TcArf1: a Trypanosoma cruzi ADP-ribosylation factor

Sá-Freire, Alexandre de; Nepomuceno-Silva, José Luciano; Paixão, Julio Cesar; Mendonça, S. M.; Melo, Luiz Dione Barbosa de; Lopes, Ulisses Gazos

doi:10.1007/s00436-003-0952-0

Cited by 11 publications

(10 citation statements)

References 34 publications

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“…In contrast to the substitution observed in the N‐myristoylation site motif, the Ld ARF1 showed 100% sequence identity/similarity in the four GTP‐binding/hydrolysis consensus motifs (32) characteristically present in each of the ARFs examined in this study. It is of interest to note that among the nine different ARF homologs examined here, the Ld ARF 1 showed its highest level of similarity/identity to an ARF recently reported in a related organism, T. cruzi (19).…”

Section: Discussionmentioning

confidence: 67%

“…To assess the significance of the putative LdARF1 homology, we deduced the amino acid sequence and compared this sequence to the nonredundant protein database (GenBank) using the program BlastP (18). The best homology (76% identity and 89% similarity) was obtained with the Trypanosoma cruzi ARF amino acid sequence (19). From the BlastP analysis, the ARFs found to have the greatest homology were: Trypanosoma cruzi ARF (GenBank™ AAF82562), Giardia lamblia ARF (GenBank™ S29008) has 69% identity and 84% similarity, Xenopus laevis ARF (GenBank™ P51643) has 69% identity and 84% similarity, Oryza sativa ARF1 (GenBank™ AAB65432) has 67% identity and 84% similarity, Mus musculus ARF1 (GenBank™ NP 031502) has 69% identity and 84% similarity, Homo sapiens ARF1 (GenBank™ NP 001649) has 69% identity and 84% similarity, Drosophila melanogaster ARF1 (GenBank™ A49520) has 69% identity and 84% similarity, Caenorhabditis elegans ARF (GenBank™ T32978) has 68% identity and 85% similarity and Arabidopsis thaliana ARF1 (GenBank™ NP182239) has 67% identity and 84% similarity.…”

Section: Resultsmentioning

confidence: 99%

“…In that regard, homologs of Rab‐2, ‐4, ‐5, ‐11 and ‐18 been described in the African bloodstream trypanosome, Trypanosoma brucei (22–25) and Rab‐7 homologs have been reported in both Leishmania and in Trypanosoma cruzi (26,27). Moreover, on the basis of sequence homology alone, an ARF‐homolog was recently described in T. cruzi (19). In addition, a few ARF‐like (ARLs) proteins have been reported in Leishmania (28,29).…”

Section: Discussionmentioning

confidence: 99%

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LdARF1 in Trafficking and Structural Maintenance of the trans‐Golgi Cisternal Network in the Protozoan Pathogen Leishmania donovani

Gerald

Engel

Ghedin

et al. 2004

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Adenosine diphosphate ribosylation factors (ARFs) are small guanosine-5 0 -triphosphatases that are essential in vesicular trafficking and in the maintenance of the Golgi network. In this report, we identified a homolog of the mammalian ARF1 in the human pathogenic protozoan parasite, Leishmania donovani (Ld). Ld ARF1 is a 549 bp gene encoding a 183-amino acid deduced protein of $ 20 kDa. We demonstrated by Southern blot analysis that there are at least two copies of ARF1 in the Ld genome. Moreover, Northern blot analysis revealed that Ld ARF1 is expressed on a 1.35 kb transcript in both the insect vector (promastigotes) and mammalian host (amastigotes) forms of this parasite. Fluorescent microscopy studies using Ld promastigotes episomally transfected with an ARF1::GFP (green fluorescent protein) chimeric construct showed that such chimeras appeared to localize to the Golgi region of these organisms. This observation was verified by immunoelectron microscopy using an anti-GFP antibody. Such studies also revealed that Ld ARF1::GFP chimeras localized to trans-Golgi vesicles, the flagellar pocket/reservoir and other vesicles located between the trans-Golgi network and flagellar pocket in these apically polarized cells. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching experiments revealed both the dynamic binding and releasing activity of Ld ARF1 from the Golgi network in these parasites. Further, episomal expression of a constitutively active (''on'') ARF1 (Q71L mutation) resulted in the aberrant swelling and distended-structure of the trans-Golgi cisternae in these cells. These results show that Ld ARF1 is transiently associated with the Golgi network and plays a role in the structural maintenance of this organelle in these important human pathogens.

