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

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

Primers for dsRNA synthesis and qRT-PCR.

<p>T7 promoter is underlined.</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

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