The midgut transcriptome of Phlebotomus (Larroussius) perniciosus, a vector of Leishmania infantum: comparison of sugar fed and blood fed sand flies

<p>Abstract</p> <p>Background</p> <p>Parasite-vector interactions are fundamental in the transmission of vector-borne diseases such as leishmaniasis. <it>Leishmania </it>development in the vector sand fly is confined to the digestive tract, where sand fly midgut molecules interact with the parasites. In this work we sequenced and analyzed two midgut-specific cDNA libraries from sugar fed and blood fed female <it>Phlebotomus perniciosus </it>and compared the transcript expression profiles.</p> <p>Results</p> <p>A total of 4111 high quality sequences were obtained from the two libraries and assembled into 370 contigs and 1085 singletons. Molecules with putative roles in blood meal digestion, peritrophic matrix formation, immunity and response to oxidative stress were identified, including proteins that were not previously reported in sand flies. These molecules were evaluated relative to other published sand fly transcripts. Comparative analysis of the two libraries revealed transcripts differentially expressed in response to blood feeding. Molecules up regulated by blood feeding include a putative peritrophin (<it>PperPer1</it>), two chymotrypsin-like proteins (<it>PperChym1 </it>and <it>PperChym2</it>), a putative trypsin (<it>PperTryp3</it>) and four putative microvillar proteins (<it>PperMVP1</it>, <it>2</it>, <it>4 </it>and <it>5</it>). Additionally, several transcripts were more abundant in the sugar fed midgut, such as two putative trypsins (<it>PperTryp1 </it>and <it>PperTryp2</it>), a chymotrypsin (<it>PperChym3</it>) and a microvillar protein (<it>PperMVP3</it>). We performed a detailed temporal expression profile analysis of the putative trypsin transcripts using qPCR and confirmed the expression of blood-induced and blood-repressed trypsins. Trypsin expression was measured in <it>Leishmania infantum</it>-infected and uninfected sand flies, which identified the <it>L. infantum</it>-induced down regulation of <it>PperTryp3 </it>at 24 hours post-blood meal.</p> <p>Conclusion</p> <p>This midgut tissue-specific transcriptome provides insight into the molecules expressed in the midgut of <it>P. perniciosus</it>, an important vector of visceral leishmaniasis in the Old World. Through the comparative analysis of the libraries we identified molecules differentially expressed during blood meal digestion. Additionally, this study provides a detailed comparison to transcripts of other sand flies. Moreover, our analysis of putative trypsins demonstrated that <it>L. infantum </it>infection can reduce the transcript abundance of trypsin <it>PperTryp3 </it>in the midgut of <it>P. perniciosus</it>.</p

Trypsin-Like Serine Proteases in Lutzomyia longipalpis – Expression, Activity and Possible Modulation by Leishmania infantum chagasi

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 BArNA. 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

Shotgun Sequencing Analysis of Trypanosoma cruzi I Sylvio X10/1 and Comparison with T. cruzi VI CL Brener

Trypanosoma cruzi is the causative agent of Chagas disease, which affects more than 9 million people in Latin America. We have generated a draft genome sequence of the TcI strain Sylvio X10/1 and compared it to the TcVI reference strain CL Brener to identify lineage-specific features. We found virtually no differences in the core gene content of CL Brener and Sylvio X10/1 by presence/absence analysis, but 6 open reading frames from CL Brener were missing in Sylvio X10/1. Several multicopy gene families, including DGF, mucin, MASP and GP63 were found to contain substantially fewer genes in Sylvio X10/1, based on sequence read estimations. 1,861 small insertion-deletion events and 77,349 nucleotide differences, 23% of which were non-synonymous and associated with radical amino acid changes, further distinguish these two genomes. There were 336 genes indicated as under positive selection, 145 unique to T. cruzi in comparison to T. brucei and Leishmania. This study provides a framework for further comparative analyses of two major T. cruzi lineages and also highlights the need for sequencing more strains to understand fully the genomic composition of this parasite

Antiprogestin mifepristone inhibits the growth of cancer cells of reproductive and non-reproductive origin regardless of progesterone receptor expression

