Chavez, Daniela et al. (2017) International Worm Meeting "Sex, Secretion, Muscle, and Males."

Stanfield, Gillian, Chavez, Daniela

[International Worm Meeting, 2017]

To successfully complete the journey to fertilize an oocyte, sperm rely on extracellular cues to direct their development and migration. In the final stages of maturation, during a process called sperm activation, sperm become polarized and develop a pseudopod used to crawl towards oocytes. Establishment of an extracellular signaling environment that spatially and temporally regulates activation is critical to fertility, as males with prematurely activated sperm are infertile. Key components of this regulation are the serine protease TRY-5 and a protease inhibitor, SWM-1. TRY-5 is a seminal fluid protein that is stored in the vas deferens and transferred to hermaphrodites, where it activates sperm. In the absence of SWM-1, TRY-5 spreads into the seminal vesicle, where sperm are stored, resulting in premature activation. We are investigating how TRY-5 is regulated by SWM-1 to ensure that sperm gain motility at the right place and time. We have discovered that somatic tissues create the sperm signaling environment that precisely controls the location and timing of activation. By analyzing a swm-1 transcriptional reporter and a SWM-1::mCherry knock-in, we determined that swm-1 is expressed by body wall muscle cells and cuboidal cells in the vas deferens while SWM-1 protein is present not only in the vas deferens but also in the seminal vesicle, surrounding sperm. SWM-1 is also present in the pseudocoelom, where it is taken up into coelomocytes. Surprisingly, tissue-specific expression suggests that muscle-derived SWM-1 is sufficient to rescue the premature activation of swm-1 mutants, but that vas deferens-derived SWM-1 fails to rescue. Our data suggest a model in which SWM-1 is secreted from muscle into the pseudocoelom and then taken up by the gonad where it attenuates TRY-5. Indeed, removing the secretion signal from SWM-1 caused it to accumulate in muscle, where it no longer rescues activation. These data show that we have identified a soma-to-germline signal critical to fertility and have determined that muscle is a key regulator of sperm cell success. Interestingly, uptake of proteins from the pseudocoelom is not specific to SWM-1, as extragonadal sources of mCherry and GFP also enter the gonad. Furthermore, vas deferens-specific mCherry is found in the pseudocoelom, thus, exchange of proteins into and out of the gonad is apparently not limited to SWM-1, and could be a general phenomenon. In ongoing work, we seek to determine whether swm-1 plays a second role within the hermaphrodite to promote reproductive success. SWM-1 is present in hermaphrodites, where it has a small but measurable effect on sperm activation. Additional SWM-1 is transferred in seminal fluid, but its role in this context is unknown. We are analyzing whether absence of SWM-1, in either the hermaphrodite or seminal fluid, affects sperm functions including migration, fertilization, or competition.

Violeta Chavez et al. (2005) International Worm Meeting "C. elegans Pathogen Defense Mechanisms"

Danielle A Garsin, Violeta Chavez

[International Worm Meeting, 2005]

Caenorhabditis elegans has proved a useful model host for the elucidation of host/pathogen interactions, and both immune response and microbial infection strategies have been investigated. Previously, it has been shown that the insulin-like signaling pathway mediates resistance to bacterial pathogens in C. elegans, particularly Gram-positives such as Enterococcus faecalis and Staphylococcus aureus. The pathway consists of an insulin-like receptor called DAF-2, activating a PI3-kinase signaling pathway, which leads to the downregulation of a forkhead transcription factor called DAF-16. A mutation in the DAF-2 gene increases DAF-16 activity resulting in phenotypes such as increased lifespan, stress resistance and pathogen resistance. DAF-16 has been shown to upregulate genes encoding putative antimicrobials such as lysozyme and saposin and genes encoding enzymes involved in the resolution of oxidative stress such as catalase and superoxide dismutase. We present data showing that many of the putative antimicrobials do indeed play a role in pathogen defense. Additionally we propose that C. elegans may produce an oxidative burst as a pathogen defense mechanism based on data generated while exploring the role of oxidative stress factors. The details of this new hypothesis and the supporting data will be presented.

Chavez, Daniela et al. (2011) International Worm Meeting "A sperm competition mutant with defects in sperm motility."

