ES) products and is produced by the sporocyst during infection. The presence of SmLeish interferes with the migration of Biomphalaria glabrata haemocytes, and causes them to present a phenotype that is less capable of sporocyst encapsulation. Knockdown of SmLeish in S. mansoni miracidia prior to exposure to susceptible B. glabrata reduces miracidia penetration success, causes a delay in reaching patent infection, and lowers cercaria output from infected snails. [ABSTRACT FROM AUTHOR]

Despite causing considerable damage to host tissue at the onset of parasitism, invasive helminths establish remarkably persistent infections in both animals and plants. Secretions released by these obligate parasites during host invasion are thought to be crucial for their persistence in infection. Helminth secretions are complex mixtures of molecules, most of which have unknown molecular targets and functions in host cells or tissues. Although the habitats of animal- and plant-parasitic helminths are very distinct, their secretions share the presence of a structurally conserved group of proteins called venom allergen-like proteins (VALs). Helminths abundantly secrete VALs during several stages of parasitism while inflicting extensive damage to host tissue. The tight association between the secretion of VALs and the onset of parasitism has triggered a particular interest in this group of proteins, as improved knowledge on their biological functions may assist in designing novel protection strategies against parasites in humans, livestock, and important food crops. [ABSTRACT FROM AUTHOR]

Kinetoplastid parasites—trypanosomes and leishmanias—infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5'-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear granules. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, the results indicate that mRNA processing is a primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing, and the EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Molecular modeling results suggested that inhibition of CPSF3 by AN7973 is feasible. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition and resistance after CPSF3 expression did not, however, always correlate and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. It is therefore possible that the modes of action of oxaboroles that target trypanosome mRNA processing might extend beyond CPSF3 inhibition. [ABSTRACT FROM AUTHOR]

Naegleria fowleri is a free-living amoeba that can also act as an opportunistic pathogen causing severe brain infection, primary amebic meningoencephalitis (PAM), in humans. The high mortality rate of PAM (exceeding 97%) is attributed to (i) delayed diagnosis, (ii) lack of safe and effective anti-N. fowleri drugs, and (iii) difficulty of delivering drugs to the brain. Our work addresses identification of new molecular targets that may link anti-Naegleria drug discovery to the existing pharmacopeia of brain-penetrant drugs. Using inhibitors with known mechanism of action as molecular probes, we mapped the sterol biosynthesis pathway of N. fowleri by GC-MS analysis of metabolites. Based on this analysis, we chemically validated two enzymes downstream to CYP51, sterol C24-methyltransferase (SMT, ERG6) and sterol Δ8−Δ7 -isomerase (ERG2), as potential therapeutic drug targets in N. fowleri. The sterol biosynthetic cascade in N. fowleri displayed a mixture of canonical features peculiar to different domains of life: lower eukaryotes, plants and vertebrates. In addition to the cycloartenol→ergosterol biosynthetic route, a route leading to de novo cholesterol biosynthesis emerged. Isotopic labeling of the de novo-synthesized sterols by feeding N. gruberi trophozoites on the U13C-glucose-containing growth medium identified an exogenous origin of cholesterol, while 7-dehydrocholesterol (7DHC) had enriched 13C-content, suggesting a dual origin of this metabolite both from de novo biosynthesis and metabolism of scavenged cholesterol. Sterol homeostasis in Naegleria may be orchestrated over the course of its life-cycle by a “switch” between ergosterol and cholesterol biosynthesis. By demonstrating the growth inhibition and synergistic effects of the sterol biosynthesis inhibitors, we validated new, potentially druggable, molecular targets in N. fowleri. The similarity of the Naegleria sterol Δ8−Δ7 -isomerase to the human non-opioid σ1 receptor, implicated in human CNS conditions such as addiction, amnesia, pain and depression, provides an incentive to assess structurally diverse small-molecule brain-penetrant drugs targeting the human receptor for anti-Naegleria activity. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) and the malaria parasite Plasmodium use the membrane protein CD81 to invade human liver cells. Here we mapped 33 host protein interactions of CD81 in primary human liver and hepatoma cells using high-resolution quantitative proteomics. In the CD81 protein network, we identified five proteins which are HCV entry factors or facilitators including epidermal growth factor receptor (EGFR). Notably, we discovered calpain-5 (CAPN5) and the ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene B (CBLB) to form a complex with CD81 and support HCV entry. CAPN5 and CBLB were required for a post-binding and pre-replication step in the HCV life cycle. Knockout of CAPN5 and CBLB reduced susceptibility to all tested HCV genotypes, but not to other enveloped viruses such as vesicular stomatitis virus and human coronavirus. Furthermore, Plasmodium sporozoites relied on a distinct set of CD81 interaction partners for liver cell entry. Our findings reveal a comprehensive CD81 network in human liver cells and show that HCV and Plasmodium highjack selective CD81 interactions, including CAPN5 and CBLB for HCV, to invade cells. [ABSTRACT FROM AUTHOR]

The sleeping sickness parasite Trypanosoma brucei has a complex life cycle, alternating between a mammalian host and the tsetse fly vector. A tightly controlled developmental programme ensures parasite transmission between hosts as well as survival within them and involves strict regulation of mitochondrial activities. In the glucose-rich bloodstream, the replicative ‘slender’ stage is thought to produce ATP exclusively via glycolysis and uses the mitochondrial F1FO-ATP synthase as an ATP hydrolysis-driven proton pump to generate the mitochondrial membrane potential (ΔΨm). The ‘procyclic’ stage in the glucose-poor tsetse midgut depends on mitochondrial catabolism of amino acids for energy production, which involves oxidative phosphorylation with ATP production via the F1FO-ATP synthase. Both modes of the F1FO enzyme critically depend on FO subunit a, which is encoded in the parasite’s mitochondrial DNA (kinetoplast or kDNA). Comparatively little is known about mitochondrial function and the role of kDNA in non-replicative ‘stumpy’ bloodstream forms, a developmental stage essential for disease transmission. Here we show that the L262P mutation in the nuclear-encoded F1 subunit γ that permits survival of ‘slender’ bloodstream forms lacking kDNA (‘akinetoplastic’ forms), via FO-independent generation of ΔΨm, also permits their differentiation into stumpy forms. However, these akinetoplastic stumpy cells lack a ΔΨm and have a reduced lifespan in vitro and in mice, which significantly alters the within-host dynamics of the parasite. We further show that generation of ΔΨm in stumpy parasites and their ability to use α-ketoglutarate to sustain viability depend on F1-ATPase activity. Surprisingly, however, loss of ΔΨm does not reduce stumpy life span. We conclude that the L262P γ subunit mutation does not enable FO-independent generation of ΔΨm in stumpy cells, most likely as a consequence of mitochondrial ATP production in these cells. In addition, kDNA-encoded genes other than FO subunit a are important for stumpy form viability. [ABSTRACT FROM AUTHOR]

