達卡產氣單胞菌會造成壞死性筋膜炎等較為嚴重的感染症，在南臺灣以及世界各地都有這樣的案例，然而在宿主對於達卡產氣單胞菌感染下之反應機制仍所知甚寡。先前的研究已經發現秀麗隱桿線蟲之激酶RIOK-1在達卡產氣單胞菌的感染中，扮演著抑制p38 MAPK先天免疫路徑的重要角色。在本研究中我們將從已知的線蟲激酶資料庫中，以RNA干擾方法抑制目標基因表現，以搜尋其他影響線蟲先天免疫路徑的激酶。我們發現抑制參與在細胞極性化以及胚胎發育過程中的重要激酶PAR-1表現時，竟使得線蟲對於達卡產氣單胞菌具有抵抗能力。PAR-1基因剔除之線蟲同樣具有抵抗達卡產氣單胞菌之感染。進一步以顯微鏡觀察與測量發現PAR-1基因剔除之線蟲相較於野生種體型略小，並在產卵數量以及孵化率都有顯著降低的情形。另外我們也發現PAR-1基因剔除之線蟲對於綠膿桿菌同樣具有抵抗能力。藉由即時定量聚合酶連鎖反應，我們得知線蟲PAR-1激酶的表現量會在達卡產氣單胞菌感染下有所增加。而在利用遺傳學上位基因調控分析方法以及網路工具的分析下，我們判斷出PAR-1激酶位於多條先天免疫訊息傳遞路徑之上游，包含p38 MAPK或FSHR-1等，而在PAR-1激酶剔除線蟲，其p38 MAPK免疫路徑竟是處於活化狀態，進一步經由即時定量聚合酶連鎖反應發現，剔除PAR-1之後，上述這些先天免疫訊息傳遞路徑下游之抗菌胜肽基因表現量皆有所提升。總結本研究，我們證實線蟲之PAR-1激酶除了調控生殖發育等過程之外，同時作為一重要先天免疫抑制因子。

Aeromonas dhakensis, which causes severe infection such as necrotizing fasciitis, is prevalent in southern Taiwan and worldwide. However, the mechanism for how hosts resist A. dhakensis infection remains unclear. In our previous study, kinase RIOK-1 was found to be a repressor of the p38 MAPK innate immune pathway in Caenorhabditis elegans upon A. dhakensis infection. In this study, we screened an established kinase library which contains 304 kinase genes of C. elegans to discover other kinases that are involved in innate immunity against A. dhakensis infection. We used RNA interference (RNAi) to knock down the expression of kinases in C. elegans infected with A. dhakensis and observed C. elegans’ survival. In the screening, knockdown of kinase par-1, which has been reported to maintain cell polarity and embryonic pattern, conferred a longer survival in C. elegans against A. dhakensis infection. The par-1 mutant worm also demonstrated resistance to A. dhakensis. In addition, par-1 mutant worms were smaller than wild-type individuals. The hatching rate and progeny size of the par-1 mutant strain were both reduced compared to wild-type worms. The par-1 mutant worms were also resistant to Pseudomonas aeruginosa infection. By using qRT-PCR, we found the expression level of par-1 in wild-type worms was up-regulated under A. dhakensis infection. With genetic epistasis analysis and aid of online predictive tools, we discovered that par-1 lays on the upstream of multiple immune pathways such as p38 MAPK and fshr-1. Moreover, par-1 mutant worms had an increased activity of p38 MAPK. We also found the expression levels of antimicrobial peptides encoded by downstream genes regulated by these innate immune pathways were increased in the par-1 mutant strain. Overall, we discovered that PAR-1 kinase not only regulates reproductive development processes but also acts as an important innate immune suppressor in C. elegans.

1 Chen, P. L., Lamy, B. & Ko, W. C. Aeromonas dhakensis, an Increasingly Recognized Human Pathogen. Front Microbiol 7, 793, doi:10.3389/fmicb.2016.00793 (2016).
2 Chen, P. L. et al. Virulence diversity among bacteremic Aeromonas isolates: ex vivo, animal, and clinical evidences. PLoS One 9, e111213, doi:10.1371/journal.pone.0111213 (2014).
3 Janda, J. M. & Abbott, S. L. The genus Aeromonas: taxonomy, pathogenicity, and infection. Clin Microbiol Rev 23, 35-73, doi:10.1128/CMR.00039-09 (2010).
4 Chen, P. L. et al. Matrix-assisted laser desorption ionization-time of flight mass spectrometry can accurately differentiate Aeromonas dhakensis from A. hydrophila, A. caviae, and A. veronii. J Clin Microbiol 52, 2625-2628, doi:10.1128/JCM.01025-14 (2014).
