Functional characterization of the translation initiation factor eIF4E of Echinococcus granulosus.

The eukaryotic initiation factor 4E (eIF4E) specifically recognizes the 5' mRNA cap, a rate-limiting step in the translation initiation process. Although the 7-methylguanosine cap (MMGcap) is the most common 5' cap structure in eukaryotes, the trans-splicing process that occurs in several organism groups, including nematodes and flatworms, leads to the addition of a trimethylguanosine cap (TMGcap) to some RNA transcripts. In some helminths, eIF4E can have a dual capacity to bind both MMGcap and TMGcap. In the present work, we evaluated the distribution of eIF4E protein sequences in platyhelminths and we showed that only one gene coding for eIF4E is present in most parasitic flatworms. Based on this result, we cloned the Echinococcus granulosus cDNA sequence encoding eIF4E in Escherichia coli, expressed the recombinant eIF4E as a fusion protein to GST, and tested its ability to capture mRNAs through the 5' cap using pull-down assay and qPCR. Our results indicate that the recombinant eIF4E was able to bind preferentially 5'-capped mRNAs compared with rRNAs from total RNA preparations of E. granulosus. By qPCR, we observed an enrichment in MMG-capped mRNA compared with TMG-capped mRNAs among Eg-eIF4E-GST pull-down RNAs. Eg-eIF4E structural model using the Schistosoma mansoni eIF4E as template showed to be well preserved with only a few differences between chemically similar amino acid residues at the binding sites. These data showed that E. granulosus eIF4E can be used as a potential tool to study full-length 5'-capped mRNA, besides being a potential drug target against parasitic flatworms.

First description of Echinococcus ortleppi and cystic echinococcosis infection status in Chile.

Cystic echinococcosis (CE), a parasitic disease caused by the cestode Echinococcus granulosus sensu lato (s.l.), is a worldwide zoonotic infection. Although endemic in Chile, information on the molecular characteristics of CE in livestock remains scarce. Therefore we aimed to describe the status of infection with E. granulosus s.l. in cattle from central Chile and also to contribute to the study of the molecular epidemiology of this parasite. According to our results, the prevalence of CE is 18.84% in cattle, similar to previous reports from Chile, suggesting that the prevalence in Santiago Metropolitan area has not changed in the last 30 years. Most of the cysts were found only in lungs (51%), followed by concurrent infection in liver and lungs (30%), and only liver (19%). Molecular characterization of the genetic diversity and population structure of E. granulosus s.l. from cattle in central Chile was performed using a section of the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene. E. granulosus sensu stricto (s.s.) (G1-G3 genotypes) was confirmed by RFLP-PCR to be the dominant species affecting cattle (284 samples/290 samples); we also report for the first time in Chile the presence of E. ortleppi (G5 genotype) (2 samples/61 samples). The Chilean E. granulosus s.s. parsimony network displayed 1 main haplotype. Additional studies using isolates from many locations across Chile and different intermediate hosts will provide more data on the molecular structure of E. granulosus s.s. within this region. Likewise, investigations of the importance of E. ortleppi in human infection in Chile deserve future attention.

Supporting data for comparative proteomic analysis of Listeria monocytogenes ATCC 7644 exposed to a sublethal concentration of nisin.

Here we provide the LC-MS/MS data from a comparative analysis of Listeria monocytogenes ATCC 7644 treated and non-treated with a sublethal concentration of nisin (10(-3) mg/mL). Protein samples were analyzed by multidimensional protein identification technology (MudPIT) approach, in an off-line configuration. The raw MS/MS data allowed the detection of 49,591 spectra which resulted in 576 protein identifications. After Scaffold validation, 179 proteins were identified with high confidence. A label-free quantitative analysis based of normalized spectral abundance factor (NSAF) was used and 13 proteins were found differentially expressed between nisin-treated and non-treated cells. Gene ontology analysis of differentially expressed proteins revealed that most of them are correlated to metabolic process, oxidative stress response mechanisms and molecular binding. A detailed analysis and discussion of these data may be found in Miyamoto et al. [1].