Trypanosoma brucei, the agents of African trypanosomiasis, undergo density-dependent differentiation in the mammalian bloodstream to prepare for transmission by tsetse flies. This involves the generation of cell-cycle arrested, quiescent, stumpy forms from proliferative slender forms. The signalling pathway responsible for the quorum sensing response has been catalogued using a genome-wide selective screen, providing a compendium of signalling protein kinases phosphatases, RNA binding proteins and hypothetical proteins. However, the ordering of these components is unknown. To piece together these components to provide a description of how stumpy formation arises we have used an extragenic suppression approach. This exploited a combinatorial gene knockout and overexpression strategy to assess whether the loss of developmental competence in null mutants of pathway components could be compensated by ectopic expression of other components. We have created null mutants for three genes in the stumpy induction factor signalling pathway (RBP7, YAK, MEKK1) and evaluated complementation by expression of RBP7, NEK17, PP1-6, or inducible gene silencing of the proposed differentiation inhibitor TbTOR4. This indicated that the signalling pathway is non-linear. Phosphoproteomic analysis focused on one pathway component, a putative MEKK, identified molecules with altered expression and phosphorylation profiles in MEKK1 null mutants, including another component in the pathway, NEK17. Our data provide a first molecular dissection of multiple components in a signal transduction cascade in trypanosomes. [ABSTRACT FROM AUTHOR]

Epigenetic mechanisms and chromatin structure play an important role in development. Their impact is therefore expected to be strong in parasites with complex life cycles and multiple, strikingly different, developmental stages, i.e. developmental plasticity. Some studies have already described how the chromatin structure, through histone modifications, varies from a developmental stage to another in a few unicellular parasites. However, this, to our knowledge, has never been done before in multicellular metazoan parasites. We used chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) to characterize the profile of two histone post-translational modifications (trimethylation on lysine 4 of histone H3, H3K4me3, and trimethylation on lysine 27 of histone H3 H3K27me3) over five developmental stages (miracidium, primary sporocyst, cercaria, schistosomulum, adult) of the human blood fluke Schistosoma mansoni. While H3K4me3 profiles remain relatively constant, H3K27 trimethylation and bivalent methylation show strong variation. Inhibitors (A366 and GSK343) of H3K27 histone methyltransferase activity in S. mansoni efficiently blocked miracidium to sporocyst transition indicating that H3K27 trimethylation is required for life cycle progression. As S. mansoni is a multicellular parasite that significantly affects both the health and economy of endemic areas, a better understanding of fluke developmental processes within the definitive host will likely highlight novel disease control strategies. Towards this goal, we also studied H4K20me1 in female cercariae and adults. In particular, we found that bivalent trimethylation of H3K4 and H3K27 at the transcription start site of genes is a landmark of the cercarial stage. In cercariae, H3K27me3 presence and strong enrichment in H4K20me1 over long regions (10–100 kb) is associated with development related genes. Here, we provide a broad overview of the chromatin structure of a metazoan parasite throughout its most important lifecycle stages. The five developmental stages studied here present distinct chromatin structures, indicating that histone methylation plays an important role during development. Hence, components of the histone methylation (and demethylation) machinery may provide suitable Schistosomiasis control targets. [ABSTRACT FROM AUTHOR]

Within the liver a single Plasmodium parasite transforms into thousands of blood-infective forms to cause malaria. Here, we use RNA-sequencing to identify host genes that are upregulated upon Plasmodium berghei infection of hepatocytes with the hypothesis that host pathways are hijacked to benefit parasite development. We found that expression of aquaporin-3 (AQP3), a water and glycerol channel, is significantly induced in Plasmodium-infected hepatocytes compared to uninfected cells. This aquaglyceroporin localizes to the parasitophorous vacuole membrane, the compartmental interface between the host and pathogen, with a temporal pattern that correlates with the parasite’s expansion in the liver. Depletion or elimination of host AQP3 expression significantly reduces P. berghei parasite burden during the liver stage and chemical disruption by a known AQP3 inhibitor, auphen, reduces P. falciparum asexual blood stage and P. berghei liver stage parasite load. Further use of this inhibitor as a chemical probe suggests that AQP3-mediated nutrient transport is an important function for parasite development. This study reveals a previously unknown potential route for host-dependent nutrient acquisition by Plasmodium which was discovered by mapping the transcriptional changes that occur in hepatocytes throughout P. berghei infection. The dataset reported may be leveraged to identify additional host factors that are essential for Plasmodium liver stage infection and highlights Plasmodium’s dependence on host factors within hepatocytes. [ABSTRACT FROM AUTHOR]

