Increasing in cysteine proteinase B expression and enzymatic activity during in vitro differentiation of Leishmania (Viannia) braziliensis: First evidence of modulation during morphological transition

Gomes, Cinthia Bernardes; Souza-Silva, Franklin; Charret, Karen dos Santos; Pereira, Bernardo Acácio Santini; Finkelstein, Léa C.; Santos-de-Souza, Raquel; Côrtes, Luzia Monteiro de Castro; Pereira, Mirian Cláudia de Souza; Oliveira, Francisco Odencio Rodrigues de; Alves, Carlos Roberto

doi:10.1016/j.biochi.2016.11.015

Cited by 18 publications

(20 citation statements)

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“…Some cysteine protease actions can be affected by the differential regulation of these enzymes genes, since they are originated in the same transcriptional unit suggesting large differences in the expression pattern in L. ( L. ) infantum [ 40 ]. As previously suggested, the increase of temperature can be an inductor control factor of cpb genes expression influencing regulatory elements that accelerate RNA degradation [ 41 ].…”

Section: Discussionmentioning

confidence: 99%

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Detection of Metalloproteases and Cysteine Proteases RNA Transcripts ofLeishmania (Leishmania) infantumin Ear Edge Skin of Naturally Infected Dogs

Veloso

Cardoso

Charret

et al. 2020

BioMed Research International

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Leishmania spp. proteases have been proposed as virulence factors contributing to adaptive success these parasites to the mammalian hosts. Since these enzymes are poorly studied in naturally infected dogs, this work aims to show the differences in metalloprotease and cysteine proteases gene expression in ear edge skin of dogs naturally infected by Leishmania (Leishmania) infantum. A cohort of dogs (n=20) naturally infected by L. (L.) infantum was clinically classified as asymptomatic, oligosymptomatic, and polysymptomatic and the parasite load range estimated. The analysis of proteases expression by RT-PCR in the ear edge skin was also assessed, suggesting more transcripts of proteases in cDNA samples from polysymptomatic dogs than oligosymptomatic and asymptomatic ones. Metalloprotease RT-PCR assays yielded products (202 bp) in all assessed cDNA dog samples. In contrast, cysteine proteases transcripts (227 bp) had shown to be better detected in cDNA samples of polysymptomatic dogs, compared with cDNA samples from asymptomatic and oligosymptomatic dogs. Predictive in silico assays suggested that secondary structures of metalloproteasee mRNAs can be more stable than cysteine proteases at the skin temperature of dogs. Evidence is presented that during natural infection of dogs by L. (L.) infantum, this parasite produces transcripts of metalloprotease and cysteine protease RNA in the skin from asymptomatic, oligosymptomatic, and polysymptomatic dogs.

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Section: Discussionmentioning

confidence: 99%

“…Additional assays are necessary to prove the two-dimensional mRNA structure in the regulation of these genes. It is known that parasite mRNA structure changes can be a mechanism for expression and activity control of its proteases, as previously proposed by in vitro L.(V.) braziliensis differentiation due to temperature changes [ 41 ].…”

Section: Discussionmentioning

confidence: 99%

Detection of Metalloproteases and Cysteine Proteases RNA Transcripts ofLeishmania (Leishmania) infantumin Ear Edge Skin of Naturally Infected Dogs

Veloso

Cardoso

Charret

et al. 2020

BioMed Research International

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“…Even though intracellular amastigote is the "gold standard" model to test susceptibility, axenic amastigotes have been previously used to evaluate response towards Sb V , which was found to be stage and strain-speci c in L. (L.) donovani [47] and L. (L.) infantum [48]. Furthermore, it is important to remember that assays with intracellular amastigotes are di cult to standardize and strongly depend on the type of host cell and the medium used [40,49], which might bias the nal results. Therefore, axenic…”

Section: Discussionmentioning

confidence: 99%

“…amastigotes are a feasible model to characterize in vitro phenotypes since they maintain similar morphology, metabolic and virulence genes expression pro les as intracellular amastigotes [49][50][51][52].…”

Section: Discussionmentioning

confidence: 99%

Serine Proteases Profiles of Leishmania (Viannia) Braziliensis Clinical Isolates With Distinct Susceptibilities to Antimony

