Von Stetina, S. E. et al. (2019) International Worm Meeting "Testing local CRISPR gene drives using C. elegans."

Esvelt, K., Normandin, A, Min, J., Von Stetina, S. E.

[International Worm Meeting, 2019]

CRISPR-mediated genetic engineering has the potential to help humanity in a multitude of ways, such as improved gene therapies, the ability to easily mimic disease-causing mutations in model organisms, and the potential to use CRISPR-driven gene drives to reduce vector-borne diseases. Gene drives function by inserting all necessary CRISPR machinery, plus the payload, into genome of the organism. When a CRISPR-carrying organism mates with a wild-type, super-Mendelian inheritance is observed, as all the progeny become homozygous CRISPR-carrying organisms. However, with nothing to stop CRISPR-activity, the potential exists for every organism within a species to become edited, a very undesirable outcome. The Sculpting Evolution group is dedicated to solving this problem by inventing and testing self-limiting gene drives, as well as immunizing reversal drives. We have dubbed one form of self-exhausting drive a daisy drive1, as the elements required to drive are separated into modules (example: parts A, B, C) that depend on each other for complete drive. Part C drives B which drives A, but nothing drives C. This means that when C is diluted out, B is no longer driven. When B is diluted out, C is no longer driven and the gene drive stops. Our mathematical models predict that the introduced drives quickly spread throughout a local population, but in the absence of additional modified animals the population will revert back to wild-type. We are developing daisy drives in C. elegans to test if the biology matches the mathematical models. Nematodes are the only metazoan where a billion organisms over multiple generations could be tested in a reasonable amount of time, and thus determine if our theory can become reality. Our preliminary results suggest that germline expressed SpCas9 has insufficient activity to sustain a drive at C. elegansrearing temperatures. We have redesigned our daisy drive constructs to use the Cas12a/Cpf1 nuclease from Lachnospiraceae bacterium ND2006 (LbCas12a), which is reported to be active at lower temperatures than SpCas9 or AsCas12a2,3. We are currently generating strains that express germline LbCas12a and the daisy drive elements, using fluorescent proteins to mark the daisy elements. I will present negative data regarding Cas9 activity and gene drive in worms, our current data on LbCas12a activity and an update on generating functional daisy drives in worms. Our future ambitions are to use community-guided science to identify problems where daisy drives could be helpful (i.e. culling mosquito-borne diseases or rat infestations), and with the community's help come up with viable solutions using our tested ideas. 1. Noble et al, 2016, bioRxiv 2. Malzahn et al, 2019, BMC Biol. 3. Liu et al, 2019, NAR

Kaushal NA et al. (1982) J Immunol "Identification and characterization of excretory-secretory products of Brugia malayi, adult filarial parasites."

Kaushal NA, Ottesen EA, Nash TE, Hussain R

[J Immunol, 1982]

Although E-S antigens may be particularly important for both the pathogenesis and immunodiagnosis of helminth infections, little is known about the immunochemistry or functional roles in human filarial infections. In the present paper, we have done some initial identification and characterization of E-S products of adult Brugia malayi by employing a combination of sensitive biochemical and immunochemical techniques. E-S products, collected by incubating B. malayi adults in vitro in a defined protein-free medium, were radiolabeled with 125I. SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography of labeled E-S products revealed 11 protein bands in the m.w. range of 10,000 to 70,000. Comparison of radiolabeled E-S products and adult somatic antigen (B.m.A) in SDS-PAGE indicated many common bands, and crossed immunoelectrophoresis and competitive Staph-A RIA confirmed the presence of most E-S antigens in B.m.A. Of the 11 E-S bands, two appeared to be derived from the surface of the adult worms and microfilariae as shown by SDS-PAGE and autoradiography of lodogen surface-labeled parasites; the presence of two host proteins in E-S was detected by crossed-line immunoelectrophoresis. The E-S antigens were highly immunogenic when tested both with rabbit antiserum raised against B.m.A and with a serum pool of patients with natural filarial infection.

Musset-Bilal F et al. (1994) Worm Breeder's Gazette "Characterization of the C. elegans Basement Membrane-Associated Protein SPARC"

Musset-Bilal F, Schwarzbauer JE, Ryan CS

[Worm Breeder's Gazette, 1994]

Characterization of the C. elegans Basement Membrane- Associated Frederique Musset-Bilal, Carol S. Ryan, and Jean E. Schwarzbauer Department of Molecular Biology, Princeton University, Princeton NJ 08544

Bakaev VV et al. (1998) Worm Breeder's Gazette "An Attempt to Slow Aging in C. elegans. 10. The Slight Positive Effect of C. elegans Larvae Extract"

Bakaev VV, Ivlev-Duntau AP

[Worm Breeder's Gazette, 1998]

C. elegans larvae (1-2 days old) were collected from separate stock, frozen and after 12 hours or more time, thawed, centrifuged and mixed with S medium containing E. coli (1:1), this mixture was used as a medium for worms in experimental group. The medium for the control group was prepared by mixing S medium containing E. coli with S medium without E. coli (1:1). Three adult animals (3-5 days old) were kept in microtitre wells containing 0.5 ml of liquid medium (with E. coli and with or without larvae extract) during 12 hours, then they were transferred in next wells everyday (one worm in one well). Dilutions of larvae extract (1:10, 1:100 and 1:1000) were applied on third day. This investigation was carried out in temperature +24C and in the darkness. The obtained results are presented in the following table.

