Bma-LAD-2, an Intestinal Cell Adhesion Protein, as a Potential Therapeutic Target for Lymphatic Filariasis

Abstract

Lymphatic filariasis is a debilitating disease that afflicts over 70 million people worldwide. It is caused by the parasitic nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori. Despite substantial success, efforts to eliminate LF will likely require more time and resources than predicted. Identifying new drug and vaccine targets in adult filariae could help elimination efforts. This study's aim was to evaluate intestinal proteins in adult Brugia malayi worms as possible therapeutic targets. Using short interfering RNA (siRNA), we successfully targeted four candidate gene transcripts: Bma-Serpin, Bma-ShTK, Bma-Reprolysin, and Bma-LAD-2. Of those, Bma-LAD-2, an immunoglobulin superfamily cell adhesion molecule (IgSF CAM), was determined to be essential for adult worm survival. We observed a 70.42% knockdown in Bma-LAD-2 transcript levels 1 day post-siRNA incubation and an 87.02% reduction in protein expression 2 days post-siRNA incubation. This inhibition of Bma-LAD-2 expression resulted in an 80% decrease in worm motility over 6 days, a 93.43% reduction in microfilaria release (Mf) by day 6 post-siRNA incubation, and a dramatic decrease in (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. Transmission electron microscopy revealed the loss of microvilli and unraveling of mitochondrial cristae in the intestinal epithelium of Bma-LAD-2 siRNA-treated worms. Strikingly, Bma-LAD-2 siRNA-treated worms exhibited an almost complete loss of pseudocoelomic fluid. A luciferase immunoprecipitation system assay did not detect anti-Bma-LAD-2 IgE in the serum of 30 LF patients, indicating that LF exposure does not result in IgE sensitization to this antigen. These results indicate that Bma-LAD-2 is an essential protein for adult Brugia malayi and may be an effective therapeutic target. IMPORTANCE Brugia malayi is a parasitic nematode that can cause lymphatic filariasis, a debilitating disease prevalent in tropical and subtropical countries. Significant progress has been made toward eliminating the disease. However, complete eradication may require new therapeutics such as drugs or a vaccine that kill adult filariae. In this study, we identified an immunoglobulin superfamily cell adhesion molecule (Bma-LAD-2) as a potential drug and vaccine candidate. When we knocked down Bma-LAD-2 expression, we observed a decrease in worm motility, fecundity, and metabolism. We also visualized the loss of microvilli, destruction of the mitochondria in the intestinal epithelium, and loss of pseudocoelomic fluid contents after Bma-LAD-2 siRNA treatment. Finally, we demonstrated that serum from filaria-infected patients does not contain preexisting IgE to Bma-LAD-2, which indicates that this antigen would be safe to administer as a vaccine in populations where the disease is endemic.

Keywords: Brugia malayi; Brugia pahangi; Brugia timori; Wuchereria bancrofti; filaria; filariasis; helminth; neglected tropical diseases; siRNA; vaccine.

FIG 1

Molecular organization of Bma-LAD-2 extracellular domain. (A) Bma-LAD-2 monomer. Schematic domain organization (top) and model of monomer structure (bottom) assembled based on sequence similarity and available crystal structures of homologous proteins as described in Materials and Methods. (B) Putative structure of Bma-LAD-2 dimer. Expanded view shows the dimer interface (indicated by red arrows) with Ig domains as labeled.

FIG 2

Phylogenetic tree of Bma-LAD-2 and orthologs from other helminths. The amino acid sequence of Bma-LAD-2 (WormBase gene ID WBGene00227085) has high level of relatedness to other filarial species and is evolutionarily distant from cats, dogs, and humans. Based on sequence alignment using multiple-sequence comparison by log expectation (MUSCLE), the phylogenetic tree was constructed by the maximum likelihood method. The phylogenetic scale represents genetic change as defined by the average number of nucleotide substitutions per site. The numbers at each branch represent the bootstrap value out of 100.

FIG 3

cy3-labeled Bma-LAD-2 siRNA entry into the intestinal tract of female B. malayi adult worms. B. malayi adult female worms soaked in cy3-labeled siRNA (red) for 24 h and visualized at magnifications of 40× (A) and 100× (B). As a negative control, worms were cultured in medium only for 24 h and visualized at magnifications of 40× (C) and 100× (D). Worms were counterstained with DAPI (blue).