5 Puthucheary, S. D., Puah, S. M. & Chua, K. H. Molecular characterization of clinical isolates of Aeromonas species from Malaysia. PLoS One 7, e30205, doi:10.1371/journal.pone.0030205 (2012).
6 Martinez-Murcia, A. J. et al. Aeromonas aquariorum sp. nov., isolated from aquaria of ornamental fish. Int J Syst Evol Microbiol 58, 1169-1175, doi:10.1099/ijs.0.65352-0 (2008).
7 Alperi, A. et al. Aeromonas taiwanensis sp. nov. and Aeromonas sanarellii sp. nov., clinical species from Taiwan. Int J Syst Evol Microbiol 60, 2048-2055, doi:10.1099/ijs.0.014621-0 (2010).
8 Aravena-Roman, M., Harnett, G. B., Riley, T. V., Inglis, T. J. & Chang, B. J. Aeromonas aquariorum is widely distributed in clinical and environmental specimens and can be misidentified as Aeromonas hydrophila. J Clin Microbiol 49, 3006-3008, doi:10.1128/JCM.00472-11 (2011).
9 Chen, P. L. et al. A comparative study of clinical Aeromonas dhakensis and Aeromonas hydrophila isolates in southern Taiwan: A. dhakensis is more predominant and virulent. Clin Microbiol Infect 20, O428-434, doi:10.1111/1469-0691.12456 (2014).
10 Dal Peraro, M. & van der Goot, F. G. Pore-forming toxins: ancient, but never really out of fashion. Nat Rev Microbiol 14, 77-92, doi:10.1038/nrmicro.2015.3 (2016).
11 Chen, P. L. et al. Aeromonas stool isolates from individuals with or without diarrhea in southern Taiwan: Predominance of Aeromonas veronii. J Microbiol Immunol Infect 48, 618-624, doi:10.1016/j.jmii.2014.08.007 (2015).
12 Chen, P. L. et al. A Disease Model of Muscle Necrosis Caused by Aeromonas dhakensis Infection in Caenorhabditis elegans. Front Microbiol 7, 2058, doi:10.3389/fmicb.2016.02058 (2016).
13 Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71-94 (1974).
14 Sterken, M. G., Snoek, L. B., Kammenga, J. E. & Andersen, E. C. The laboratory domestication of Caenorhabditis elegans. Trends Genet 31, 224-231, doi:10.1016/j.tig.2015.02.009 (2015).
15 Loer, C. M. & Kenyon, C. J. Serotonin-deficient mutants and male mating behavior in the nematode Caenorhabditis elegans. J Neurosci 13, 5407-5417 (1993).
16 Corsi, A. K. A biochemist's guide to Caenorhabditis elegans. Anal Biochem 359, 1-17, doi:10.1016/j.ab.2006.07.033 (2006).
17 Berks, M. The C. elegans genome sequencing project. C. elegans Genome Mapping and Sequencing Consortium. Genome Res 5, 99-104, doi:10.1101/gr.5.2.99 (1995).
18 Kamath, R. S. et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231-237, doi:10.1038/nature01278 (2003).
19 Kim, D. H. et al. A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity. Science 297, 623-626, doi:10.1126/science.1073759 (2002).
20 Oh, S. W. et al. JNK regulates lifespan in Caenorhabditis elegans by modulating nuclear translocation of forkhead transcription factor/DAF-16. Proc Natl Acad Sci U S A 102, 4494-4499, doi:10.1073/pnas.0500749102 (2005).
21 Shin, H., Kaplan, R. E. W., Duong, T., Fakieh, R. & Reiner, D. J. Ral Signals through a MAP4 Kinase-p38 MAP Kinase Cascade in C. elegans Cell Fate Patterning. Cell Rep 24, 2669-2681 e2665, doi:10.1016/j.celrep.2018.08.011 (2018).
22 Le Roith, D. & Zick, Y. Recent advances in our understanding of insulin action and insulin resistance. Diabetes Care 24, 588-597, doi:10.2337/diacare.24.3.588 (2001).
23 Lapierre, L. R. et al. The TFEB orthologue HLH-30 regulates autophagy and modulates longevity in Caenorhabditis elegans. Nat Commun 4, 2267, doi:10.1038/ncomms3267 (2013).
24 Engelmann, I. & Pujol, N. Innate immunity in C. elegans. Adv Exp Med Biol 708, 105-121, doi:10.1007/978-1-4419-8059-5_6 (2010).
25 Avery, L. & Shtonda, B. B. Food transport in the C. elegans pharynx. J Exp Biol 206, 2441-2457, doi:10.1242/jeb.00433 (2003).
26 Avery, L. & Horvitz, H. R. Effects of starvation and neuroactive drugs on feeding in Caenorhabditis elegans. J Exp Zool 253, 263-270, doi:10.1002/jez.1402530305 (1990).
