Grote A et al. (2020) PLoS Negl Trop Dis "Prediction pipeline for discovery of regulatory motifs associated with Brugia malayimolting."

Voronin D, Lustigman S, Geber A, Unnasch TR, Ghedin E, Grote A, Liu C, Li Y, Welch L

[PLoS Negl Trop Dis, 2020]

Filarial nematodes can cause debilitating diseases in humans. They have complicated life cycles involving an insect vector and mammalian hosts, and they go through a number of developmental molts. While whole genome sequences of parasitic worms are now available, very little is known about transcription factor (TF) binding sites and their cognate transcription factors that play a role in regulating development. To address this gap, we developed a novel motif prediction pipeline, Emotif Alpha, that integrates ten different motif discovery algorithms, multiple statistical tests, and a comparative analysis of conserved elements between the filarial worms Brugia malayi and Onchocerca volvulus, and the free-living nematode Caenorhabditis elegans. We identified stage-specific TF binding motifs in B. malayi, with a particular focus on those potentially involved in the L3-L4 molt, a stage important for the establishment of infection in the mammalian host. Using an in vitro molting system, we tested and validated three of these motifs demonstrating the accuracy of the motif prediction pipeline.

Grote A et al. (2017) PLoS Negl Trop Dis "Defining Brugia malayi and Wolbachia symbiosis by stage-specific dual RNA-seq."

Ghedin E, Grote A, Unnasch TR, Voronin D, Ding T, Lustigman S, Twaddle A

[PLoS Negl Trop Dis, 2017]

BACKGROUND: Filarial nematodes currently infect up to 54 million people worldwide, with millions more at risk for infection, representing the leading cause of disability in the developing world. Brugia malayi is one of the causative agents of lymphatic filariasis and remains the only human filarial parasite that can be maintained in small laboratory animals. Many filarial nematode species, including B. malayi, carry an obligate endosymbiont, the alpha-proteobacteria Wolbachia, which can be eliminated through antibiotic treatment. Elimination of the endosymbiont interferes with development, reproduction, and survival of the worms within the mamalian host, a clear indicator that the Wolbachia are crucial for survival of the parasite. Little is understood about the mechanism underlying this symbiosis. METHODOLOGY/ PRINCIPLE FINDINGS: To better understand the molecular interplay between these two organisms we profiled the transcriptomes of B. malayi and Wolbachia by dual RNA-seq across the life cycle of the parasite. This helped identify functional pathways involved in this essential symbiotic relationship provided by the co-expression of nematode and bacterial genes. We have identified significant stage-specific and gender-specific differential expression in Wolbachia during the nematode's development. For example, during female worm development we find that Wolbachia upregulate genes involved in ATP production and purine biosynthesis, as well as genes involved in the oxidative stress response. CONCLUSIONS/ SIGNIFICANCE: This global transcriptional analysis has highlighted specific pathways to which both Wolbachia and B. malayi contribute concurrently over the life cycle of the parasite, paving the way for the development of novel intervention strategies.

Phillip Grote et al. (2001) International C. elegans Meeting "Specification of the Sexually Dimorphic Programmed Cell Death of the CEM Neurons"

Phillip Grote, Claudia Huber, BARBARA CONRADT

[International C. elegans Meeting, 2001]

