Publications by authors named "Yara Maria Traub-Csekö"

10 Publications

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The gene expression of Leishmania infantum chagasi inside Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil.

Mem Inst Oswaldo Cruz 2021 8;116:e200571. Epub 2021 Mar 8.

Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Biologia Molecular de Parasitas e Vetores, Rio de Janeiro, RJ, Brasil.

Leishmania infantum chagasi is the causative agent and Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the Americas. We investigated the expression of Leishmania genes within L. longipalpis after artificial infection. mRNAs from genes involved in sugar and amino acid metabolism were upregulated at times of high parasite proliferation inside the insect. mRNAs from genes involved in metacyclogenesis had higher expression in late stages of infection. Other modulated genes of interest were involved in immunomodulation, purine salvage pathway and protein recycling. These data reveal aspects of the adaptation of the parasite to the microenvironment of the vector gut and reflect the preparation for infection in the vertebrate.
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http://dx.doi.org/10.1590/0074-02760200571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949195PMC
March 2021

Proteomic analysis of exosomes derived from procyclic and metacyclic-like cultured Leishmania infantum chagasi.

J Proteomics 2020 09 14;227:103902. Epub 2020 Jul 14.

Laboratório de Biologia Molecular de Parasitas e Vetores, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brazil. Electronic address:

Leishmania infantum chagasi is the primary etiological agent of visceral leishmaniasis in Latin America, a lethal disease that afflicts hundreds of thousands of people worldwide every year. Previous studies have shown that the parasite releases microvesicles known as exosomes, which prolong and exacerbate infection in the vertebrate vector. However, little is known of their role in the insect vector, the sand fly Lutzomyia longipalpis. Exosomes were isolated from cultured L. i. chagasi in logarithmic (procyclic) (LOG) and stationary phase (metacyclic-like) (STAT) growth stages, which are the parasite stages found in the vector, and submitted to proteomic analysis. Our studies showed that exosomes from LOG and STAT L. i. chagasi display discrete protein profiles. The presence of approximately 50 known virulence factors was detected, including molecules for immunomodulation and evasion (GP63, EF1α, Oligopeptidase), increased pathogenicity (Casein kinase, KMP-11, Cysteine Peptidase and BiP) and parasite protection (Peroxidoxin). Additionally, the majority of ontological terms were associated with both exosome phases, and no substantial ontological enrichment was observed associated with any of the two exosomal stages. We demonstrated that LOG exosomes show a marked increase in protein number and abundance, including many virulence factors, compared to STAT L. i. chagasi exosomes. SIGNIFICANCE: The knowledge of the role of Leishmania exosomes on leishmaniasis opened up a new world of potential and complexity regarding our understanding of the disease. In Brazil the majority of visceral leishmaniasis cases are caused by the parasite Leishmania infantum chagasi and transmitted by the vector Lutzomyia longipalpis. While Leishmania exosomes were found to play an active role in the mammalian host, little is understood about their effects on the sand fly, or how they might impact on the insect infection by the parasite. For this reason, we isolated exosomes from two developmental stages of L. i. chagasi that occur within the insect with a view to identifying and describing the alterations they undergo. We have identified many hundreds of proteins within both exosome phases and have developed a structure by which to examine potential candidates. Our findings regarding the composition of the exosome proteome raise many questions regarding their function and provide compelling evidence that exosomes play an active role in the parasite's development within the sand fly.
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http://dx.doi.org/10.1016/j.jprot.2020.103902DOI Listing
September 2020

TGF-β Has a Role in Survival in the Vector.

Front Cell Infect Microbiol 2019 27;9:71. Epub 2019 Mar 27.

Laboratório de Biologia Molecular de Parasitas e Vetores, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil.

