Aedes cadherin receptor that mediates Bacillus thuringiensis Cry11A toxicity is essential for mosquito development.

Aedes cadherin (AaeCad, AAEL024535) has been characterized as a receptor for Bacillus thuringiensis subsp. israelensis (Bti) Cry11A toxins. However, its role in development is still unknown. In this study, we modified the cadherin gene using ZFN and TALEN. Even though we obtained heterozygous deletions, no homozygous mutants were viable. Because ZFN and TALEN have lower off-targets than CRISPR/Cas9, we conclude the cadherin gene is essential for Aedes development. In contrast, in lepidopteran insects loss of a homologous cadherin does not appear to be lethal, since homozygous mutants are viable. To analyze the role of AaeCad in vivo, we tagged this protein with EGFP using CRISPR-Cas9-mediated homologous recombination and obtained a homozygous AaeCad-EGFP line. Addition of Aedes Rad51 mRNA enhanced the rate of recombination. We then examined AaeCad protein expression in most tissues and protein dynamics during mosquito development. We observe that AaeCad is expressed in larval and adult midgut-specific manner and its expression pattern changed during the mosquito development. Confocal images showed AaeCad has high expression in larval caecae and posterior midgut, and also in adult midgut. Expression of AaeCad is observed primarily in the apical membranes of epithelial cells, and not in cell-cell junctions. The expression pattern observed suggests AaeCad does not appear to play a role in these junctions. However, we cannot exclude its role beyond cell-cell adhesion in the midgut. We also observed that Cry11A bound to the apical side of larval gastric caecae and posterior midgut cells exactly where AaeCad-EGFP was expressed. Their co-localization suggests that AaeCad is indeed a receptor for the Cry11A toxin. Using this mosquito line we also observed that low doses of Cry11A toxin caused the cells to slough off membranes, which likely represents a defense mechanism, to limit cell damage from Cry11A toxin pores formed in the cell membrane

Functional characterization of a glutamate/aspartate transporter from the mosquito Aedes aegypti

Glutamate elicits a variety of effects in insects, including inhibitory and excitatory signals at both neuromuscular junctions and brain. Insect glutamatergic neurotransmission has been studied in great depth especially from the standpoint of the receptor-mediated effects, but the molecular mechanisms involved in the termination of the numerous glutamatergic signals have only recently begun to receive attention. In vertebrates, glutamatergic signals are terminated by Na^+/K^+-dependent high-affinity excitatory amino acid transporters (EAAT), which have been cloned and characterized extensively. Cloning and characterization of a few insect homologues have followed, but functional information for these homologues is still limited. Here we report a study conducted on a cloned mosquito EAAT homologue isolated from the vector of the dengue virus, Aedes aegypti. The deduced amino acid sequence of the protein, AeaEAAT, exhibits 40–50% identity with mammalian EAATs, and 45–50% identity to other insect EAATs characterized thus far. It transports l-glutamate as well as l- and d-aspartate with high affinity in the micromolar range, and demonstrates a substrate-elicited anion conductance when heterologously expressed in Xenopus laevis oocytes, as found with mammalian homologues. Analysis of the spatial distribution of the protein demonstrates high expression levels in the adult thorax, which is mostly observed in the thoracic ganglia. Together, the work presented here provides a thorough examination of the role played by glutamate transport in Ae. aegypti

Functional characterization of Aedes aegypti alkaline phosphatase ALP1 involved in the toxicity of Cry toxins from Bacillus thuringiensis subsp. israelensis and jegathesan