Sexual dimorphism in the nervous system of C. elegans is created by differential numbers of cell divisions, by differential cell fates or by programmed cell death (PCD). The hermaphrodite specific neurons (HSNs), two motoneurons necessary for egg laying, and the male-specific cephalic companion cells (CEMs), four sensory neurons of so far unknown function, are the only neurons that are sex-specific as a result of sexually dimorphic PCD. The HSNs and CEMs are born in both sexes; however, the HSNs are subsequently eliminated by PCD in males and the CEMs in hermaphrodites (1). The survival of the HSNs in hermaphrodites is specified by a direct interaction between the terminal, global regulator of somatic sexual fate, the Zn finger DNA binding protein TRA-1A ( tra , tra nsformer) (2), and the egl-1 locus ( egl , eg g- l aying defective), which encodes the primary activator of PCD in somatic tissues (3). The TRA-1A protein, which acts to promote female development in somatic tissues, binds to a TRA-1A binding site in the egl-1 locus thereby causing the repression of egl-1 transcription in the HSNs and HSN survival in hermaphrodites (3). To determine the role of the tra-1 and egl-1 genes in the specification of the death of the CEMs in hermaphrodites, we analyzed the survival of the CEMs in hermaphrodites (XX) homozygous mutant for loss-of-function (lf) mutations in either tra-1 or egl-1 . We found that both genes are required for the death of the CEMs in hermaphrodites. Furthermore, an egl-1 (lf) mutation blocks the ability of a tra-1 gain-of-function (gf) mutation to induce the CEMs to undergo PCD in males (X0). This suggests that TRA-1A acts as an activator of egl-1 in the CEMs. The TRA-1A binding site required for the ability of TRA-1A to repress egl-1 transcription in the HSNs, is, however, not required for the ability of TRA-1A to induce the CEMs to die. This indicates that TRA-1A acts as an indirect activator of egl-1 transcription or through a different binding site within the egl-1 locus. To determine how TRA-1A might specify the death of the CEMs and to identify additional factors required for this process, we are using two approaches. First, we are determining the region of the egl-1 locus that is required to rescue the death of the CEMs in egl-1 (lf) hermaphrodites. So far, we have identified a 690 bp region 3 kb downstream of the egl-1 transcription unit that is required to cause CEM death in hermaphrodites. Second, using a CEM-specific GFP reporter ( pkd-2 :: gfp ) (4), we performed a screen for mutations that cause the CEMs to survive in hermaphrodites and have identified at least two new loci that can mutate to cause CEM survival in hermaphrodites. (1) Sulston, JE and Horvitz, HR (1977) Dev Biol , 56(1):110-156 (2) Zarkower, D and Horvitz, HR (1992) Cell , 70(2):237-249 (3) Conradt, B and Horvitz, HR (1999) Cell , 98(3):317-327 (4) Barr, MM and Sternberg, PW (1999) Nature , 401(6751):386-389

Phillip Grote et al. (2004) East Coast Worm Meeting "The genes tra-4 and mog-7 are necessary to ensure hermaphrodite development in the soma and in the germline"

Phillip Grote, Claudia Huber, BARBARA CONRADT

[East Coast Worm Meeting, 2004]

During C. elegans development, the decision to develop into a hermaphrodite (XX) or a male (XO) is determined by the ratio of the number of X chromosomes to the number of sets of autosomes (X :A ratio). In the soma the signal induced by this ratio is transmitted through a cascade of interacting genes, which determines the activity of the most downstream factor of this cascade, the transcription factor TRA-1A ( tra , tra nsformer). The same genes are involved in determining sexual fate in the germline; however, additional genes are required for this process. The determination of the sexual fate in the germline is a complex regulatory process because self-fertilizing hermaphrodites produce sperm during the last larval stage (L4) but have to switch to the production of oocytes after becoming adults. Strong loss-of-function (lf) mutations in genes required for feminization ( tra genes) can lead to the development of completely transformed animals: XX animals develop into phenotypic males. Weak lf mutations in tra genes can cause a weak masculinization: XX animals lack the hermaphrodite-specific neurons (HSNs), have the male-specific cephalic companion neurons (CEMs) and show additional defects indicative of masculinization. In a forward genetic screen originally designed to identify factors involved in the regulation of the sexually dimorphic death of the CEMs, we identified a gene, which when mutated causes a weak masculinization of XX animals, suggesting that it is required for feminization. We therefore named this gene tra-4 . tra-4 encodes a protein with seven C2H2 type zinc-finger domains, which are known to interact with DNA and/or RNA. Furthermore we identified a homologue of tra-4 in the C. elegans genome . Hermaphrodites homozygous for a lf mutation in the tra-4 homologue fail to switch from sperm production to oocyte production after the L4 stage. We therefore refer to this gene as mog-7 ( mog , m ale- o nly g onad). We are currently investigating the roles of tra-4 and mog-7 in the regulation of sex determination in the soma and in the germline, respectively. We propose that tra-4 is necessary for female development in the soma and that mog-7 fulfills a similar function in the germline. Furthermore, while tra4 and mog-7 seem to play similar but independent roles in sex determination, they appear to have a redundant function in an essential process, since tra-4 (lf) ; mog-7 (lf) double mutants are non-viable.

