Luck AN et al. (2016) FASEB J "Heme acquisition in the parasitic filarial nematode Brugia malayi."

Hamza I, Voronin D, Slatko BE, Yuan X, Foster JM, Luck AN

[FASEB J, 2016]

Nematodes lack a heme biosynthetic pathway and must acquire heme from exogenous sources. Given the indispensable role of heme, this auxotrophy may be exploited to develop drugs that interfere with heme uptake in parasites. Although multiple heme-responsive genes (HRGs) have been characterized within the free-living nematode Caenorhabditis elegans, we have undertaken the first study of heme transport in Brugia malayi, a causative agent of lymphatic filariasis. Through functional assays in yeast, as well as heme analog, RNAi, and transcriptomic experiments, we have shown that the heme transporter HRG-1 (BmHRG-1) is indeed functional in B. malayi In addition, BmHRG-1 localizes both to the endocytic compartments and cell membrane when expressed in yeast cells. Transcriptomic sequencing revealed that BmHRG-1, BmHRG-2, and BmMRP-5 (all orthologs of HRGs in C. elegans) are down-regulated in heme-treated B. malayi, as compared to non-heme-treated control worms. Likely because of short gene lengths, multiple exons, other HRGs in B. malayi (BmHRG-3, -4, -5, and -6) remain unidentified. Although the precise mechanisms of heme homeostasis in a nematode with the ability to acquire heme remains unknown, this study clearly demonstrates that the filarial nematode B. malayi is capable of transporting exogenous heme.-Luck, A. N., Yuan, X., Voronin, D., Slatko, B. E., Hamza, I., Foster, J. M. Heme acquisition in the parasitic filarial nematode Brugia malayi.

Libro S et al. (2016) Symbiosis "Characterization of innate immunity genes in the parasitic nematode Brugia malayi."

Foster JM, Libro S, Slatko BE

[Symbiosis, 2016]

The filarial nematode Brugia malayi is one of the causative agents of lymphatic filariasis, a neglected tropical disease that affects 120 million people worldwide. The limited effectiveness of available anthelmintics and the absence of a vaccine have prompted extensive research on the interaction between Brugia and its obligate bacterial endosymbiont, Wolbachia. Recent studies suggest that Wolbachia is able to manipulate its nematode host immunity but relatively little is known about the immune system of filarial nematodes. Therefore, elucidation of the mechanisms underlying the immune system of B. malayi may be useful for understanding how the symbiotic relationship is maintained and help in the identification of new drug targets. In order to characterize the main genetic pathways involved in B. malayi immunity, we exposed adult female worms to two bacterial lysates (Escherichia coli and Bacillus amyloliquefaciens), dsRNA and dsDNA. We performed transcriptome sequencing of worms exposed to each immune elicitor at two different timepoints. Gene expression analysis of untreated and immune-challenged worms was performed to characterize gene expression patterns associated with each type of immune stimulation. Our results indicate that different immune elicitors produced distinct expression patterns in B. malayi, with changes in the expression of orthologs of well-characterized C. elegans immune pathways such as insulin, TGF-, and p38 MAPK pathways, as well as C-type lectins and several stress-response genes.

Landmann F et al. (2012) Parasit Vectors "Efficient in vitro RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, Brugia malayi."

Foster JM, Landmann F, Slatko BE, Sullivan W

[Parasit Vectors, 2012]

BACKGROUND: RNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes. The method has been widely used in model organisms, such as the free-living nematode Caenorhabditis elegans, where it has been deployed in genome-wide high throughput screens to identify genes involved in many cellular and developmental processes. However, RNAi techniques have not translated efficiently to animal parasitic nematodes that afflict humans, livestock and companion animals across the globe, creating a dependency on data tentatively inferred from C. elegans. RESULTS: We report improved and effective in vitro RNAi procedures we have developed using heterogeneous short interfering RNA (hsiRNA) mixtures that when coupled with optimized immunostaining techniques yield detailed analysis of cytological defects in the human parasitic nematode, Brugia malayi. The cellular disorganization observed in B. malayi embryos following RNAi targeting the genes encoding -tubulin, and the polarity determinant protein, PAR-1, faithfully phenocopy the known defects associated with gene silencing of their C. elegans orthologs. Targeting the B. malayi cell junction protein, AJM-1 gave a similar but more severe phenotype than that observed in C. elegans. Cellular phenotypes induced by our in vitro RNAi procedure can be observed by immunofluorescence in as little as one week. CONCLUSIONS: We observed cytological defects following RNAi targeting all seven B. malayi transcripts tested and the phenotypes mirror those documented for orthologous genes in the model organism C. elegans. This highlights the reliability, effectiveness and specificity of our RNAi and immunostaining procedures. We anticipate that these techniques will be widely applicable to other important animal parasitic nematodes, which have hitherto been mostly refractory to such genetic analysis.

