Biology subjects

Tan, C.-H.

Publications and source records attributed to Tan, C.-H..

9 recordsLinked to original sources

bioRxiv2025

Evolution of parasitism-related traits in nematodes

The abundant resources provided by the host provide an evolutionary rationale for parasitism and drive the metabolic and developmental divergence of parasitic and free-living animals. Two evolutionarily distant nematode genera, Steinernema and Heterorhabditis, independently evolved an entomopathogenic lifestyle, in which they invade insects and kill them with the assistance of specifically associated symbiotic pathogenic bacteria. It had been generally assumed that the worms, being bacterivores, feed on their symbiotic bacteria, which rapidly reproduce as they consume the insect host. The evolutionary adaptations of entomopathogenic nematodes to a parasitic lifestyle developmentally, and the symbiotic relationships of entomopathogenicity, remain largely unknown. We developed an axenic culture medium that supports robust and sustained growth of Steinernema hermaphroditum, with finite control of nutrients available to the nematodes. We found that, uniquely among the nematodes tested, S. hermaphroditum hatchlings cannot endure in a nutrient-poor environment; this ability is impaired but still present in Heterorhabditis bacteriophora. Similarly, the ability to forage for food is completely lost in H. bacteriophora hatchlings and severely compromised in S. hermaphroditum. We reasoned that these traits were lost because they are unnecessary to obligate parasites that always hatch in a resource-rich host. We further found that Steinernema and, to a limited extent, Heterorhabditis nematodes can successfully invade, develop, and reproduce inside a living insect host independent of their symbiotic bacteria, apparently feeding on the hemolymph, and emerge carrying bacteria found within, explaining the evolutionary origins of entomopathogenic nematodes. HighlightsO_LIA simple but robust axenic culturing method for the emerging model nematode Steinernema hermaphroditum and other nematode parasites of invertebrates. C_LIO_LINematode adaptation to parasitism is associated with changes in modes of feeding. C_LIO_LIConvergent evolution led to the loss of hatchling survival traits in entomopathogenic nematodes. C_LIO_LIEntomopathogenic nematodes evolved from parasitoid ancestors and likely acquired their bacterial symbionts from their hosts. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=146 HEIGHT=200 SRC="FIGDIR/small/678730v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@880785org.highwire.dtl.DTLVardef@a5cbcforg.highwire.dtl.DTLVardef@e78671org.highwire.dtl.DTLVardef@2b9d99_HPS_FORMAT_FIGEXP M_FIG C_FIG

evolutionary biology↗

bioRxiv2025

Genomes of the entomopathogenic nematode Steinernema hermaphroditum and its associated bacteria

As an entomopathogenic nematode (EPN), Steinernema hermaphroditum parasitizes insect hosts and harbors symbiotic Xenorhabdus griffinae bacteria. In contrast to other Steinernematids, S. hermaphroditum has hermaphroditic genetics, offering the experimental scope found in Caenorhabditis elegans. To enable biological analysis of S. hermaphroditum, we have assembled and analyzed its reference genome. This genome assembly has five chromosomal scaffolds and 83 unassigned scaffolds totaling 90.7 Mb, with 19,426 protein-coding genes having a BUSCO completeness of 88.0%. Its autosomes show higher densities of strongly conserved genes in their centers, as in C. elegans, but repetitive elements are evenly distributed along all chromosomes, rather than with higher arm densities as in C. elegans. Either when comparing protein motif frequencies between nematode species or when analyzing gene family expansions during nematode evolution, we observed two categories of genes preferentially associated with the origin of Steinernema or S. hermaphroditum: orthologs of venom genes in S. carpocapsae or S. feltiae; and some types of chemosensory G protein-coupled receptors, despite the tendency of parasitic nematodes to have reduced numbers of chemosensory genes. Three-quarters of venom orthologs occurred in gene clusters, with the larger clusters comprising functionally diverse pathogenicity islands rather than paralogous repeats of a single venom gene. While assembling the genome of S. hermaphroditum, we coassembled bacterial genomes, finding sequence data for not only the known symbiont, X. griffinae, but also for eight other bacterial genera. All eight genera have previously been observed to be associated with Steinernema species or the EPN Heterorhabditis, and may constitute a "second bacterial circle" of EPNs. The genome assemblies of S. hermaphroditum and its associated bacteria will enable use of these organisms as a model system for both entomopathogenicity and symbiosis.

genomics↗

bioRxiv2023

Single tissue proteomics in Caenorhabditis elegans reveals proteins resident in intestinal lysosome-related organelles