27 Elkabti, A. B., Issi, L. & Rao, R. P. Caenorhabditis elegans as a Model Host to Monitor the Candida Infection Processes. J Fungi (Basel) 4, doi:10.3390/jof4040123 (2018).
28 Guo, S. & Kemphues, K. J. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81, 611-620, doi:10.1016/0092-8674(95)90082-9 (1995).
29 Krebs, E. G. Historical perspectives on protein phosphorylation and a classification system for protein kinases. Philos Trans R Soc Lond B Biol Sci 302, 3-11, doi:10.1098/rstb.1983.0033 (1983).
30 Corbellino, M. et al. The role of human herpesvirus 8 and Epstein-Barr virus in the pathogenesis of giant lymph node hyperplasia (Castleman's disease). Clin Infect Dis 22, 1120-1121, doi:10.1093/clinids/22.6.1120 (1996).
31 Scheeff, E. D. & Bourne, P. E. Structural evolution of the protein kinase-like superfamily. PLoS Comput Biol 1, e49, doi:10.1371/journal.pcbi.0010049 (2005).
32 Manning, G., Whyte, D. B., Martinez, R., Hunter, T. & Sudarsanam, S. The protein kinase complement of the human genome. Science 298, 1912-1934, doi:10.1126/science.1075762 (2002).
33 Plowman, G. D., Sudarsanam, S., Bingham, J., Whyte, D. & Hunter, T. The protein kinases of Caenorhabditis elegans: a model for signal transduction in multicellular organisms. Proc Natl Acad Sci U S A 96, 13603-13610, doi:10.1073/pnas.96.24.13603 (1999).
34 Manning, G., Plowman, G. D., Hunter, T. & Sudarsanam, S. Evolution of protein kinase signaling from yeast to man. Trends Biochem Sci 27, 514-520, doi:10.1016/s0968-0004(02)02179-5 (2002).
35 Muhlrad, P. J. & Ward, S. Spermiogenesis initiation in Caenorhabditis elegans involves a casein kinase 1 encoded by the spe-6 gene. Genetics 161, 143-155 (2002).
36 Vowels, J. J. & Thomas, J. H. Genetic analysis of chemosensory control of dauer formation in Caenorhabditis elegans. Genetics 130, 105-123 (1992).
37 Schlesinger, A., Shelton, C. A., Maloof, J. N., Meneghini, M. & Bowerman, B. Wnt pathway components orient a mitotic spindle in the early Caenorhabditis elegans embryo without requiring gene transcription in the responding cell. Genes Dev 13, 2028-2038, doi:10.1101/gad.13.15.2028 (1999).
38 Popovici, C., Roubin, R., Coulier, F., Pontarotti, P. & Birnbaum, D. The family of Caenorhabditis elegans tyrosine kinase receptors: similarities and differences with mammalian receptors. Genome Res 9, 1026-1039, doi:10.1101/gr.9.11.1026 (1999).
39 Chen, Y. W., Ko, W. C., Chen, C. S. & Chen, P. L. RIOK-1 Is a Suppressor of the p38 MAPK Innate Immune Pathway in Caenorhabditis elegans. Front Immunol 9, 774, doi:10.3389/fimmu.2018.00774 (2018).
40 Fuchs, S. Y., Adler, V., Pincus, M. R. & Ronai, Z. MEKK1/JNK signaling stabilizes and activates p53. Proc Natl Acad Sci U S A 95, 10541-10546, doi:10.1073/pnas.95.18.10541 (1998).
41 Singh, V. & Aballay, A. Regulation of DAF-16-mediated Innate Immunity in Caenorhabditis elegans. J Biol Chem 284, 35580-35587, doi:10.1074/jbc.M109.060905 (2009).
42 Pukkila-Worley, R. & Ausubel, F. M. Immune defense mechanisms in the Caenorhabditis elegans intestinal epithelium. Curr Opin Immunol 24, 3-9, doi:10.1016/j.coi.2011.10.004 (2012).
43 Kyriakis, J. M. & Avruch, J. Mammalian MAPK signal transduction pathways activated by stress and inflammation: a 10-year update. Physiol Rev 92, 689-737, doi:10.1152/physrev.00028.2011 (2012).
44 Xu, A., Shi, G., Liu, F. & Ge, B. Caenorhabditis elegans mom-4 is required for the activation of the p38 MAPK signaling pathway in the response to Pseudomonas aeruginosa infection. Protein Cell 4, 53-61, doi:10.1007/s13238-012-2080-z (2013).
45 Kim, D. H. et al. Integration of Caenorhabditis elegans MAPK pathways mediating immunity and stress resistance by MEK-1 MAPK kinase and VHP-1 MAPK phosphatase. Proc Natl Acad Sci U S A 101, 10990-10994, doi:10.1073/pnas.0403546101 (2004).