<p>Abstract</p> <p>Background</p> <p>Mifepristone (MF) has been largely used in reproductive medicine due to its capacity to modulate the progesterone receptor (PR). The study of MF has been expanded to the field of oncology; yet it remains unclear whether the expression of PR is required for MF to act as an anti-cancer agent. Our laboratory has shown that MF is a potent inhibitor of ovarian cancer cell growth. In this study we questioned whether the growth inhibitory properties of MF observed in ovarian cancer cells would translate to other cancers of reproductive and non-reproductive origin and, importantly, whether its efficacy is related to the expression of cognate PR.</p> <p>Methods</p> <p>Dose-response experiments were conducted with cancer cell lines of the nervous system, breast, prostate, ovary, and bone. Cultures were exposed to vehicle or increasing concentrations of MF for 72 h and analysed for cell number and cell cycle traverse, and hypodiploid DNA content characteristic of apoptotic cell death. For all cell lines, expression of steroid hormone receptors upon treatment with vehicle or cytostatic doses of MF for 24 h was studied by Western blot, whereas the activity of the G1/S regulatory protein Cdk2 in both treatment groups was monitored <it>in vitro </it>by the capacity of Cdk2 to phosphorylate histone H1.</p> <p>Results</p> <p>MF growth inhibited all cancer cell lines regardless of tissue of origin and hormone responsiveness, and reduced the activity of Cdk2. Cancer cells in which MF induced G1 growth arrest were less susceptible to lethality in the presence of high concentrations of MF, when compared to cancer cells that did not accumulate in G1. While all cancer cell lines were growth inhibited by MF, only the breast cancer MCF-7 cells expressed cognate PR.</p> <p>Conclusions</p> <p>Antiprogestin MF inhibits the growth of different cancer cell lines with a cytostatic effect at lower concentrations in association with a decline in the activity of the cell cycle regulatory protein Cdk2, and apoptotic lethality at higher doses in association with increased hypodiploid DNA content. Contrary to common opinion, growth inhibition of cancer cells by antiprogestin MF is not dependent upon expression of classical, nuclear PR.</p

The population genetics of Trypanosoma cruzi revisited in the light of the predominant clonal evolution model

Comparing the population structure of Trypanosoma cruzi with that of other pathogens, including par-asitic protozoa, fungi, bacteria and viruses, shows that the agent of Chagas disease shares typical traitswith many other species, related to a predominant clonal evolution (PCE) pattern: statistically signif-icant linkage disequilibrium, overrepresented multilocus genotypes, near-clades (genetic subdivisionssomewhat blurred by occasional genetic exchange/hybridization) and "Russian doll" patterns (PCE isobserved, not only at the level of the whole species, but also, within the near-clades). Moreover, T. cruzipopulation structure exhibits linkage with the diversity of several strongly selected genes, with geneexpression profiles, and with some major phenotypic traits. We discuss the evolutionary significance ofthese results, and their implications in terms of applied research (molecular epidemiology/strain typing,analysis of genes of interest, vaccine and drug design, immunological diagnosis) and of experimentalevolution. Lastly, we revisit the long-term debate of describing new species within the T. cruzi taxon

Analyses of 32 Loci Clarify Phylogenetic Relationships among Trypanosoma cruzi Lineages and Support a Single Hybridization prior to Human Contact

Trypanosoma cruzi is the protozoan parasite that causes Chagas disease, a major health problem in Latin America. The genetic diversity of this parasite has been traditionally divided in two major groups: T. cruzi I and II, which can be further divided in six major genetic subdivisions (subgroups TcI-TcVI). T. cruzi I and II seem to differ in important biological characteristics, and are thought to represent a natural division relevant for epidemiological studies and development of prophylaxis. Having a correct reconstruction of the evolutionary history of T. cruzi is essential for understanding the potential connection between the genetic and phenotypic variability of T. cruzi with the different manifestations of Chagas disease. Here we present results from a comprehensive phylogenetic analysis of T. cruzi using more than 26 Kb of aligned sequence data. We show strong evidence that T. cruzi II (TcII-VI) is not a natural evolutionary group but a paraphyletic lineage and that all major lineages of T. cruzi evolved recently (<3 million years ago [mya]). Furthermore, the sequence data is consistent with one major hybridization event having occurred in this species recently (< 1 mya) but well before T. cruzi entered in contact with humans in South America

Tsetse GmmSRPN10 has anti-complement activity and is important for successful establishment of trypanosome infections in the fly midgut

The complement cascade in mammalian blood can damage the alimentary tract of haematophagous arthropods. As such, these animals have evolved their own repertoire of complement-inactivating factors, which are inadvertently exploited by blood-borne pathogens to escape complement lysis. Unlike the bloodstream stages, the procyclic (insect) stage of Trypanosoma brucei is highly susceptible to complement killing, which is puzzling considering that a tsetse takes a bloodmeal every 2–4 days. In this study, we identified four tsetse (Glossina morsitans morsitans) serine protease inhibitors (serpins) from a midgut expressed sequence tag (EST) library (GmmSRPN3, GmmSRPN5, GmmSRPN9 and GmmSRPN10) and investigated their role in modulating the establishment of a T. brucei infection in the midgut. Although not having evolved in a common blood-feeding ancestor, all four serpins have an active site sharing remarkable homology with the human complement C1-inhibitor serpin, SerpinG1. RNAi knockdown of individual GmmSRPN9 and GmmSRPN10 genes resulted in a significant decreased rate of infection by procyclic form T. brucei. Furthermore, recombinant GmmSRPN10 was both able to inhibit the activity of human complement-cascade serine proteases, C1s and Factor D, and to protect the in vitro killing of procyclic trypanosomes when incubated with complement-activated human serum. Thus, the secretion of serpins, which may be part of a bloodmeal complement inactivation system in tsetse, is used by procyclic trypanosomes to evade an influx of fresh trypanolytic complement with each bloodmeal. This highlights another facet of the complicated relationship between T. brucei and its tsetse vector, where the parasite takes advantage of tsetse physiology to further its chances of propagation and transmission