The nematode intestine is the primary site for nutrient uptake and storage as well as the synthesis of biomolecules; lysosome-related organelles known as gut granules are important for many of these functions. Aspects of intestine biology are not well understood, including the export of the nutrients it imports and the molecules it synthesizes, as well as the complete functions and protein content of the gut granules. Here, we report a mass spectrometry-based proteomic analysis of the intestine of the Caenorhabditis elegans and of its gut granules. Overall, we identified approximately 5,000 proteins each in the intestine and the gonad and showed that most of these proteins can be detected in samples extracted from a single worm, suggesting the feasibility of individual-level genetic analysis using proteomes. Comparing proteomes and published transcriptomes of the intestine and the gonad, we identified proteins that appear to be synthesized in the intestine and then transferred to the gonad. To identify gut granule proteins, we compared the proteome of individual intestines deficient in gut granules to the wild-type. The identified gut granule proteome includes proteins known to be exclusively localized to the granules and additional putative novel gut granule proteins. We selected two of these putative gut granule proteins for validation via immunohistochemistry, and our successful confirmation of both suggests that our strategy was effective in identifying the gut granule proteome. Our results demonstrate the practicability of single tissue mass-spectrometry- based proteomic analysis in small organisms and in its utility for making discoveries. Significance statementWe show that tissue-specific proteomic analysis is achievable and can be done efficiently at an individual level in a small nematode, with resolution sufficient for genetic analysis on a single animal basis. With data collected from single animals, we produced high-quality sets of proteins that described the proteomes of the gonad and the intestine. Comparison of these proteomes with the organs transcriptomes improved our understanding of interorgan protein transport. We applied single-tissue proteomic to describe the proteome of the gut granules in the nematode intestine, a specialized lysosome-related organelle with important functions but which is not well characterized, identifying proteins not previously known to be associated with LROs and verifying two by subcellular localization.

cell biology↗

bioRxiv2023

Molecular identification of a peroxidase gene controlling body size in the entomopathogenic nematode Steinernema hermaphroditum

The entomopathogenic nematode Steinernema hermaphroditum was recently rediscovered and is being developed as a genetically tractable experimental system for the study of previously unexplored biology, including parasitism of its insect hosts and mutualism with its bacterial endosymbiont Xenorhabdus griffiniae. Through whole-genome re-sequencing and genetic mapping we have for the first time molecularly identified the gene responsible for a mutationally defined phenotypic locus in an entomopathogenic nematode. In the process we observed an unexpected mutational spectrum following EMS mutagenesis in this species. We find that the ortholog of the essential C. elegans peroxidase gene skpo-2 controls body size and shape in S. hermaphroditum. We confirmed this identification by inactivating the gene using CRISPR-Cas9. We propose that the identification of skpo-2 will accelerate gene targeting in other Steinernema entomopathogenic nematodes used commercially in pest control, as skpo-2 is X-linked and males hemizygous for loss of its function can mate, making skpo-2 an easily recognized and maintained marker for use in co-CRISPR.

genetics↗

bioRxiv2023

Fluorescence dynamics of lysosomal-related organelle flashing in the intestinal cells of Caenorhabditis elegans

The lysosome-related organelles ("gut granules") in the intestinal cells of many nematodes, including Caenorhabditis elegans, play an important role in metabolic and signaling processes, but they have not been fully characterized. We report here a previously undescribed phenomenon in which the autofluorescence of these granules displays a "flash" phenomenon in which fluorescence decreases are preceded by sharp increases in fluorescence intensity that expand into the surrounding area when the granules are stimulated with blue light. Autofluorescent granules are present in the intestinal cells of all six nematode species examined, with differences in morphology and distribution pattern. Five species exhibit the flash phenomenon: Panagrellus redivivus (Clade IV), Steinernema hermaphroditum (Clade IV), C. elegans (Clade V), Oscheius tipulae (Clade V), and Pristionchus pacificus (Clade V). The reaction of the granules to blue light stimulation greatly differs among different developmental stages and may also be dependent on physiological conditions. In addition, even within the same animal, the sensitivity of individual granules differs, with some of the variation associated with other characteristics of the granules, such as their overall location within the intestine. We hypothesize that the differences in response to blue light indicate the existence of different sub-populations of gut granules in nematode intestines, and the visually spectacular dynamic fluorescence phenomenon we describe might provide a handle on their eventual characterization.

cell biology↗

bioRxiv2023

LINKIN-associated proteins necessary for tissue integrity during collective cell migration