Lefoulon E et al. (2019) Sci Rep "Author Correction: Large Enriched Fragment Targeted Sequencing (LEFT-SEQ) Applied to Capture of Wolbachia Genomes."

Voland L, Lefoulon E, Vaisman N, Foster JM, Frydman HM, Slatko BE, Sun L

[Sci Rep, 2019]

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Landmann F et al. (2014) PLoS Negl Trop Dis "Co-evolution between an endosymbiont and its nematode host: Wolbachia asymmetric posterior localization and AP polarity establishment."

Foster JM, Slatko BE, Michalski ML, Sullivan W, Landmann F

[PLoS Negl Trop Dis, 2014]

While bacterial symbionts influence a variety of host cellular responses throughout development, there are no documented instances in which symbionts influence early embryogenesis. Here we demonstrate that Wolbachia, an obligate endosymbiont of the parasitic filarial nematodes, is required for proper anterior-posterior polarity establishment in the filarial nematode B. malayi. Characterization of pre- and post-fertilization events in B. malayi reveals that, unlike C. elegans, the centrosomes are maternally derived and produce a cortical-based microtubule organizing center prior to fertilization. We establish that Wolbachia rely on these cortical microtubules and dynein to concentrate at the posterior cortex. Wolbachia also rely on PAR-1 and PAR-3 polarity cues for normal concentration at the posterior cortex. Finally, we demonstrate that Wolbachia depletion results in distinct anterior-posterior polarity defects. These results provide a striking example of endosymbiont-host co-evolution operating on the core initial developmental event of axis determination.

Scott AL et al. Parasite Immunol "Filarial and Wolbachia genomics."

Slatko BE, Nutman TB, Scott AL, McReynolds LA, Foster JM, Poole CB, Ghedin E

[Parasite Immunol]

Filarial nematode parasites, the causative agents for a spectrum of acute and chronic diseases including lymphatic filariasis and river blindness, threaten the well-being and livelihood of hundreds of millions of people in the developing regions of the world. The 2007 publication on a draft assembly of the 95-Mb genome of the human filarial parasite Brugia malayi- representing the first helminth parasite genome to be sequenced - has been followed in rapid succession by projects that have resulted in the genome sequencing of six additional filarial species, seven nonfilarial nematode parasites of animals and nearly 30 plant parasitic and free-living species. Parallel to the genomic sequencing, transcriptomic and proteomic projects have facilitated genome annotation, expanded our understanding of stage-associated gene expression and provided a first look at the role of epigenetic regulation of filarial genomes through microRNAs. The expansion in filarial genomics will also provide a significant enrichment in our knowledge of the diversity and variability in the genomes of the endosymbiotic bacterium Wolbachia leading to a better understanding of the genetic principles that govern filarial-Wolbachia mutualism. The goal here is to provide an overview of the trends and advances in filarial and Wolbachia genomics.

Lefoulon E et al. (2019) Sci Rep "Large Enriched Fragment Targeted Sequencing (LEFT-SEQ) Applied to Capture of Wolbachia Genomes."

Frydman HM, Vaisman N, Lefoulon E, Sun L, Foster JM, Slatko BE

[Sci Rep, 2019]

Symbiosis is a major force of evolutionary change, influencing virtually all aspects of biology, from population ecology and evolution to genomics and molecular/biochemical mechanisms of development and reproduction. A remarkable example is Wolbachia endobacteria, present in some parasitic nematodes and many arthropod species. Acquisition of genomic data from diverse Wolbachia clades will aid in the elucidation of the different symbiotic mechanisms(s). However, challenges of de novo assembly of Wolbachia genomes include the presence in the sample of host DNA: nematode/vertebrate or insect. We designed biotinylated probes to capture large fragments of Wolbachia DNA for sequencing using PacBio technology (LEFT-SEQ: Large Enriched Fragment Targeted Sequencing). LEFT-SEQ was used to capture and sequence four Wolbachia genomes: the filarial nematode Brugia malayi, wBm, (21-fold enrichment), Drosophila mauritiana flies (2 isolates), wMau (11-fold enrichment), and Aedes albopictus mosquitoes, wAlbB (200-fold enrichment). LEFT-SEQ resulted in complete genomes for wBm and for wMau. For wBm, 18 single-nucleotide polymorphisms (SNPs), relative to the wBm reference, were identified and confirmed by PCR. A limit of LEFT-SEQ is illustrated by the wAlbB genome, characterized by a very high level of insertion sequences elements (ISs) and DNA repeats, for which only a 20-contig draft assembly was achieved.

