Montage of some of the major human helminth parasites, their developmental stages, and disease pathology.

<p>(A) Microfilaria of <i>Brugia malayi</i> in a thick blood smear, stained with Giemsa (<a href="http://www.dpd.cdc.gov/dpdx/html/frames/a-f/filariasis/body_Filariasis_mic1.htm" target="_blank">http://www.dpd.cdc.gov/dpdx/html/frames/a-f/filariasis/body_Filariasis_mic1.htm</a>); the microfilaria is about 250 µm in length. (B) Patient with lymphedema of the left leg due to lymphatic filariasis (<a href="http://www.cdc.gov/ncidod/dpd/parasites/lymphaticfilariasis/index.htm" target="_blank">http://www.cdc.gov/ncidod/dpd/parasites/lymphaticfilariasis/index.htm</a>). (C) Hookworm egg passed in the stool of an infected person; the microscopic egg, barrel-shaped with a thin wall, is about 70×40 µm in dimension. (D) longitudinal section through an adult hookworm attached to wall of small intestine, ingesting host blood and mucosal wall. The parasite is about 1 cm in length. (E) Eggs of <i>Schistosoma mansoni</i>. The egg is about 150×50 µm in dimension; the lateral spine is diagnostic for <i>S. mansoni</i> in comparison to the other human schistosome species. Fibrotic responses to schistosome eggs trapped in the intestines, liver, and other organs of the infected person are the cause of the schistosomiasis pathology and morbidity. (F) A pair of adult worms of the blood fluke <i>Schistosoma mansoni</i>; the more slender female worm resides in the gynecophoral canal of the thicker male. The worms are about 1.5 cm in length, and live for many years (<a href="http://www.dpd.cdc.gov/dpdx/HTML/ImageLibrary/Schistosomiasis_il.htm" target="_blank">http://www.dpd.cdc.gov/dpdx/HTML/ImageLibrary/Schistosomiasis_il.htm</a> ).</p

Human parasitic helminths (and their close relatives) with genome sequencing projects completed or underway.

a<p>WUGC, Washington University's Genome Center.</p>b<p>Phylogeny based on Blaxter et al. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000538#pntd.0000538-Blaxter1" target="_blank">[47]</a>.</p><p>BI, Broad Institute; CNHGC, Chinese National HGC; SI, Sanger Institute; SNUCM, Seoul National University College of Medicine; TIGR, The Institute for Genomic Research (now JCVI).</p

Expression of aldolase in <i>B</i>. <i>malayi</i>.

<p>(A) <i>Bma-aldolase-1</i> and <i>Bma-aldolase-2</i> are overexpressed in adult female worms as compared to microfilaria (Mf). Relative gene expression between females and Mf was calculated using the ΔΔCt method. The median value of the Mf group was set to 100% and the increase in expression in the female worms was calculated as a percentage of the control. (B) Western blot analysis shows the expression of BMA-ALDO-1 and BMA-ALDO-2 proteins in Mf (lane 1), in 6-day doxycycline-treated Mf (lane 2), in adult females (lane 3), and in 6-day doxycycline-treated adult female worms (lane 4). Reduction of <i>Bma-aldolase-2</i> (C) and <i>Bma-aldolase-1</i> (D) expression in doxycycline (3- and 6-day, grey bars) treated female worms as compared to control untreated worm samples (transparent bars). The median value of the control group was set to 100% and the reduction in expression in the treated groups was calculated as a percentage of the control. *: <i>p</i> < 0.001, NS: not significant.</p

Decreased fitness of <i>Wolbachia</i> leads to increased glucose and glycogen amounts in the <i>B</i>. <i>malayi</i> female worms, and regulates the expression of genes that are part of the glycogen metabolic pathway.

<p>Increase in glucose and glycogen levels (A) observed in 6-day doxycycline-treated <i>B</i>. <i>malayi</i> female worms (grey bars) as compared to control worms (transparent bars). The Y axis shows μg of glucose (or glycogen) per mg of total protein in 1 ml of soluble worm extract (*: <i>p</i> < 0.01, and **: <i>p</i> = 0.02 between control and treated samples). (B) Similar glucose and glycogen levels are found in 6-day doxycycline-treated <i>A</i>. <i>viteae</i> (<i>Wolbachia</i>-free filariae) female worms (grey bars) and in control <i>A</i>. <i>viteae</i> worms (transparent bars). (C) and (D) Optical Density levels obtained from MTT assay performed on 6-day doxycycline-treated (grey bars) and control (transparent bar) <i>B</i>. <i>malayi</i> (C) and <i>A</i>. <i>viteae</i> (D). Black bars (C and D) represent OD levels measured in dead worms. (E) Percent of relative gene expression of the following genes: glycogen synthase (GS), glycogen synthase phosphatase-3 (GSK-3), glycogen phosphorylase (GP), and hexokinase (HK) in 3-day doxycycline-treated (3d) and 6-day doxycycline-treated (6d) <i>B</i>. <i>malayi</i> female worms (grey bars) and in control samples (transparent bars). The median value of the control group was set to 100% and the reduction or increase in the expression levels of these genes in the treated groups was calculated as a percentage of the control. *: <i>p</i> < 0.001, as determined by comparing the values between doxycycline-treated and control worms for each experimental treatment group. NS: not significant.</p

Co-localization of <i>Wolbachia</i>, aldolase and glycogen in the lateral cord of <i>B</i>. <i>malayi</i>.

