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Jeremy C. Mottram - One of the best experts on this subject based on the ideXlab platform.
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An Essential Signal Peptide Peptidase Identified in an RNAi Screen of Serine Peptidases of Trypanosoma brucei
PLOS ONE, 2015Co-Authors: Catherine X. Moss, Koen Augustyns, Elaine Brown, Alana Hamilton, Pieter Van Der Veken, Jeremy C. MottramAbstract:The serine peptidases of Trypanosoma brucei have been viewed as potential drug targets. In particular, the S9 prolyl oligopeptidase subfamily is thought to be a good avenue for drug discovery. This is based on the finding that some S9 peptidases are secreted and active in the mammalian bloodstream, and that they are a class of enzyme against which drugs have successfully been developed. We collated a list of all serine peptidases in T. brucei, identifying 20 serine peptidase genes, of which nine are S9 peptidases. We screened all 20 serine peptidases by RNAi to determine which, if any, are essential for bloodstream form T. brucei Survival. All S9 serine peptidases were dispensable for Parasite Survival in vitro, even when pairs of similar genes, coding for oligopeptidase B or prolyl oligopeptidase, were targeted simultaneously. We also found no effect on Parasite Survival in an animal host when the S9 peptidases oligopeptidase B, prolyl oligopeptidase or dipeptidyl peptidase 8 were targeted. The only serine peptidase to emerge from the RNAi screen as essential was a putative type-I signal peptide peptidase (SPP1). This gene was essential for Parasite Survival both in vitro and in vivo. The growth defect conferred by RNAi depletion of SPP1 was rescued by expression of a functional peptidase from an RNAi resistant SPP1 gene. However, expression of catalytically inactive SPP1 was unable to rescue cells from the SPP1 depleted phenotype, demonstrating that SPP1 serine peptidase activity is necessary for T. brucei Survival.
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Leishmania Inhibitor of Serine Peptidase 2 Prevents TLR4 Activation by Neutrophil Elastase Promoting Parasite Survival in Murine Macrophages
Journal of immunology (Baltimore Md. : 1950), 2010Co-Authors: Marilia S. Faria, Jeremy C. Mottram, Flavia C. G. Reis, Ricardo Luiz Azevedo-pereira, Lesley S. Morrison, Ana Paula C. A. LimaAbstract:Leishmania major is a protozoan Parasite that causes skin ulcerations in cutaneous leishmaniasis. In the mammalian host, the Parasite resides in professional phagocytes and has evolved to avoid killing by macrophages. We identified L. major genes encoding inhibitors of serine peptidases (ISPs), which are orthologs of bacterial ecotins, and found that ISP2 inhibits trypsin-fold S1A family peptidases. In this study, we show that L. major mutants deficient in ISP2 and ISP3 (Δisp2/3) trigger higher phagocytosis by macrophages through a combined action of the complement type 3 receptor, TLR4, and unregulated activity of neutrophil elastase (NE), leading to Parasite killing. Whereas all three components are required to mediate enhanced Parasite uptake, only TLR4 and NE are necessary to promote Parasite killing postinfection. We found that the production of superoxide by macrophages in the absence of ISP2 is the main mechanism controlling the intracellular infection. Furthermore, we show that NE modulates macrophage infection in vivo, and that the lack of ISP leads to reduced Parasite burdens at later stages of the infection. Our findings support the hypothesis that ISPs function to prevent the activation of TLR4 by NE during the Leishmania-macrophage interaction to promote Parasite Survival and growth.
Pradeep Das - One of the best experts on this subject based on the ideXlab platform.
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LdIscU is a [2Fe-2S] scaffold protein which interacts with LdIscS and its expression is modulated by Fe-S proteins in Leishmania donovani.
International journal of biological macromolecules, 2018Co-Authors: Krishn Pratap Singh, Pradeep Das, Shadab Anwar, Amir Zaidi, Kuljit Singh, Samudrala Gourinath, Vahab AliAbstract:The pathogenicity of protozoan Parasites is frequently attributed to their ability to circumvent the deleterious effects of ROS and Fe-S clusters are among their susceptible targets with paramount importance for Parasite Survival. The biogenesis of Fe-S clusters is orchestrated by ISC system; the sulfur donor IscS and scaffold protein IscU being its core components. However, among protozoan Parasites including Leishmania, no information is available regarding biochemical aspect of IscU, its interaction partners and regulation. Here, we show that Leishmania donovani IscU homolog, LdIscU, readily assembles [2Fe-2S] clusters and, interestingly, follows Michaelis-Menten enzyme kinetics. It is localized in the mitochondria of the Parasite and interacts with LdIscS to form a stable complex. Additionally, LdIscU and Fe-S proteins activity is significantly upregulated in resistant isolates and during stationary growth stage indicating an association between them. The differential expression of LdIscU modulated by Fe-S proteins demand suggests its potential role in Parasite Survival and drug resistance. Thus, our study provides novel insight into the Fe-S scaffold protein of a protozoan Parasite.
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Leishmania donovani Inhibitor of Serine Peptidases 2 Mediated Inhibition of Lectin Pathway and Upregulation of C5aR Signaling Promote Parasite Survival inside Host.