46 Zeke, A., Misheva, M., Remenyi, A. & Bogoyevitch, M. A. JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships. Microbiol Mol Biol Rev 80, 793-835, doi:10.1128/MMBR.00043-14 (2016).
47 Engstrom, W., Ward, A. & Moorwood, K. The role of scaffold proteins in JNK signalling. Cell Prolif 43, 56-66, doi:10.1111/j.1365-2184.2009.00654.x (2010).
48 Han, M. & Sternberg, P. W. let-60, a gene that specifies cell fates during C. elegans vulval induction, encodes a ras protein. Cell 63, 921-931, doi:10.1016/0092-8674(90)90495-z (1990).
49 Beitel, G. J., Clark, S. G. & Horvitz, H. R. Caenorhabditis elegans ras gene let-60 acts as a switch in the pathway of vulval induction. Nature 348, 503-509, doi:10.1038/348503a0 (1990).
50 Vlahopoulos, S. A. Aberrant control of NF-kappaB in cancer permits transcriptional and phenotypic plasticity, to curtail dependence on host tissue: molecular mode. Cancer Biol Med 14, 254-270, doi:10.20892/j.issn.2095-3941.2017.0029 (2017).
51 Meffert, M. K., Chang, J. M., Wiltgen, B. J., Fanselow, M. S. & Baltimore, D. NF-kappa B functions in synaptic signaling and behavior. Nat Neurosci 6, 1072-1078, doi:10.1038/nn1110 (2003).
52 Powell, J. R., Kim, D. H. & Ausubel, F. M. The G protein-coupled receptor FSHR-1 is required for the Caenorhabditis elegans innate immune response. Proc Natl Acad Sci U S A 106, 2782-2787, doi:10.1073/pnas.0813048106 (2009).
53 Estes, K. A., Dunbar, T. L., Powell, J. R., Ausubel, F. M. & Troemel, E. R. bZIP transcription factor zip-2 mediates an early response to Pseudomonas aeruginosa infection in Caenorhabditis elegans. Proc Natl Acad Sci U S A 107, 2153-2158, doi:10.1073/pnas.0914643107 (2010).
54 Wu, Y. & Griffin, E. E. Regulation of Cell Polarity by PAR-1/MARK Kinase. Curr Top Dev Biol 123, 365-397, doi:10.1016/bs.ctdb.2016.11.001 (2017).
55 Macfarlane, S. R., Seatter, M. J., Kanke, T., Hunter, G. D. & Plevin, R. Proteinase-activated receptors. Pharmacol Rev 53, 245-282 (2001).
56 Goransson, O. et al. Regulation of the polarity kinases PAR-1/MARK by 14-3-3 interaction and phosphorylation. J Cell Sci 119, 4059-4070, doi:10.1242/jcs.03097 (2006).
57 Wu, C. J. et al. Genome sequence of a novel human pathogen, Aeromonas aquariorum. J Bacteriol 194, 4114-4115, doi:10.1128/JB.00621-12 (2012).
58 Chou, T. C. et al. Enterohaemorrhagic Escherichia coli O157:H7 Shiga-like toxin 1 is required for full pathogenicity and activation of the p38 mitogen-activated protein kinase pathway in Caenorhabditis elegans. Cell Microbiol 15, 82-97, doi:10.1111/cmi.12030 (2013).
59 Miller, E. V., Grandi, L. N., Giannini, J. A., Robinson, J. D. & Powell, J. R. The Conserved G-Protein Coupled Receptor FSHR-1 Regulates Protective Host Responses to Infection and Oxidative Stress. PLoS One 10, e0137403, doi:10.1371/journal.pone.0137403 (2015).
60 Cohen, L. B. & Troemel, E. R. Microbial pathogenesis and host defense in the nematode C. elegans. Curr Opin Microbiol 23, 94-101, doi:10.1016/j.mib.2014.11.009 (2015).
61 Kato, T. et al. Isolation of a novel human gene, MARKL1, homologous to MARK3 and its involvement in hepatocellular carcinogenesis. Neoplasia 3, 4-9, doi:10.1038/sj.neo.7900132 (2001).
62 Ansar, M. et al. Visual impairment and progressive phthisis bulbi caused by recessive pathogenic variant in MARK3. Hum Mol Genet 27, 2703-2711, doi:10.1093/hmg/ddy180 (2018).
63 Liu, X., Yu, J., Song, S., Yue, X. & Li, Q. Protease-activated receptor-1 (PAR-1): a promising molecular target for cancer. Oncotarget 8, 107334-107345, doi:10.18632/oncotarget.21015 (2017).
64 Adjei, A. A. Blocking oncogenic Ras signaling for cancer therapy. J Natl Cancer Inst 93, 1062-1074, doi:10.1093/jnci/93.14.1062 (2001).