362 research outputs found
Distinct roles in autophagy and importance in infectivity of the two ATG4 cysteine peptidases of leishmania major
No full textMacroautophagy in Leishmania, which is important for the cellular remodeling required during differentiation, relies upon the hydrolytic activity of two ATG4 cysteine peptidases (ATG4.1 and ATG4.2). We have investigated the individual contributions of each ATG4 to Leishmania major by generating individual gene deletion mutants (Δatg4.1 and Δatg4.2); double mutants could not be generated, indicating that ATG4 activity is required for parasite viability. Both mutants were viable as promastigotes and infected macrophages in vitro and mice, but Δatg4.2 survived poorly irrespective of infection with promastigotes or amastigotes, whereas this was the case only when promastigotes of Δatg4.1 were used. Promastigotes of Δatg4.2 but not Δatg4.1 were more susceptible than wild type promastigotes to starvation and oxidative stresses, which correlated with increased reactive oxygen species levels and oxidatively damaged proteins in the cells as well as impaired mitochondrial function. The antioxidant N-acetylcysteine reversed this phenotype, reducing both basal and induced autophagy and restoring mitochondrial function, indicating a relationship between reactive oxygen species levels and autophagy. Deletion of ATG4.2 had a more dramatic effect upon autophagy than did deletion of ATG4.1. This phenotype is consistent with a reduced efficiency in the autophagic process in Δatg4.2, possibly due to ATG4.2 having a key role in removal of ATG8 from mature autophagosomes and thus facilitating delivery to the lysosomal network. These findings show that there is a level of functional redundancy between the two ATG4s, and that ATG4.2 appears to be the more important. Moreover, the low infectivity of Δatg4.2 demonstrates that autophagy is important for the virulence of the parasite
Rationale for simplifying the strength formulae for the design of multi-row bolted connections failing in net tension
No full textHart-Smith [1] developed a set of closed form strength formulae for a semi-empirical approach to determine the net tension strength of multi-row bolted connections with composite materials. Mottram [2] showed that, for a pultruded fibre reinforced polymer material, the approach to be reliable (and conservative) for the configuration comprising two rows with a single bolt per row. This led to the formulae being developed into clauses in an American pre-standard for Load and Resistance Factor Design (LRFD) of Pultruded Fiber-Reinforced Polymer (FRP) Structures [3]. Because the expressions in the Hart-Smith formulae are not simple, the message coming from the practitioners, on the ASCE/SEI Fiber Composites And Polymers Standards committee (FCAPS) tasked with developing the pre-standard [3] into a standard, is that they would not use them when designing bolted connections. Taking stock of the specified geometries, bolt details and design parameters permitted by the pre-standard [3] the author conducted an analytical
parametric study using the Hart-Smith formulae with the aim of establishing simplified forms that could be routinely used in the design office. Presented in this paper is the provenance to this code-specific work when the connection has more than a single row of bolts. A presentation is given to what has been lost, in terms of calculated net tension strength, by providing the
simplified strength formula in the mandatory part to the standard. To enable the designer to be able to take full advantage of the Hart-Smith design approach [1, 2], the ‘complicated’ formulae and their accompanying mandatory-style text are to be found in an appendix with the standard’s commentary [3]
Trypanosoma brucei peptidase inhibitors.Immunolocalization, secretion and potential use as targets for therapy
No full textAs peptidases encontram-se envolvidas em diversas funções biológicas possuindo um papel importante na patogenicidade de várias infecções parasitárias. Em mamíferos, a actividade peptidica é controlada por inibidores endógenos, como as cistatinas e as serpinas. Os genes que codificam para inibidores homólogos às cistatinas e serpinas de mamíferos, encontram-se ausentes do genoma de tripanossomatídeos. A ecotina é uma proteína de Escherichia coli, capaz de inibir uma grande variedade de peptidases serínicas da família S1A, tais como a tripsina. Existem duas proteínas do tipo da ecotina em T. brucei, ISP1 e ISP2. A ausência de peptidases sensíveis à acção dos ISPs no genoma de Trypanosoma brucei, sugere que estes tenham como alvo as peptidases serínicas do hospedeiro. Linhas celulares de T. brucei deficientes em ISP1 (Δisp1), ISP2 (Δisp2) e em ambos os ISPs (Δisp1/2) foram produzidas com sucesso e a ausência dos inibidores comprovada com o uso de anticorpos monoclonais específicos contra o ISP1 e o ISP2, que reconhecem a proteina alvo em extractos de proteína total de parasitas selvagens mas não em extractos de parasitas mutantes. O efeito da ausência dos ISPs nas células foi avaliado in vitro e in vivo, verificando-se que a delecção individual de cada ISP não produz qualquer efeito nos parasitas que