Phillip Grote et al. (2002) European Worm Meeting "Using a Combination of Two Recombinases to Create Targeted Single- copy Genomic Insertion in C. elegans"

Matthias Lauth, Michael Meyer, BARBARA CONRADT, Phillip Grote

[European Worm Meeting, 2002]

The most commonly used technique to create transgenic C. elegans animals has several disadvantages for a number of applications, such as promotor studies or studies on gene regulation. Extrachromosomal arrays created by microinjection contain, for instance, the transgene at a high copy number (1). This can cause silencing and/or misregulation of the transgene. The ideal technique for creating transgenic animals (at least for some applications) would be a technique that allows the insertion of a single copy of a transgene at a specific chromosomal location.

Phillip Grote et al. (2003) International Worm Meeting "tra-4 is necessary to ensure complete hermaphrodite development and encodes a zinc finger DNA binding protein"

BARBARA CONRADT, Phillip Grote, Claudia Huber

[International Worm Meeting, 2003]

During C. elegans development, the decision to develop a female (XX) or male (XO) soma is determined by the ratio of the number of X chromosomes to the number of sets of autosomes (X:A ratio). The resulting signal is transmitted through a cascade of interacting genes and determines the activity of the most downstream factor of this cascade, the transcription factor TRA-1A (TRA, transformer). Strong loss-of-function (lf) mutations in the tra-1 gene lead to a complete transformation of the XX soma into a male soma while weak tra-1(lf) mutations result in a partial masculinization of the XX soma. A partially masculinized XX soma overall resembles a wild-type XX soma, however, frequently the hermaphrodite-specific HSN neurons are absent and the male-specific CEM neurons are present. The HSNs and CEMs are the only neurons in C. elegans that are sex-specific as a result of programmed cell death. The specification of the cell death fate of these cells therefore seems to be particularly sensitive to changes in tra-1 dosage. In a screen for mutants, in which the CEMs inappropriately survive in XX animals, we identified mutations in a previously not described gene, referred to as tra-4, which is required for complete feminization of the XX soma. bc45, a lf mutation in the tra-4 gene, causes a partial masculinization of the XX soma: 73% of the HSNs undergo programmed cell death and 80% of the CEMs escape programmed cell death. In addition, in tra-4(bc45) L1 hermaphrodites the B cells and the ventral left coelomocytes have a male appearance or position, respectively, indicating weak masculinization. Double mutant analyses suggest that tra-4 acts upstream of fem-1, fem-2, and fem-3 (fem, feminization of XX and XO animals), and downstream of her-1 (her, hermaphrodization of XO animals) and possibly tra-2. tra-4 might therefore be a negative regulator of the fem genes. The tra-4 locus encodes a protein with predicted nuclear localization signals and C2H2 type Zinc finger domains, which indicates a role in transcriptional regulation. bc45 is a missense mutation leading to the exchange of a conserved amino acid in one of the C2H2 DNA-binding motives, suggesting that bc45 might not be a null allele. The analysis of the expression pattern of a functional Ptra-4GFP::TRA-4 construct indicates that tra-4 is expressed in most if not all somatic nuclei at all developmental stages in animals of both sexes. Using various approaches, we are currently searching for targets of the TRA-4 protein.

Phillip Grote et al. (2002) West Coast Worm Meeting "Using a Combination of Two Recombinases to Create Targeted Single-copy Genomic Insertion in C. elegans"

Matthias Lauth, Michael Meyer, Phillip Grote, BARBARA CONRADT

[West Coast Worm Meeting, 2002]

The most commonly used technique to create transgenic C. elegans animals has several disadvantages for a number of applications, such as promotor studies or studies on gene regulation. Extrachromosomal arrays created by microinjection contain, for instance, the transgene at a high copy number (1). This can cause silencing and/or misregulation of the transgene. The ideal technique for creating transgenic animals (at least for some applications) would be a technique that allows the insertion of a single copy of a transgene at a specific chromosomal location.