Vega NM et al. (2013) Proc Natl Acad Sci U S A "Salmonella typhimurium intercepts Escherichia coli signaling to enhance antibiotic tolerance."

Klempner MS, Collins JJ, Samuels AN, Vega NM, Allison KR

[Proc Natl Acad Sci U S A, 2013]

Bacterial communication plays an important role in many population-based phenotypes and interspecies interactions, including those in host environments. These interspecies interactions may prove critical to some infectious diseases, and it follows that communication between pathogenic bacteria and commensal bacteria is a subject of growing interest. Recent studies have shown that Escherichia coli uses the signaling molecule indole to increase antibiotic tolerance throughout its population. Here, we show that the intestinal pathogen Salmonella typhimurium increases its antibiotic tolerance in response to indole, even though S. typhimurium does not natively produce indole. Increased antibiotic tolerance can be induced in S. typhimurium by both exogenous indole added to clonal S. typhimurium populations and indole produced by E. coli in mixed-microbial communities. Our data show that indole-induced tolerance in S. typhimurium is mediated primarily by the oxidative stress response and, to a lesser extent, by the phage shock response, which were previously shown to mediate indole-induced tolerance in E. coli. Further, we find that indole signaling by E. coli induces S. typhimurium antibiotic tolerance in a Caenorhabditis elegans model for gastrointestinal infection. These results suggest that the intestinal pathogen S. typhimurium can intercept indole signaling from the commensal bacterium E. coli to enhance its antibiotic tolerance in the host intestine.

Aballay A et al. (2000) Curr Biol "Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans."

Aballay A, Yorgey P, Ausubel FM

[Curr Biol, 2000]

Genetic analysis of host-pathogen interactions has been hampered by the lack of genetically tractable models of such interactions. We showed previously that the human opportunistic pathogen Pseudomonas aeruginosa kills Caenorhabditis elegans, that P. aeruginosa and C. elegans genes can be identified that affect this killing, and that most of these P. aeruginosa genes are also important for mammalian pathogenesis. Here, we show that Salmonella typhimurium as well as other Salmonella enterica serovars including S. enteritidis and S. dublin can also kill C. elegans. When C. elegans is placed on a lawn of S. typhimurium, the bacteria accumulate in the lumen of the worm intestine and the nematodes die over the course of several days. This killing requires contact with live bacterial cells. The worms die with similar kinetics when placed on a lawn of S. typhimurium for a relatively short time (3-5 hours) before transfer to a lawn of E. coli. After the transfer to E. coli, a high titer of S. typhimurium persists in the C. elegans intestinal lumen for the rest of the worms' life. Furthermore, feeding for 5 hours on a 1:1000 mixture of S. typhimurium and E. coli followed by transfer to 100% E. coli, also led to death after several days. This killing correlated with an increase in the titer of S. typhimurium in the C. elegans lumen, which reached 10,000 bacteria per worm. These data indicate that, in contrast to P. aeruginosa, a small inoculum of S. typhimurium can proliferate in the C. elegans intestine and establish a persistent infection. S. typhimurium mutated in the PhoP/PhoQ signal transduction system caused significantly less killing of C. elegans.

Okimoto R et al. (1994) J Mol Evol "The mitochondrial ribosomal RNA genes of the nematodes Caenorhabditis elegans and Ascaris suum: consensus secondary-structure models and conserved nucleotide sets for phylogenetic analysis."

Wolstenholme DR, Okimoto R, Macfarlane JL

[J Mol Evol, 1994]

The small- and large-subunit mitochondrial ribosomal RNA genes (mt-s-rRNA and mt-1-rRNA) of the nematode worms Caenorhabditis elegans and Ascaris suum encode the smallest rRNAs so far reported for metazoa. These size reductions correlate with the previously described, smaller, structurally anomalous mt-tRNAs of C. elegans and A. suum. Using primer extension analysis, the 5' end nucleotides of the mt-s-rRNA and mt-1-rRNA genes were determined to be adjacent to the 3' end nucleotides of the tRNA(Glu) and tRNA(His) genes, respectively. Detailed, consensus secondary-structure models were constructed for the mt-s-rRNA genes and the 3' 64% of mt-1-rRNA genes of the two nematodes. The mt-s-rRNA secondary-structure model bears a remarkable resemblance to the previously defined universal core structure of E. coli 16S rRNA: most of the nucleotides that have been classified as variable or semiconserved in the E. coli model appear to have been eliminated from the C. elegans and A. suum sequences. Also, the secondary structure model constructed for the 3' 64% of the mt-1-rRNA is similar to the corresponding portion of the previously defined E. coli 23S rRNA core secondary structure. The proposed C. elegans/A. suum mt-s-rRNA and mt-1-rRNA models include all of the secondary-structure element-forming sequences that in E. coli rRNAs contain nucleotides important for A-site and P-site (but not E-site) interactions with tRNAs. Sets of apparently homologous sequences within the mt-s-rRNA and mt-1-rRNA core structures, derived by alignment of the C. elegans and A. suum mt-rRNAs to the corresponding mt-rRNAs of other eukaryotes, and E. coli rRNAs were used in maximum-likelihood analyses. The patterns of divergence of metazoan phyla obtained show considerable agreement with the most prevalent metazoan divergence patterns derived

