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The nematode Caenorhabditis elegans is an invaluable host model for studying infections caused by various pathogens, including microsporidia. Microsporidia represent the first natural pathogens identified in C. elegans, revealing the previously unknown Nematocida genus of microsporidia. Following this discovery, the utilization of nematodes as a model host has rapidly expanded our understanding of microsporidia biology and has provided key insights into the cell and molecular mechanisms of antimicrosporidia defenses. Here, we first review the isolation history, morphological characteristics, life cycles, tissue tropism, genetics, and host immune responses for the four most well-characterized Nematocida species that infect C. elegans. We then highlight additional examples of microsporidia that infect related terrestrial and aquatic nematodes, including parasitic nematodes. To conclude, we assess exciting potential applications of the nematode-microsporidia system while addressing the technical advances necessary to facilitate future growth in this field., (© 2024 International Society of Protistologists.)

The maize ligule is an epidermis-derived structure that arises from the preligule band (PLB) at a boundary between the blade and sheath. A hinge-like auricle also develops immediately distal to the ligule and contributes to blade angle. Here, we characterize the stages of PLB and early ligule development in terms of topography, cell area, division orientation, cell wall rigidity and auxin response dynamics. Differential thickening of epidermal cells and localized periclinal divisions contributed to the formation of a ridge within the PLB, which ultimately produces the ligule fringe. Patterns in cell wall rigidity were consistent with the subdivision of the PLB into two regions along a distinct line positioned at the nascent ridge. The proximal region produces the ligule, while the distal region contributes to one epidermal face of the auricles. Although the auxin transporter PIN1 accumulated in the PLB, observed differential auxin transcriptional response did not underlie the partitioning of the PLB. Our data demonstrate that two zones with contrasting cellular properties, the preligule and preauricle, are specified within the ligular region before ligule outgrowth., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Although the type-I interferon (IFN-I) response is considered vertebrate-specific, recent findings about the Intracellular Pathogen Response (IPR) in nematode Caenorhabditis elegans indicate that there are similarities between these two transcriptional immunological programs. The IPR is induced during infection with natural intracellular fungal and viral pathogens of the intestine and promotes resistance against these pathogens. Similarly, the IFN-I response is induced by viruses and other intracellular pathogens and promotes resistance against infection. Whether the IPR and the IFN-I response evolved in a divergent or convergent manner is an unanswered and exciting question, which could be addressed by further studies of immunity against intracellular pathogens in C. elegans and other simple host organisms. Here we highlight similar roles played by RIG-I-like receptors, purine metabolism enzymes, proteotoxic stressors, and transcription factors to induce the IPR and IFN-I response, as well as the similar consequences of these defense programs on organismal development., (© 2023 The Authors. BioEssays published by Wiley Periodicals LLC.)

The immune system continually battles against pathogen-induced pressures, which often leads to the evolutionary expansion of immune gene families in a species-specific manner. For example, the pals gene family expanded to 39 members in the Caenorhabditis elegans genome, in comparison to a single mammalian pals ortholog. Our previous studies have revealed that two members of this family, pals-22 and pals-25, act as antagonistic paralogs to control the Intracellular Pathogen Response (IPR). The IPR is a protective transcriptional response, which is activated upon infection by two molecularly distinct natural intracellular pathogens of C. elegans-the Orsay virus and the fungus Nematocida parisii from the microsporidia phylum. In this study, we identify a previously uncharacterized member of the pals family, pals-17, as a newly described negative regulator of the IPR. pals-17 mutants show constitutive upregulation of IPR gene expression, increased immunity against intracellular pathogens, as well as impaired development and reproduction. We also find that two other previously uncharacterized pals genes, pals-20 and pals-16, are positive regulators of the IPR, acting downstream of pals-17. These positive regulators reverse the effects caused by the loss of pals-17 on IPR gene expression, immunity, and development. We show that the negative IPR regulator protein PALS-17 and the positive IPR regulator protein PALS-20 colocalize inside and at the apical side of intestinal epithelial cells, which are the sites of infection for IPR-inducing pathogens. In summary, our study demonstrates that several pals genes from the expanded pals gene family act as ON/OFF switch modules to regulate a balance between organismal development and immunity against natural intracellular pathogens in C. elegans., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lažetić et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