Cell adhesion plays essential roles in almost every aspect of metazoan biology. LINKIN (Human: ITFG1, Caenorhabditis elegans: lnkn-1) is a conserved transmembrane protein that has been identified to be necessary for tissue integrity during migration. In C. elegans, loss of lnkn-1 results in the detachment of the lead migratory cell from the rest of the developing male gonad. Previously, three interactors of ITFG1/lnkn-1 - RUVBL1/ruvb-1, RUVBL2/ruvb-2, and alpha-tubulin - were identified by immunoprecipitation-mass spectrometry (IP-MS) analysis using human HEK293T cells and then validated in the nematode male gonad. The ITFG1-RUVBL1 interaction has since been independently validated in a breast cancer cell line model that also implicates the involvement of the pair in metastasis. Here, we showed that epitope-tagged ITFG1 localized to the cell surface of MDA-MB-231 breast cancer cells. Using IP-MS analysis, we identified a new list of potential interactors of ITFG1. Loss-of-function analysis of their C. elegans orthologs found that three of the interactors - ATP9A/tat-5, NME1/ndk-1, and ANAPC2/apc-2 - displayed migratory detachment phenotypes similar to that of lnkn-1. Taken together with the other genes whose reduction-of-function phenotype is similar to that of lnkn-1 (notably cohesion and condensin), suggests the involvement of membrane remodeling and chromosome biology in LINKIN-dependent cell adhesion and supports the hypothesis for a structural role of chromosomes in post-mitotic cells.

developmental biology↗

bioRxiv2021

Lysosome-related organelles contain an expansion compartment that mediates delivery of zinc transporters to promote homeostasis

Lysosome-related organelles play evolutionarily conserved roles in zinc storage, but mechanisms that control zinc flow in and out are not well understood. In C. elegans intestinal cells, the CDF-2 transporter stores zinc in these organelles during excess. Here we identify ZIPT-2.3 as the transporter that releases zinc during deficiency. The expression levels of CDF-2 and ZIPT-2.3 are reciprocally regulated in zinc excess and deficiency, establishing a fundamental mechanism of homeostasis. Super-resolution microscopy demonstrated these organelles are composed of a spherical acidified compartment and a hemispherical expansion compartment. The expansion compartment inflates during zinc excess and deficiency by vesicle fusion delivering zinc transporters. These results identify an unexpected structural feature of lysosome-related organelles that facilitates rapid transitions in the composition of zinc transporters to mediate homeostasis.

cell biology↗

bioRxiv2021

The entomopathogenic nematode Steinernema hermaphroditum is a self-fertilizing hermaphrodite and a genetically tractable system for the study of parasitic and mutualistic symbiosis

Entomopathogenic nematodes, including Heterorhabditis and Steinernema, are parasitic to insects and contain mutualistically symbiotic bacteria in their intestines (Photorhabdus and Xenorhabdus, respectively) and therefore offer opportunities to study both mutualistic and parasitic symbiosis. The establishment of genetic tools in entomopathogenic nematodes has been impeded by limited genetic tractability, inconsistent growth in vitro, variable cryopreservation, and low mating efficiency. We obtained the recently described Steinernema hermaphroditum strain CS34 and optimized its in vitro growth, with a rapid generation time on a lawn of its native symbiotic bacteria Xenorhabdus griffiniae. We developed a simple and efficient cryopreservation method. Previously, S. hermaphroditum isolated from insect hosts was described as first-generation hermaphroditic and second-generation gonochoristic. We discovered that CS34, when grown in vitro, produced consecutive generations of autonomously reproducing hermaphrodites accompanied by rare males. We performed mutagenesis screens in S. hermaphroditum that produced mutant lines with visible and heritable phenotypes. Genetic analysis of the mutants demonstrated that this species reproduces by self-fertilization rather than parthenogenesis and that its sex is determined chromosomally. Genetic mapping has thus far identified markers on the X chromosome and three of four autosomes. We report that S. hermaphroditum CS34 is the first consistently hermaphroditic entomopathogenic nematode and is suitable for genetic model development to study naturally occurring mutualistic symbiosis and insect parasitism.

genetics↗

bioRxiv2021

TRPM2 ion channels steer neutrophils towards a source of hydrogen peroxide

Neutrophils must navigate accurately towards pathogens in order to destroy invaders and thus defend our bodies against infection. Here we show that hydrogen peroxide, a potent neutrophil chemoattractant, guides chemotaxis by activating calcium-permeable TRPM2 ion channels and thus generating an intracellular leading-edge calcium "pulse". The thermal sensitivity of TRPM2 activation means that chemotaxis towards hydrogen peroxide is strongly promoted by small temperature elevations, suggesting that an important function of fever may be to enhance neutrophil chemotaxis by facilitating calcium influx through TRPM2. Chemotaxis towards conventional chemoattractants such as LPS, CXCL2 and C5a does not depend on TRPM2 but is driven in a similar way by leading-edge calcium pulses. Other proposed initiators of neutrophil movement, such as PI3K, Rac and lyn, influence chemotaxis by modulating the amplitude of calcium pulses. We propose that intracellular leading-edge calcium pulses are universal drivers of the motile machinery involved in neutrophil chemotaxis.

immunology↗