Grote P et al. (2006) Dev Cell "The PLZF-like protein TRA-4 cooperates with the Gli-like transcription factor TRA-1 to promote female development in C. elegans."

Conradt B, Grote P

[Dev Cell, 2006]

The Gli-like transcription factor TRA-1 of C. elegans promotes female development by repressing the transcription of male-specific genes. We have found that tra-1 interacts with tra-4, a previously uncharacterized gene that encodes a protein similar to the human proto-oncoprotein and transcriptional repressor PLZF. In this context, the TRA-4 protein functions with NASP-1, a C. elegans homolog of the mammalian histone chaperone NASP, and the histone deacetylase HDA-1. We also found that tra-4 is a member of the synMuv B group of genes, many of which encode homologs of components of the Drosophila Myb-Muv B transcriptional repressor complex, and that several synMuv B genes also promote female development. Based on these results, we propose that male-specific genes are repressed in C. elegans hermaphrodites by the combined action of TRA-1/Gli, a complex composed of TRA-4/PLZF-like, NASP, and HDA-1/HDAC, and synMuv B proteins. Similar interactions may function in sex determination and developmental regulation in other species.

Liu C et al. (2020) PLoS Negl Trop Dis "CRISPR-mediated Transfection of Brugia malayi."

Unnasch TR, Grote A, Liu C, Ghedin E

[PLoS Negl Trop Dis, 2020]

The application of reverse genetics in the human filarial parasites has lagged due to the difficult biology of these organisms. Recently, we developed a co-culture system that permitted the infective larval stage of Brugia malayi to be transfected and efficiently develop to fecund adults. This was exploited to develop a piggyBac transposon-based toolkit that can be used to produce parasites with transgene sequences stably integrated into the parasite genome. However, the piggyBac system has generally been supplanted by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based technology, which allows precise editing of a genome. Here we report adapting the piggyBac mediated transfection system of B. malayi for CRISPR mediated knock-in insertion into the parasite genome. Suitable CRISPR insertion sites were identified in intergenic regions of the B. malayi genome. A dual reporter piggybac vector was modified, replacing the piggyBac inverted terminal repeat regions with sequences flanking the insertion site. B. malayi molting L3 were transfected with a synthetic guide RNA, the modified plasmid and the CAS9 nuclease. The transfected parasites were implanted into gerbils and allowed to develop into adults. Progeny microfilariae were recovered and screened for expression of a secreted luciferase reporter encoded in the plasmid. Approximately 3% of the microfilariae were found to secrete luciferase; all contained the transgenic sequences inserted at the expected location in the parasite genome. Using an adaptor mediated PCR assay, transgenic microfilariae were examined for the presence of off target insertions; no off-target insertions were found. These data demonstrate that CRISPR can be used to modify the genome of B. malayi, opening the way to precisely edit the genome of this important human filarial parasite.

Foster JM et al. (2020) Nat Commun "Sex chromosome evolution in parasitic nematodes of humans."

Rogers MB, Tsai YC, Michalski ML, Grote A, Chung M, Tracey A, Ghedin E, Geber A, Foster JM, Libro S, Welch L, Cotton JA, Paulini M, Twaddle A, Korlach J, Lustigman S, Teigen L, Berriman M, Li Y, Holroyd N, Mattick J, Dunning Hotopp JC, Clark TA

[Nat Commun, 2020]

Sex determination mechanisms often differ even between related species yet the evolution of sex chromosomes remains poorly understood in all but a few model organisms. Some nematodes such as Caenorhabditis elegans have an XO sex determination system while others, such as the filarial parasite Brugia malayi, have an XY mechanism. We present a complete B. malayi genome assembly and define Nigon elements shared with C. elegans, which we then map to the genomes of other filarial species and more distantly related nematodes. We find a remarkable plasticity in sex chromosome evolution with several distinct cases of neo-X and neo-Y formation, X-added regions, and conversion of autosomes to sex chromosomes from which we propose a model of chromosome evolution across different nematode clades. The phylum Nematoda offers a new and innovative system for gaining a deeper understanding of sex chromosome evolution.