Correlation of meta 1 expression with culture stage, cell morphology and infectivity in Leishmania (Leishmania) amazonensis promastigotes

Santos, Marcos Gonzaga dos; Silva, Maria Fernanda Laranjeira da; Zampieri, Ricardo Andrade; Lafraia, Rafaella Marino; Flöeter-Winter, Lucile Maria

doi:10.1590/s0074-02762011000200012

Cited by 8 publications

(7 citation statements)

References 16 publications

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“…Moreover, during the stationary phase promastigotes with small bodies and large flagella could be clearly identified by optic microscopy (data not shown); these were confirmed by flow cytometry (Figure 2D) as FSC lo PI - cells [33,34]. Based on this typical morphology of metacyclic promastigotes, we analyzed metacyclogenesis throughout the 6 days of culture by quantifying the expression of specific genes which are upregulated ( Meta-1 and SHERP ) or downregulated ( histone H4 ) during the process [25,33,35] (Figure 3 and Additional file 5: Figure S4). Despite the observed interstrain variations on the fold modifications of each gene, all of the strains showed a dramatic decrease in histone H4 expression consistent with the cell cycle analysis.…”

Section: Resultsmentioning

confidence: 82%

Characterization of the biology and infectivity of Leishmania infantum viscerotropic and dermotropic strains isolated from HIV+ and HIV- patients in the murine model of visceral leishmaniasis

et al. 2013

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BackgroundLeishmaniasis is a group of diseases with a variety of clinical manifestations. The form of the disease is highly dependent on the infective Leishmania species and the immunological status of the host. The infectivity of the parasite strain also plays an important role in the progression of the infection. The aim of this work is to understand the influence of the natural infectivity of Leishmania strains in the outcome of visceral leishmaniasis.MethodsIn this study we have characterized four strains of L. infantum in terms of molecular typing, in vitro cultivation and differentiation. Two strains were isolated from HIV+ patients with visceral leishmaniasis (Bibiano and E390M), one strain was isolated from a cutaneous lesion in an immunocompetent patient (HL) and another internal reference strain causative of visceral leishmaniasis (ST) also from an immunocompetent patient was used for comparison. For this objective, we have compared their virulence by in vitro and in vivo infectivity in a murine model of visceral leishmaniasis.ResultsMolecular typing unraveled a new k26 sequence attributed to MON-284 zymodeme and allowed the generation of a molecular signature for the identification of each strain. In vitro cultivation enabled the production of promastigotes with comparable growth curves and metacyclogenesis development. The HL strain was the most infective, showing the highest parasite loads in vitro that were corroborated with the in vivo assays, 6 weeks post-infection in BALB/c mice. The two strains isolated from HIV+ patients, both belonging to two different zymodemes, revealed different kinetics of infection.ConclusionDifferences in in vitro and in vivo infectivity found in the murine model were then attributed to intrinsic characteristics of each strain. This work is supported by other studies that present the parasite’s inherent features as factors for the multiplicity of clinical manifestations and severity of leishmaniasis.

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Section: Resultsmentioning

confidence: 82%

Characterization of the biology and infectivity of Leishmania infantum viscerotropic and dermotropic strains isolated from HIV+ and HIV- patients in the murine model of visceral leishmaniasis

et al. 2013

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“…The mRNA expression and arginine uptake were evaluated along the growth curve by maintaining the parasites at log phase by sub-culturing them every 24 h with the same initial cell ratio (5×10 5 parasites/mL), as previously described [44].…”

Section: Methodsmentioning

confidence: 99%

“…A 7300 System (Applied Biosystems, USA) was used to run the qRT-PCR. The primers used to amplify GAPDH (internal control), arginase and SSUrRNA are described elsewhere [44].…”

Section: Methodsmentioning

confidence: 99%

Axenic Leishmania amazonensis Promastigotes Sense both the External and Internal Arginine Pool Distinctly Regulating the Two Transporter-Coding Genes