Frontiers in immunology, 2018Co-Authors: Sudha Verma, Abhishek Mandal, Yousuf Ansari, Ajay Kumar, Kumar Abhishek, Ayan Kumar Ghosh, Ashish Kumar, Vinod Kumar, Sushmita Das, Pradeep DasAbstract:Leishmania donovani, the causative agent of Indian visceral leishmaniasis has to face several barriers of the immune system inside the mammalian host for its Survival. The complement system is one of the first barriers and consists of a well-balanced network of proteases including S1A family serine proteases (SPs). Inhibitor of serine peptidases (ISPs) is considered as inhibitor of S1A family serine peptidases and is reported to be present in trypanosomes, including Leishmania. In our previous study, we have deciphered the role of ISPs [LdISP1 and L. donovani inhibitor of serine peptidases 2 (LdISP2)] in the Survival of L. donovani inside the sandfly midgut. However, the role of theses ISPs in the Survival of L. donovani inside mammalian host still remains elusive. In the present study, we have deciphered the inhibitory effect of LdISPs on the host complement S1A serine peptidases, such as C1r/C1s and MASP1/MASP2. Our study suggested that although both rLdISP1 and rLdISP2 inferred strong interaction with C1complex and MBL-associated serine proteases (MASPs) but rLdISP2 showed the stronger inhibitory effect on MASP2 than rLdISP1. Moreover, we found that rLdISP2 significantly reduces the formation of C3, C5 convertase, and membrane attacking complex (MAC) by lectin pathway (LP) resulting in significant reduction in serum mediated lysis of the Parasites. The role of LdISP2 on neutrophil elastase-mediated C5aR signaling was also evaluated. Notably, our results showed that infection of macrophages with ISP2-overexpressed Leishmania Parasites significantly induces the expression of C5aR both at the transcript and translational level. Simultaneously, infection with ISP2KD Parasites results in downregulation of host PI3K/AKT phosphorylation and increased in IL-12 production. Taken together, our findings clearly suggest that LdISP2 promotes Parasite Survival inside host by inhibiting MAC formation and complement-mediated lysis via LP and by upregulation of C5aR signaling.
Haddijatou Mbye - One of the best experts on this subject based on the ideXlab platform.
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tolerance of gambian plasmodium falciparum to dihydroartemisinin and lumefantrine detected by ex vivo Parasite Survival rate assay
Antimicrobial Agents and Chemotherapy, 2020Co-Authors: Haddijatou Mbye, Fatoumata Bojang, Aminata Seedy Jawara, Bekai Njie, Nuredin Ibrahim Mohammed, Joseph Okebe, Umberto Dalessandro, Alfred AmambuangwaAbstract:Monitoring of Plasmodium falciparum sensitivity to antimalarial drugs in Africa is vital for malaria elimination. However, the commonly used ex vivo/in vitro 50% inhibitory concentration (IC50) test gives inconsistent results for several antimalarials, while the alternative ring-stage Survival assay (RSA) for artemisinin derivatives has not been widely adopted. Here, we applied an alternative two-color flow cytometry-based Parasite Survival rate assay (PSRA) to detect ex vivo antimalarial tolerance in P. falciparum isolates from The Gambia. The PSRA infers Parasite viability by quantifying reinvasion of uninfected cells following 3 consecutive days of drug exposure (10-fold the IC50 of drug for field isolates). The drug Survival rate is obtained for each isolate from the slope of the growth/death curve. We obtained Parasite Survival rates of 41 isolates for dihydroartemisinin (DHA) and lumefantrine (LUM) out of 51 infections tested by ring-stage Survival assay (RSA) against DHA. We also determined the genotypes for known drug resistance genetic loci in the P. falciparum genes Pfdhfr, Pfdhps, Pfmdr, Pfcrt, and Pfk13 The PSRA results determined for 41 Gambian isolates showed faster killing and lower variance after treatment with DHA than after treatment with LUM, despite a strong correlation between the two drugs. Four and three isolates were tolerant to DHA and LUM, respectively, with continuous growth during drug exposure. Isolates with the PfMDR1-Y184F mutant variant showed increased LUM Survival, though the results were not statistically significant. Sulfadoxine/pyrimethamine (SP) resistance markers were fixed, while all other antimalarial variants were prevalent in more than 50% of the population. The PSRA detected ex vivo antimalarial tolerance in Gambian P. falciparum This calls for its wider application and for increased vigilance against resistance to artemisinin combination therapies (ACTs) in this population.
Boris Striepen - One of the best experts on this subject based on the ideXlab platform.