revelam um crescimento normal em cultura e padrões de infectividade em murganhos idênticos aos de parasitas selvagens. Em contraste, os parasitas Δisp1/2, embora possuam um crescimento normal in vitro com ausência de alterações morfológicas grosseiras, são caracterizados por um alteração na mobilidade e pela acumulação de micro-vesiculas na região da bolsa flagelar, consistente com um defeito no processo de endocitose/exocitose. Adicionalmente, por imunofluorescência foi possível localizar os ISPs no citoplasma e na perto da bolsa flagelar. Em conjunto, estes dados sugerem uma função intracelular, independente da actividade inibitória dos ISPs e provavelmente associada ao flagelo ou à bolsa flagelar. O papel biológico dos ISPs na interação parasita-hospedeiro foi também avaliada através da infecção de murganhos com os parasitas Δisp1/2.A monitorização da infecção demonstrou que a deleção de ambos os inibidores resulta em sobrevivência prolongada dos murganhos com cargas parasitárias reduzidas, sugerindo que os ISPs possuem um papel importante na sobrevivência do parasita. Em contraste com os resultados obtidos em Leishmania que atribuem diferentes funções a ISP1 e ISP2, este estudo sugere que existe uma redundância funcional dos mesmos em T. brucei, sendo necessária a presença de ambos para que o parasita estabeleça eficientemente a infecção no hospedeiro mamifero. Foram também realizados estudos de imuno-protecção em murganhos de modo a determinar o potencial dos ISPs como alvos terapêuticos. A imunização com ISP recombinante e o tratamento com anticorpos específicos contra ISP1 e ISP2 não produzem qualquer efeito protector ou neutralizante da infecção de murganhos, sugerindo que estes inibidores não constituem bons candidatos para o desenvolvimento de uma vacina ou terapia baseada em anticorpos.Peptidases are involved in several biological functions playing an important role in the pathogenicity of many parasitic infections. In mammals, one way in which the activity of these peptidases is controlledisby interaction with natural inhibitors, such as cystatins and serpins. In trypanosomatids, the genes encoding forendogenousinhibitors homologous to mammalian cystatins and serpins, are absent. Ecotin is an Escherichia coliprotein capable of inhibiting a wide range of serine peptidases from S1A family such as trypsin. Ecotin orthologues can be found in a restricted range of bacterial pathogens and also in trypanosomatid parasitic protozoa.In Trypanosomabruceitwo proteins of 19.7 kDa and 17.8 kDa were identified as ecotin-like inhibitors and given the generic name of Inhibitors of Serine Peptidases (ISP). The absence of peptidases predicted to be sensitive to the action of ISPs in T. bruceisuggests that these inhibitors are targeting the host’s serine peptidases, although their exact biological role was unknown. T. bruceimutant cell linesdeficientin ISP1 (Δisp1),ISP2 (Δisp2) and both ISPs (Δisp1/2) weresuccessfully generatedby targeted gene disruption. This was confirmed using monoclonal antibodies generated specifically against ISP1 and ISP2 that recognize the target protein in wild type and re-expressor cell lysates but not in the respective mutant cell line. Theeffect of ISP deletion in the parasiteswasdeterminedboth in culture andin micein vivo. The deletion of ISPgenes individually was shown to have no effect on the parasites in vitroor in vivo. However, different results were obtained for the simultaneous deletion of ISP1and ISP2. Although Δisp1/2parasites have normal growth and morphology in culture, they exhibit a motility defect and are characterized by the accumulation of small vesicles outside the flagellar pocket consistent with an endocytosis/exocytosis defect. Furthermore, immunofluorescence shows that both ISPs are localized in the cytosol and in small punctuated structures near the flagellar pocket region. These data suggests that ISPs have an intracellular function independent of their inhibitory activityand probably associated with the flagellar pocket.The biological role of ISP1 and ISP2 in the host-parasite interaction was also evaluated by infecting mice with Δisp1/2parasites. Parasitemia monitoring shows that the deletion of both ISPsresults in prolonged host survival with reduced or undetectable parasite loads, suggesting an important role for these inhibitors in the parasite’s survival. In contrast with recent findings of ISPs functionin Leishmania, that assigns different biological roles to ISP1 and ISP2, the present study reveals some functional redundancy of ISPs in T. brucei,with both inhibitors being required for the parasite to infect the mammalian host efficiently. Finally, immuno-protection studies were made, to assess the potential of ISPs as targets for therapy. Immunization with recombinant ISPs and treatment with antibodies specific against ISP1 and ISP2 had no protective or neutralizing effecton T. bruceiinfected mice, revealing that these inhibitors are poor candidates for an anti-disease vaccine or for antibody-based therapy
Inhibitor of serine peptidase 2 enhances Leishmania major survival in the skin through control of monocytes and monocyte-derived cells