Zabala-Peñafiel¹,

Dias-Lopes²,

Cysne-Finkelstein

et al. 2020

Preprint

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Background: Glucantime® (SbV) is considered the first-line treatment against American Tegumentary Leishmaniasis in South America, though increased parasite resistance towards it have been reported and hampered its effectiveness. In this context, subtilisins serine proteases have been related to parasites’ susceptibility to drugs such as SbV and derivatives, through modulation of the parasite detoxification system. However, little is known about parasites causing ATL and their distinct responses towards this treatment.Methods: The study was conducted using Leishmania (Viannia) braziliensis clinical isolates from patients that presented clinical cure or Responders (R) and relapse/therapeutic failure or Non-responders (NR). Twelve clinical isolates were used to assess their in vitro susceptibility to SbIII and SbV, serine proteases activity, and expression of subtilisins-like and tryparedoxin-peroxidase (TXNPx) transcripts. Results: SbIII was able to better distinguish axenic amastigotes from each clinical group. These isolates were also assessed for serine protease activity, using z-FR-AMC as substrate and detecting distinct enzyme profiles with specific inhibitors. TLCK inhibited almost 100% of activity in both promastigotes and axenic amastigotes while AEBSF inhibited around 70%. PMSF showed low inhibition of specific isolates (35%). Gathering all the quantitative data, we performed principal component analysis and then used the K-means algorithm to cluster the isolates. This analysis yielded one cluster with only one isolate (R isolate), one homogeneous cluster (NR isolates), and three heterogeneous clusters (R and NR isolates). Additionally, gene transcripts of subtilisins and TXNPx were detected in promastigotes and axenic amastigotes from both groups. Conclusions: Cluster analysis showed that there is a phenotypic heterogeneity among the isolates, however, exploration of in vitro phenotypes based on SbIII and serine proteases profiles can aid in the characterization and better understanding of L. (V.) braziliensis clinical isolates.

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“…The severity of the disease is explained by the high virulence of its etiological agents, which results from the action of a set of functional molecules with intense biological activity in their hosts. The role of protease inhibitors in the development of pathogenic mechanisms was investigated [7]. These enzymes can be divided into endopeptidases and exopeptidases, depending on the site of hydrolysis.…”

Section: Introductionmentioning

confidence: 99%

Exploring Leishmania infantum cathepsin as a new molecular marker for phylogenetic relationships and visceral leishmaniasis diagnosis

et al. 2019

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BackgroundLeishmania infantum, the etiological agent of visceral leishmaniasis, is a neglected zoonosis that requires validation and standardization of satisfactory diagnostic methodologies. Thus, the aim of the present study was to evaluate the effectiveness of cathepsin L-like protease as a target for making molecular diagnoses and as a phylogenetic marker enabling to understand the intraspecies variations and evolutionary history of L. infantum in Brazil.MethodsWe used 44 isolates of L. infantum. The cathepsin L-like gene fragments were amplified, sequenced, manually aligned and analyzed using inference methods. The sequences generated were used to search and design oligonucleotide primers to be used in reactions specific to the target parasite.ResultsThe cathepsin L-like gene did not show any intraspecies variability among the isolates analyzed. The pair of primers proposed amplified the target deoxyribonucleic acid (DNA) of L. infantum isolates and were effective for DNA amplification at concentrations of as low as 10− 11 ng/μl. The proposed marker did not present cross-reactions with other hemoparasites. When used for making the diagnosis in a panel of clinical samples from dogs, a positivity rate of 49.03% (102/208) was obtained, versus 14.42% (30/208) for a ribosomal internal transcribed spacer (ITS) marker. In samples from sandflies, the rate was 6.25% and from humans, 14.28%.ConclusionsThe results described in this work allow us to infer that CatLeish-PCR is a sensitive and specific marker for use in diagnostic trials of L. infantum and in clinical and epidemiological surveys.

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Increasing in cysteine proteinase B expression and enzymatic activity during in vitro differentiation of Leishmania (Viannia) braziliensis: First evidence of modulation during morphological transition

Cited by 18 publications

References 50 publications

Detection of Metalloproteases and Cysteine Proteases RNA Transcripts ofLeishmania (Leishmania) infantumin Ear Edge Skin of Naturally Infected Dogs

Detection of Metalloproteases and Cysteine Proteases RNA Transcripts ofLeishmania (Leishmania) infantumin Ear Edge Skin of Naturally Infected Dogs

Serine Proteases Profiles of Leishmania (Viannia) Braziliensis Clinical Isolates With Distinct Susceptibilities to Antimony

Exploring Leishmania infantum cathepsin as a new molecular marker for phylogenetic relationships and visceral leishmaniasis diagnosis

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