Pukkila-Worley R et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Antifungal immune specificity in Caenorhabditis elegans"

Mylonakis E, Ausubel F M, Pukkila-Worley R

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

C. albicans is found in the intestine and genitourinary tract of virtually all humans, yet this opportunist can invade host tissues and cause life-threatening infection in susceptible individuals. Here we establish a novel system for the study of antifungal innate immunity using the nematode Caenorhabditis elegans. We found that the laboratory reference strain Candida albicans DAY185 established an intestinal infection in the nematode on solid media, whereas heat-killed yeast are avirulent. Furthermore, we show that the PMK-1 p38 mitogen-activated protein kinase pathway is required for normal resistance to C. albicans infection. To generate hypotheses regarding immune recognition of fungal infection in the nematode, we generated whole-genome transcriptional profiles of nematodes exposed to nonpathogenic, heat-killed C. albicans. Interestingly, we found that, despite being non-pathogenic, heat-killed C. albicans induces a specific transcriptional response in nematodes involving 134 genes (~0.6% of the genome). 55% of these genes are also induced during infection of the nematode with live C. albicans and include antimicrobial peptides such as abf-2, a protein previously shown to have direct anti-Candida activity. We study further the regulation of four antimicrobial peptide genes, abf-2, fipr-22, cnc-4 and cnc-7 during infection with C. albicans and show that their induction is dependent on PMK-1. Thus, we propose that cellular factors possessed by heat-killed C. albicans, so-called Pathogen-Associated Molecular Patterns (PAMPs), are detected by the nematode during infection with C. albicans and may directly stimulate the PMK-1 pathway. Strikingly, we also found that both heat-killed and live C. albicans cause the downregulation of antibacterial immune response genes. These data provide the first genome-wide analysis in C. elegans during infection with a pathogenic fungus and provide insights into the mechanisms of antifungal immune specificity in the nematode.

Pukkila-Worley R et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Characterization of Small Molecule Anti-Infectives that Cure C. elegans of an Intestinal Bacterial Infection"

Larkins-Ford J, Ausubel F, Kirienko N, Moy T, Pukkila-Worley R, Connery A

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Because C. elegans is small enough to fit into the wells of a 384-well microtiter plate and because various pathogens cause persistent lethal infections in the C. elegans intestine, we have been able to develop screens for compounds that ""cure"" C. elegans of a bacterial infection. We have also developed C. elegans as an automated high throughput whole-animal chemical screening platform. This involves a COPAS BioSort robot to distribute worms to microtiter wells and automated image acquisition and analysis software that distinguishes live from dead worms. We performed a pilot screen of ~37,000 compounds at the Broad Institute and identified substances that promoted the survival of worms infected with the bacterium Enterococcus faecalis. Interestingly, a subset of the identified compounds appear to act differently than traditional antibiotics in that they improve worm survival at concentrations that do not inhibit the growth of the free living pathogen. Thus, this screening methodology allows the identification of new classes of anti-infective compounds that either act in the worm by stimulating innate immunity or that act in the pathogen by inhibiting virulence mechanisms. Some of the hits activate C. elegans innate immune response genes in a PMK-1-dependent manner. PMK-1 is a MAPK that plays a key role in C. elegans basal immunity and is homologous to the mammalian p38 MAPK that plays a key role in innate immunity.

Anderson, S.M. et al. (2019) International Worm Meeting "The fatty acid oleate is required for innate immune activation and pathogen defense in Caernohabditis elegans."

Peterson, N.D., Soukas, A.A., Anderson, S.M., Pukkila-Worley, R., Cheesman, H.K.

[International Worm Meeting, 2019]

Fatty acids affect a number of physiological processes, in addition to forming the building blocks of membranes and body fat stores. In this study, we uncover a role for the monounsaturated fatty acid oleate in the innate immune response of the nematode Caenorhabditis elegans. From an RNAi screen for regulators of innate immune defenses, we identified the two stearoyl-coenzyme A desaturases that synthesize oleate in C. elegans. We show that the synthesis of oleate is necessary for the pathogen-mediated induction of immune defense genes. Accordingly, C. elegans deficient in oleate production are hypersusceptible to infection with diverse human pathogens, which can be rescued by the addition of exogenous oleate. However, oleate is not sufficient to drive protective immune activation. Together, these data add to the known health-promoting effects of monounsaturated fatty acids, and suggest an ancient link between nutrient stores, metabolism, and host susceptibility to bacterial infection.