Despite the increasing number of studies concerning insect immunity, immune responses in the presence of infection has not been widely investigated. The few available studies analyzed the role of the Toll and IMD pathways involved in response against and microbial infections. Nevertheless, effector molecules responsible for controlling sand fly infections have not been identified. In the present study we investigated the role a signal transduction pathway, the Transforming Growth Factor-beta (TGF-β) pathway, on the interrelation between and . We identified an homolog belonging to the multifunctional cytokine TGF-β gene family (-β), which is closely related to the activin/inhibin subfamily and potentially involved in responses to infections. We investigated this gene expression through the insect development and in adult flies infected with . Our results showed that -β was expressed in all developmental stages and was upregulated at the third day post infection, when protein levels were also higher as compared to uninfected insects. At this point blood digestion is finished and parasites are in close contact with the insect gut. In addition, we investigated the role of LlTGF-β on infection by using either gene silencing by RNAi or pathway inactivation by addition of the TGF-β receptor inhibitor SB431542. The blockage of the LlTGF-β pathway increased significantly antimicrobial peptides expression and nitric oxide levels in the insect gut, as expected. Both methods led to a decreased infection. Our results show that inactivation of the TGF-β signal transduction pathway reduce survival, therefore suggesting that under natural conditions the parasite benefits from the insect LlTGF-β pathway, as already seen in infection of mosquitoes.
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http://dx.doi.org/10.3389/fcimb.2019.00071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445956PMC
December 2019

Leishmania, microbiota and sand fly immunity.

Parasitology 2018 09 20;145(10):1336-1353. Epub 2018 Jun 20.

Laboratório de Biologia Molecular de Parasitas e Vetores,Instituto Oswaldo Cruz-Fiocruz,Av. Brasil 4365,Rio de Janeiro,RJ,Brazil.

In this review, we explore the state-of-the-art of sand fly relationships with microbiota, viruses and Leishmania, with particular emphasis on the vector immune responses. Insect-borne diseases are a major public health problem in the world. Phlebotomine sand flies are proven vectors of several aetiological agents including viruses, bacteria and the trypanosomatid Leishmania, which are responsible for diseases such as viral encephalitis, bartonellosis and leishmaniasis, respectively. All metazoans in nature coexist intimately with a community of commensal microorganisms known as microbiota. The microbiota has a fundamental role in the induction, maturation and function of the host immune system, which can modulate host protection from pathogens and infectious diseases. We briefly review viruses of public health importance present in sand flies and revisit studies done on bacterial and fungal gut contents of these vectors. We bring this information into the context of sand fly development and immune responses. We highlight the immunity mechanisms that the insect utilizes to survive the potential threats involved in these interactions and discuss the recently discovered complex interactions among microbiota, sand fly, Leishmania and virus. Additionally, some of the alternative control strategies that could benefit from the current knowledge are considered.
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http://dx.doi.org/10.1017/S0031182018001014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137379PMC
September 2018

Identification of Secreted Proteins Involved in Nonspecific dsRNA-Mediated Lutzomyia longipalpis LL5 Cell Antiviral Response.

Viruses 2018 01 18;10(1). Epub 2018 Jan 18.

Laboratório de Biologia Molecular de Parasitas e Vetores, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, Rio de Janeiro 21040-360, RJ, Brazil.

Hematophagous insects transmit infectious diseases. Sand flies are vectors of leishmaniasis, but can also transmit viruses. We have been studying immune responses of , the main vector of visceral leishmaniasis in the Americas. We identified a non-specific antiviral response in LL5 embryonic cells when treated with non-specific double-stranded RNAs (dsRNAs). This response is reminiscent of interferon response in mammals. We are investigating putative effectors for this antiviral response. Secreted molecules have been implicated in immune responses, including interferon-related responses. We conducted a mass spectrometry analysis of conditioned medium from LL5 cells 24 and 48 h after dsRNA or mock treatment. We identified 304 proteins. At 24 h, 19 proteins had an abundance equal or greater than 2-fold change, while the levels of 17 proteins were reduced when compared to control cells. At the 48 h time point, these numbers were 33 and 71, respectively. The two most abundant secreted peptides at 24 h in the dsRNA-transfected group were phospholipid scramblase, an interferon-inducible protein that mediates antiviral activity, and forskolin-binding protein (FKBP), a member of the immunophilin family, which mediates the effect of immunosuppressive drugs. The transcription profile of most candidates did not follow the pattern of secreted protein abundance.
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http://dx.doi.org/10.3390/v10010043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5795456PMC
January 2018

Alternative splicing originates different domain structure organization of Lutzomyia longipalpis chitinases.