Malaria transmission remains high in Sub-Saharan Africa despite large-scale implementation of malaria control interventions. A comprehensive understanding of the transmissibility of infections to mosquitoes may guide the design of more effective transmission reducing strategies. The impact of P. falciparum sexual stage immunity on the infectious reservoir for malaria has never been studied in natural settings. Repeated measurements were carried out at start-wet, peak-wet and dry season, and provided data on antibody responses against gametocyte/gamete antigens Pfs48/45 and Pfs230 as anti-gametocyte immunity. Data on high and low-density infections and their infectiousness to anopheline mosquitoes were obtained using quantitative molecular methods and mosquito feeding assays, respectively. An event-driven model for P. falciparum sexual stage immunity was developed and fit to data using an agent based malaria model infrastructure. We found that Pfs48/45 and Pfs230 antibody densities increased with increasing concurrent gametocyte densities; associated with 55–70% reduction in oocyst intensity and achieved up to 44% reduction in proportions of infected mosquitoes. We showed that P. falciparum sexual stage immunity significantly reduces transmission of microscopic (p < 0.001) but not submicroscopic (p = 0.937) gametocyte infections to mosquitoes and that incorporating sexual stage immunity into mathematical models had a considerable impact on the contribution of different age groups to the infectious reservoir of malaria. Human antibody responses to gametocyte antigens are likely to be dependent on recent and concurrent high-density gametocyte exposure and have a pronounced impact on the likelihood of onward transmission of microscopic gametocyte densities compared to low density infections. Our mathematical simulations indicate that anti-gametocyte immunity is an important factor for predicting and understanding the composition and dynamics of the human infectious reservoir for malaria. [ABSTRACT FROM AUTHOR]

Tachyzoite to bradyzoite development in Toxoplasma is marked by major changes in gene expression resulting in a parasite that expresses a new repertoire of surface antigens hidden inside a modified parasitophorous vacuole called the tissue cyst. The factors that control this important life cycle transition are not well understood. Here we describe an important transcriptional repressor mechanism controlling bradyzoite differentiation that operates in the tachyzoite stage. The ApiAP2 factor, AP2IV-4, is a nuclear factor dynamically expressed in late S phase through mitosis/cytokinesis of the tachyzoite cell cycle. Remarkably, deletion of the AP2IV-4 locus resulted in the expression of a subset of bradyzoite-specific proteins in replicating tachyzoites that included tissue cyst wall components BPK1, MCP4, CST1 and the surface antigen SRS9. In the murine animal model, the mis-timing of bradyzoite antigens in tachyzoites lacking AP2IV-4 caused a potent inflammatory monocyte immune response that effectively eliminated this parasite and prevented tissue cyst formation in mouse brain tissue. Altogether, these results indicate that suppression of bradyzoite antigens by AP2IV-4 during acute infection is required for Toxoplasma to successfully establish a chronic infection in the immune-competent host. [ABSTRACT FROM AUTHOR]

The malaria-causing blood stage of Plasmodium falciparum requires extracellular pantothenate for proliferation. The parasite converts pantothenate into coenzyme A (CoA) via five enzymes, the first being a pantothenate kinase (PfPanK). Multiple antiplasmodial pantothenate analogues, including pantothenol and CJ-15,801, kill the parasite by targeting CoA biosynthesis/utilisation. Their mechanism of action, however, remains unknown. Here, we show that parasites pressured with pantothenol or CJ-15,801 become resistant to these analogues. Whole-genome sequencing revealed mutations in one of two putative PanK genes (Pfpank1) in each resistant line. These mutations significantly alter PfPanK activity, with two conferring a fitness cost, consistent with Pfpank1 coding for a functional PanK that is essential for normal growth. The mutants exhibit a different sensitivity profile to recently-described, potent, antiplasmodial pantothenate analogues, with one line being hypersensitive. We provide evidence consistent with different pantothenate analogue classes having different mechanisms of action: some inhibit CoA biosynthesis while others inhibit CoA-utilising enzymes. [ABSTRACT FROM AUTHOR]

Due to their remarkable parasitocidal activity, artemisinins represent the key components of first-line therapies against Plasmodium falciparum malaria. However, the decline in efficacy of artemisinin-based drugs jeopardizes global efforts to control and ultimately eradicate the disease. To better understand the resistance phenotype, artemisinin-resistant parasite lines were derived from two clones of the 3D7 strain of P. falciparum using a selection regimen that mimics how parasites interact with the drug within patients. This long term in vitro selection induced profound stage-specific resistance to artemisinin and its relative compounds. Chemosensitivity and transcriptional profiling of artemisinin-resistant parasites indicate that enhanced adaptive responses against oxidative stress and protein damage are associated with decreased artemisinin susceptibility. This corroborates our previous findings implicating these cellular functions in artemisinin resistance in natural infections. Genomic characterization of the two derived parasite lines revealed a spectrum of sequence and copy number polymorphisms that could play a role in regulating artemisinin response, but did not include mutations in pfk13, the main marker of artemisinin resistance in Southeast Asia. Taken together, here we present a functional in vitro model of artemisinin resistance that is underlined by a new set of genetic polymorphisms as potential genetic markers. [ABSTRACT FROM AUTHOR]

The article examines the sexual behavior of the parasite Trichomonas vaginalis. Sexually reproducing parasites create genetic variation as a result of creating new combinations of genes. Trichomonas vaginalis population genetics indicates the power of the parasite to undergo genetic exchange of some kind.

Circadian rhythms enable organisms to synchronise the processes underpinning survival and reproduction to anticipate daily changes in the external environment. Recent work shows that daily (circadian) rhythms also enable parasites to maximise fitness in the context of ecological interactions with their hosts. Because parasite rhythms matter for their fitness, understanding how they are regulated could lead to innovative ways to reduce the severity and spread of diseases. Here, we examine how host circadian rhythms influence rhythms in the asexual replication of malaria parasites. Asexual replication is responsible for the severity of malaria and fuels transmission of the disease, yet, how parasite rhythms are driven remains a mystery. We perturbed feeding rhythms of hosts by 12 hours (i.e. diurnal feeding in nocturnal mice) to desynchronise the host’s peripheral oscillators from the central, light-entrained oscillator in the brain and their rhythmic outputs. We demonstrate that the rhythms of rodent malaria parasites in day-fed hosts become inverted relative to the rhythms of parasites in night-fed hosts. Our results reveal that the host’s peripheral rhythms (associated with the timing of feeding and metabolism), but not rhythms driven by the central, light-entrained circadian oscillator in the brain, determine the timing (phase) of parasite rhythms. Further investigation reveals that parasite rhythms correlate closely with blood glucose rhythms. In addition, we show that parasite rhythms resynchronise to the altered host feeding rhythms when food availability is shifted, which is not mediated through rhythms in the host immune system. Our observations suggest that parasites actively control their developmental rhythms. Finally, counter to expectation, the severity of disease symptoms expressed by hosts was not affected by desynchronisation of their central and peripheral rhythms. Our study at the intersection of disease ecology and chronobiology opens up a new arena for studying host-parasite-vector coevolution and has broad implications for applied bioscience. [ABSTRACT FROM AUTHOR]

The article reports on the administration of host metabolism by pathogens. It mentions its aim in promoting and progressing infection and transmission as part of its strategy of immune evasion. An overview of the pathogens Plasmodium falciparum, Bordetella pertussis and Trypanosoma cruzi which are known of inducing malaria, whooping cough and Chagas disease is also presented.