Foster, K.J. et al. (2019) International Worm Meeting "Olfactory neuron protein promotes intestinal immune homeostasis to ensure evolutionary fitness."

Liu, P., Pukkila-Worley, R., Cheesman, H.K., Foster, K.J., Peterson, N.D.

[International Worm Meeting, 2019]

Innate immune homeostasis is critically important for the health of metazoans, including C. elegans. Here, we demonstrate that control of innate immune activation via a signaling axis between sensory neurons and the intestine is required for reproductive fitness. We show that an olfactory neuron protein functions in the cell non-autonomous suppression of the canonical p38 MAP kinase PMK-1 immune pathway in the intestine. Immune de-repression in loss-of-function mutants causes constitutive activation of the p38 MAP PMK-1, hyperinduction of immune effector transcription and resistance to bacterial infection. Importantly, we demonstrate that regulation of the p38 MAPK PMK-1 pathway is required for healthy nematode development and reproduction. These data demonstrate that olfactory neurons regulate the p38 MAPK PMK-1 immune pathway in the intestine and indicate that maintenance of immune homeostasis is an evolutionarily ancient requirement for growth and reproduction of free-living organisms.

Nhan, J. et al. (2019) International Worm Meeting "Transcriptional redirection of activated SKN-1/NRF2 abates the negative metabolic outcomes of a perceived pathogen infection."

Curran, S., Yen, C.A., Ruter, D., Naresh, N.U., Dalton, H., Pukkila-Worley , R., Haynes, C., Nhan, J., Anderson, S.

[International Worm Meeting, 2019]

Optimal health requires continuous transcriptional fidelity of gene expression. SKN-1/NRF2 is a cytoprotective transcription factor in C. elegans that regulates the expression of cellular defenses during stress, including: nutrient deprivation, redox imbalance, and xenobiotic and pathogen exposure. Constitutive activation of SKN-1 results in pleiotropic outcomes, including shortened lifespan and protective redistribution of somatic fat to the germline. We measured lipid distribution between the soma and germ tissues after manipulation of SKN-1 activity. Modulating the epigenetic landscape refines SKN-1 activity away from innate immunity targets and alleviates negative metabolic outcomes. Similarly, paraquat exposure redirects SKN-1 activity toward oxidative stress responses and away from pathogen response genes, which restores lipid distribution across tissues. Lastly, activating p38 MAPK signaling is sufficient to drive SKN-1-dependent loss of somatic fat. These data reveal a coordination of organismal metabolic homeostasis with pathogen responses and identifies mechanisms for counteracting the pleiotropic consequences of aberrant transcriptional activity.

Siddiqui, Shahid S. et al. (2013) International Worm Meeting "Antimicrobial compound screens using C. elegans model system for periodontal pathogens -."

Siddiqui, Shahid S., Al-Beyari, Mohammad, Faskhani, Fathy A.

[International Worm Meeting, 2013]

