Expression of five acetylcholine receptor subunit genes in Brugia malayi adult worms.

Acetylcholine receptors (AChRs) are required for body movement in parasitic nematodes and are targets of "classical" anthelmintic drugs such as levamisole and pyrantel and of newer drugs such as tribendimidine and derquantel. While neurotransmission explains the effects of these drugs on nematode movement, their effects on parasite reproduction are unexplained. The levamisole AChR type (L-AChRs) in Caenorhabditis elegans is comprised of five subunits: Cel-UNC-29, Cel-UNC-38, Cel-UNC-63, Cel-LEV-1 and Cel-LEV-8. The genome of the filarial parasite Brugia malayi contains nine AChRs subunits including orthologues of Cel-unc-29, Cel-unc-38, and Cel-unc-63. We performed in situ hybridization with RNA probes to localize the expression of five AChR genes (Bm1_35890-Bma-unc-29, Bm1_20330-Bma-unc-38, Bm1_38195-Bma-unc-63, Bm1_48815-Bma-acr-26 and Bm1_40515-Bma-acr-12) in B. malayi adult worms. Four of these genes had similar expression patterns with signals in body muscle, developing embryos, spermatogonia, uterine wall adjacent to stretched microfilariae, wall of V as deferens, and lateral cord. Three L-AChR subunit genes (Bma-unc-29, Bma-unc-38 and Bma-unc-63) were expressed in body muscle, which is a known target of levamisole. Bma-acr-12 was co-expressed with these levamisole subunit genes in muscle, and this suggests that its protein product may form receptors with other alpha subunits. Bma-acr-26 was expressed in male muscle but not in female muscle. Strong expression signals of these genes in early embryos and gametes in uterus and testis suggest that AChRs may have a role in nervous system development of embryogenesis and spermatogenesis. This would be consistent with embryotoxic effects of drugs that target these receptors in filarial worms. Our data show that the expression of these receptor genes is tightly regulated with regard to localization in adult worms and developmental stage in embryos and gametes. These results may help to explain the broad effects of drugs that target AChRs in filarial worms.

High level expression of a glutamate-gated chloride channel gene in reproductive tissues of Brugia malayi may explain the sterilizing effect of ivermectin on filarial worms.

Glutamate-gated chloride channels (GluCl) are targets for avermectin/milbemycin (A/M) anthelmintics such as ivermectin that cause paralysis of somatic and pharyngeal muscles in gastrointestinal nematodes. Ivermectin is useful for onchocerciasis control programs because of its activity against microfilariae that often cause ocular disease and severe dermatitis. However, mechanisms responsible for reduced microfilaria production by adult worms following ivermectin treatment are poorly understood. We synthesized subunit-specific RNA probes for the Brugia malayi GluCl gene avr-14 (BmAVR-14) to localize expression of this gene in adult filarial worms. Both subunits of BmAVR-14 exhibited very similar expression patterns. In female worms, strong expression signals were detected in the ovary, developing embryos and lateral hypodermal chords, with moderate expression in the uterus wall adjacent to stretched microfilariae. These genes were also highly expressed in adult male worms (in spermatogonia, in the wall of the vas deferens, and in the lateral chords, but not in mature spermatozoa). In addition, avr-14 was highly expressed in somatic muscles adjacent to the terminal end of the vas deferens which contains mature sperm. These results show that avr-14 is highly expressed in B. malayi developing embryos and reproductive tissues, and they provide evidence for the involvement of GluCl in gamete production and embryogenesis in filarial worms. This may explain the observed suppression of microfilaria (Mf) production by female worms following treatment with avermectin/milbemycin anthelmintics.

Transcription profiling reveals stage- and function-dependent expression patterns in the filarial nematode Brugia malayi.