et al. 2011

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Leishmania (L.) amazonensis uses arginine to synthesize polyamines to support its growth and survival. Here we describe the presence of two gene copies, arranged in tandem, that code for the arginine transporter. Both copies show similar Open Reading Frames (ORFs), which are 93% similar to the L. (L.) donovani AAP3 gene, but their 5′ and 3′ UTR's have distinct regions. According to quantitative RT-PCR, the 5.1 AAP3 mRNA amount was increased more than 3 times that of the 4.7 AAP3 mRNA along the promastigote growth curve. Nutrient deprivation for 4 hours and then supplemented or not with arginine (400 µM) resulted in similar 4.7 AAP3 mRNA copy-numbers compared to the starved and control parasites. Conversely, the 5.1 AAP3 mRNA copy-numbers increased in the starved parasites but not in ones supplemented with arginine (p<0.05). These results correlate with increases in amino acid uptake. Both Meta1 and arginase mRNAs remained constant with or without supplementation. The same starvation experiment was performed using a L. (L.) amazonensis null knockout for arginase (arg -) and two other mutants containing the arginase ORF with (arg -/ARG) or without the glycosomal addressing signal (arg -/argΔSKL). The arg - and the arg -/argΔSKL mutants did not show the same behavior as the wild-type (WT) parasite or the arg -/ARG mutant. This can be an indicative that the internal pool of arginine is also important for controlling transporter expression and function. By inhibiting mRNA transcription or/and mRNA maturation, we showed that the 5.1 AAP3 mRNA did not decay after 180 min, but the 4.7 AAP3 mRNA presented a half-life decay of 32.6 +/− 5.0 min. In conclusion, parasites can regulate amino acid uptake by increasing the amount of transporter-coding mRNA, possibly by regulating the mRNA half-life in an environment where the amino acid is not present or is in low amounts.

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“…Transcriptomic and proteomic data compiled in S2 Table indicate stage-specific expression for additional Class I and II "CEST-like" molecules in trypanosomatids [50, 63, 66]. Interestingly, META proteins were shown to be involved in Leishmania growth and infectivity [58, 64, 65]. Our present results show a somehow related sub-cellular localization and growth phenotype for TCLP 1.…”

Section: Discussionmentioning

confidence: 55%

“…In the same line, it was reported that META1, a conserved Leishmania virulence factor display distant homology and structural similarity to Shigella MxiM, an outer membrane protein required for TIIISS-mediated effector translocation [58, 61]. Interestingly, META1 and other related molecules display several structural and functional features similar to TCLP 1 [62–65]. On one hand, their expression profiles are not homogeneous along the parasite life-cycle, suggesting they are under certain developmental regulation program.…”

Section: Discussionmentioning

confidence: 96%

A Novel Trypanosoma cruzi Protein Associated to the Flagellar Pocket of Replicative Stages and Involved in Parasite Growth

Durante¹,

Camara²,

Buscaglia³

2015

PLoS ONE

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The flagellar pocket constitutes an active and strategic site in the body of trypanosomatids (i.e. parasitic protozoa that cause important human and/or livestock diseases), which participates in several important processes such as cell polarity, morphogenesis and replication. Most importantly, the flagellar pocket is the unique site of surface protein export and nutrient uptake in trypanosomatids, and thus constitutes a key portal for the interaction with the host. In this work, we identified and characterized a novel Trypanosoma cruzi protein, termed TCLP 1, that accumulates at the flagellar pocket area of parasite replicative forms, as revealed by biochemical, immuno-cytochemistry and electron microscopy techniques. Different in silico analyses revealed that TCLP 1 is the founding member of a family of chimeric molecules restricted to trypanosomatids bearing, in addition to eukaryotic ubiquitin-like and protein-protein interacting domains, a motif displaying significant structural homology to bacterial multi-cargo chaperones involved in the secretion of virulence factors. Using the fidelity of an homologous expression system we confirmed TCLP 1 sub-cellular distribution and showed that TCLP 1-over-expressing parasites display impaired survival and accelerated progression to late stationary phase under starvation conditions. The reduced endocytic capacity of TCLP 1-over-expressors likely underlies (at least in part) this growth phenotype. TCLP 1 is involved in the uptake of extracellular macromolecules required for nutrition and hence in T. cruzi growth. Due to the bacterial origin, sub-cellular distribution and putative function(s), we propose TCLP 1 and related orthologs in trypanosomatids as appealing therapeutic targets for intervention against these health-threatening parasites.

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Correlation of meta 1 expression with culture stage, cell morphology and infectivity in Leishmania (Leishmania) amazonensis promastigotes

Abstract: The parasitic protozoan Leishmania (Leishmania)

Cited by 8 publications

References 16 publications

Characterization of the biology and infectivity of Leishmania infantum viscerotropic and dermotropic strains isolated from HIV+ and HIV- patients in the murine model of visceral leishmaniasis

Characterization of the biology and infectivity of Leishmania infantum viscerotropic and dermotropic strains isolated from HIV+ and HIV- patients in the murine model of visceral leishmaniasis

Axenic Leishmania amazonensis Promastigotes Sense both the External and Internal Arginine Pool Distinctly Regulating the Two Transporter-Coding Genes

A Novel Trypanosoma cruzi Protein Associated to the Flagellar Pocket of Replicative Stages and Involved in Parasite Growth

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