Bakaev VV (1996) Worm Breeder's Gazette "AN ATTEMPT TO SLOW AGING IN C. elegans. 3.THE EFFECT OF EXTRACT FROM YOUNG WORMS DURING POSTREPRODUCTIVE PERIOD"

Bakaev VV

[Worm Breeder's Gazette, 1996]

C. elegans (1 - 2 days old) were collected from separate stock, frozen and after 12 hours or more time, thawed, filtrated and mixed with S medium containing E. coli (1:1), this mixture was used as a medium for worms in experimental group. The medium for control group was prepared by mixing S medium containing E. coli with S medium without E. coli (1:1). Three adult animals (3 - 5 days old) were kept in microtitre wells containing 0,75 ml of liquid medium (with E. coli and with or without young worms extract) during 12 hours, then they were discarded and newborn larvae were transferred in next wells every day (one worm in one well). The medium with extract from young worms was used in experimental group beginning from 12th day, that is practically in postreproductive period of nematode life span. A number of progeny was calculated every day. This investigation was carried out in temperature +21C and in the darkness. The obtained results are presented in the following table.

Yuen, Grace J. et al. (2013) International Worm Meeting "Enterococcus infection of C. elegans as a model of innate immunity."

Yuen, Grace J., Ausubel, Frederick M.

[International Worm Meeting, 2013]

Enterococcus is a Gram-positive commensal that is also an important opportunistic pathogen. Most human enterococcal infections are caused by either E. faecalis or E. faecium. Our laboratory has modeled Enterococcus infection in C. elegans using both species. We have previously shown that infection with either species leads to gut distention, but only E. faecalis is able to establish a persistent and lethal infection in the nematode. We now provide evidence that at least three canonical C. elegans immune signaling pathways are important for survival during infection with E. faecalis and E. faecium. While the lifespan of wild-type worms is unaffected by E. faecium infection, mutations in the PMK-1, FSHR-1, and BAR-1 immune signaling pathways lead to an immunocompromised phenotype. This new finding suggests that an active host response is required to keep E. faecium infection "in check" in the worm intestine. To further characterize the C. elegans host response to Enterococcus infections, we used genome-wide transcriptional profiling of nematodes feeding on E. faecalis and E. faecium, as well as two controls, heat-killed E. coli and live Bacillus subtilis, a non-pathogenic Gram-positive. We found that relative to B. subtilis, E. faecalis and E. faecium caused the upregulation of 249 and 166 genes, respectively, of which 105 genes were common to both, comprising the Enterococcus gene signature. Shared by both Enterococcus infection signatures were genes relating to oxidation/reduction, acyl-CoA dehydrogenase/oxidase activity, fatty acid metabolism, and C-type lectins. Additionally, the Enterococcus infection gene signature is fairly distinct from the P. aeruginosa, S. aureus, and C. albicans infection signatures. Furthermore, of the 91 genes that are upregulated in E. faecalis (more virulent) relative to E. faecium (less virulent), 22 are shared with the 77 genes upregulated in worms infected with virulent Microbacterium nematophilum relative to avirulent M. nematophilum (O'Rourke et al., 2006), suggesting that these genes may comprise a "virulence response signature." Studies are underway in understanding the biology of Enterococcus infection in C. elegans and identifying novel Enterococcus-activated pathways.

Budde MW et al. (2010) Mol Biol Cell "Hydrogen sulfide increases hypoxia-inducible factor-1 activity independently of von Hippel-Lindau tumor suppressor-1 in C. elegans."

Roth MB, Budde MW

[Mol Biol Cell, 2010]

Rapid alteration of gene expression in response to environmental changes is essential for normal development and behavior. The transcription factor hypoxia-inducible factor (HIF)-1 is well known to respond to alterations in oxygen availability. In nature, low oxygen environments are often found to contain high levels of hydrogen sulfide (H(2)S). Here, we show that Caenorhabditis elegans can have mutually exclusive responses to H(2)S and hypoxia, both involving HIF-1. Specifically, H(2)S results in HIF-1 activity throughout the hypodermis, whereas hypoxia causes HIF-1 activity in the gut as judged by a reporter for HIF-1 activity. C. elegans require hif-1 to survive in room air containing trace amounts of H(2)S. Exposure to H(2)S results in HIF-1 nuclear localization and transcription of HIF-1 targets. The effects of H(2)S on HIF-1 reporter activity are independent of von Hippel-Lindau tumor suppressor (VHL)-1, whereas VHL-1 is required for hypoxic regulation of HIF-1 reporter activity. Because H(2)S is naturally produced by animal cells, our results suggest that endogenous H(2)S may influence HIF-1 activity.