Jen Daub et al. (2001) International C. elegans Meeting "Mapping the genome(s) of the human nematode parasite Brugia malayi"

Mike Quail, Mark Blaxter, Jenna Ware, Jeremy Foster, Jen Daub, Ibrahim Kamal, Barton Slatko, The Filarial Genome Project, Bart Barrell, Neil Hall, Mehul Ganatra, David Guiliano

[International C. elegans Meeting, 2001]

The human-infective filarial nematode parasite Brugia malayi contains three genomes : the mitochondrial genome, the genome of the rickettsia-like Wolbachia endosymbiont and the nuclear genome. We have sequenced the mitochondrial genome of Brugia malayi and compared it to the other sequenced nematode mitochondria. The genome is, as expected, very similar to that of Onchocerca volvulus , and is remarkably different from C. elegans in gene order and sequence. Phylogenetic anlysis of nematode mitochondrial DNAs conflict with phylogenies derived from nuclear genes. Most filarial species harbour an bacterium that is believed to be in mutualistic symbiosis with the nematode. The bacteria are closely related to the Wolbachia endosymbionts of arthropods. The genome of the Wolbachia endosymbiont is being mapped and sequenced by a consortium headed by Barton Slatko at New England Biolabs. The nuclear genome of B. malayi is estimated to be 100 Mbp with an expected gene number comparable to C. elegans . To date, the Filarial Genome Project has produced 22,441 ESTs from 11 different cDNA libraries from various stages of the life cycle. These are estimated to represent ~8000 different genes. As a prelude to whole genome sequencing we are now in the process of constructing a physical map for the nuclear genome. A BAC library is being end sequenced, and BAC-derived end-probes hybridised to the gridded library to create contigs in a sampling-without replacement strategy. A number of EST clones have also been hybridised. In the course of this mapping program we have defined the distribution of sattelite repeats in the library, and have identified at least two families of retrotransposon-like elements.

Mhashilkar AS et al. (2016) Int J Parasitol "Phenotypic and molecular analysis of the effect of 20-hydroxyecdysone on the human filarial parasite Brugia malayi."

Slatko BE, Moorhead AR, Unnasch TR, Zaky W, Adapa SR, Jiang RH, Luck AN, Williams SA, Mhashilkar AS

[Int J Parasitol, 2016]

A homologue of the ecdysone receptor has been identified and shown to be responsive to 20-hydroxyecdysone in Brugia malayi. However, the role of this master regulator of insect development has not been delineated in filarial nematodes. Gravid adult female B. malayi cultured in the presence of 20-hydroxyecdysone produced significantly more microfilariae and abortive immature progeny than control worms, implicating the ecdysone receptor in regulation of embryogenesis and microfilarial development. Transcriptome analyses identified 30 genes whose expression was significantly up-regulated in 20-hydroxyecdysone-treated parasites compared with untreated controls. Of these, 18% were identified to be regulating transcription. A comparative proteomic analysis revealed 932 proteins to be present in greater amounts in extracts of 20-hydroxyecdysone-treated adult females than in extracts prepared from worms cultured in the absence of the hormone. Of the proteins exhibiting a greater than two-fold difference in the 20-hydroxyecdysone-treated versus untreated parasite extracts, 16% were involved in transcriptional regulation. RNA interference (RNAi) phenotype analysis of Caenorhabditis elegans orthologs revealed that phenotypes involved in developmental processes associated with embryogenesis were significantly over-represented in the transcripts and proteins that were up-regulated by exposure to 20-hydroxyecdysone. Taken together, the transcriptomic, proteomic and phenotypic data suggest that the filarial ecdysone receptor may play a role analogous to that in insects, where it serves as a regulator of egg development.

Wu B et al. (2013) Proc Natl Acad Sci U S A "Interdomain lateral gene transfer of an essential ferrochelatase gene in human parasitic nematodes."

Kumar S, Ford L, Taylor MJ, Dailey HA, Slatko BE, Landmann F, Jiang D, Foster JM, Carlow CK, Novelli J, Wu B

[Proc Natl Acad Sci U S A, 2013]

Lateral gene transfer events between bacteria and animals highlight an avenue for evolutionary genomic loss/gain of function. Herein, we report functional lateral gene transfer in animal parasitic nematodes. Members of the Nematoda are heme auxotrophs, lacking the ability to synthesize heme; however, the human filarial parasite Brugia malayi has acquired a bacterial gene encoding ferrochelatase (BmFeCH), the terminal step in heme biosynthesis. BmFeCH, encoded by a 9-exon gene, is a mitochondrial-targeted, functional ferrochelatase based on enzyme assays, complementation, and inhibitor studies. Homologs have been identified in several filariae and a nonfilarial nematode. RNAi and ex vivo inhibitor experiments indicate that BmFeCH is essential for viability, validating it as a potential target for filariasis control.