Presently three major groups of proteins from Aedes aegypti, cadherin, alkaline phosphatases (ALP) and aminopeptidases N (APN), have been identified as Cry11Aa toxin receptors. To further characterize their role on toxicity, transgenic mosquitoes with silenced Aedes cadherin expression were previously generated and the role of cadherin in mediating the toxicity of four different mosquitocidal toxins (Cry11Aa, Cry11Ba, Cry4Aa and Cry4Ba) was demonstrated. Here, we investigated the role of another reported Cry11Aa receptor, ALP1. As with Aedes cadherin, this protein is localized in the apical cell membrane of distal and proximal gastric caecae and the posterior midgut. We also successfully generated transgenic mosquitoes that knockdowned ALP1 transcript levels using an inducible Aedes heat shock promoter, Hsp70A driving dsALP1RNA. Four different mosquitocidal toxins were used for larval bioassays against this transgenic mosquito. Bioassay results show thatCry11Aa toxicity to these transgenic larvae following a heat shock decreased (4.4 fold) and Cry11Ba toxicity is slightly attenuated. But Cry4Aa and Cry4Ba toxicity to ALP1 silenced larvae is unchanged. Without heat shock, toxicity of all four toxins does not change, suggesting this heat shock promoter is heat-inducible. Notably, transgenic mosquitoes with ALP1 knockdown are about 3.7 times less resistant to Cry11Aa toxin than those with Aedes cadherin knockdown. These results demonstrate that the ALP1 is an important secondary receptor for Cry11Aa and Cry11Ba, but it might not be involved in Cry4Aa and Cry4Ba toxicity

Transcriptional cellular responses in midgut tissue of Aedes aegypti larvae following intoxication with Cry11Aa toxin from Bacillus thuringiensis.

BackgroundAlthough much is known about the mechanism of action of Bacillus thuringiensis Cry toxins, the target tissue cellular responses to toxin activity is less understood. Previous transcriptomic studies indicated that significant changes in gene expression occurred during intoxication. However, most of these studies were done in organisms without a sequenced and annotated reference genome. A reference genome and transcriptome is available for the mosquito Aedes aegypti, and its importance as a disease vector has positioned its biological control as a primary health concern. Through RNA sequencing we sought to determine the transcriptional changes observed during intoxication by Cry11Aa in A. aegypti and to analyze possible defense and recovery mechanisms engaged after toxin ingestion.ResultsIn this work the changes in the transcriptome of 4(th) instar A. aegypti larvae exposed to Cry11Aa toxin for 0, 3, 6, 9, and 12 h were analyzed. A total of 1060 differentially expressed genes after toxin ingestion were identified with two bioconductoR packages: DESeq2 and EdgeR. The most important transcriptional changes were observed after 9 or 12 h of toxin exposure. GO enrichment analysis of molecular function and biological process were performed as well as Interpro protein functional domains and pBLAST analyses. Up regulated processes include vesicular trafficking, small GTPase signaling, MAPK pathways, and lipid metabolism. In contrast, down regulated functions are related to transmembrane transport, detoxification mechanisms, cell proliferation and metabolism enzymes. Validation with RT-qPCR showed large agreement with Cry11Aa intoxication since these changes were not observed with untreated larvae or larvae treated with non-toxic Cry11Aa mutants, indicating that a fully functional pore forming Cry toxin is required for the observed transcriptional responses.ConclusionsThis study presents the first transcriptome of Cry intoxication response in a fully sequenced insect, and reveals possible conserved cellular processes that enable larvae to contend with Cry intoxication in the disease vector A. aegypti. We found some similarities of the mosquito responses to Cry11Aa toxin with previously observed responses to other Cry toxins in different insect orders and in nematodes suggesting a conserved response to pore forming toxins. Surprisingly some of these responses also correlate with transcriptional changes observed in Bti-resistant and Cry11Aa-resistant mosquito larvae

The transporter-like protein inebriated mediates hyperosmotic stimuli through intracellular signaling

We cloned the inebriated homologue MasIne from Manduca sexta and expressed it in Xenopus laevis oocytes. MasIne is homologous to neurotransmitter transporters but no transport was observed with a number of putative substrates. Oocytes expressing MasIne respond to hyperosmotic stimulation by releasing intracellular Ca(2+), as revealed by activation of the endogenous Ca(2+)-activated Cl(-) current. This Ca(2+) release requires the N-terminal 108 amino acid residues of MasIne and occurs via the inositol trisphosphate pathway. Fusion of the N terminus to the rat gamma-aminobutyric acid transporter (rGAT1) also renders rGAT1 responsive to hyperosmotic stimulation. Immunohistochemical analyses show that MasIne and Drosophila Ine have similar tissue distribution patterns, suggesting functional identity. Inebriated is expressed in tissues and cells actively involved in K(+) transport, which suggests that it may have a role in ion transport, particularly of K(+). We propose that stimulation of MasIne releases intracellular Ca(2+) in native tissues, activating Ca(2+)-dependent K(+) channels, and leading to K(+) transport

