Fig 1.
Phylogenetic comparison of Bm-UGT.
Based on the B. malayi cDNA sequence alignment, there is a high level of relatedness to other filarial species for Bm-UGT and vast evolutionary distance to cat, dog, and human UGT molecules. The sequence alignment was generated using MUltiple Sequence Comparison by Log-Expectation (MUSCLE) performed by software SeaView. The tree was constructed based on the maximum likelihood method. The phylogenetic scale indicates genetic change as measured by the number of nucleotide substitutions per site.
Table 1.
Stage specific expression of Bm-UGT.
Bm-ugt transcript is expressed in both sexes of the B. malayi adult filaria as well as the L3 larval stage. Protein expression of Bm-UGT is consistent with this transcript profile.
Fig 2.
Structural analysis of filarial B. malayi intestinal UDP-glucuronosyltransferase (Bm-UGT).
(A) Schematic representation of the Bm-UGT protein domains. (B) Structure model of the UDP-glucuronosyltransferase generated using SWISS-MODEL and PDB ID: 5NLM (UDP-glucosyltransferase from Polygonum tinctorium). The structure has a N-terminal domain (purple) and a co-factor binding domain (grey) with linker residues 468–492 modelled (right). (C) Sequence alignment of UGT2B7 C-terminal domain (PDB ID: 2O6L) with the filarial Bm-UGT C-terminal domain. Putative co-factor binding residues are indicated based on sequence and structural homology. (D) Structure of the co-factor binding domain is shown with transparent surface representation and inferred UDPGA-interacting residues shown in stick representation and labeled. The UDPGA location is modelled based on the plant glycosyltransferase Medicago truncatula UGT71G1—UDP-glucose structure (PDB ID: 2ACW).
Fig 3.
Cy3-labeled Bm-UGT siRNA enters the intestinal tracts of female B malayi adult worms.
B. malayi adult worms incubated with Cy3-labeled siRNA (red) for 24 hrs and visualized at magnifications of (A) 100x and (B) 200x. Worms incubated in media alone for 24 hrs and visualized at magnifications of (C) 100x and (D) 200x. Worms were counterstained with DAPI (blue).
Fig 4.
Bm-UGT siRNA incubation reduces target transcript and protein levels in adult B. malayi worms.
Adult female worms were incubated with Bm-UGT siRNA, scrambled siRNA, or media alone. (A) Target mRNA levels (n = 3) in the specific siRNA and scrambled siRNA treated groups were measured by RT-qPCR relative to the media control normalized to Bm-gapdh, (Day 1, p = 0.0002; Day 3, p<0.0001; Day 6, p = 0.0034). p values were generated using an ordinary one-way ANOVA followed by Tukey’s multiple comparison test. These experiments were successfully repeated twice and the data shown is from a single representative experiment; mean ± SEM. (B) Bm-UGT expression (n = 5) was evaluated 24 hrs post-siRNA incubation using anti-Bm-UGT peptide antibodies. (C) Western blot quantification was performed using the ImageStudioLite software. Depicted are the signal intensities of anti-UGT normalized to beta-actin.
Fig 5.
Bm-UGT expression knockdown decreases worm motility, microfilariae release, and metabolism.
Adult female worms (n = 5) were incubated with Bm-UGT siRNA, scrambled siRNA, or media alone. Bm-UGT knockdown in female B. malayi adult worms caused reductions in (A) motility (n = 5; Day 1, p = 0.0006; Day 3, p = 0.0007; Day 6, p = 0.0004), (B) microfilaria count per worm (n = 5; Day 1, p = 0.0048; Day 3, p = 0.0096; Day 6, p = 0.0347) per 24-hr period, and (C) metabolism (n = 2; Day 1, p = 0.0148; Day 3, p = 0,0373; Day 6, p = 0.0243) as measured by MTT reduction relative to the media control at timepoints 1, 3, and 6 days post-siRNA treatment. For motility (A) and microfilaria release (B), p values were generated using an ordinary one-way ANOVA followed by Tukey’s multiple comparison test. For metabolism (C), p values were generated by an unpaired t-test. These experiments were successfully repeated twice and the data shown is from a single representative experiment; mean + SEM.
Fig 6.
UGT inhibitors exhibit macrofilaricidal activity in vitro.
Motility scores of adult B. malayi adult worms after incubation with various concentrations of (A) sulfinpyrazone (n = 5 for each group; 2500 μM, p<0.0001; 1000 μM, p<0.0001; 200 μM, p<0.0001; 100 μM, ns) and (B) probenecid (n = 5 for each group; 5000 μM, p<0.0001; 500 μM, p = 0.0004; 250 μM, ns; 40 μM, ns). For (A) and (B), the area under the curve (AUC) was calculated using the PRISM 7.0 software. Using these AUC values, a two-way ANOVA was performed to determine significance followed by Tukey’s multiple comparison test. Percent survival of B. malayi microfilariae incubated with (C) sulfinpyrazone (2500 μM, p<0.0001; 200 μM, p = 0.0005) and (D) probenecid (5000 μM, p<0.0001; 500 μM, ns). Percent survival was calculated from the first 100 Mf observed. The generated AUC values were analyzed using an ordinary one-way ANOVA to determine significance followed by Tukey’s multiple comparison test. These experiments were successfully repeated twice.
Fig 7.
UGT inhibitors exhibit synergy with albendazole in vitro.
Motility scores of adult B. malayi adult worms after incubation with 10 μM albendazole and (A) 40 μM sulfinpyrazone (n = 4, p = 0.0001) or (B) 100 μM probenecid (n = 4, p = 0.0022). For (A) and (B), the generated AUC values were analyzed by two-way ANOVA to determine significance followed by Tukey’s multiple comparison test. These experiments were successfully repeated three times.
Fig 8.
Serum of filaria-infected individuals does not contain detectable IgG or IgE against Bm-UGT.
Filarial patient serum was incubated with a Bm-UGT-luciferase fusion protein. No detectable (A) IgG and (B) IgE was measured using the LIPS assay. The Bm-UGT peptide rabbit polyclonal antibodies served as a positive control for the detection of IgG. MF = microfilaremic, CP = chronic pathology, EN = endemic normal, BB = blood bank donors, TPE = tropical pulmonary eosinophilic, and Peptide Ab = Bm-UGT peptide polyclonal antibodies.