The Homeodomain Protein Ladybird Late Regulates Synthesis of Milk Proteins during Pregnancy in the Tsetse Fly (<i>Glossina morsitans</i>)

<div><p>Regulation of tissue and development specific gene expression patterns underlies the functional specialization of organs in multi-cellular organisms. In the viviparous tsetse fly (<i>Glossina</i>), the female accessory gland is specialized to generate nutrients in the form of a milk-like secretion to support growth of intrauterine larva. Multiple milk protein genes are expressed specifically in the female accessory gland and are tightly linked with larval development. Disruption of milk protein synthesis deprives developing larvae of nutrients and results in extended larval development and/or in abortion. The ability to cause such a disruption could be utilized as a tsetse control strategy. Here we identify and delineate the regulatory sequence of a major milk protein gene (<i>milk gland protein</i> 1:<i>mgp1</i>) by utilizing a combination of molecular techniques in tsetse, <i>Drosophila</i> transgenics, transcriptomics and <i>in silico</i> sequence analyses. The function of this promoter is conserved between tsetse and <i>Drosophila</i>. In transgenic <i>Drosophila</i> the <i>mgp1</i> promoter directs reporter gene expression in a tissue and stage specific manner orthologous to that of <i>Glossina</i>. Analysis of the minimal required regulatory region of <i>mgp1</i>, and the regulatory regions of other <i>Glossina</i> milk proteins identified putative homeodomain protein binding sites as the sole common feature. Annotation and expression analysis of <i>Glossina</i> homeodomain proteins identified <i>ladybird late</i> (<i>lbl</i>) as being accessory gland/fat body specific and differentially expressed between lactating/non-lactating flies. Knockdown of <i>lbl</i> in tsetse resulted in a significant reduction in transcript abundance of multiple milk protein genes and in a significant loss of fecundity. The role of Lbl in adult reproductive physiology is previously unknown. These results suggest that Lbl is part of a conserved reproductive regulatory system that could have implications beyond tsetse to other vector insects such as mosquitoes. This system is critical for tsetse fecundity and provides a potential target for development of a reproductive inhibitor.</p></div

RNA-seq identification of lactation associated homeodomain genes.

<p>RNA–seq statistics comparing the differential expression of putative tsetse homeodomain proteins between transcript datasets from lactating and non-lactating flies. The fold change between samples was tested for significance by Kal's Z-test analysis.</p

Schematic of the 124<i>mgp1</i> regulatory sequence required for tissue and stage specific transgene expression.

<p>This schematic represents a scale model of <i>in silico</i> predicted homeodomain (DHOM) binding sites. The associated table lists the number of DHOM binding sites predicted within the 500 bp upstream sequence from the 11 other milk protein genes and the associated p-value of these findings.</p

Pre and post parturition timecourse of <i>mgp1</i> transcript and protein levels in <i>Glossina</i>.

<p><b>A.</b> Quantitative PCR analysis of <i>mgp1</i> transcript levels just prior to parturition and in 24 hour periods after parturition. Pregnant <i>Glossina</i> flies carrying 3^rd instar larvae were collected and staged by time of parturition within 24 hour windows. The values represent the mean <i>mgp1</i> transcript level from 3 individual flies at each point. Error bars represent standard error. All qPCR data was normalized to tubulin. Statistical notation: a = not significantly different than pregnant; b = significantly different from pregnant (P-value <0.05 by students <i>t</i>-test); c = significantly different from pregnant (P-value <0.01 by students <i>t</i>-test). <b>B.</b> Western blot analysis of MGP1 levels in flies from the same samples as described in A. Values represent quantification of the mean signal intensity from 3 individuals for each point and are normalized to tubulin levels. Statistical notation is as described above.</p

<i>mgp1</i> driven reporter expression in transgenic <i>Drosophila</i>.

