Real-time qPCR confirmation of cell death-related genes in <i>Culex</i> treated with Bin toxin at 6, 12, or 18 h.

<p>Bar charts represent -fold changes in caspase-1, caspase-3, and cytochrome c gene expression levels relative to levels in the non-treated <i>C</i>. <i>quinquefasciatus</i> larval gut. Error bars indicate the standard error of the mean from four biological replicates by Student’s <i>t</i>-test. Numeric data is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175473#pone.0175473.s003" target="_blank">S3 Table</a>.</p

Expression of dengue virus and Zika virus NS2B-NS3pro constructs alter cellular fatty acids, but co-expression with a Zika virus virus-like particle is detrimental to virus-like particle expression

Abstract Objective Studies have shown that Flavivirus infection remodels the host cell to favour viral replication. In particular, the host cell lipid profile is altered, and it has been proposed that this process alters membrane fluidity to allow wrapping of the outer structural proteins around the viral nucleocapsid. We investigated whether expression of the Zika virus (ZIKV) and dengue virus (DENV) protease induced alterations in the cellular lipid profile, and subsequently whether co-expression of these proteases with VLP constructs was able to improve VLP yield. Results Our results showed that both ZIKV and DENV proteases induced alterations in the lipid profile, but that both active and inactive proteases induced many of the same changes. Neither co-transfection of protease and VLP constructs nor bicistronic vectors allowing expression of both protease and VLP separated by a cell cleavable linker improved VLP yield, and indeed many of the constructs showed significantly reduced VLP production. Further work in developing improved VLP expression platforms is required

Molecular analysis of <i>Culex quinquefasciatus</i> larvae responses to <i>Lysinibacillus sphaericus</i> Bin toxin

<div><p><i>Lysinibacillus sphaericus</i> produces the mosquito larvicidal binary toxin consisting of BinA and BinB, which are both required for toxicity against <i>Culex</i> and <i>Anopheles</i> larvae. The molecular mechanisms behind Bin toxin-induced damage remain unexplored. We used whole-genome microarray-based transcriptome analysis to better understand how <i>Culex</i> larvae respond to Bin toxin treatment at the molecular level. Our analyses of <i>Culex quinquefasciatus</i> larvae transcriptome changes at 6, 12, and 18 h after Bin toxin treatment revealed a wide range of transcript signatures, including genes linked to the cytoskeleton, metabolism, immunity, and cellular stress, with a greater number of down-regulated genes than up-regulated genes. Bin toxin appears to mainly repress the expression of genes involved in metabolism, the mitochondrial electron transport chain, and the protein transporter of the outer/inner mitochondrial membrane. The induced genes encode proteins linked to mitochondrial-mediated apoptosis and cellular detoxification including autophagic processes and lysosomal compartments. This study is, to our knowledge, the first microarray analysis of Bin toxin-induced transcriptional responses in <i>Culex</i> larvae, providing a basis for an in-depth understanding of the molecular nature of Bin toxin-induced damage.</p></div

Functional classification of the Bin toxin intoxication transcriptome.

<p>The significantly changed transcripts in <i>Culex</i> after Bin toxin treatment for 6, 12, or 18 h were subsequently classified into 11 functional groups: 1) cytoskeletal and structural function (CST); 2) chemosensory reception (CSR); 3) blood and sugar food digestive (DIG); 4) diverse functions (DIV); 5) immunity (IMM); 6) metabolism (MET); 7) proteolysis (PRT); 8) redox, stress, and mitochondrion (RSM); 9) replication, transcription, and translation (RTT); 10) transport (TRP); and 11) unknown function (UNK). Bin toxin treatment responsive gene expression data is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175473#pone.0175473.s002" target="_blank">S2 Table</a>.</p

Highly Effective Broad Spectrum Chimeric Larvicide That Targets Vector Mosquitoes Using a Lipophilic Protein

Abstract Two mosquitocidal bacteria, Bacillus thuringiensis subsp. israelensis (Bti) and Lysinibacillus sphaericus (Ls) are the active ingredients of commercial larvicides used widely to control vector mosquitoes. Bti’s efficacy is due to synergistic interactions among four proteins, Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa, whereas Ls’s activity is caused by Bin, a heterodimer consisting of BinA, the toxin, and BinB, a midgut-binding protein. Cyt1Aa is lipophilic and synergizes Bti Cry proteins by increasing midgut binding. We fused Bti’s Cyt1Aa to Ls’s BinA yielding a broad-spectrum chimeric protein highly mosquitocidal to important vector species including Anopheles gambiae, Culex quinquefasciatus, and Aedes aegypti, the latter an important Zika and Dengue virus vector insensitive to Ls Bin. Aside from its vector control potential, our bioassay data, in contrast to numerous other reports, provide strong evidence that BinA does not require conformational interactions with BinB or microvillar membrane lipids to bind to its intracellular target and kill mosquitoes