BACKGROUND: Brugia malayi is a nematode parasite that causes lymphatic filariasis, a disfiguring and disabiling tropical disease. Although a first draft genome sequence was released in 2007, very little is understood about transcription programs that govern developmental changes required for the parasite's development and survival in its mammalian and insect hosts. RESULTS: We used a microarray with probes that represent some 85% of predicted genes to generate gene expression profiles for seven parasite life cycle stages/sexes. Approximately 41% of transcripts with detectable expression signals were differentially expressed across lifecycle stages. Twenty-six percent of transcripts were exclusively expressed in a single parasite stage, and 27% were expressed in all stages studied. K-means clustering of differentially expressed transcripts revealed five major transcription patterns that were associated with parasite lifecycle stages or gender. Examination of known stage-associated transcripts validated these data sets and suggested that newly identified stage or gender-associated transcripts may exercise biological functions in development and reproduction. The results also indicate that genes with similar transcription patterns were often involved in similar functions or cellular processes. For example, nuclear receptor family gene transcripts were upregulated in gene expression pattern four (female-enriched) while protein kinase gene family transcripts were upregulated in expression pattern five (male-enriched). We also used pair-wise comparisons to identify transcriptional changes between life cycle stages and sexes. CONCLUSIONS: Analysis of gene expression patterns of lifecycle in B. malayi has provided novel insights into the biology of filarial parasites. Proteins encoded by stage-associated and/or stage-specific transcripts are likely to be critically important for key parasite functions such as establishment and maintenance of infection, development, reproduction, and survival in the host. Some of these may be useful targets for vaccines or new drug treatments for filariasis.

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

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.

Transcriptomes and pathways associated with infectivity, survival and immunogenicity in Brugia malayi L3.

BACKGROUND: Filarial nematode parasites cause serious diseases such as elephantiasis and river blindness in humans, and heartworm infections in dogs. Third stage filarial larvae (L3) are a critical stage in the life cycle of filarial parasites, because this is the stage that is transmitted by arthropod vectors to initiate infections in mammals. Improved understanding of molecular mechanisms associated with this transition may provide important leads for development of new therapies and vaccines to prevent filarial infections. This study explores changes in gene expression associated with the transition of Brugia malayi third stage larvae (BmL3) from mosquitoes into mammalian hosts and how these changes are affected by radiation. Radiation effects are especially interesting because irradiated L3 induce partial immunity to filarial infections. The underlying molecular mechanisms responsible for the efficacy of such vaccines are unkown. RESULTS: Expression profiles were obtained using a new filarial microarray with 18, 104 64-mer elements. 771 genes were identified as differentially expressed in two-way comparative analyses of the three L3 types. 353 genes were up-regulated in mosquito L3 (L3i) relative to cultured L3 (L3c). These genes are important for establishment of filarial infections in mammalian hosts. Other genes were up-regulated in L3c relative to L3i (234) or irradiated L3 (L3ir) (22). These culture-induced transcripts include key molecules required for growth and development. 165 genes were up-regulated in L3ir relative to L3c; these genes encode highly immunogenic proteins and proteins involved in radiation repair. L3ir and L3i have similar transcription profiles for genes that encode highly immunogenic proteins, antioxidants and cuticle components. CONCLUSION: Changes in gene expression that normally occur during culture under conditions that support L3 development and molting are prevented or delayed by radiation. This may explain the enhanced immunogenicity of L3ir. Gene Ontology and KEGG analyses revealed altered pathways between L3 types. Energy and "immune pathways" are up-regulated and may be needed for L3i invasion and survival, while growth and development are priorities for L3c. This study has improved our understanding of molecules involved in parasite invasion and immune evasion, potential targets of protective immunity, and molecules required for parasite growth and development.

Brugia malayi: effects of radiation and culture on gene expression in infective larvae.