Stanfield, Gillian, Chavez, Daniela

[International Worm Meeting, 2011]

Although males are not necessary for reproduction in C. elegans, if mating occurs then male sperm win: cross progeny are produced to the exclusion of self progeny. We would like to understand the mechanistic basis for male sperm precedence. It has been shown previously that precedence is due to an intrinsic characteristic of male sperm cells, that it requires motility, and that it correlates with the larger size of male sperm. Using a genetic screen for males with reduced precedence, we identified a mutant, me69, that has defects in sperm usage under conditions of competition with wild-type sperm. me69 male sperm show reduced precedence when they compete with wild-type hermaphrodite sperm. However, me69 hermaphrodites have normal self fertility, arguing against a generalized spermatogenesis defect. Interestingly, me69 sperm show a normal size distribution. me69 male sperm show motility defects; after transfer to the hermaphrodite, sperm accumulate poorly in the spermatheca. We are analyzing their migration in more detail to determine whether this difference arises from poor targeting, poor retention, or some other alteration in motility. We have identified a candidate gene encoding a divergent kinase that is expressed in sperm. A deletion mutant shows migration and precedence defects similar to those of me69. We are determining the localization of this kinase and investigating the possibility that either its levels or localization may be different in males vs. hermaphrodites.

Chavez, Daniela et al. (2015) International Worm Meeting "Regulation of C. elegans Sperm Motility By Protease Signaling."

Chavez, Daniela, Stanfield, Gillian

[International Worm Meeting, 2015]

C. elegans sperm undergo rapid cellular morphogenesis to develop into motile, fertilization-competent cells. During this process, termed sperm activation, cells reorganize their cytoskeleton to generate a pseudopod used to crawl towards oocytes. Sperm activation is regulated by the serine protease, TRY-5, and the serine protease inhibitor, SWM-1. TRY-5 is a seminal fluid protease transferred to hermaphrodites during mating and has the characteristics of a male sperm activation factor. In swm-1 mutant males, sperm become prematurely activated and are inefficiently transferred to hermaphrodites during mating, resulting in reduced male fertility. These previous studies suggest a model in which SWM-1 delays sperm activation by inhibiting TRY-5 protease activity from cleaving sperm cell membrane factors to initiate activation. To test this model, we have sought to determine the relationship between SWM-1 and TRY-5. It was previously shown that TRY-5 is expressed in the male somatic gonad in cells of the seminal vesicle, valve and vas deferens. We now have shown that swm-1 is co-expressed with try-5 in a subset of vas deferens cuboidal cells and that SWM-1::mCherry and TRY-5::GFP co-localize in vesicles within these cells. We also have found that swm-1 is expressed in extra-gonadal somatic cells near the seminal vesicle, and is not expressed in the valve. These data are consistent with SWM-1's inhibiting TRY-5 activity, but also suggest an alternative model where SWM-1 indirectly inhibits TRY-5, perhaps by regulating the release of TRY-5 vesicles. To further dissect the relationship of SWM-1 and TRY-5, we are analyzing the functional domains of the proteins. SWM-1 is predicted to have two trypsin inhibitor-like (TIL) domains, and previous studies have suggested that these domains may have distinct functions. To directly test the role of SWM-1 TIL domains in regulating sperm activation, we are generating precise endogenous gene edits of the predicted reactive site residues of SWM-1. Similarly, we are testing the predicted catalytic residues of TRY-5 for their role in activation. In ongoing studies, we are testing our models for SWM-1 regulation of TRY-5 activity or release by expressing swm-1 and try-5 in specific tissues and visualizing the dynamics of vesicle release from the vas deferens during mating.

Chavez, Daniela et al. (2013) International Worm Meeting "Timing is everything: dissecting the male sperm activation pathway."

Smith, Joseph, Snow, Angela, Stanfield, Gillian, Chavez, Daniela

[International Worm Meeting, 2013]

C. elegans sperm undergo rapid morphological changes to develop into polarized cells capable of migrating to and fertilizing an oocyte. During this process, termed sperm activation, the cells reorganize their cytoskeleton to generate a pseudopod, allowing them to crawl. Sperm activation is regulated by a serine protease, TRY-5, and a serine protease inhibitor, SWM-1. swm-1 mutant males contain prematurely activated spermatozoa, which are transferred inefficiently to hermaphrodites. Thus, SWM-1 is required for the temporal regulation of sperm activation and is essential to male fertility. TRY-5 is transferred in seminal fluid to a hermaphrodite and has the characteristics of a male sperm activation signal. Therefore, our model for male sperm activation is that SWM-1 directly or indirectly inhibits TRY-5 from activating spermatids within the male. Examination of the relationship between TRY-5 and SWM-1 is key to testing this model and understanding how sperm activation is regulated. To gain insight into the male sperm activation pathway, we are performing experiments to identify the location of SWM-1 expression and to analyze the functional domains of SWM-1 and TRY-5. We have previously shown TRY-5 to be expressed in the male somatic gonad in cells of the seminal vesicle, valve, and vas deferens. Similarly, we have found HA-tagged SWM-1 to be expressed in and likely secreted from a subset of vas deferens cuboidal cells, near the site of TRY-5 expression. In swm-1 mutants, TRY-5 becomes localized to the seminal vesicle. Therefore, SWM-1 and TRY-5 localization is consistent with our model in which SWM-1 inhibits TRY-5 from prematurely activating sperm. SWM-1 has two trypsin inhibitor-like (TIL) domains. We are genetically testing the necessity and sufficiency of each TIL domain for sperm activation. Preliminary results suggest that the C-terminal TIL domain plays a greater role in inhibiting sperm activation than the N-terminal TIL domain. Additionally, we are testing necessity of the catalytic domain of TRY-5 and we have preliminary evidence that it is required for activity in vivo. In the future, we will further investigate the function of SWM-1 and TRY-5 using biochemical analysis.