Mem Inst Oswaldo Cruz 2018 Feb;113(2):96-101

Laboratório de Biologia Molecular de Parasitos e Vetores, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz-Fiocruz, Rio de Janeiro, RJ, Brasil.

BACKGROUND The insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation. OBJECTIVES To determine the occurrence of alternative splicing in chitinases of L. longipalpis. METHODS We sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA. FINDINGS We showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae. MAIN CONCLUSIONS We show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.
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http://dx.doi.org/10.1590/0074-02760170179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5722264PMC
February 2018

The sandfly Lutzomyia longipalpis LL5 embryonic cell line has active Toll and Imd pathways and shows immune responses to bacteria, yeast and Leishmania.

Parasit Vectors 2016 Apr 20;9:222. Epub 2016 Apr 20.

Laboratório de Biologia Molecular de Parasitas e Vetores, Instituto Oswaldo Cruz - Fiocruz, Av. Brasil 4365, 21045-900, Rio de Janeiro, RJ, Brazil.

Background: Lutzomyia longipalpis is the main vector of visceral leishmaniasis in Latin America. Sandfly immune responses are poorly understood. In previous work we showed that these vector insects respond to bacterial infections by modulating a defensin gene expression and activate the Imd pathway in response to Leishmania infection. Aspects of innate immune pathways in insects (including mosquito vectors of human diseases) have been revealed by studying insect cell lines, and we have previously demonstrated antiviral responses in the L. longipalpis embryonic cell line LL5.

Methods: The expression patterns of antimicrobial peptides (AMPs) and transcription factors were evaluated after silencing the repressors of the Toll pathway (cactus) and Imd pathway (caspar). AMPs and transcription factor expression patterns were also evaluated after challenge with heat-killed bacteria, heat-killed yeast, or live Leishmania.

Results: These studies showed that LL5 cells have active Toll and Imd pathways, since they displayed an increased expression of AMP genes following silencing of the repressors cactus and caspar, respectively. These pathways were also activated by challenges with bacteria, yeast and Leishmania infantum chagasi.

Conclusions: We demonstrated that L. longipalpis LL5 embryonic cells respond to immune stimuli and are therefore a good model to study the immunological pathways of this important vector of leishmaniasis.
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http://dx.doi.org/10.1186/s13071-016-1507-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4839078PMC
April 2016

Disruption of the peritrophic matrix by exogenous chitinase feeding reduces fecundity in Lutzomyia longipalpis females.

Mem Inst Oswaldo Cruz 2012 Jun;107(4):543-5

Laboratório de Biologia Molecular de Parasitos e Vetores, Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro, RJ, Brazil.

Lutzomyia longipalpis is the most important vector of visceral leishmaniasis in Brazil. When female sandflies feed on blood, a peritrophic matrix (PM) is formed around the blood bolus. The PM is secreted by midgut cells and composed of proteins, glycoproteins and chitin microfibrils. The PM functions as both a physical barrier against pathogens present in the food bolus and blood meal digestion regulator. Previous studies of mosquitoes and sandflies have shown that the absence of a PM, resulting from adding an exogenous chitinase to the blood meal, accelerates digestion. In the present study, we analysed biological factors associated with the presence of a PM in L. longipalpis females. Insects fed blood containing chitinase (BCC) accelerated egg-laying relative to a control group fed blood without chitinase. However, in the BCC-fed insects, the number of females that died without laying eggs was higher and the number of eggs laid per female was lower. The eggs in both groups were viable and generated adults. Based on these data, we suggest that the absence of a PM accelerates nutrient acquisition, which results in premature egg production and oviposition; however, the absence of a PM reduces the total number of eggs laid per female. Reduced fecundity in the absence of a PM may be due to inefficient nutrient conversion or the loss of the protective role of the PM.
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http://dx.doi.org/10.1590/s0074-02762012000400016DOI Listing
June 2012

Trypsin-like serine proteases in Lutzomyia longipalpis--expression, activity and possible modulation by Leishmania infantum chagasi.