No full textLeishmania major is the causative agent of the neglected tropical disease, cutaneous leishmaniasis. In the mouse, protective immunity to Leishmania is associated with inflammatory responses. Here, we assess the dynamics of the inflammatory responses at the lesion site during experimental long-term, low-dose intradermal infection of the ear, employing noninvasive imaging and genetically modified L. major. Significant infiltrates of neutrophils and monocytes occurred at 1–4 d and 2–4 wk, whereas dermal macrophage and dendritic cell (DC) numbers were only slightly elevated in the first days. Quantitative whole-body bioluminescence imaging of myeloperoxidase activity and the quantification of parasite loads indicated that the Leishmania virulence factor, inhibitor of serine peptidase 2 (ISP2), is required to modulate phagocyte activation and is important for parasite survival at the infection site. ISP2 played a role in the control of monocyte, monocyte-derived macrophage, and monocyte-derived DC (moDC) influx, and was required to reduce iNOS expression in monocytes, monocyte-derived cells, and dermal DCs; the expression of CD80 in moDCs; and levels of IFN-γ in situ. Our findings indicate that the increased survival of L. major in the dermis during acute infection is associated with the down-regulation of inflammatory monocytes and monocyte-derived cells via ISP2.—Goundry, A., Romano, A., Lima, A. P. C. A., Mottram, J. C., Myburgh, E. Inhibitor of serine peptidase 2 enhances Leishmania major survival in the skin through control of monocytes and monocyte-derived cells
Tracking autophagy during proliferation and differentiation of trypanosoma brucei
No full textAutophagy is a lysosome-dependent degradation mechanism that sequesters target cargo into autophagosomal vesicles. The Trypanosoma brucei genome contains apparent orthologues of several autophagy-related proteins including an ATG8 family. These ubiquitin-like proteins are required for autophagosome membrane formation, but our studies show that ATG8.3 is atypical. To investigate the function of other ATG proteins, RNAi compatible T. brucei were modified to function as autophagy reporter lines by expressing only either YFP-ATG8.1 or YFP-ATG8.2. In the insect procyclic lifecycle stage, independent RNAi down-regulation of ATG3 or ATG7 generated autophagy-defective mutants and confirmed a pro-survival role for autophagy in the procyclic form nutrient starvation response. Similarly, RNAi depletion of ATG5 or ATG7 in the bloodstream form disrupted autophagy, but did not impede proliferation. Further characterisation showed bloodstream form autophagy mutants retain the capacity to undergo the complex cellular remodelling that occurs during differentiation to the procyclic form and are equally susceptible to dihydroxyacetone-induced cell death as wild type parasites, not supporting a role for autophagy in this cell death mechanism. The RNAi reporter system developed, which also identified TOR1 as a negative regulator controlling YFP-ATG8.2 but not YFP-ATG8.1 autophagosome formation, will enable further targeted analysis of the mechanisms and function of autophagy in the medically relevant bloodstream form of T. brucei
ATG5 is essential for ATG8-dependent autophagy and mitochondrial homeostasis in Leishmania major
No full textMacroautophagy has been shown to be important for the cellular remodelling required for Leishmania differentiation. We now demonstrate that L. major contains a functional ATG12-ATG5 conjugation system, which is required for ATG8-dependent autophagosome formation. Nascent autophagosomes were found commonly associated with the mitochondrion. L. major mutants lacking ATG5 (Δatg5) were viable as promastigotes but were unable to form autophagosomes, had morphological abnormalities including a much reduced flagellum, were less able to differentiate and had greatly reduced virulence to macrophages and mice. Analyses of the lipid metabolome of Δatg5 revealed marked elevation of phosphatidylethanolamines (PE) in comparison to wild type parasites. The Δatg5 mutants also had increased mitochondrial mass but reduced mitochondrial membrane potential and higher levels of reactive oxygen species. These findings indicate that the lack of ATG5 and autophagy leads to perturbation of the phospholipid balance in the mitochondrion, possibly through ablation of membrane use and conjugation of mitochondrial PE to ATG8 for autophagosome biogenesis, resulting in a dysfunctional mitochondrion with impaired oxidative ability and energy generation. The overall result of this is reduced virulence
Comparative study of the ability of <i>Leishmania mexicana</i> promastigotes and amastigotes to alter macrophage signalling and functions