Violeta Chavez et al. (2004) West Coast Worm Meeting "DAF-16 Regulated Genes Mediate Resistance to Pathogens"

Violeta Chavez, Danielle A Garsin

[West Coast Worm Meeting, 2004]

It has been previously shown that the insulin signaling pathway mediates resistance to bacterial pathogens in Caenorhabditis elegans , particularly Gram-positives such as Enterococcus faecalis and Staphylococcus aureus . The pathway consists of an insulin-like receptor called DAF-2, activating a PI3-kinase signaling pathway, which leads to the downregulation of a forkhead transcription factor called DAF-16. When DAF-16 activity is increased, by a mutation in DAF-2 for example, the resulting phenotypes include increased lifespan, stress resistance and pathogen resistance. DAF-16 has been shown to upregulate genes that resolve oxidative stress such as catalase and superoxide dismutase. A crucial question is, are the observed oxidative stress resistance and pathogen resistance occurring by the same mechanism? Gram-positives such as E. faecalis and S. aureus are known to produce reactive oxygen species such as hydrogen peroxide and superoxides. It has been shown in different pathogen assays that hydrogen peroxide produced by Streptococcus pyogenes and pyocyanides produced by Pseudomonas aeruginosa can kill C. elegans , presumably by causing oxidative stress. Under our assay conditions, we present evidence that oxidative stress is indeed one mechanism by which C. elegans are killed, but that it is not the only factor. We also show that the resistance of the daf-2 mutant is also mediated by the upregulation of antimicrobial factors.

Violeta Chavez et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Elucidation of a Conserved Innate Immune Defense Mechanism in Caenorhabditis elegans"

Violeta Chavez, Danielle Garsin

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Caenorhabditis elegans has recently been developed as a model system to study both pathogen virulence mechanisms and host defense responses. We have shown that C. elegans produces reactive oxygen species (ROS) in response to exposure to Enterococcus faecalis. We have also shown evidence of oxidative stress and an upregulation of stress response after exposure to the pathogen. As in mammalian immune cells, production of ROS appeared to be occurring via an NADPH oxidase, as addition of an NADPH oxidase inhibitor reduced ROS production. Here we show that reducing expression of a NADPH oxidase, specifically a dual oxidase, in the intestine and the hypodermis increases susceptibility to pathogenic bacteria. This dual oxidase has previously been localized to the hypodermis, but we show that it is additionally localized to the intestine of C. elegans. We hypothesize that the generation of ROS by a dual oxidase is a highly conserved innate immune defense mechanismthat may apply to the mucosal surfaces of more complex animals.

Violeta Chavez et al. (2007) International Worm Meeting "Oxidative stress enzymes are required for DAF-16 mediated immunity due to generation of reactive oxygen species."

Violeta Chavez, Luis Vega, Akiko Mohri-Shiomi, Arash Maadani, Danielle A. Garsin

[International Worm Meeting, 2007]

Caenorhabditis elegans has recently been developed as a model for microbial pathogenesis, yet little is known about its immunological defenses. Previous work implicated insulin signaling in mediating pathogen resistance in a manner dependent on the transcriptional regulator DAF-16, but the mechanism has not been elucidated. We present evidence that C. elegans, like mammalian phagocytes, produces reactive oxygen species (ROS) in response to pathogens. Signs of oxidative stress occur in the intestine - the site of the host-pathogen interface - suggesting that ROS release is localized to this tissue. Evidence includes the accumulation of lipofuscin, a pigment resulting from oxidative damage at this site. In addition, SOD-3, a superoxide dismutase regulated by DAF-16, is induced in intestinal tissue after exposure to pathogenic bacteria. Moreover, we show that the oxidative stress response genes sod-3 and ctl-2 are required for DAF-16-mediated resistance to E. faecalis using a C. elegans killing assay. We propose a model, whereby C. elegans responds to pathogens by producing ROS in the intestine while simultaneously inducing a DAF-16-dependent oxidative stress response to protect adjacent tissues. Because insulin signaling mutants over-produce oxidative stress response enzymes, the model provides an explanation for their increased resistance to pathogens.