<p>Tissue and stage specific expression of <i>mgp1</i> driven <i>ß-gal</i> and <i>egfp</i> reporter genes. All qPCR analyses normalized to Drosophila <i>tub</i>. Error bars represent standard error. Letter groups (a,b,c) represent significant statistical differences of P-value <0.05 by students t-test. <b>A.</b> Accessory gland specific staining of transgenic Drosophila reproductive tissue (<i>mgp1- β-gal-2.0</i>). <b>B.</b> Florescent microscopy of accessory gland specific egfp expression in transgenic Drosophila (<i>mgp1-egfp-509</i>). <b>C.</b> qPCR analysis of sex/stage specific <i>egfp</i> levels in transgenic line <i>mgp1-egfp-509</i>. Samples represent the average of three groups of 20 flies. <b>D.</b> qPCR analysis of <i>egfp</i> levels in 3–5 day old mated adult females from the <i>mgp1-egfp-509</i>, <i>mgp1-egfp-236</i>, <i>mgp1-egfp-112</i> or <i>mgp1-egfp-13</i> lines. Samples represent three groups of 20 flies. <b>E.</b> 3–5 day old transgenic Drosophila (509 bp-<i>mgp1/egfp</i>) were reared on control or nutrient deficient media. Data represent the mean eggs/day from five groups of 60 flies. Statistical notation: a = statistical difference between minimal and control media (P-value <0.05 by students t-test). <b>F.</b> qPCR analysis of <i>egfp</i> levels in <i>mgp1-egfp-509</i> flies reared on control or minimal media. Data represents the mean <i>egfp</i> transcript level from three groups of 20 flies. Statistical notation: as in E.</p

Tissue specificity and siRNA analysis of the <i>lbl</i> gene in <i>Glossina</i>.

<p>All qPCR analyses normalized to Tsetse <i>tub</i>. Error bars represent standard error. <b>A.</b> qPCR analysis of <i>lbl</i> tissue specificity. Samples represent 3 replicates of tissues isolated from 5 individuals. Statistical notation: a = significant difference between groups (P-value <0.05 by students <i>t</i>-test). <b>B.</b> qPCR analysis of tsetse <i>lbl</i> abundance 4 days post <i>sigfp</i> (control) or <i>silbl</i> injection. Statistical notation: a = significant difference between groups (P-value <0.05 by students <i>t</i>-test). <b>C.</b> Relative levels of <i>asmase</i>, <i>mgp1</i> and <i>mgp2</i> following injection of <i>sigfp</i> (control) or <i>silbl</i>. Statistical notation: a = statistical difference from control (P-value <0.01 by students <i>t</i>-test). All flies were injected 5–6 days after adult emergence and tested at 11–13 days for <i>lbl</i> suppression and 16–17 days for <i>asmase</i>, <i>mgp1</i> and <i>mgp2</i> interference.</p

Fecundity effects of <i>lbl</i> knockdown.

<p>2 groups of 50 flies were injected with either <i>sigfp</i> or <i>silbl</i> at 7 days post eclosion and monitored daily for mortality and pupal deposition. <b>A.</b> Rate of larval deposition per fly per day after injection of either <i>sigfp</i> or <i>silbl</i>. Error bars represent standard error. Statistical notation: a = statistical difference between groups (P-value <0.05 by students t-test). <b>B.</b> Cumulative count of larval deposition post injection over the course of the first gonotrophic cycle.</p

Cysteine Cathepsin Inhibitors as Anti-Ebola Agents

The recent Ebola virus outbreak in western Africa highlights the need for novel therapeutics that target Ebola virus and other filoviruses. Filoviruses require processing by host cell-derived cysteine cathepsins for productive infection. Here we report the generation of a focused library of cysteine cathepsin inhibitors and subsequent screening to identify compounds with potent activity against viral entry and replication. Our top compounds show highly potent and broad-spectrum activity against cysteine cathepsins and were able to effectively block entry of Ebola and Marburg viruses. These agents are promising leads for development as antifilovirus therapeutics

Cysteine Cathepsin Inhibitors as Anti-Ebola Agents

The recent Ebola virus outbreak in western Africa highlights the need for novel therapeutics that target Ebola virus and other filoviruses. Filoviruses require processing by host cell-derived cysteine cathepsins for productive infection. Here we report the generation of a focused library of cysteine cathepsin inhibitors and subsequent screening to identify compounds with potent activity against viral entry and replication. Our top compounds show highly potent and broad-spectrum activity against cysteine cathepsins and were able to effectively block entry of Ebola and Marburg viruses. These agents are promising leads for development as antifilovirus therapeutics

Total read number generated by Illumina RNA-seq and following quality control measures including symbiont removal and elimination of low quality reads.

<p>Total read number generated by Illumina RNA-seq and following quality control measures including symbiont removal and elimination of low quality reads.</p