PLoS One 2010 May 18;5(5):e10697. Epub 2010 May 18.

Laboratório de Biologia Molecular de Parasitas e Vetores, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil.

Background: Midgut enzymatic activity is one of the obstacles that Leishmania must surpass to succeed in establishing infection. Trypsins are abundant digestive enzymes in most insects. We have previously described two trypsin cDNAs of L. longipalpis: one (Lltryp1) with a bloodmeal induced transcription pattern, the other (Lltryp2) with a constitutive transcription pattern. We have now characterized the expression and activity of trypsin-like proteases of Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil.

Methodology And Principal Findings: In order to study trypsin expression profiles we produced antibodies against peptides specific for Lltryp1 and Lltryp2. The anti-Lltryp1-peptide antibody revealed a band of 28 kDa between 6 and 48 hours. The anti-Lltryp2 peptide antibody did not evidence any band. When proteinaceous substrates (gelatin, hemoglobin, casein or albumin) were co-polymerized in polyacrylamide gels, insect midguts obtained at 12 hours after feeding showed a unique proteolytic pattern for each substrate. All activity bands were strongly inhibited by TLCK, benzamidine and 4-amino-benzamidine, indicating that they are trypsin-like proteases. The trypsin-like activity was also measured in vitro at different time points after ingestion of blood or blood containing Leishmania infantum chagasi, using the chromogenic substrate BArhoNA. L. longipalpis females fed on blood infected with L. i. chagasi had lower levels of trypsin activity after 12 and 48 hours than non-infected insects, suggesting that the parasite may have a role in this modulation.

Conclusions And Significance: Trypsins are important and abundant digestive enzymes in L. longipalpis. Protein production and enzymatic activity followed previously identified gene expression of a blood modulated trypsin gene. A decrease of enzymatic activity upon the parasite infection, previously detected mostly in Old World vectors, was detected for the first time in the natural vector-parasite pair L. longipalpis-L. i. chagasi.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0010697PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872664PMC
May 2010

Constitutive and blood meal-induced trypsin genes in Lutzomyia longipalpis.

Arch Insect Biochem Physiol 2007 Oct;66(2):53-63

Laboratório de Biologia Molecular de Tripanosomatídeos e Flebotomíneos, Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro, Brasil.

Trypsins constitute some of the most abundant midgut digestive proteases expressed by hematophagous insects upon blood feeding. In addition to their role in the digestion of the blood meal, these proteases also have been implicated in the ability of certain pathogens to infect their natural vector. In sand flies, digestive proteases including trypsins were associated with early killing of Leishmania and are believed to play a role in the species-specificity dictating sand fly vectorial capacity. Our group is involved in studies of midgut digestive proteases in the sand fly Lutzomyia longipalpis, the principal vector of visceral leishmaniasis in Brazil. Here we report on the identification of two cDNAs, Lltryp1 and Lltryp2, which code for putative midgut trypsins in L. longipalpis. Analyses of RNA abundance using semi-quantitative RT-PCR show a different pattern of expression between the two genes. Lltryp1 expression remains undetected until blood feeding and reaches a peak at 12 h post-blood meal (PBM), returning to pre-blood meal levels at 72 h PBM. Additionally, Lltryp1 expression is undetected during larval development. Lltryp2, on the other hand, is constitutively expressed as high levels in the non-blood fed female, but is reduced upon blood feeding. At the end of the digestive cycle, Lltryp2 regains its pre-blood meal levels. This cDNA also is present in all developmental stages and in adult males. This pattern of expression is reminiscent of what is seen in mosquitoes and Old World sand flies, but has characteristics that are unique to L. longipalpis.
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http://dx.doi.org/10.1002/arch.20198DOI Listing
October 2007