The obligate intracellular parasite Toxoplasma gondii exploits cells of the immune system to disseminate. Upon T. gondii-infection, γ–aminobutyric acid (GABA)/GABAA receptor signaling triggers a hypermigratory phenotype in dendritic cells (DCs) by unknown signal transduction pathways. Here, we demonstrate that calcium (Ca2+) signaling in DCs is indispensable for T. gondii-induced DC hypermotility and transmigration in vitro. We report that activation of GABAA receptors by GABA induces transient Ca2+ entry in DCs. Murine bone marrow-derived DCs preferentially expressed the L-type voltage-dependent Ca2+ channel (VDCC) subtype Cav1.3. Silencing of Cav1.3 by short hairpin RNA or selective pharmacological antagonism of VDCCs abolished the Toxoplasma-induced hypermigratory phenotype. In a mouse model of toxoplasmosis, VDCC inhibition of adoptively transferred Toxoplasma-infected DCs delayed the appearance of cell-associated parasites in the blood circulation and reduced parasite dissemination to target organs. The present data establish that T. gondii-induced hypermigration of DCs requires signaling via VDCCs and that Ca2+ acts as a second messenger to GABAergic signaling via the VDCC Cav1.3. The findings define a novel motility-related signaling axis in DCs and unveil that interneurons and DCs share common GABAergic motogenic pathways. T. gondii employs GABAergic non-canonical pathways to induce host cell migration and facilitate dissemination. [ABSTRACT FROM AUTHOR]

Targeting of Toxoplasma gondii by autophagy is an effective mechanism by which host cells kill the protozoan. Thus, the parasite must avoid autophagic targeting to survive. Here we show that the mammalian cytoplasmic molecule Focal Adhesion Kinase (FAK) becomes activated during invasion of host cells. Activated FAK appears to accompany the formation of the moving junction (as assessed by expression the parasite protein RON4). FAK activation was inhibited by approaches that impaired β1 and β3 integrin signaling. FAK caused activation of Src that in turn mediated Epidermal Growth Factor Receptor (EGFR) phosphorylation at the unique Y845 residue. Expression of Src-resistant Y845F EGFR mutant markedly inhibited ROP16-independent activation of STAT3 in host cells. Activation of FAK, Y845 EGFR or STAT3 prevented activation of PKR and eIF2α, key stimulators of autophagy. Genetic or pharmacologic inhibition of FAK, Src, EGFR phosphorylation at Y845, or STAT3 caused accumulation of the autophagy protein LC3 and LAMP-1 around the parasite and parasite killing dependent on autophagy proteins (ULK1 and Beclin 1) and lysosomal enzymes. Parasite killing was inhibited by expression of dominant negative PKR. Thus, T. gondii activates a FAK→Src→Y845-EGFR→STAT3 signaling axis within mammalian cells, thereby enabling the parasite to survive by avoiding autophagic targeting through a mechanism likely dependent on preventing activation of PKR and eIF2α. [ABSTRACT FROM AUTHOR]

The article offers information about circadian rhythm and its impact to the behavior of parasites and other living organisms. Topics discussed include an overview of how circadian rhythm work, the parameters that govern the circadian rhythm including photosynthesis in plants and the sleep-wake cycle in animals, and the effect of the circadian rhythm to physiological systems of living organisms.

The article focuses on heme, an essential cofactor for aerobic organisms. Its biological functions mediated by hemoproteins and in blood stages, malaria parasites consume most of the hemoglobin inside the infected erythrocytes, forming nontoxic hemozoin crystals from large quantities of heme released during digestion. The article mentions that heme de novo biosynthetic pathway in the human malaria parasite Plasmodium falciparum has been considered essential and potential drug target.

A complete understanding of the mechanisms underlying the acquisition of protective immunity is crucial to improve vaccine strategies to eradicate malaria. However, it is still unclear whether recognition of damage signals influences the immune response to Plasmodium infection. Adenosine triphosphate (ATP) accumulates in infected erythrocytes and is released into the extracellular milieu through ion channels in the erythrocyte membrane or upon erythrocyte rupture. The P2X7 receptor senses extracellular ATP and induces CD4 T cell activation and death. Here we show that P2X7 receptor promotes T helper 1 (Th1) cell differentiation to the detriment of follicular T helper (Tfh) cells during blood-stage Plasmodium chabaudi malaria. The P2X7 receptor was activated in CD4 T cells following the rupture of infected erythrocytes and these cells became highly responsive to ATP during acute infection. Moreover, mice lacking the P2X7 receptor had increased susceptibility to infection, which correlated with impaired Th1 cell differentiation. Accordingly, IL-2 and IFNγ secretion, as well as T-bet expression, critically depended on P2X7 signaling in CD4 T cells. Additionally, P2X7 receptor controlled the splenic Tfh cell population in infected mice by promoting apoptotic-like cell death. Finally, the P2X7 receptor was required to generate a balanced Th1/Tfh cell population with an improved ability to transfer parasite protection to CD4-deficient mice. This study provides a new insight into malaria immunology by showing the importance of P2X7 receptor in controlling the fine-tuning between Th1 and Tfh cell differentiation during P. chabaudi infection and thus in disease outcome. [ABSTRACT FROM AUTHOR]