Third-stage infective larvae (L3i) of Brugia malayi are developmentally arrested in mosquitoes but must quickly adapt to a new environment when they enter mammalian hosts to initiate infections. These changes can be studied by in vitro culture of L3 (L3c) under conditions that permit molting of L3-L4. Irradiated L3 (L3ir) have stunted growth and limited lifespan in mammalian hosts, and they induce high levels of immunity to challenge infections in animal models. This study explored differences in gene expression in L3i, L3c and L3ir by expressed sequence tag EST generation and qRT-PCR. 2506 ESTs generated from cDNA libraries constructed from L3i, L3c and L3ir were grouped into 1309 gene clusters. Despite extensive prior sampling from B. malayi (>22,000 ESTs in dbEST), 73% of the L3 clusters described here are novel. Sixty-three percentage of the clusters have homology to proteins from other species including 187 specific to nematodes and 141 that have to date only been described in non-nematode species. The transcript levels of 62 candidates for up- or down-regulation in L3i, L3c and L3ir based on EST frequencies were evaluated by qRT-PCR. Twenty-eight were confirmed to have > or = 3-fold differences in expression. Genes coding for proteins believed to be involved in establishment of infection, host adaptation and targets of protective immunity were confirmed to have higher expression in L3i than in L3c. Some of the genes that were down-regulated in L3c were highly expressed in L3ir. This study provides an improved description of the adaptations that accompany the transition from L3i to L3c and the special ability of L3ir to induce protective immunity.

Profiling of gender-regulated gene transcripts in the filarial nematode Brugia malayi by cDNA oligonucleotide array analysis.

Microarray technology permits high-throughput comparisons of gene expression in different parasite stages or sexes and has been used widely. We report the first use of this technology for analysis of gene expression in filarial male and female worms. The slide array (comprised of 65-mer oligos representing 3569 EST clusters) was spotted with sequences selected from the extensive Brugia malayi EST database (). Arrays were hybridized with Cy dye labeled male and female cDNA. The experimental design included both biological and technical (dye-flip) replicates. The data were normalized for background and probe intensity, and the relative abundance of hybridized cDNA for each spot was determined. Genes showing two-fold or greater differences with P<0.05 were considered gender-regulated candidates. One thousand one hundred and seventy of 2443 clusters (48%) with signals above threshold in at least one sex were considered as gender-regulated gene candidates. This included 520 and 650 clusters up-regulated in male and female worms, respectively. Fifty of 53 (94%) gender-regulated candidate genes identified by microarray analysis were confirmed by real-time RT-PCR. Approximately 61% of gender-regulated genes had significant similarity to known genes in other organisms such as Caenorhabditis elegans. Many C. elegans homologues of these genes have been reported to have reproductive phenotypes (sterility or abnormal embryo development) by RNA interference. This study has provided the first broad view of gender-regulated gene expression in B. malayi; this should lead to improved understanding of reproduction in filarial nematodes. More generally, this approach holds great promise as a means of studying stage-specific or tissue-specific gene expression in parasitic nematodes.

Quantitative analysis of gender-regulated transcripts in the filarial nematode Brugia malayi by real-time RT-PCR.

Improved understanding of the biology of reproduction in filarial worms may lead to identification of new targets for drugs or vaccines. Real-time RT-PCR is increasingly being adopted for RNA quantification and genetic analysis. Candidate gender-regulated genes were selected from genes identified in prior studies by differential display RT-PCR and by electronic selection of the Brugia malayi expression sequence tag (EST) database for clusters with possible gender-specific expression (four or more transcripts in male cDNA library ESTs but none in female ESTs or vice versa). Expression of candidate genes in male and female worms was compared by real-time reverse transcription PCR with sequence-specific primers. Double stranded DNA product was measured by SYBR Green I fluorescence; melting curves and agarose gel electrophoresis were used to verify the specificity of results. Relative gene expression results were normalized by parallel studies with internal control genes that were shown to be equally expressed in male and female worms (beta actin 2B, histone H3, NADH dehydroxygenase subunit 1) and calculated by the comparative C(t) method. Nineteen of 31 candidate genes were verified to have reproducible, gender-biased expression with fold differences between 5 and >30,000. These included several well-known genes (for example, genes encoding major sperm protein and a microfilaria sheath protein) and many novel genes. This paper reports the first large scale use of real time RT-PCR to quantitate and study gene expression in a nematode parasite. Our results represent an important step toward improved understanding of the molecular biology of reproduction in filarial nematodes.