Chavez, Ivan et al. (2021) International Worm Meeting "Skin-penetrating nematodes exhibit life-stage-specific interactions with host-associated and environmental bacteria"

Chavez, Ivan, Bryant, Astra, Assie, Adrien, Samuel, Buck, Hallem, Elissa, Brown, Taylor

[International Worm Meeting, 2021]

Skin-penetrating nematodes of the genus Strongyloides infect over 600 million people, posing a major global health burden. Their life cycle includes both a parasitic and free-living generation. During the parasitic generation, infective third-stage larvae (iL3s) actively engage in host seeking. During the free-living generation, the nematodes develop and reproduce on host feces. At different points of their life cycle, Strongyloides species encounter bacteria from various ecological niches. However, the microbial interactions between Strongyloides and bacteria remain uncharacterized. We first investigated the microbiome of the human parasite Strongyloides stercoralis using 16S-based amplicon sequencing. We found that S. stercoralis free-living adults have a distinct microbiome, suggesting that they selectively associate with specific fecal bacteria. We then investigated the behavioral responses of S. stercoralis and the closely related rat parasite Strongyloides ratti to an ecologically diverse panel of bacteria. We found that S. stercoralis and S. ratti showed similar responses to bacteria. The responses of both nematodes to bacteria varied dramatically across life stages: free-living adults were strongly attracted to most of the bacteria tested, while iL3s were attracted specifically to soil bacteria. The behavioral responses to bacteria were dynamic, consisting of distinct short- and long-term behaviors. Finally, a comparison of the growth and reproduction of S. stercoralis free-living adults on different bacteria revealed that the bacterium Proteus mirabilis inhibits S. stercoralis egg hatching, greatly decreasing parasite viability. Our results identify bacteria that serve as key sensory cues for directing movement, as well as bacteria that decrease the parasite's reproductive fitness.

Chavez, Sindy et al. (2021) International Worm Meeting "Characterization of a potential gene interaction between spr-5, met-2, and mep-1 in determining germline versus soma in C. elegans"

Chavez, Sindy, Katz, David, Birol, Onur, Brockett, Jovan, Schmeichel, Karen, Carpenter, Brandon

[International Worm Meeting, 2021]

In C. elegans, histone modifying enzymes aid in the activation and repression of genes that are required to distinguish germline from soma. For example, SPR-5 is a histone demethylase that removes the activating histone modification H3K4me1/2 and MET-2 is a histone methyltransferase that adds the repressive histone modification H3K9me2. Together, these two histone modifying enzymes work synergistically to establish a totipotent ground state by shutting down the transcription of germline specific genes that were expressed in the germline of the parents. Previous studies have shown that spr-5;met-2 double mutants produce progeny that experience a severe developmental delay at the L2 larval stage. Transcriptomic analysis of these animals indicated that the delay is largely due to the inappropriate expression of germline expressed genes in somatic tissues. Here we explore the possibility that the chromatin state established in the early embryo by SPR-5 and MET-2 is reinforced in the later embryo by ATP-dependent chromatin remodeling activity. MEP-1 is a component of the ATP-dependent epigenetic deacetylase complex, MEC. Similar to spr-5;met-2 double mutants, mep-1 mutants fail to suppress germline genes in somatic tissues, as indicated by the misexpression of PGL-1 and GLH-3 germline proteins in the soma of L1 larvae. We tested a potential interaction between these two pathways by subjecting spr-5, met-2 and spr-5;met-2 mutants to mep-1 RNAi. mep-1 knockdown when combined with any of these mutant backgrounds, results in an exacerbated developmental delay phenotype. The "triple" mutant is most severe, displaying a full L1 arrest. To fully understand the molecular basis of this synergy, we performed RNA-sequencing on these arrested L1 larvae versus controls. We find very little evidence of new expression changes induced in the triple mutants compared to controls. Instead, we find that expression changes in the spr-5;met-2 double mutants are exacerbated in triple mutants. This suggests that MEP-1 specifically reinforces the repressed chromatin state established by SPR-5 and MET-2 reprogramming at fertilization. These results help establish how chromatin modifying enzymes work together to establish developmental cell fates.