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Section: Discussionmentioning

confidence: 67%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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LdARF1 in Trafficking and Structural Maintenance of the trans‐Golgi Cisternal Network in the Protozoan Pathogen Leishmania donovani

Gerald

Engel

Ghedin

et al. 2004

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“…A different ARF gene (LinJ.04.0470) is down-regulated. The ARF1 has been characterized in T. cruzi and in L. donovani, where it is involved in coatomer assembly in budding vessicles in the secretory pathway and endocytosis [ 50 , 51 ]. The profilin may be involved in the actin microfilament polymerization machinery [ 52 ].…”

Section: Resultsmentioning

confidence: 99%

In vitro infectivity and differential gene expression of Leishmania infantum metacyclic promastigotes: negative selection with peanut agglutinin in culture versus isolation from the stomodeal valve of Phlebotomus perniciosus

et al. 2016

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BackgroundLeishmania infantum is the protozoan parasite responsible for zoonotic visceral leishmaniasis in the Mediterranean basin. A recent outbreak in humans has been reported in this area. The life cycle of the parasite is digenetic. The promastigote stage develops within the gut of phlebotomine sand flies, whereas amastigotes survive and multiply within phagolysosomes of mammalian host phagocytes. The major vector of L. infantum in Spain is Phlebotomus perniciosus. The axenic culture model of promastigotes is generally used because it is able to mimic the conditions of the natural environment (i.e. the sand fly vector gut). However, infectivity decreases with culture passages and infection of laboratory animals is frequently required. Enrichment of the stationary phase population in highly infective metacyclic promastigotes is achieved by negative selection with peanut agglutinin (PNA), which is possible only in certain Leishmania species such as L. major and L. infantum. In this study, in vitro infectivity and differential gene expression of cultured PNA-negative promastigotes (Pro-PNA−) and metacyclic promastigotes isolated from the sand fly anterior thoracic midgut (Pro-Pper) have been compared.ResultsIn vitro infectivity is about 30 % higher in terms of rate of infected cells and number of amastigotes per infected cell in Pro-Pper than in Pro-PNA−. This finding is in agreement with up-regulation of a leishmanolysin gene (gp63) and genes involved in biosynthesis of glycosylinositolphospholipids (GIPL), lipophosphoglycan (LPG) and proteophosphoglycan (PPG) in Pro-Pper. In addition, differences between Pro-Pper and Pro-PNA− in genes involved in important cellular processes (e.g. signaling and regulation of gene expression) have been found.ConclusionsPro-Pper are significantly more infective than peanut lectin non-agglutinating ones. Therefore, negative selection with PNA is an appropriate method for isolating metacyclic promastigotes in stationary phase of axenic culture but it does not allow reaching the in vitro infectivity levels of Pro-Pper. Indeed, GIPL, LPG and PPG biosynthetic genes together with a gp63 gene are up-regulated in Pro-Pper and interestingly, the correlation coefficient between both transcriptomes in terms of transcript abundance is R2 = 0.68. This means that the correlation is sufficiently high to consider that both samples are physiologically comparable (i.e. the experiment was correctly designed and performed) and sufficiently low to conclude that important differences in transcript abundance have been found. Therefore, the implications of axenic culture should be evaluated case-by-case in each experimental design even when the stationary phase population in culture is enriched in metacyclic promastigotes by negative selection with PNA.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2672-8) contains supplementary material, which is available to authorized users.

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“…Aiming to characterize Ras superfamily genes in the kinetoplastid parasite T. cruzi, we have cloned several cDNA fragments corresponding to Ras-related genes (Nepomuceno-Silva et al, 2001;Sá-Freire et al, 2003). One of these clones shared discreet similarity to Rab genes.…”

Section: Cloning Of a Trypanosomatid Ras-related Genementioning

confidence: 99%