The repeat region of the Plasmodium falciparum circumsporozoite protein (CSP) is a major vaccine antigen because it can be targeted by parasite neutralizing antibodies; however, little is known about this interaction. We used isothermal titration calorimetry, X-ray crystallography and mutagenesis-validated modeling to analyze the binding of a murine neutralizing antibody to Plasmodium falciparum CSP. Strikingly, we found that the repeat region of CSP is bound by multiple antibodies. This repeating pattern allows multiple weak interactions of single FAB domains to accumulate and yield a complex with a dissociation constant in the low nM range. Because the CSP protein can potentially cross-link multiple B cell receptors (BCRs) we hypothesized that the B cell response might be T cell independent. However, while there was a modest response in mice deficient in T cell help, the bulk of the response was T cell dependent. By sequencing the BCRs of CSP-repeat specific B cells in inbred mice we found that these cells underwent somatic hypermutation and affinity maturation indicative of a T-dependent response. Last, we found that the BCR repertoire of responding B cells was limited suggesting that the structural simplicity of the repeat may limit the breadth of the immune response. [ABSTRACT FROM AUTHOR]

Although the importance of humoral immunity to malaria has been established, factors that control antibody production are poorly understood. Follicular helper T cells (Tfh cells) are pivotal for generating high-affinity, long-lived antibody responses. While it has been proposed that expansion of antigen-specific Tfh cells, interleukin (IL) 21 production and robust germinal center formation are associated with protection against malaria in mice, whether Tfh cells are found during Plasmodium vivax (P. vivax) infection and if they play a role during disease remains unknown. Our goal was to define the role of Tfh cells during P. vivax malaria. We demonstrate that P. vivax infection triggers IL-21 production and an increase in Tfh cells (PD-1+ICOS+CXCR5+CD45RO+CD4+CD3+). As expected, FACS-sorted Tfh cells, the primary source of IL-21, induced immunoglobulin production by purified naïve B cells. Furthermore, we found that P. vivax infection alters the B cell compartment and these alterations were dependent on the number of previous infections. First exposure leads to increased proportions of activated and atypical memory B cells and decreased frequencies of classical memory B cells, whereas patients that experienced multiple episodes displayed lower proportions of atypical B cells and higher frequencies of classical memory B cells. Despite the limited sample size, but consistent with the latter finding, the data suggest that patients who had more than five infections harbored more Tfh cells and produce more specific antibodies. P. vivax infection triggers IL-21 production by Tfh that impact B cell responses in humans. [ABSTRACT FROM AUTHOR]

The combination of drug resistance, lack of an effective vaccine, and ongoing conflict and poverty means that malaria remains a major global health crisis. Understanding metabolic pathways at all parasite life stages is important in prioritising and targeting novel anti-parasitic compounds. The unusual heme synthesis pathway of the rodent malaria parasite, Plasmodium berghei, requires eight enzymes distributed across the mitochondrion, apicoplast and cytoplasm. Deletion of the ferrochelatase (FC) gene, the final enzyme in the pathway, confirms that heme synthesis is not essential in the red blood cell stages of the life cycle but is required to complete oocyst development in mosquitoes. The lethality of FC deletions in the mosquito stage makes it difficult to study the impact of these mutations in the subsequent liver stage. To overcome this, we combined locus-specific fluorophore expression with a genetic complementation approach to generate viable, heterozygous oocysts able to produce a mix of FC expressing and FC deficient sporozoites. These sporozoites show normal motility and can invade liver cells, where FC deficient parasites can be distinguished by fluorescence and phenotyped. Parasites lacking FC exhibit a severe growth defect within liver cells, with development failure detectable in the early to mid stages of liver development in vitro. FC deficient parasites could not complete liver stage development in vitro nor infect naïve mice, confirming liver stage arrest. These results validate the heme pathway as a potential target for prophylactic drugs targeting liver stage parasites. In addition, we demonstrate that our simple genetic approach can extend the phenotyping window beyond the insect stages, opening considerable scope for straightforward reverse genetic analysis of genes that are dispensable in blood stages but essential for completing mosquito development. [ABSTRACT FROM AUTHOR]

Toxoplasma is an obligate intracellular parasite that replicates in mammalian cells within a parasitophorous vacuole (PV) that does not fuse with any host organelles. One mechanism developed by the parasite for nutrient acquisition is the attraction of host organelles to the PV. Here, we examined the exploitation of host lipid droplets (LD), ubiquitous fat storage organelles, by Toxoplasma. We show that Toxoplasma replication is reduced in host cells that are depleted of LD, or impaired in TAG lipolysis or fatty acid catabolism. In infected cells, the number of host LD and the expression of host LD-associated genes (ADRP, DGAT2), progressively increase until the onset of parasite replication. Throughout infection, the PV are surrounded by host LD. Toxoplasma is capable of accessing lipids stored in host LD and incorporates these lipids into its own membranes and LD. Exogenous addition of oleic acid stimulates LD biogenesis in the host cell and results in the overaccumulation of neutral lipids in very large LD inside the parasite. To access LD-derived lipids, Toxoplasma intercepts and internalizes within the PV host LD, some of which remaining associated with Rab7, which become wrapped by an intravacuolar network of membranes (IVN). Mutant parasites impaired in IVN formation display diminished capacity of lipid uptake from host LD. Moreover, parasites lacking an IVN-localized phospholipase A2 are less proficient in salvaging lipids from host LD in the PV, suggesting a major contribution of the IVN for host LD processing in the PV and, thus lipid content release. Interestingly, gavage of parasites with lipids unveils, for the first time, the presence in Toxoplasma of endocytic-like structures containing lipidic material originating from the PV lumen. This study highlights the reliance of Toxoplasma on host LD for its intracellular development and the parasite’s capability in scavenging neutral lipids from host LD. [ABSTRACT FROM AUTHOR]