Chronic periodontal disease (PD) is an inflammatory condition that is highly widespread. Periodontal disease is a result of sub-gingival plaque pathogens, leading to alveolar bone loss. PD is also associated with diabetes, obesity, cardiovascular disease, respiratory disease, rheumatic arthritis and pre-term birth. The mechanism of periodontal infection is poorly understood due to the complexity of microbial factors, and highly complex inflammatory and anti-inflammatory signaling between pathogens and the host. New microbial pathogens are emerging that are resistant to conventional antibiotics; thus there is a need for drugs that are new class of antibiotics, and novel compounds that reduce pathogenicity of invading microbes. Pioneering studies in Frederick Ausubel's Laboratory (Harvard Medical School) have shown the effective use of C. elegans as a simple and powerful genetic model to screen for antimicrobial compounds, such as the discovery of RPW-24 that protects C. elegans from bacterial infections by modulating the p38 MAPK, and ATF7 transcription regulatory pathways [Pukkila-Worley et al. 2012]. We have previously used C. elegans to screen chemicals by using the nematode egg laying assay, and have identified novel compounds that affect egg laying behavior in a high throughput screen of commercially available chemical library [Siddiqui et al., C. elegans meeting UCLA, 2011]. We are screening chemical libraries in C. elegans for novel compounds that may inhibit the growth of oral pathogens such as Pseudomonas aeruginosa, found in the periodontal infections; and look for modulators of NSY-1, SEK-1, PMK-1 Mitogen Activated Protein (MAP) Kinase pathway that is comparable to the mammalian ASK-1(MAP kinase kinase kinase)/MKK3/6 (MAP kinase kinase) p38 MAP kinase signaling pathway. To validate the discovery of novel compounds in C. elegans high throughput screens for periodontal pathogens, we will use in vitro PLC (periodontal ligament cell) cell culture screens. Results will be presented on the efficacy of screened compounds on oral pathogens specific to chronic periodontal disease.

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Kohn RE, Rand JB, McManus JR, Moulder GL, Duke A, Barstead RJ, Duerr JS

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

A V Bicknell et al. (2002) European Worm Meeting "The C. elegans F47F2.1 gene encodes a cyclic AMP-dependent protein kinase (PK-A) catalytic subunit"

A V Bicknell, M J Fisher, M Tabish, R A Clegg, H H Rees

[European Worm Meeting, 2002]

PK-A mediates all known effects of cyclic AMP on cellular activity in eukaryotes. The holoenzyme is an inactive tetramer of two regulatory (R) subunits and two catalytic (C) subunits. Following binding of cyclic AMP to the R subunits, dissociation of active C-subunits occurs. In mammals, , and isoforms of C-subunit, encoded by different genes, have been identified.

Angelo RG et al. (1997) International C. elegans Meeting "DISCOVERY OF A POTENTIAL ANCHOR/SCAFFOLD PROTEIN FOR C. ELEGANS PROTEIN KINASE A (PKA)"

Angelo RG, Rubin CS

[International C. elegans Meeting, 1997]

C. elegans PKA is composed exclusively of catalytic and regulatory (R) subunits encoded by the kin-1 and kin-2 genes. Since C. elegans lacks other PKA isoforms, this enzyme must disseminate signals carried by cAMP to all cell compartments. Approximately 60% of total R (PKA) is in the particulate fraction of disrupted C. elegans, indicating that protein/protein interactions may diversify PKA signaling by anchoring the kinase at specific intracellular locations. Co-localization of PKA with upstream activators and/or downstream effectors can create target sites for cAMP action. To understand the mechanism of PKA localization in C. elegans, a cDNA expression library was screened with recombinant radiolabeled-R (0.5 nM) to obtain high-affinity binding proteins. A cDNA encoding a novel, 143 kDa A kinase anchor protein (AKAP1) was retrieved and sequenced. The corresponding gene (rap-1) is located in LGII and contains 17 exons. AKAP1 is an acidic protein (pI=4.4) that is unrelated to previously characterized proteins. C. elegans R is avidly bound by both soluble and immobilized fragments of AKAP1. Competition binding studies indicate that C. elegans R is a preferred ligand, whereas mammalian RII and RI isoforms are only weakly sequestered. Scatchard analysis yielded a Kd value of ~10 nM for the R/AKAP1 complex. Deletion mutagenesis, coupled with protein expression in E. coli and in vitro assays, demonstrated that residues 235 to 255 govern high-affinity binding of R by AKAP1. A hydrophobic surface generated by amino acids with branched aliphatic side chains may be a key determinant of R binding activity. Site directed mutagenesis will pinpoint essential residues involved in PKA binding. Studies aimed at identifying the AKAP1 binding site on R are also in progress. High-affinity anti-AKAP1 IgGs are being used to determine (a) whether AKAP1 expression is developmentally-regulated and cell- specific and (b) the relationship between R (PKA) and AKAP1 in vivo.