Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity

Toledo, Juliano Simões de; Ferreira, Tiago Luiz; Defina, Tânia Paula Aquino; Dossin, Fernando de M.; Beattie, Kenneth A.; Lamont, Douglas J.; Cloutier, Serge; Papadopoulou, Barbara; Schenkman, Sérgio; Cruz, Ângela K.

doi:10.1016/j.biocel.2010.06.013

Cited by 5 publications

(7 citation statements)

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“…We have previously shown that Leishmania spp. parasites overexpressing a spliced‐leader (SL) RNA ectopic array display a sixfold downregulation of a putative ortholog of PRMT7 (Toledo et al ., and unpublished results). Analysis of homologous genes in other trypanosomatids revealed that the Trypanosoma brucei syntenic ortholog (Tb927.7.5490) was described as an active Tb PRMT7 in a comprehensive investigation (Fisk et al ., ).…”

Section: Resultsmentioning

confidence: 54%

“…Constructs bearing the myc‐RBPs were generated in the same manner as HA‐ Lmj PRMT7. All DNA constructs used in this work were confirmed by Sanger sequencing according to protocols optimized by our group (Toledo et al ., ).…”

Section: Methodsmentioning

confidence: 97%

“…Total RNA was extracted from 4 × 10 8 promastigotes using TRIzol, according to the manufacturer's instructions (Invitrogen), and was resolved in 2.2 M formaldehyde/1.8% agarose gels. The DNA and RNA gels were transferred to Hybond‐N+ nylon membranes (GE‐Healthcare) and submitted to hybridization following standard protocols (Toledo et al ., ). Radiolabeled DNA probes were prepared using the random priming method (Feinberg and Vogelstein, ) with 30 μCi α[ 32 P]dCTP (PerkinElmer, Waltham, MA, USA) and Ready‐To‐Go DNA labeling beads (‐dCTP) (GE‐Healthcare).…”

Section: Methodsmentioning

confidence: 97%

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Altered expression of an RBP‐associated arginine methyltransferase 7 in Leishmania major affects parasite infection

Ferreira

Alves-Ferreira

Defina

et al. 2014

Molecular Microbiology

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SummaryProtein arginine methylation is a widely conserved post-translational modification performed by arginine methyltransferases (PRMTs). However, its functional role in parasitic protozoa is still under-explored. The Leishmania major genome encodes five PRMT homologs, including PRMT7. Here we show that LmjPRMT7 expression and arginine monomethylation are tightly regulated in a lifecycle stage-dependent manner. LmjPRMT7 levels are higher during the early promastigote logarithmic phase, negligible at stationary and late-stationary phases and rise once more post-differentiation to intracellular amastigotes. Immunofluorescence and co-immunoprecipitation studies demonstrate that LmjPRMT7 is a cytosolic protein associated with several RNA-binding proteins (RBPs) from which Alba20 is monomethylated only in LmjPRMT7-expressing promastigote stages. In addition, Alba20 protein levels are significantly altered in stationary promastigotes of the LmjPRMT7 knockout mutant. Considering RBPs are well-known mammalian PRMT substrates, our data suggest that arginine methylation via LmjPRMT7 may modulate RBP function during Leishmania spp. lifecycle progression. Importantly, genomic deletion of the LmjPRMT7 gene leads to an increase in parasite infectivity both in vitro and in vivo, while lesion progression is significantly reduced in LmjPRMT7-overexpressing parasites. This study is the first to describe a role of Leishmania protein arginine methylation in host-parasite interactions.