Profilin is an actin monomer binding protein that provides ATP-actin for incorporation into actin filaments. In contrast to higher eukaryotic cells with their large filamentous actin structures, apicomplexan parasites typically contain only short and highly dynamic microfilaments. In apicomplexans, profilin appears to be the main monomer-sequestering protein. Compared to classical profilins, apicomplexan profilins contain an additional arm-like β-hairpin motif, which we show here to be critically involved in actin binding. Through comparative analysis using two profilin mutants, we reveal this motif to be implicated in gliding motility of Plasmodium berghei sporozoites, the rapidly migrating forms of a rodent malaria parasite transmitted by mosquitoes. Force measurements on migrating sporozoites and molecular dynamics simulations indicate that the interaction between actin and profilin fine-tunes gliding motility. Our data suggest that evolutionary pressure to achieve efficient high-speed gliding has resulted in a unique profilin-actin interface in these parasites. [ABSTRACT FROM AUTHOR]

Interactions between early developing Schistosoma mansoni larval stages and the hemolymph of its snail intermediate host represent the first molecular encounter with the snail’s immune system. To gain a more comprehensive understanding of this early parasite-host interaction, biotinylated sporocyst tegumental membrane (Mem) proteins and larval transformation proteins (LTP) were affixed to streptavidin-agarose beads and used as affinity matrices to enrich for larval-reactive plasma proteins from susceptible (NMRI) and resistant (BS-90) strains of the snail Biomphalaria glabrata. Nano-LC/MS-MS proteomic analyses of isolated plasma proteins revealed a diverse array of 94 immune-and nonimmune-related plasma proteins. Included among the immune-related subset were pattern recognition receptors (lectins, LPS-binding protein, thioester-containing proteins-TEPs), stress proteins (HSP60 and 70), adhesion proteins (dermatopontins), metalloproteases (isintegrin nd etalloproteinase (ADAM), ADAM-related Zn proteinases), cytotoxins (biomphalysin) and a Ca2+-binding protein (neo-calmodulin). Variable immunoglobulin and lectin domain (VIgL) gene family members, including fibrinogen-related proteins (FREPs), galectin-related proteins (GREPs) and C-type lectin-related proteins (CREPs), were the most prevalent of larval-reactive immune lectins present in plasma. FREPs were highly represented, although only a subset of FREP subfamilies (FREP 2, 3 and 12) were identified, suggesting potential selectivity in the repertoire of plasma lectins recognizing larval glycoconjugates. Other larval-binding FREP-like and CREP-like proteins possessing a C-terminal fibrinogen-related domain (FReD) or C-type lectin binding domain, respectively, and an Ig-fold domain also were identified as predicted proteins from the B. glabrata genome, although incomplete sequence data precluded their placement into specific FREP/CREP subfamilies. Similarly, a group of FReD-containing proteins (angiopoeitin-4, ficolin-2) that lacked N-terminal Ig-fold(s) were identified as a distinct group of FREP-like proteins, separate from the VIgL lectin family. Finally, differential appearance of GREPs in BS-90 plasma eluates, and others proteins exclusively found in eluates of the NMRI strain, suggested snail strain differences in the expression of select larval-reactive immune proteins. This hypothesis was supported by the finding that differential gene expression of the GREP in BS-90 and ADAM in NMRI snail strains generally correlated with their patterns of protein expression. In summary, this study is the first to provide a global comparative proteomic analysis of constitutively expressed plasma proteins from susceptible and resistant B. glabrata strains capable of binding early-expressed larval S. mansoni proteins. Identified proteins, especially those exhibiting differential expression, may play a role in determining immune compatibility in this snail host-parasite system. A complete listing of raw peptide data are available via ProteomeXchange using identifier PXD004942. [ABSTRACT FROM AUTHOR]

Cell cycle of Entamoeba histolytica, the etiological agent of amoebiasis, follows a novel pathway, which includes nuclear division without the nuclear membrane disassembly. We report a nuclear localized Ca2+-binding protein from E. histolytica (abbreviated hereafter as EhCaBP6), which is associated with microtubules. We determined the 3D solution NMR structure of EhCaBP6, and identified one unusual, one canonical and two non-canonical cryptic EF-hand motifs. The cryptic EF-II and EF-IV pair with the Ca2+-binding EF-I and EF-III, respectively, to form a two-domain structure similar to Calmodulin and Centrin proteins. Downregulation of EhCaBP6 affects cell proliferation by causing delays in transition from G1 to S phase, and inhibition of DNA synthesis and cytokinesis. We also demonstrate that EhCaBP6 modulates microtubule dynamics by increasing the rate of tubulin polymerization. Our results, including structural inferences, suggest that EhCaBP6 is an unusual CaBP involved in regulating cell proliferation in E. histolytica similar to nuclear Calmodulin. [ABSTRACT FROM AUTHOR]

The kinetoplast (k), the uniquely packaged mitochondrial DNA of trypanosomatid protists is formed by a catenated network of minicircles and maxicircles that divide and segregate once each cell cycle. Although many proteins involved in kDNA replication and segregation are now known, several key steps in the replication mechanism remain uncharacterized at the molecular level, one of which is the nabelschnur or umbilicus, a prominent structure which in the mammalian parasite Trypanosoma brucei connects the daughter kDNA networks prior to their segregation. Here we characterize an M17 family leucyl aminopeptidase metalloprotease, termed TbLAP1, which specifically localizes to the kDNA disk and the nabelschur and represents the first described protein found in this structure. We show that TbLAP1 is required for correct segregation of kDNA, with knockdown resulting in delayed cytokinesis and ectopic expression leading to kDNA loss and decreased cell proliferation. We propose that TbLAP1 is required for efficient kDNA division and specifically participates in the separation of daughter kDNA networks. [ABSTRACT FROM AUTHOR]