Defense against intracellular infection has been extensively studied in vertebrate hosts, but less is known about invertebrate hosts; specifically, the transcription factors that induce defense against intracellular intestinal infection in the model nematode Caenorhabditis elegans remain understudied. Two different types of intracellular pathogens that naturally infect the C. elegans intestine are the Orsay virus, which is an RNA virus, and microsporidia, which comprise a phylum of fungal pathogens. Despite their molecular differences, these pathogens induce a common host transcriptional response called the intracellular pathogen response (IPR). Here we show that zip-1 is an IPR regulator that functions downstream of all known IPR-activating and regulatory pathways. zip-1 encodes a putative bZIP transcription factor, and we show that zip-1 controls induction of a subset of genes upon IPR activation. ZIP-1 protein is expressed in the nuclei of intestinal cells, and is at least partially required in the intestine to upregulate IPR gene expression. Importantly, zip-1 promotes resistance to infection by the Orsay virus and by microsporidia in intestinal cells. Altogether, our results indicate that zip-1 represents a central hub for triggers of the IPR, and that this transcription factor has a protective function against intracellular pathogen infection in C. elegans., (© 2022. The Author(s).)

One of the strongest drivers in evolution is the struggle to survive a host-pathogen battle. This pressure selects for diversity among the factors directly involved in this battle, including virulence factors deployed by pathogens, their corresponding host targets, and host immune factors. A logical outcome of this diversification is that over time, the sequence of many immune factors will not be evolutionarily conserved across a broad range of species. Thus, while universal sequence conservation is often hailed as the hallmark of the importance of a particular gene, the immune system does not necessarily play by these rules when defending against co-evolving pathogens. This loss of sequence conservation is in contrast to many signaling pathways in development and basic cell biology that are not targeted by pathogens. In addition to diversification, another consequence of host-pathogen battles can be an amplification in gene number, thus leading to large gene families that have sequence relatively specific to a particular strain, species, or clade. Here we highlight this general theme across a variety of pathogen virulence factors and host immune factors. We summarize the wide range and number across species of these expanded, lineage-specific host-pathogen factors including ubiquitin ligases, nucleotide-binding leucine-rich repeat receptors, GTPases, and proteins without obvious biochemical function but that nonetheless play key roles in immunity., (© 2020 Federation of European Biochemical Societies.)

Endocytosis, the process by which cells internalize plasma membrane and associated cargo, is regulated extensively by posttranslational modifications. Previous studies suggested the potential involvement of scores of protein kinases in endocytic control, of which only a few have been validated in vivo. Here we show that the conserved NIMA-related kinases NEKL-2/NEK8/9 and NEKL-3/NEK6/7 (the NEKLs) control clathrin-mediated endocytosis in C. elegans. Loss of NEKL-2 or NEKL-3 activities leads to penetrant larval molting defects and to the abnormal localization of trafficking markers in arrested larvae. Using an auxin-based degron system, we also find that depletion of NEKLs in adult-stage C. elegans leads to gross clathrin mislocalization and to a dramatic reduction in clathrin mobility at the apical membrane. Using a non-biased genetic screen to identify suppressors of nekl molting defects, we identified several components and regulators of AP2, the major clathrin adapter complex acting at the plasma membrane. Strikingly, reduced AP2 activity rescues both nekl mutant molting defects as well as associated trafficking phenotypes, whereas increased levels of active AP2 exacerbate nekl defects. Moreover, in a unique example of mutual suppression, NEKL inhibition alleviates defects associated with reduced AP2 activity, attesting to the tight link between NEKL and AP2 functions. We also show that NEKLs are required for the clustering and internalization of membrane cargo required for molting. Notably, we find that human NEKs can rescue molting and trafficking defects in nekl mutant worms, suggesting that the control of intracellular trafficking is an evolutionarily conserved function of NEK family kinases., Competing Interests: The authors have declared that no competing interests exist.

Natural genetic variation can determine the outcome of an infection, and often reflects the co-evolutionary battle between hosts and pathogens. We previously found that a natural variant of the nematode Caenorhabditis elegans from Hawaii (HW) has increased resistance against natural microsporidian pathogens in the Nematocida genus, when compared to the standard laboratory strain of N2. In particular, HW animals can clear infection, while N2 animals cannot. In addition, HW animals have lower levels of initial colonization of Nematocida inside intestinal cells, compared to N2. Here we investigate how this natural variation in resistance relates to autophagy. We found that there is much better targeting of autophagy-related machinery to parasites under conditions where they are cleared. In particular, ubiquitin targeting to Nematocida cells correlates very well with their subsequent clearance in terms of timing, host strain and age, as well as species of Nematocida. Furthermore, clearance correlates with targeting of the LGG-2/LC3 autophagy protein to parasite cells, with HW animals having much more efficient targeting of LGG-2 to parasite cells than N2 animals. Surprisingly, however, we found that LGG-2 is not required to clear infection. Instead, we found that LGG-2/LC3 regulates Nematocida colonization inside intestinal cells. Interestingly, LGG-2/LC3 regulates intracellular colonization only in the HW strain, and not in N2. Altogether these results demonstrate that there is natural genetic variation in an LGG-2-dependent process that regulates microsporidia colonization inside intestinal cells, although not microsporidia clearance., Competing Interests: The authors have declared that no competing interests exist.