The Trypanosoma rangeli trypomastigote surfaceome reveals novel proteins and targets for specific diagnosis.

Sympatric distribution and sharing of hosts and antigens by Trypanosoma rangeli and Trypanosoma cruzi, the etiological agent of Chagas' disease, often incur in misdiagnosis and improper epidemiological inferences. Many secreted and surface proteins (SP) have been described as important antigens shared by these species. This work describes the T. rangeli surfaceome obtained by gel-free (LC-ESI-MS/MS) and gel-based (GeLC-ESI-MS/MS) proteomic approaches, and immunoblotting analyses and the comparison of these SP with T. cruzi. A total of 138 T. rangeli proteins and 343 T. cruzi proteins were obtained, among which, 42 and 157 proteins were exclusively identified in T. rangeli or T. cruzi trypomastigotes, respectively. Immunoblotting assays using sera from experimentally infected mice revealed a distinct band pattern for each species. MS/MS analysis of T. rangeli exclusive bands revealed two unique GP63-related proteins and flagellar calcium-binding protein. Also, a ~32kDa band composed of 12 distinct proteins was exclusively recognized by anti-T. cruzi serum. This highly sensitive proteomic assessment of surface proteins characterized the T. rangeli surfaceome, revealing several differences and similarities between these two parasites. The study reports new T. rangeli-specific proteins with promising use in differential diagnosis from T. cruzi. BIOLOGICAL SIGNIFICANCE: In this manuscript, we report the first proteomic analysis of the T. rangeli surface (surfaceome), a non-pathogenic parasite occurring in sympatry with T. cruzi, the etiological agent of Chagas disease. This comparative proteomic analysis was performed using high-throughput in-gel and gel-free proteomic approaches combined with immunoblotting, allowing us to identify new T. rangeli-specific proteins with promising use in differential serodiagnosis, among several other protein not previously reported for this taxon. Additionally, cross-recognition assays showed that T. cruzi surface proteins were recognized by heterologous serum (anti-T. rangeli) that strengthens the possibility of misdiagnosis of Chagas disease in humans and other mammals. Thus, this work provides new insights to understand the serological cross-reactivity between T. cruzi and T. rangeli, as well as, the identification of targets for specific T. rangeli diagnosis as revealed by the comparative surfaceome analysis. We strongly believe that this research is of importance to the readers of Journal of Proteomics since it provides new potential markers for diagnosis of both T. cruzi and T. rangeli parasites increasing the spectrum of specific targets for unambiguous diagnosis of T. rangeli and T. cruzi infections, besides describing new approaches to assess the trypanosomatids proteome.

Variable number of tandem aminoacid repeats in adhesion-related CDS products in Mycoplasma hyopneumoniae strains.

The Mycoplasma hyopneumoniae genome contains at least 22 regions with a variable number of tandem nucleotide repeats (VNTRs) within coding DNA sequences (CDSs). In this work, the VNTR-containing CDSs were analysed in order to evaluate their degree of variation, possible correlations with antigenic properties, and their potential to be used as a basis for a strain typing PCR assay. We have analysed the VNTRs in five M. hyopneumoniae strains (J, 7448, 7422, PMS, and 232), based on published genomic sequences and on amplified and sequenced DNA segments. These VNTRs are distributed among 12 genes, most of which encode putative surface proteins, including known adhesins. The number of repeat units in any of the VNTRs is highly variable among the analysed strains, but they are, without exception, translationally in frame, and, therefore, code for a variable number of aminoacid repeats (VNTARs). These VNTARs determine putative structural, physicochemical and antigenic variations in the corresponding proteins, with potential implications for aspects associated to M. hyopneumoniae pathogenicity, such as cell adhesion and interactions with the host immune system. Considering that the characterized VNTARs are relatively stable, at least in vitro, and their sizes are strain-specific, we have developed a VNTR-based PCR assay for M. hyopneumoniae strain identification, useful for enzootic pneumonia (EP) diagnosis, strain typing, and distinction of circulating field isolates from vaccine strains in animals vaccinated against EP.