Investigation of HIV-1 Gag binding with RNAs and Lipids using Atomic Force Microscopy

Atomic Force Microscopy was utilized to study the morphology of Gag, {\Psi}RNA, and their binding complexes with lipids in a solution environment with 0.1{\AA} vertical and 1nm lateral resolution. TARpolyA RNA was used as a RNA control. The lipid used was phospha-tidylinositol-(4,5)-bisphosphate (PI(4,5)P2). The morphology of specific complexes Gag-{\Psi}RNA, Gag-TARpolyA RNA, Gag-PI(4,5)P2 and PI(4,5)P2-{\Psi}RNA-Gag were studied. They were imaged on either positively or negatively charged mica substrates depending on the net charges carried. Gag and its complexes consist of monomers, dimers and tetramers, which was confirmed by gel electrophoresis. The addition of specific {\Psi}RNA to Gag is found to increase Gag multimerization. Non-specific TARpolyA RNA was found not to lead to an increase in Gag multimerization. The addition PI(4,5)P2 to Gag increases Gag multimerization, but to a lesser extent than {\Psi}RNA. When both {\Psi}RNA and PI(4,5)P2 are present Gag undergoes comformational changes and an even higher degree of multimerization

DNA-encoded nucleosome occupancy is associated with transcription levels in the human malaria parasite Plasmodium falciparum.

BackgroundIn eukaryotic organisms, packaging of DNA into nucleosomes controls gene expression by regulating access of the promoter to transcription factors. The human malaria parasite Plasmodium falciparum encodes relatively few transcription factors, while extensive nucleosome remodeling occurs during its replicative cycle in red blood cells. These observations point towards an important role of the nucleosome landscape in regulating gene expression. However, the relation between nucleosome positioning and transcriptional activity has thus far not been explored in detail in the parasite.ResultsHere, we analyzed nucleosome positioning in the asexual and sexual stages of the parasite's erythrocytic cycle using chromatin immunoprecipitation of MNase-digested chromatin, followed by next-generation sequencing. We observed a relatively open chromatin structure at the trophozoite and gametocyte stages, consistent with high levels of transcriptional activity in these stages. Nucleosome occupancy of genes and promoter regions were subsequently compared to steady-state mRNA expression levels. Transcript abundance showed a strong inverse correlation with nucleosome occupancy levels in promoter regions. In addition, AT-repeat sequences were strongly unfavorable for nucleosome binding in P. falciparum, and were overrepresented in promoters of highly expressed genes.ConclusionsThe connection between chromatin structure and gene expression in P. falciparum shares similarities with other eukaryotes. However, the remarkable nucleosome dynamics during the erythrocytic stages and the absence of a large variety of transcription factors may indicate that nucleosome binding and remodeling are critical regulators of transcript levels. Moreover, the strong dependency between chromatin structure and DNA sequence suggests that the P. falciparum genome may have been shaped by nucleosome binding preferences. Nucleosome remodeling mechanisms in this deadly parasite could thus provide potent novel anti-malarial targets

A novel minimal in vitro system for analyzing HIV-1 Gag mediated budding

A biomimetic minimalist model membrane was used to study the mechanism and kinetics of cell-free in vitro HIV-1 Gag budding from a giant unilamellar vesicle (GUV). Real time interaction of Gag, RNA and lipid leading to the formation of mini-vesicles was measured using confocal microscopy. Gag forms resolution limited punctae on the GUV lipid membrane. Introduction of the Gag and urea to a GUV solution containing RNA led to the budding of mini-vesicles on the inside surface of the GUV. The GUV diameter showed a linear decrease in time due to bud formation. Both bud formation and decrease in GUV size were proportional to Gag concentration. In the absence of RNA, addition of urea to GUVs incubated with Gag also resulted in subvesicle formation but exterior to the surface. These observations suggest the possibility that clustering of GAG proteins leads to membrane invagination even in the absence of host cell proteins. The method presented here is promising, and allows for systematic study of the dynamics of assembly of immature HIV and help classify the hierarchy of factors that impact the Gag protein initiated assembly of retroviruses such as HIV.Comment: 27 pages, 9 Figures and 0 Table