No full textLeishmania alternates between two morphologically different stages: promastigotes and amastigotes. While the majority of reports focused on how the promastigote form can alter macrophage signalling and function, fewer reports investigated signalling alterations mediated by amastigotes, and comparative studies are lacking. In this study, we performed a comparison between the ability of both forms of the parasite to alter macrophage signalling and functions. Here, we show that promastigotes and amastigotes were both able to rapidly activate host protein tyrosine phosphatases (PTPs), importantly the Src-homology 2 domain-containing PTP (SHP-1). However, we found that PTP-1B is specifically activated by promastigote but not amastigote infection and that lmcpb-/- promastigotes were no longer able to activate PTP-1B. We also show a similarity in the way promastigotes and amastigotes inactivate the transcription factors (TFs) STAT-1{alpha} and AP-1, but differences in the modulation of NF-{kappa}B with promastigotes cleaving the p65 subunit generating a smaller p35 subunit while amastigotes fully degrading the p65 subunit with no p35 production. Importantly, we show that the cysteine proteinase LmCPb plays a key role in the alteration of NF-{kappa}B, STAT-1{alpha}, and AP-1 by promastigote and amastigote infections, ultimately leading to the inability of these TFs to translocate to the nucleus in response to IFN-{gamma} stimulation and thus contributing to the ability of both parasite forms to effectively block IFN-{gamma}-mediated NO production in macrophages
Studies on the energy metabolism of Leishmania mexicana mexicana
No full textSIGLEAvailable from British Library Document Supply Centre- DSC:D66243/86 / BLDSC - British Library Document Supply CentreGBUnited Kingdo
Identification of semicarbazones, thiosemicarbazones and triazine nitriles as inhibitors of Leishmania mexicana cysteine protease CPB
No full textCysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas' disease and leishmaniasis. Homology modeling of the mature Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput screening of a compound library against this enzyme and bovine cathepsin B in a counter assay identified four novel inhibitors, containing the warhead-types semicarbazone, thiosemicarbazone and triazine nitrile, that can be used as leads for antiparasite drug design. Covalent docking experiments confirmed the SARs of these lead compounds in an effort to understand the structural elements required for specific inhibition of CPB2.8. This study has provided starting points for the design of selective and highly potent inhibitors of L. mexicana cysteine protease CPB that may also have useful efficacy against other important cysteine proteases
Divergent cytochrome c maturation system in kinetoplastid protists
No full textIn eukaryotes, heme attachment through two thioether bonds to mitochondrial cytochromes c and c 1 is catalyzed by either multisubunit cytochrome c maturation system I or holocytochrome c synthetase (HCCS). The former was inherited from the alphaproteobacterial progenitor of mitochondria; the latter is a eukaryotic innovation for which prokaryotic ancestry is not evident. HCCS provides one of a few exemplars of de novo protein innovation in eukaryotes, but structure-function insight of HCCS is limited. Uniquely, euglenozoan protists, which include medically relevant kinetoplastids Trypanosoma and Leishmania parasites, attach heme to mitochondrial c-type cytochromes by a single thioether linkage. Yet the mechanism is unknown, as genes encoding proteins with detectable similarity to any proteins involved in cytochrome c maturation in other taxa are absent. Here, a bioinformatics search for proteins conserved in all hemoprotein-containing kinetoplastids identified kinetoplastid cytochrome c synthetase (KCCS), which we reveal as essential and mitochondrial and catalyzes heme attachment to trypanosome cytochrome c. KCCS has no sequence identity to other proteins, apart from a slight resemblance within four short motifs suggesting relatedness to HCCS. Thus, KCCS provides a novel resource for studying eukaryotic cytochrome c maturation, possibly with wider relevance, since mutations in human HCCS leads to disease. Moreover, many examples of mitochondrial biochemistry are different in euglenozoans compared to many other eukaryotes; identification of KCCS thus provides another exemplar of extreme, unusual mitochondrial biochemistry in an evolutionarily divergent group of protists. IMPORTANCE Cytochromes c are essential proteins for respiratory and photosynthetic electron transfer. They are posttranslationally modified by covalent attachment of a heme cofactor. Kinetoplastids include important tropical disease-causing parasites; many aspects of their biology differ from other organisms, including their mammalian or plant hosts. Uniquely, kinetoplastids produce cytochromes c with a type of heme attachment not seen elsewhere in nature and were the only cytochrome c-bearing taxa without evidence of protein machinery to attach heme to the apocyto-chrome. Using bioinformatics, biochemistry, and molecular genetics, we report how kinetoplastids make their cytochromes c. Unexpectedly, they use a highly diverged version of an enzyme used for heme-protein attachment in many eukaryotes. Mutations in the human enzyme lead to genetic disease. Identification of kinetoplastid cytochrome c synthetase, thus, solves an evolutionary unknown, provides a possible target for antiparasite drug development, and an unanticipated resource for studying the mechanistic basis of a human genetic disease. </p
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