<p>(A) and (B), Immunogold labelling showing the localization of aldolase on <i>Wolbachia</i> (W) surface and within glycogen (gly). (C) Transmission electron micrograph showing <i>Wolbachia</i> (W) embedded within the granules of glycogen (gly) in the cytoplasm of the lateral chord. Bar = 1 μm.</p

Schematic representation of the role <i>w</i>Bm may play in regulating the metabolism of glucose and glycogen in <i>B</i>. <i>malayi</i>.

<p>(A) <i>w</i>Bm require pyruvate for intracellular glucose and energy production. Parasite glycolytic enzymes (GE) are clustered to the bacterial surface in a complex with wBm00432, which can support the conversion of the intracellular glucose into pyruvate. Pyruvate is then utilized by the bacteria either for energy production or gluconeogenesis. (B) Bacteria change the expression of host genes that are involved in the glycogen metabolic pathway. A decline in bacterial fitness due to antibiotic treatment increases glucose levels and decreases utilization of glycogen, resulting in an increase in glycogen stores as well. Abbreviations: TCA: tricarboxylic acid cycle, GS: glycogen synthase, GP: glycogen phosphorylase, GSK-3: glycogen synthase phosphatase-3, HK: hexokinase, G-6-P: glucose-6-phosphate.</p

Primers for dsRNA synthesis and qRT-PCR.

<p>T7 promoter is underlined.</p

Silencing of <i>Bma-aldolase-2</i> decreases <i>Wolbachia</i> load in adult female worms and induces apoptosis in developing embryos.

<p>(A) qPCR analysis of the number (#) of <i>wsp</i> copies per adult female worm at the end of the RNAi experiments. Significant reduction of <i>Wolbachia</i> load was only detected in females treated with siRNA specifically targeting <i>Bma-aldolase-2</i> (siALDO-2) as compared to RNAi-control (worms were treated with siRNA-GFP; <i>p</i> < 0.01) or untreated worms (<i>p</i> < 0.01). (B) TUNEL assay for developing embryos extracted from control (left) or treated with siRNA specific to <i>Bma-aldolase-2</i> (right). Green represents apoptotic nuclei and all nuclei were also co-stained with a DNA-specific dye (propidium iodide, red), magnification 60X. (C) Number of apoptotic embryos extracted from females and number of microfilaria (Mf) released from control females and females after treatment with siRNAs. The increase in the proportion of apoptotic embryos in worms treated with siRNA specifically targeting <i>Bma-aldolase-2</i> (siALDO-2) was significant as compared to the RNAi-control (<i>p</i> < 0.05) or untreated (<i>p</i> < 0.05) worms. The number of Mf released by worms treated with siRNA specifically targeting <i>Bma-aldolase-2</i> (siALDO-2) was about 50% of those released by RNAi-control (<i>p</i> < 0.05) or untreated (<i>p</i> < 0.05) worms.</p

Defining <i>Brugia malayi</i> and <i>Wolbachia</i> symbiosis by stage-specific dual RNA-seq

<div><p>Background</p><p>Filarial nematodes currently infect up to 54 million people worldwide, with millions more at risk for infection, representing the leading cause of disability in the developing world. <i>Brugia malayi</i> is one of the causative agents of lymphatic filariasis and remains the only human filarial parasite that can be maintained in small laboratory animals. Many filarial nematode species, including <i>B</i>. <i>malayi</i>, carry an obligate endosymbiont, the alpha-proteobacteria <i>Wolbachia</i>, which can be eliminated through antibiotic treatment. Elimination of the endosymbiont interferes with development, reproduction, and survival of the worms within the mamalian host, a clear indicator that the <i>Wolbachia</i> are crucial for survival of the parasite. Little is understood about the mechanism underlying this symbiosis.</p><p>Methodology/ Principle findings</p><p>To better understand the molecular interplay between these two organisms we profiled the transcriptomes of <i>B</i>. <i>malayi</i> and <i>Wolbachia</i> by dual RNA-seq across the life cycle of the parasite. This helped identify functional pathways involved in this essential symbiotic relationship provided by the co-expression of nematode and bacterial genes. We have identified significant stage-specific and gender-specific differential expression in <i>Wolbachia</i> during the nematode’s development. For example, during female worm development we find that <i>Wolbachia</i> upregulate genes involved in ATP production and purine biosynthesis, as well as genes involved in the oxidative stress response.</p><p>Conclusions/ Significance</p><p>This global transcriptional analysis has highlighted specific pathways to which both <i>Wolbachia</i> and <i>B</i>. <i>malayi</i> contribute concurrently over the life cycle of the parasite, paving the way for the development of novel intervention strategies.</p></div