Comparative and functional triatomine genomics reveals reductions and expansions in insecticide resistance-related gene families.

BACKGROUND: Triatomine insects are vectors of Trypanosoma cruzi, a protozoan parasite that is the causative agent of Chagas' disease. This is a neglected disease affecting approximately 8 million people in Latin America. The existence of diverse pyrethroid resistant populations of at least two species demonstrates the potential of triatomines to develop high levels of insecticide resistance. Therefore, the incorporation of strategies for resistance management is a main concern for vector control programs. Three enzymatic superfamilies are thought to mediate xenobiotic detoxification and resistance: Glutathione Transferases (GSTs), Cytochromes P450 (CYPs) and Carboxyl/Cholinesterases (CCEs). Improving our knowledge of key triatomine detoxification enzymes will strengthen our understanding of insecticide resistance processes in vectors of Chagas' disease. METHODS AND FINDINGS: The discovery and description of detoxification gene superfamilies in normalized transcriptomes of three triatomine species: Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis is presented. Furthermore, a comparative analysis of these superfamilies among the triatomine transcriptomes and the genome of Rhodnius prolixus, also a triatomine vector of Chagas' disease, and other well-studied insect genomes was performed. The expression pattern of detoxification genes in R. prolixus transcriptomes from key organs was analyzed. The comparisons reveal gene expansions in Sigma class GSTs, CYP3 in CYP superfamily and clade E in CCE superfamily. Moreover, several CYP families identified in these triatomines have not yet been described in other insects. Conversely, several groups of insecticide resistance related enzymes within each enzyme superfamily are reduced or lacking in triatomines. Furthermore, our qRT-PCR results showed an increase in the expression of a CYP4 gene in a T. infestans population resistant to pyrethroids. These results could point to an involvement of metabolic detoxification mechanisms on the high levels of pyrethroid resistance detected in triatomines from the Gran Chaco ecoregion. CONCLUSIONS AND SIGNIFICANCE: Our results help to elucidate the potential insecticide resistance mechanisms in vectors of Chagas' disease and provide new relevant information for this field. This study shows that metabolic resistance might be a contributing cause of the high pyrethroid resistance observed in wild T. infestans populations from the Gran Chaco ecoregion, area in which although subjected to intense pyrethroid treatments, vector control has failed. This study opens new avenues for further functional studies on triatomine detoxification mechanisms.

Cap binding-independent recruitment of eIF4E to cytoplasmic foci.

Eukaryotic translation initiation factor 4E (eIF4E) is required for cap-dependent initiation. In addition, eIF4E occurs in cytoplasmic foci such as processing bodies (PB) and stress granules (SG). We examined the role of key functional amino acid residues of eIF4E in the recruitment of this protein to cytoplasmic foci. We demonstrate that tryptophan residues required for mRNA cap recognition are not required for the recruitment of eIF4E to SG or PB. We show that a tryptophan residue required for protein-protein interactions is essential for the accumulation of eIF4E in granules. Moreover, we show, by the analysis of two Drosophila eIF4E isoforms, that the tryptophan residue is the common feature for eIF4E for the transfer of active mRNA from polysomes to other ribonucleoprotein particles in the cytoplasm. This residue resides in a putative interaction domain different than the eIF4E-BP domain. We conclude that protein-protein interactions rather than interactions with the mRNA are essential for the recruitment of eIF4E and for a putative nucleation function.

Metagenomic analysis of taxa associated with Lutzomyia longipalpis, vector of visceral leishmaniasis, using an unbiased high-throughput approach.