RING-between-RING (RBR) E3 ubiquitin ligases are implicated in various developmental processes, and mutations in genes encoding RBR proteins HHARI/ARIH1 and Parkin are associated with human diseases. Here we show by phylogenetic analysis that the ARI1 family has undergone a dramatic expansion within the Caenorhabditis clade in recent history, a characteristic shared by some genes involved in germline development. We then examined the effects of deleting all ARI1 family members in the nematode Caenorhabditis elegans, which to our knowledge represents the first complete knockout of ARI1 function in a metazoan. Hermaphrodites that lacked or had strongly reduced ARI1 activity had low fecundity and were partially defective in initiation of oocyte differentiation. We provide evidence that the C. elegans ARI1s likely function downstream or in parallel to FBF-1 and FBF-2, two closely related RNA-binding proteins that are required for the switch from spermatogenesis to oogenesis during late larval development. Previous studies have shown that the E2 enzymes UBC-18/UBCH7 and UBC-3/CDC34 can functionally collaborate with ARI1 family members. Our data indicated that UBC-18, but not UBC-3, specifically cooperates with the ARI1s in germline development. These findings provide new insights into the functions of RING-between-RING proteins and Ariadne E3s during development.

Molting is an essential process in the nematode Caenorhabditis elegans during which the epidermal apical extracellular matrix, termed the cuticle, is detached and replaced at each larval stage. The conserved NIMA-related kinases NEKL-2/NEK8/NEK9 and NEKL-3/NEK6/NEK7, together with their ankyrin repeat partners, MLT-2/ANKS6, MLT-3/ANKS3, and MLT-4/INVS, are essential for normal molting. In nekl and mlt mutants, the old larval cuticle fails to be completely shed, leading to entrapment and growth arrest. To better understand the molecular and cellular functions of NEKLs during molting, we isolated genetic suppressors of nekl molting-defective mutants. Using two independent approaches, we identified CDC-42, a conserved Rho-family GTPase, and its effector protein kinase, SID-3/ACK1. Notably, CDC42 and ACK1 regulate actin dynamics in mammals, and actin reorganization within the worm epidermis has been proposed to be important for the molting process. Inhibition of NEKL-MLT activities led to strong defects in the distribution of actin and failure to form molting-specific apical actin bundles. Importantly, this phenotype was reverted following cdc-42 or sid-3 inhibition. In addition, repression of CDC-42 or SID-3 also suppressed nekl-associated defects in trafficking, a process that requires actin assembly and disassembly. Expression analyses indicated that components of the NEKL-MLT network colocalize with both actin and CDC-42 in specific regions of the epidermis. Moreover, NEKL-MLT components were required for the normal subcellular localization of CDC-42 in the epidermis as well as wild-type levels of CDC-42 activation. Taken together, our findings indicate that the NEKL-MLT network regulates actin through CDC-42 and its effector SID-3. Interestingly, we also observed that downregulation of CDC-42 in a wild-type background leads to molting defects, suggesting that there is a fine balance between NEKL-MLT and CDC-42-SID-3 activities in the epidermis.

Molting is an essential developmental process for the majority of animal species on Earth. During the molting process, which is a specialized form of extracellular matrix (ECM) remodeling, the old apical ECM, or cuticle, is replaced with a new one. Many of the genes and pathways identified as important for molting in nematodes are highly conserved in vertebrates and include regulators and components of vesicular trafficking, steroid-hormone signaling, developmental timers, and hedgehog-like signaling. In this review, we discuss what is known about molting, with a focus on studies in Caenorhabditis elegans . We also describe the key structural elements of the cuticle that must be released, newly synthesized, or remodeled for proper molting to occur.

