Gender-associated genes in filarial nematodes are important for reproduction and potential intervention targets

Abstract

Background: A better understanding of reproductive processes in parasitic nematodes may lead to development of new anthelmintics and control strategies for combating disabling and disfiguring neglected tropical diseases such as lymphatic filariasis and onchocerciasis. Transcriptomatic analysis has provided important new insights into mechanisms of reproduction and development in other invertebrates. We have performed the first genome-wide analysis of gender-associated (GA) gene expression in a filarial nematode to improve understanding of key reproductive processes in these parasites.

Methodology/principal findings: The Version 2 Filarial Microarray with 18,104 elements representing ∼85% of the filarial genome was used to identify GA gene transcripts in adult Brugia malayi worms. Approximately 19% of 14,293 genes were identified as GA genes. Many GA genes have potential Caenorhabditis elegans homologues annotated as germline-, oogenesis-, spermatogenesis-, and early embryogenesis- enriched. The potential C. elegans homologues of the filarial GA genes have a higher frequency of severe RNAi phenotypes (such as lethal and sterility) than other C. elegans genes. Molecular functions and biological processes associated with GA genes were gender-segregated. Peptidase, ligase, transferase, regulator activity for kinase and transcription, and rRNA and lipid binding were associated with female GA genes. In contrast, catalytic activity from kinase, ATP, and carbohydrate binding were associated with male GA genes. Cell cycle, transcription, translation, and biological regulation were increased in females, whereas metabolic processes of phosphate and carbohydrate metabolism, energy generation, and cell communication were increased in males. Significantly enriched pathways in females were associated with cell growth and protein synthesis, whereas metabolic pathways such as pentose phosphate and energy production pathways were enriched in males. There were also striking gender differences in environmental information processing and cell communication pathways. Many proteins encoded by GA genes are secreted by Brugia malayi, and these encode immunomodulatory molecules such as antioxidants and host cytokine mimics. Expression of many GA genes has been recently reported to be suppressed by tetracycline, which blocks reproduction in female Brugia malayi. Our localization of GA transcripts in filarial reproductive organs supports the hypothesis that these genes encode proteins involved in reproduction.

Conclusions/significance: Genome-wide expression profiling coupled with a robust bioinformatics analysis has greatly expanded our understanding of the molecular biology of reproduction in filarial nematodes. This study has highlighted key molecules and pathways associated with reproductive and other biological processes and identified numerous potential candidates for rational drug design to target reproductive processes.

Figure 1. The most significant KEGG pathways were identified in gender-associated genes.

KEGG pathway analyses were applied to filarial proteins encoded by filarial gender-associated genes. Values for each category are reported as percentages of functionally annotated proteins.

Figure 2. Functional categories of filarial gender-associated genes with specific expression pattern of potential C. elegans homologues.

Pie charts show functional annotation of each of the major gene sets based on the assigned molecular function using gene ontology (GO) annotation. A: Functional categories of the female-associated genes with potential C. elegans homologues within oogenesis and intrinsic gene set. B: Functional categories of the male-associated genes with potential C. elegans homologues within spermatogenesis gene set. The sidebars divide genes encoding binding proteins into three categories: RNA binding, nucleotide and other nucleic acid and other bindings.

Figure 3. Expression patterns for four genes as revealed by in situ hybridization.

Bm1_45785 (A–C)- female-associated; Bm1_19785 (D–F)-male-associated; Bm1_25705 (G–J)-female-associated and Bm1_35060 (K–N)-male-associated in adult B. malayi. A–C: hybridization signals were detected in morula stage embryos (C, arrows) in females with no signals in males (A,B); D–F: expression was detected in spermatocytes (D, arrows) and spermatids (E, arrow) in males, but no signal was detected in females (F); G–J: hybridization signals were detected in spermatocytes (G, arrow) in males and signals were also detected in developing oocytes (H, arrows) and morula stages embryos (I,J, arrows) in females; K–N: expression signals were present in muscle in males (K, arrows) and females (L,M,N, arrows); signals were also seen in stretched microfilariae (M,N, arrows) and in uterus epithelial cells (M,N, arrow heads) where stretched microfilariae were present. I: intestine; O: ovary. Scale bars: 20 µm.