Trypanosoma brucei, causing African sleeping-sickness, exploits quorum-sensing (QS) to generate the ‘stumpy forms’ necessary for the parasite’s transmission to tsetse-flies. These quiescent cells are generated by differentiation in the bloodstream from proliferative slender forms. Using genome-wide RNAi selection we screened for repressors of transmission stage-enriched mRNAs in slender forms, using the stumpy-elevated ESAG9 transcript as a model. This identified REG9.1, whose RNAi-silencing alleviated ESAG9 repression in slender forms and tsetse-midgut procyclic forms. Interestingly, trypanosome surface protein Family 5 and Family 7 mRNAs were also elevated, which, like ESAG9, are T. brucei specific and stumpy-enriched. We suggest these contribute to the distinct transmission biology and vector tropism of T. brucei from other African trypanosome species. As well as surface family regulation, REG9.1-depletion generated QS-independent development to stumpy forms in vivo, whereas REG9.1 overexpression in bloodstream forms potentiated spontaneous differentiation to procyclic forms in the absence of an external signal. Combined, this identifies REG9.1 as a regulator of developmental cell fate, controlling the expression of Trypanosoma brucei-specific molecules elevated during transmission. [ABSTRACT FROM AUTHOR]

The article discusses the cell biology of protozoan parasites including trypanosomatid and apicomplexan. Topics include the connection between the nucleus and lamina in nuclear positioning, the composition of the nuclear pore complex, and modifications to chromatin. It states the need to understand cell biology of infectious agents to implement their control strategies.

In this study the ‘Malaria Box’ chemical library comprising 400 compounds with antiplasmodial activity was screened for compounds that perturb the internal pH of the malaria parasite, Plasmodium falciparum. Fifteen compounds induced an acidification of the parasite cytosol. Two of these did so by inhibiting the parasite’s formate nitrite transporter (PfFNT), which mediates the H+-coupled efflux from the parasite of lactate generated by glycolysis. Both compounds were shown to inhibit lactate transport across the parasite plasma membrane, and the transport of lactate by PfFNT expressed in Xenopus laevis oocytes. PfFNT inhibition caused accumulation of lactate in parasitised erythrocytes, and swelling of both the parasite and parasitised erythrocyte. Long-term exposure of parasites to one of the inhibitors gave rise to resistant parasites with a mutant form of PfFNT that showed reduced inhibitor sensitivity. This study provides the first evidence that PfFNT is a druggable antimalarial target. [ABSTRACT FROM AUTHOR]

Mosquitoes genetically engineered to be resistant to Plasmodium parasites represent a promising novel approach in the fight against malaria. The insect immune system itself is a source of anti-parasitic genes potentially exploitable for transgenic designs. The Anopheles gambiae thioester containing protein 1 (TEP1) is a potent anti-parasitic protein. TEP1 is secreted and circulates in the mosquito hemolymph, where its activated cleaved form binds and eliminates malaria parasites. Here we investigated whether TEP1 can be used to create malaria resistant mosquitoes. Using a GFP reporter transgene, we determined that the fat body is the main site of TEP1 expression. We generated transgenic mosquitoes that express TEP1r, a potent refractory allele of TEP1, in the fat body and examined the activity of the transgenic protein in wild-type or TEP1 mutant genetic backgrounds. Transgenic TEP1r rescued loss-of-function mutations, but did not increase parasite resistance in the presence of a wild-type susceptible allele. Consistent with previous reports, TEP1 protein expressed from the transgene in the fat body was taken up by hemocytes upon a challenge with injected bacteria. Furthermore, although maturation of transgenic TEP1 into the cleaved form was impaired in one of the TEP1 mutant lines, it was still sufficient to reduce parasite numbers and induce parasite melanization. We also report here the first use of Transcription Activator Like Effectors (TALEs) in Anopheles gambiae to stimulate expression of endogenous TEP1. We found that artificial elevation of TEP1 expression remains moderate in vivo and that enhancement of endogenous TEP1 expression did not result in increased resistance to Plasmodium. Taken together, our results reveal the difficulty of artificially influencing TEP1-mediated Plasmodium resistance, and contribute to further our understanding of the molecular mechanisms underlying mosquito resistance to Plasmodium parasites. [ABSTRACT FROM AUTHOR]

The packaging and organization of genomic DNA into chromatin represents an additional regulatory layer of gene expression, with specific nucleosome positions that restrict the accessibility of regulatory DNA elements. The mechanisms that position nucleosomes in vivo are thought to depend on the biophysical properties of the histones, sequence patterns, like phased di-nucleotide repeats and the architecture of the histone octamer that folds DNA in 1.65 tight turns. Comparative studies of human and P. falciparum histones reveal that the latter have a strongly reduced ability to recognize internal sequence dependent nucleosome positioning signals. In contrast, the nucleosomes are positioned by AT-repeat sequences flanking nucleosomes in vivo and in vitro. Further, the strong sequence variations in the plasmodium histones, compared to other mammalian histones, do not present adaptations to its AT-rich genome. Human and parasite histones bind with higher affinity to GC-rich DNA and with lower affinity to AT-rich DNA. However, the plasmodium nucleosomes are overall less stable, with increased temperature induced mobility, decreased salt stability of the histones H2A and H2B and considerable reduced binding affinity to GC-rich DNA, as compared with the human nucleosomes. In addition, we show that plasmodium histone octamers form the shortest known nucleosome repeat length (155bp) in vitro and in vivo. Our data suggest that the biochemical properties of the parasite histones are distinct from the typical characteristics of other eukaryotic histones and these properties reflect the increased accessibility of the P. falciparum genome. [ABSTRACT FROM AUTHOR]

Malaria parasites (Plasmodium spp.) encounter markedly different (nutritional) environments during their complex life cycles in the mosquito and human hosts. Adaptation to these different host niches is associated with a dramatic rewiring of metabolism, from a highly glycolytic metabolism in the asexual blood stages to increased dependence on tricarboxylic acid (TCA) metabolism in mosquito stages. Here we have used stable isotope labelling, targeted metabolomics and reverse genetics to map stage-specific changes in Plasmodium berghei carbon metabolism and determine the functional significance of these changes on parasite survival in the blood and mosquito stages. We show that glutamine serves as the predominant input into TCA metabolism in both asexual and sexual blood stages and is important for complete male gametogenesis. Glutamine catabolism, as well as key reactions in intermediary metabolism and CoA synthesis are also essential for ookinete to oocyst transition in the mosquito. These data extend our knowledge of Plasmodium metabolism and point towards possible targets for transmission-blocking intervention strategies. Furthermore, they highlight significant metabolic differences between Plasmodium species which are not easily anticipated based on genomics or transcriptomics studies and underline the importance of integration of metabolomics data with other platforms in order to better inform drug discovery and design. [ABSTRACT FROM AUTHOR]

The article talks about Aspartyl Proteinases of Eukaryotic Microbial Pathogens. Pathogen–host relationship are represented by plasmepsins of P. falciparum which are major agent of malaria. Secretory Aspartyl Proteinases are produced in a secretory form by many fungal organisms and includes both primary and opportunistic pathogens.