Molting is an essential developmental process in nematodes during which the epidermal apical extracellular matrix, the cuticle, is remodeled to accommodate further growth. Using genetic approaches, we identified a requirement for three conserved ankyrin repeat-rich proteins, MLT-2/ANKS6, MLT-3/ANKS3, and MLT-4/INVS, in Caenorhabditis elegans molting. Loss of mlt function resulted in severe defects in the ability of larvae to shed old cuticle and led to developmental arrest. Genetic analyses demonstrated that MLT proteins functionally cooperate with the conserved NIMA kinase family members NEKL-2/NEK8 and NEKL-3/NEK6/NEK7 to promote cuticle shedding. MLT and NEKL proteins were specifically required within the hyp7 epidermal syncytium, and fluorescently tagged mlt and nekl alleles were expressed in puncta within this tissue. Expression studies further showed that NEKL-2-MLT-2-MLT-4 and NEKL-3-MLT-3 colocalize within largely distinct assemblies of apical foci. MLT-2 and MLT-4 were required for the normal accumulation of NEKL-2 at the hyp7-seam cell boundary, and loss of mlt-2 caused abnormal nuclear accumulation of NEKL-2 Correspondingly, MLT-3, which bound directly to NEKL-3, prevented NEKL-3 nuclear localization, supporting the model that MLT proteins may serve as molecular scaffolds for NEKL kinases. Our studies additionally showed that the NEKL-MLT network regulates early steps in clathrin-mediated endocytosis at the apical surface of hyp7, which may in part account for molting defects observed in nekl and mlt mutants. This study has thus identified a conserved NEKL-MLT protein network that regulates remodeling of the apical extracellular matrix and intracellular trafficking, functions that may be conserved across species., (Copyright © 2017 by the Genetics Society of America.)

Caenorhabditis elegans molting is a process during which the apical extracellular matrix of the epidermis, the cuticle, is remodeled through a process of degradation and re-synthesis. Using a genetic approach, we identified nekl-3 as essential for the completion of molting. NEKL-3 is highly similar to the mammalian NEK kinase family members NEK6 and NEK7. Animals homozygous for a hypomorphic mutation in nekl-3, sv3, had a novel molting defect in which the central body region, but not the head or tail, was unable to shed the old cuticle. In contrast, a null mutation in nekl-3, gk506, led to complete enclosure within the old cuticle. nekl-2, which is most similar to mammalian NEK8, was also essential for molting. Mosaic analyses demonstrated that NEKL-2 and NEKL-3 were specifically required within the large epidermal syncytium, hyp7, to facilitate molting. Consistent with this, NEKL-2 and NEKL-3 were expressed at the apical surface of hyp7 where they localized to small spheres or tubular structures. Inhibition of nekl-2, but not nekl-3, led to the mislocalization of LRP-1/megalin, a cell surface receptor for low-density lipoprotein (LDL)-binding proteins. In addition, nekl-2 inhibition led to the mislocalization of several other endosome-associated proteins. Notably, LRP-1 acts within hyp7 to facilitate completion of molting, suggesting at least one mechanism by which NEKL-2 may influence molting. Notably, our studies failed to reveal a requirement for NEKL-2 or NEKL-3 in cell division, a function reported for several mammalian NEKs including NEK6 and NEK7. Our findings provide the first genetic and in vivo evidence for a role of NEK family members in endocytosis, which may be evolutionarily conserved., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Development of the Caenorhabditis elegans foregut (pharynx) is regulated by a network of proteins that includes the Retinoblastoma protein (pRb) ortholog LIN-35; the ubiquitin pathway components UBC-18 and ARI-1; and PHA-1, a cytoplasmic protein. Loss of pha-1 activity impairs pharyngeal development and body morphogenesis, leading to embryonic arrest. We have used a genetic suppressor approach to dissect this complex pathway. The lethality of pha-1 mutants is suppressed by loss-of-function mutations in sup-35/ztf-21 and sup-37/ztf-12, which encode Zn-finger proteins, and by mutations in sup-36. Here we show that sup-36 encodes a divergent Skp1 family member that binds to several F-box proteins and the microtubule-associated protein PLT-1/τ. Like SUP-35, SUP-36 levels were negatively regulated by UBC-18-ARI-1. We also found that SUP-35 and SUP-37 physically associated and that SUP-35 could bind microtubules. Thus, SUP-35, SUP-36, and SUP-37 may function within a pathway or complex that includes cytoskeletal components. Additionally, SUP-36 may regulate the subcellular localization of SUP-35 during embryogenesis. We carried out a genome-wide RNAi screen to identify additional regulators of this network and identified 39 genes, most of which are associated with transcriptional regulation. Twenty-three of these genes acted via the LIN-35 pathway. In addition, several S-phase kinase-associated protein (Skp)1-Cullin-F-Box (SCF) components were identified, further implicating SCF complexes as part of the greater network controlling pharyngeal development.