BACKGROUND: Leishmaniasis is one of the most diverse and complex of all vector-borne diseases worldwide. It is caused by parasites of the genus Leishmania, obligate intramacrophage protists characterised by diversity and complexity. Its most severe form is visceral leishmaniasis (VL), a systemic disease that is fatal if left untreated. In Latin America VL is caused by Leishmania infantum chagasi and transmitted by Lutzomyia longipalpis. This phlebotomine sandfly is only found in the New World, from Mexico to Argentina. In South America, migration and urbanisation have largely contributed to the increase of VL as a public health problem. Moreover, the first VL outbreak was recently reported in Argentina, which has already caused 7 deaths and 83 reported cases. METHODOLOGY/PRINCIPAL FINDINGS: An inventory of the microbiota associated with insect vectors, especially of wild specimens, would aid in the development of novel strategies for controlling insect vectors. Given the recent VL outbreak in Argentina and the compelling need to develop appropriate control strategies, this study focused on wild male and female Lu. longipalpis from an Argentine endemic (Posadas, Misiones) and a Brazilian non-endemic (Lapinha Cave, Minas Gerais) VL location. Previous studies on wild and laboratory reared female Lu. longipalpis have described gut bacteria using standard bacteriological methods. In this study, total RNA was extracted from the insects and submitted to high-throughput pyrosequencing. The analysis revealed the presence of sequences from bacteria, fungi, protist parasites, plants and metazoans. CONCLUSIONS/SIGNIFICANCE: This is the first time an unbiased and comprehensive metagenomic approach has been used to survey taxa associated with an infectious disease vector. The identification of gregarines suggested they are a possible efficient control method under natural conditions. Ongoing studies are determining the significance of the associated taxa found in this study in a greater number of adult male and female Lu. longipalpis samples from endemic and non-endemic locations. A particular emphasis is being given to those species involved in the biological control of this vector and to the etiologic agents of animal and plant diseases.

Patterns of transient expression of the arenavirus nucleocapsid protein gene in transfected cells

Los genes clonados de las proteínas de nucleocápside, N, de los arenavirus Junín y LCM (choriomeningitis linfocitaria) se insertaron en el vector de expresión pKG4 regulado por el promotor tardío del virus SV40. Cuando estas construcciones se utilizaron para transfectar las líneas celulares BHK-21 (fibroblastos de hamster lactante) y CV-1 (fibroblastos de riñón de mono verde africano) se observó la expresión transiente de un polipéptido de tamaño e inmunoreactividad indistinguible de la proteína N sintetizada durante una infección viral. El análisis por inmunofluorescencia reveló un patrón de distribución intracelular semejante al observado en células infectadas. Este patrón presentó variaciones desde una tinción citoplásmica difusa hasta gránulos citoplásmicos dispersos o concentrados en la zona perinuclear. La asociación de la proteína N con gránulos basófilos es semejante a la descripta en el efecto citópático causado por los arenavirus en las células infectadas, y podría relacionarse con las características fisicoquímicas de la proteina N, que contiene numerosas secuencias de aminoácidos básicos capaces de interactuar con ácidos ribonucleicos celulares

Patterns of transient expression of the arenavirus nucleocapsid protein gene in transfected cells

Los genes clonados de las proteínas de nucleocápside, N, de los arenavirus Junín y LCM (choriomeningitis linfocitaria) se insertaron en el vector de expresión pKG4 regulado por el promotor tardío del virus SV40. Cuando estas construcciones se utilizaron para transfectar las líneas celulares BHK-21 (fibroblastos de hamster lactante) y CV-1 (fibroblastos de riñón de mono verde africano) se observó la expresión transiente de un polipéptido de tamaño e inmunoreactividad indistinguible de la proteína N sintetizada durante una infección viral. El análisis por inmunofluorescencia reveló un patrón de distribución intracelular semejante al observado en células infectadas. Este patrón presentó variaciones desde una tinción citoplásmica difusa hasta gránulos citoplásmicos dispersos o concentrados en la zona perinuclear. La asociación de la proteína N con gránulos basófilos es semejante a la descripta en el efecto citópático causado por los arenavirus en las células infectadas, y podría relacionarse con las características fisicoquímicas de la proteina N, que contiene numerosas secuencias de aminoácidos básicos capaces de interactuar con ácidos ribonucleicos celulares (AU)