Lazetic V et al. (2017) Worm "Molting in C. elegans."

Fay DS, Lazetic V

[Worm, 2017]

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.

Lazetic V et al. (2022) Nat Commun "The transcription factor ZIP-1 promotes resistance to intracellular infection in Caenorhabditis elegans."

Troemel ER, Gang SS, Cohen LB, Bhabha G, Chang YT, Wu F, Reddy KC, Lazetic V

[Nat Commun, 2022]

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.

Lazetic, V. et al. (2017) International Worm Meeting "A NimA-kinase pathway controls CDC-42 activity and actin organization in the epidermis."

Joseph, B., Fay, D., Lazetic, V.

[International Worm Meeting, 2017]

During molting the epidermal apical extracellular matrix (cuticle) is extensively remodeled. We have shown that the NimA-related kinases, NEKL-2/NEK8 and NEKL-3/NEK6/NEK7, as well as their conserved ankyrin-repeat partners, MLT-2/ANKS6, MLT-3/ANKS3 and MLT-4/INVS, are essential for molting. Both NEKLs and MLTs are expressed in the epidermal syncytia and are specifically required in hyp7 for normal molting. Our studies indicate that NEKLs and MLTs act primarily within two complexes composed of NEKL-2-MLT-2-MLT-4 and NEKL-3-MLT-3, and that the MLTs function as scaffolds to ensure proper subcellular localization of the NEKLs. To understand how the NEKL-MLT network controls molting, we identified suppressors of nekl mutant molting defects. Our screens identified CDC-42, a highly conserved Rho-family GTPase, and its upstream regulator, KIN-25/SID-3. CDC-42 is important for the establishment of cell polarity and for organization of the actin cytoskeleton. KIN-25 is the ortholog of Activated CDC42 Kinase 1, which binds to CDC42 and inhibits its GTPase activity, thereby maintaining CDC42 in an active state. Notably, molting has been proposed to require extensive reorganization of actin within the epidermis. Specifically, actin is reorganized at each molt to form a series of circumferential bundles along the apical surface of hyp7, however, the mechanisms underlying this process are unknown. We found that inhibition of NEKL-MLT activities leads to defects in the pattern of apical actin and failure to form molting-specific actin bundles. Furthermore, expression studies indicate that components of the NEKL-MLT network colocalize with actin in specific regions of the epidermis. In addition, normal localization of CDC-42 in the epidermis depends on presence of NEKL-MLT proteins, and CDC-42 partially colocalizes with MLT-2. Our findings suggest that the NEKL-MLT network may negatively regulate CDC-42 activity and that partial inhibition of CDC-42 may thus reduce defects in nekl-mlt mutants. Interestingly, we also observed that downregulation of cdc-42 on its own can lead to molting defects, suggesting that there is a tight balance between NEKL-MLT and CDC-42 activities in the epidermis. Notably, studies in mammalian cells have implicated the MLT-4 ortholog, Inversin, in the regulation of CDC42 activity and apical actin organization. In addition, it was independently shown that the mammalian orthologs of NEKL-3, NEK6 and NEK7, physically associate with CDC42, however no functional connection between these proteins has been described. For the first time, our data provide in vivo evidence for a functional link between the NEKL-MLT network and regulation of the actin cytoskeleton through the CDC42 pathway.

Lazetic, V. et al. (2019) International Worm Meeting "The role of the bZIP transcription factor ZIP-1 in the Intracellular Pathogen Response in C. elegans."

Lazetic, V., Troemel, E., Cohen, L., Reddy, K., Wu, F.

[International Worm Meeting, 2019]