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the import of proteins into the mitochondrion of toxoplasma gondii
Journal of Biological Chemistry, 2016Co-Authors: Giel G Van Dooren, Boris Striepen, Lee M Yeoh, Geoffrey I McfaddenAbstract:Outside of well characterized model eukaryotes, relatively little is known about the translocons that transport proteins across the two membranes that surround the mitochondrion. Apicomplexans are a phylum of intracellular Parasites that cause major diseases in humans and animals and are evolutionarily distant from model eukaryotes such as yeast. Apicomplexans harbor a mitochondrion that is essential for Parasite Survival and is a validated drug target. Here, we demonstrate that the apicomplexan Toxoplasma gondii harbors homologues of proteins from all the major mitochondrial protein translocons present in yeast, suggesting these arose early in eukaryotic evolution. We demonstrate that a T. gondii homologue of Tom22 (TgTom22), a central component of the translocon of the outer mitochondrial membrane (TOM) complex, is essential for Parasite Survival, mitochondrial protein import, and assembly of the TOM complex. We also identify and characterize a T. gondii homologue of Tom7 (TgTom7) that is important for Parasite Survival and mitochondrial protein import. Contrary to the role of Tom7 in yeast, TgTom7 is important for TOM complex stability, suggesting the role of this protein has diverged during eukaryotic evolution. Together, our study identifies conserved and modified features of mitochondrial protein import in apicomplexan Parasites.
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An apicoplast localized ubiquitylation system is required for the import of nuclear-encoded plastid proteins
PLoS Pathogens, 2013Co-Authors: Swati Agrawal, Giel G Van Dooren, Duk-won D Chung, Nadia Ponts, Jacques Prudhomme, Carrie F. Brooks, Elisadra M. Rodrigues, John C. Tan, Michael T. Ferdig, Boris StriepenAbstract:Apicomplexan Parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for Parasite Survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD) has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and Parasite Survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway.
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apicoplast fatty acid synthesis is essential for organelle biogenesis and Parasite Survival in toxoplasma gondii
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Jolly Mazumdar, Emma H Wilson, Kate Masek, Christopher A Hunter, Boris StriepenAbstract:Apicomplexan Parasites are the cause of numerous important human diseases including malaria and AIDS-associated opportunistic infections. Drug treatment for these diseases is not satisfactory and is threatened by resistance. The discovery of the apicoplast, a chloroplast-like organelle, presents drug targets unique to these Parasites. The apicoplast-localized fatty acid synthesis (FAS II) pathway, a metabolic process fundamentally divergent from the analogous FAS I pathway in humans, represents one such target. However, the specific biological roles of apicoplast FAS II remain elusive. Furthermore, the Parasite genome encodes additional and potentially redundant pathways for the synthesis of fatty acids. We have constructed a conditional null mutant of acyl carrier protein, a central component of the FAS II pathway in Toxoplasma gondii. Loss of FAS II severely compromises Parasite growth in culture. We show FAS II to be required for the activation of pyruvate dehydrogenase, an important source of the metabolic precursor acetyl-CoA. Interestingly, acyl carrier protein knockout also leads to defects in apicoplast biogenesis and a consequent loss of the organelle. Most importantly, in vivo knockdown of apicoplast FAS II in a mouse model results in cure from a lethal challenge infection. In conclusion, our study demonstrates a direct link between apicoplast FAS II functions and Parasite Survival and pathogenesis. Our genetic model also offers a platform to dissect the integration of the apicoplast into Parasite metabolism, especially its postulated interaction with the mitochondrion.
Rosiane A. Silva-pereira - One of the best experts on this subject based on the ideXlab platform.
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Sm16, A Schistosoma mansoni Immunomodulatory Protein, Fails to Elicit a Protective Immune Response and Does Not Have an Essential Role in Parasite Survival in the Definitive Host
Journal of immunology research, 2019Co-Authors: Wilma Patrícia De Oliveira Santos Bernardes, Juliano Michel Araujo, Gardênia Braz Carvalho, Clarice Carvalho Alves, Aline Coelho, Isabela Thamara Sabino Dutra, Sueleny Silva Ferreira Teixeira, Rosy Iara Maciel De Azambuja Ribeiro, Marina De Moraes Mourão, Rosiane A. Silva-pereiraAbstract:Sm16 is an immunomodulatory protein that seems to play a key role in the suppression of the cutaneous inflammatory response during Schistosoma mansoni penetration of the skin of definitive hosts. Therefore, Sm16 represents a potential target for protective immune responses induced by vaccination. In this work, we generated the recombinant protein rSm16 and produced polyclonal antibodies against this protein to evaluate its expression during different Parasite life-cycle stages and its location on the surface of the Parasite. In addition, we analyzed the immune responses elicited by immunization with rSm16 using two different vaccine formulations, as well as its ability to induce protection in Balb/c mice. In order to explore the biological function of Sm16 during the course of experimental infection, RNA interference was also employed. Our results demonstrated that Sm16 is expressed in cercaria and schistosomula and is located in the schistosomula surface. Despite humoral and cellular immune responses triggered by vaccination using rSm16 associated with either Freund's or alum adjuvants, immunized mice presented no reduction in either Parasite burden or Parasite egg laying. Knockdown of Sm16 gene expression in schistosomula resulted in decreased Parasite size in vitro but had no effect on Parasite Survival or egg production in vivo. Thus, our findings demonstrate that although the vaccine formulations used in this study succeeded in activating immune responses, these failed to promote Parasite elimination. Finally, we have shown that Sm16 is not vital for Parasite Survival in the definitive host and hence may not represent a suitable target for vaccine development.