Entamoeba histolytica, the highly phagocytic protozoan causative of human amoebiasis lacks the machinery to synthesize cholesterol. Here, we investigated the presence of NPC1 and NPC2 proteins in this parasite, which are involved in cholesterol trafficking in mammals. Bioinformatics analysis revealed one Ehnpc1 and two Ehnpc2 genes. EhNPC1 appeared as a transmembrane protein and both EhNPC2 as peripheral membrane proteins. Molecular docking predicted that EhNPC1 and EhNPC2 bind cholesterol and interact with each other. Genes and proteins were identified in trophozoites. Serum pulse-chase and confocal microscopy assays unveiled that after trophozoites sensed the cholesterol source, EhNPC1 and EhNPC2 were organized around the plasma membrane in a punctuated pattern. Vesicles emerged and increased in number and size and some appeared full of cholesterol with EhNPC1 or EhNPC2 facing the extracellular space. Both proteins, but mostly EhNPC2, were found out of the cell associated with cholesterol. EhNPC1 and cholesterol formed networks from the plasma membrane to the nucleus. EhNPC2 appeared in erythrocytes that were being ingested by trophozoites, co-localizing with cholesterol of erythrocytes, whereas EhNPC1 surrounded the phagocytic cup. EhNPC1 and EhNPC2 co-localized with EhSERCA in the endoplasmic reticulum and with lysobisphosphatidic acid and EhADH (an Alix protein) in phagolysosomes. Immunoprecipitation assays confirmed the EhNPC1 and EhNPC2 association with cholesterol, EhRab7A and EhADH. Serum starved and blockage of cholesterol trafficking caused a low rate of phagocytosis and incapability of trophozoites to produce damage in the mouse colon. Ehnpc1 and Ehnpc2 knockdown provoked in trophozoites a lower intracellular cholesterol concentration and a diminished rate of phagocytosis; and Ehnpc1 silencing also produced a decrease of trophozoites movement. Trafficking of EhNPC1 and EhNPC2 during cholesterol uptake and phagocytosis as well as their association with molecules involved in endocytosis strongly suggest that these proteins play a key role in cholesterol uptake. [ABSTRACT FROM AUTHOR]

The control of mosquito populations with insecticide treated bed nets and indoor residual sprays remains the cornerstone of malaria reduction and elimination programs. In light of widespread insecticide resistance in mosquitoes, however, alternative strategies for reducing transmission by the mosquito vector are urgently needed, including the identification of safe compounds that affect vectorial capacity via mechanisms that differ from fast-acting insecticides. Here, we show that compounds targeting steroid hormone signaling disrupt multiple biological processes that are key to the ability of mosquitoes to transmit malaria. When an agonist of the steroid hormone 20-hydroxyecdysone (20E) is applied to Anopheles gambiae females, which are the dominant malaria mosquito vector in Sub Saharan Africa, it substantially shortens lifespan, prevents insemination and egg production, and significantly blocks Plasmodium falciparum development, three components that are crucial to malaria transmission. Modeling the impact of these effects on Anopheles population dynamics and Plasmodium transmission predicts that disrupting steroid hormone signaling using 20E agonists would affect malaria transmission to a similar extent as insecticides. Manipulating 20E pathways therefore provides a powerful new approach to tackle malaria transmission by the mosquito vector, particularly in areas affected by the spread of insecticide resistance. [ABSTRACT FROM AUTHOR]

Entamoeba histolytica is an enteric pathogen responsible for amoebic dysentery and liver abscess. It alternates between the host-restricted trophozoite form and the infective environmentally-stable cyst stage. Throughout its lifecycle E. histolytica experiences stress, in part, from host immune pressure. Conversion to cysts is presumed to be a stress-response. In other systems, stress induces phosphorylation of a serine residue on eukaryotic translation initiation factor-2α (eIF2α). This inhibits eIF2α activity resulting in a general decline in protein synthesis. Genomic data reveal that E. histolytica possesses eIF2α (EheIF2α) with a conserved phosphorylatable serine at position 59 (Ser59). Thus, this pathogen may have the machinery for stress-induced translational control. To test this, we exposed cells to different stress conditions and measured the level of total and phospho-EheIF2α. Long-term serum starvation, long-term heat shock, and oxidative stress induced an increase in the level of phospho-EheIF2α, while short-term serum starvation, short-term heat shock, or glucose deprivation did not. Long-term serum starvation also caused a decrease in polyribosome abundance, which is in accordance with the observation that this condition induces phosphorylation of EheIF2α. We generated transgenic cells that overexpress wildtype EheIF2α, a non-phosphorylatable variant of eIF2α in which Ser59 was mutated to alanine (EheIF2α-S59A), or a phosphomimetic variant of eIF2α in which Ser59 was mutated to aspartic acid (EheIF2α-S59D). Consistent with the known functions of eIF2α, cells expressing wildtype or EheIF2α-S59D exhibited increased or decreased translation, respectively. Surprisingly, cells expressing EheIF2α-S59A also exhibited reduced translation. Cells expressing EheIF2α-S59D were more resistant to long-term serum starvation underscoring the significance of EheIF2α phosphorylation in managing stress. Finally, phospho-eIF2α accumulated during encystation in E. invadens, a model encystation system. Together, these data demonstrate that the eIF2α-dependent stress response system is operational in Entamoeba species. [ABSTRACT FROM AUTHOR]