Maintaining protein homeostasis (proteostasis) is essential for organismal health, and several different signaling pathways have been shown to aid in maintaining proteostasis. Our lab has defined a novel proteostasis pathway by analyzing the common transcriptional response to diverse intracellular pathogens, which we named the Intracellular Pathogen Response (IPR). The IPR is controlled by the antagonistic paralogs pals-22 and pals-25, which serve as a genetic switch between increased pathogen resistance and thermotolerance, and normal development. Interestingly, the IPR can be activated not just by infection, but also by heat stress and proteasome inhibitors, indicating that the IPR plays a role in response to diverse proteotoxic stressors. The IPR involves transcriptional activation of ~80 genes, some of which are predicted to encode ubiquitin ligase components. One of the most highly induced IPR genes is pals-5, which has no known biochemical function, but serves as a robust readout for the IPR activation. Using pals-5::GFP reporters, we identified ZIP-1 as a positive regulator of the IPR in two reverse genetic screens. ZIP-1 contains a bZIP domain, and is classified as a transcription factor. Our qRT-PCR and smFISH studies demonstrate that ZIP-1 controls pals-5 transcription early after IPR induction by the proteasome inhibitor bortezomib, although pals-5 transcription late after IPR induction appears independent of ZIP-1. In addition, we found that ZIP-1 is required for transcription of the putative ubiquitin ligase component skr-5, which promotes thermotolerance as part of the IPR. Moreover, we identified a set of IPR genes that are not regulated by ZIP-1. Taken together, our studies uncover three distinct types of IPR genes: a) completely ZIP-1-dependent, b) partially ZIP-1-dependent and c) ZIP-1-independent. Interestingly, we observed that loss-of-function mutations in pals-22 and zip-1 are synthetically lethal, indicating that there is a fine balance between these IPR regulators, which are important for normal development in the absence of external proteotoxic stressors. To further explore IPR regulation, we have also isolated several mutants with constitutively active IPR that are currently being characterized.

Lazetic V et al. (2023) Bioessays "Similarities in the induction of the intracellular pathogen response in Caenorhabditis elegans and the type I interferon response in mammals."

Russell AB, Troemel ER, Lazetic V, Batachari LE

[Bioessays, 2023]

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.

Lazetic V et al. (2016) Genetics "Conserved Ankyrin Repeat Proteins and Their NIMA Kinase Partners Regulate Extracellular Matrix Remodeling and Intracellular Trafficking in Caenorhabditis elegans."

Fay DS, Lazetic V

[Genetics, 2016]

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.

Lazetic V et al. (2023) PLoS Pathog "Multiple pals gene modules control a balance between immunity and development in Caenorhabditis elegans."

Blanchard MJ, Troemel ER, Lazetic V, Bui T

[PLoS Pathog, 2023]

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.

Gang SS et al. (2024) J Eukaryot Microbiol "Microsporidia: Pervasive natural pathogens of Caenorhabditis elegans and related nematodes."

Lazetic V, Gang SS

[J Eukaryot Microbiol, 2024]

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.

Balla KM et al. (2019) PLoS One "Natural variation in the roles of C. elegans autophagy components during microsporidia infection."

Balla KM, Troemel ER, Lazetic V

[PLoS One, 2019]

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.

Smith BS et al. (1990) Cryobiology "Cryopreservation of the entomogenous nematode parasite Steinernema feltiae (= Neoaplectana carpocapsae)."

Hodgson-Smith A, Popiel I, James ER, Smith BS, Minter DM

[Cryobiology, 1990]

Cryopreservation studies were conducted with the J1 juvenile and third stage infective juvenile (IJ) larval stages of the entomogenous parasitic nematode Steinernema feltiae (=Neoaplectana carpocapsae). The main parameters evaluated were (i) tolerance of the organisms to the cryoprotectants methanol, ethanediol, glycerol, and dimethyl sulfoxide; (ii) the incubation temperature and exposure period in cryoprotectant; and (iii) slow cooling (circa 1C min-1) vs rapid cooling (circa 5,100C min-1). The J1 stage was sensitive to all cryoprotectants at >20% (v/v). This sensitivity increased at 22 and 37C. Exsheathed IJ stage organisms tolerated exposure to 50% (v/v) ethanediol or Me2SO and 60% (v/v) methanol or glycerol. A proportion (3.4%) of J1s preincubated in 20% (v/v) methanol for 10 min at 0C survived slow cooling to -35C followed by plunge into liquid nitrogen. Preincubation in 60% (v/v) Me2SO for 45 sec at 0C followed by rapid cooling yielded a higher proportion (12.3%0 of surviving J1s. The infective IJ stage only survived rapid cooling. Preincubation for 20 min at 0C in either 60% (v/v) methanol or 45% glycerol, followed by rapid cooling at 5,100C min-1, yielded 30-34% viable IJs following thawing. These larvae were capable of further growth and development in vitro. The results suggest that the organisms are vitrified during the rapid cooling step. For routine maintenance of strains of S. feltiae and related