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Section: Resultsmentioning

confidence: 54%

Section: Methodsmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 97%

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Altered expression of an RBP‐associated arginine methyltransferase 7 in Leishmania major affects parasite infection

Ferreira

Alves-Ferreira

Defina

et al. 2014

Molecular Microbiology

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“…The protein extracts of logarithmic phase promastigotes (5x10 8 parasites) were obtained by precipitation with trichloroacetic acid, and the samples were subjected to 2D gel electrophoresis using Immobiline DryStrip gels (13 cm/4-7 pI) (GE HealthCare, Piscataway, New Jersey, USA). Protein extraction and 2D electrophoresis were performed as described previously [ 23 , 24 ]. A comparative analysis of the digitized proteome maps of the LbrC and LbrM isolates was performed using ImageMaster platinum v6.0 software (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

Differential Gene Expression and Infection Profiles of Cutaneous and Mucosal Leishmania braziliensis Isolates from the Same Patient

et al. 2015

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BackgroundLeishmaniasis is a complex disease in which clinical outcome depends on factors such as parasite species, host genetics and immunity and vector species. In Brazil, Leishmania (Viannia) braziliensis is a major etiological agent of cutaneous (CL) and mucosal leishmaniasis (MCL), a disfiguring form of the disease, which occurs in ~10% of L. braziliensis-infected patients. Thus, clinical isolates from patients with CL and MCL may be a relevant source of information to uncover parasite factors contributing to pathogenesis. In this study, we investigated two pairs of L. (V.) braziliensis isolates from mucosal (LbrM) and cutaneous (LbrC) sites of the same patient to identify factors distinguishing parasites that migrate from those that remain at the primary site of infection.Methodology/Principal FindingsWe observed no major genomic divergences among the clinical isolates by molecular karyotype and genomic sequencing. RT-PCR revealed that the isolates lacked Leishmania RNA virus (LRV). However, the isolates exhibited distinct in vivo pathogenesis in BALB/c mice; the LbrC isolates were more virulent than the LbrM isolates. Metabolomic analysis revealed significantly increased levels of 14 metabolites in LbrC parasites and 31 metabolites in LbrM parasites that were mainly related to inflammation and chemotaxis. A proteome comparative analysis revealed the overexpression of LbrPGF2S (prostaglandin f2-alpha synthase) and HSP70 in both LbrC isolates. Overexpression of LbrPGF2S in LbrC and LbrM promastigotes led to an increase in infected macrophages and the number of amastigotes per cell at 24–48 h post-infection (p.i.).Conclusions/SignificanceDespite sharing high similarity at the genome structure and ploidy levels, the parasites exhibited divergent expressed genomes. The proteome and metabolome results indicated differential profiles between the cutaneous and mucosal isolates, primarily related to inflammation and chemotaxis. BALB/c infection revealed that the cutaneous isolates were more virulent than the mucosal parasites. Furthermore, our data suggest that the LbrPGF2S protein is a candidate to contribute to parasite virulence profiles in the mammalian host.

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“…Nos estudos de manipulação genética, a identificação de genes ou moléculas associadas a "virulência" devem obedecer aos postulados de Koch: (i) O gene deve estar associado a patogenicidade e infecção (ii) a ausência do gene deve ocasionar a perda significativa da virulência e (iii) a restauração funcional do gene deve restaurar a patogenicidade (Beverley and Turco, 1998 (Antoniazi et al, 2000;de Toledo et al, 2009). Análise do proteoma dos transfectantes de L. major indicaram que a atenuação da virulência é decorrência do estresse molecular causado pela superexpressão de SL RNA que é acompanhado por um Introdução incremento da tradução das proteínas e de vias proteolíticas específicas (Toledo et al, 2010). Adicionalmente, uma análise do transcriptoma desse transfectante revelou uma lista de 68 genes diferencialmente expressos que, segundo a classificação em termos GO, estariam associados a processos metabólicos, vias de biossíntese, tradução, modificação de ácidos nucléicos e proteínas (Ferreira, 2010 Upregulated Genes Tabela 1.…”

Section: Estudos Sobre Virulência De Leishmania Por Genética Reversaunclassified

Investigação de genes potencialmente implicados no controle de virulência em <i>Leishmania major</i>

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Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity

Cited by 5 publications

References 75 publications

Altered expression of an RBP‐associated arginine methyltransferase 7 in Leishmania major affects parasite infection

Altered expression of an RBP‐associated arginine methyltransferase 7 in Leishmania major affects parasite infection

Differential Gene Expression and Infection Profiles of Cutaneous and Mucosal Leishmania braziliensis Isolates from the Same Patient

Investigação de genes potencialmente implicados no controle de virulência em <i>Leishmania major</i>

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