FIG 4

Treatment with Bma-LAD-2-specific siRNA reduces target transcript and protein levels in adult female B. malayi worms. Adult female filariae were cultured in Bma-LAD-2 siRNA, scrambled siRNA, or medium alone. (A) The Bma-lad-2 transcript level was reduced in specific siRNA-treated groups (n = 3) compared to the scrambled controls (n = 3) normalized to Bma-gapdh. Ordinary one-way ANOVA followed by Tukey’s multiple-comparison test was used to generate the P values. These experiments were successfully repeated twice, and the data shown are from a single representative experiment, with means + SEM. (B) Bma-LAD-2 levels were assessed 24 h post-siRNA incubation by Western blotting using anti-Bma-LAD-2 antibodies. (C) Western blot quantification was performed using the ImageStudioLite software. The signal intensities of anti-Bma-LAD-2 were normalized to those of beta-actin.

FIG 5

Bma-LAD-2 knockdown results in decreased worm motility, microfilaria release, and metabolism. Reducing Bma-LAD-2 expression in female B. malayi adult worms resulted in decreased motility (n = 5; day 1, P < 0.0001; day 6, P = 0.0004) (A), microfilaria count per worm (n = 5; day 1, P value not significant; day 6, P < 0.0001) per 24-h period (B), and metabolism (n = 2) (C) as measured by MTT reduction at 1 and 6 days post-siRNA treatment. For motility (A) and microfilaria release (B), an ordinary one-way ANOVA followed by Tukey’s multiple-comparison test was used to generate the P values. These experiments were successfully repeated twice, and the data presented are representative of a single experiment as means + SEM.

FIG 6

Intestinal tract of a female B. malayi adult worm. Image was captured by transmission electron microscopy (TEM) at 4,000×. Adult filariae were cultured in medium alone for 72 h. Microvilli (black arrowhead) line the apical surface of the intestinal epithelium. Other structures visible are apical junctions (white arrowhead), nuclei (Nu), lipid droplets (L), and mitochondria (black arrow).

FIG 7

Intestinal tract of a female B. malayi adult worm treated with Bma-LAD-2 siRNA. Image was captured by transmission electron microscopy (TEM) at 4,000×. Adult filariae were incubated with Bma-LAD-2 siRNA for 24 h and then cultured in medium alone for an additional 48 h. Microvilli (black arrowhead) are largely absent from the apical surface of the intestinal epithelium. There are some vestigial microvilli that have been invaginated by the surrounding epithelium. Other structures visible are apical junctions (white arrowhead), nuclei (Nu), and lipid droplets (L). Many mitochondria (black arrow) appear to have unraveled cristae.

FIG 8

Transmission electron microscopy (TEM) image at 30,000× of mitochondria within intestinal epithelial cells of untreated control adult B. malayi worms. Mitochondria (black arrows) appear to be normal, with a continuous, folded cristae.

FIG 9

Transmission electron microscopy (TEM) image at 30,000× of mitochondria within intestinal epithelial cells of adult B. malayi worms after incubation with BMA-LAD-2 siRNA. Mitochondria (black arrows) appear to be diminished and have misshapen cristae.

FIG 10

Adult B. malayi treated with Bma-LAD-2 siRNA exhibits loss of pseudocoelomic fluid. Cross-sectional images were captured by transmission electron microscopy (TEM) at 500×. Adult filaria treated with siRNA (A) exhibits loss of most of the pseudocoelomic fluid surrounding the intestinal and uterine tubes and appears to have fluid within the intestinal lumen. In contrast, the untreated filaria (B) has a normal distribution of pseudocoelomic fluid in the spaces around the intestine and uterine tracts. PF, pseudocoelomic fluid; IT, intestinal tube; UT, uterine tube.

FIG 11

Filarial patient serum does not contain detectable IgG or IgE against Bma-LAD-2. Serum from filaria-infected individuals was incubated with a Bma-LAD-2-luciferase fusion protein. There was no detectable IgG (A) and IgE (B) in the patient serum as measured by the LIPS assay. As a positive control for IgG binding, Bma-LAD-2 rabbit polyclonal antibodies recognized the fusion protein. HIES, hyper-IgE syndrome; MF, microfilaremic; CP, chronic pathology; EN, endemic normal; BB, blood bank donors; TPE, tropical pulmonary eosinophilic; peptide Ab, Bma-LAD-2 rabbit polyclonal antibodies; mouse antisera, serum from Litomosoides sigmodontis vaccinated mice.