東南亞已成為全球主要出產白蝦的地區，高密度水產養殖的方式導致新興的病毒性、真菌性以及細菌性的疾病爆發。細菌性疾病-急性肝胰腺壞死症(AHPND)自2009年首次於中國發現後已蔓延至世界各地，並對於蝦類產業造成重大損失。導致AHPND的病原菌為攜帶含有二元毒素 (Photorhabdus insect-related Pir toxin)基因之特殊質體pVA (69 kb)的桿狀革蘭氏陰性菌- V. parahaemolyticus。對於AHPND致病機轉以及病原菌-宿主間交互作用進一步的瞭解，將有助於建立更有效的疾病控制措施。先前研究指出，自感染AHPND後的蝦胃轉錄體樣本中發現，頂部鈉膽汁酸轉運蛋白(LvASBT)的上調現象與致毒性相關。本研究的目的在於探討膽汁酸(bile acid)以及膽汁酸轉運蛋白(LvASBT)在AHPND感染過程中扮演的角色。本研究發現bile acid含量以及LvASBT表現量在感染AHPND的蝦胃中上升。Bile acid也會促進致病性 V. parahaemolyticus生物膜(biofilm)的生成以及增加PirABvp毒素的產量及釋放。分析培養在含有膽汁酸中的致病性V. parahaemolyticus轉錄體後發現，部分參與在代謝路徑、細胞膜轉運、RND efflux pump以及細菌性分泌系統的相關基因呈現上調現象。最後根據本研究的發現，建立出膽汁酸如何幫助致病性V. parahaemolyticus在蝦胃中定殖(colonization)並遷移至肝胰線的模型。

Globally, Southeast Asia has been the major producer of the Litopenaeus vannamei. The intensive aquaculture practices are causing emergence of new viral, fungal and bacterial diseases. In 2009, the bacterial disease acute hepatopancreatic disease (AHPND) outbreak in China was first reported which eventually spread to other parts of the world and has caused immense damage to the shrimp industry. AHPND is caused by gram negative, rod-shaped Vibrio parahaemolyticus bacteria which carries a unique 69 kb pVA plasmid that carries a binary toxin gene called Photorhabdus insect-related (Pir) toxins. An improved understanding of the pathogenesis mechanism and host-pathogen interactions would help in developing effective strategies for controlling the disease. There was previously some evidence from the transcriptomic profiling of AHPND-infected shrimp stomach that virulence was associated with LvASBT (apical sodium bile acid transporter) upregulation. In this study we wanted to investigate the involvement of bile acids and their transporters during AHPND pathogenesis. We found that both bile acid levels and LvASBT expression were increased in shrimp stomach during AHPND. Crude bile acids induced biofilm formation and increased release of PirABvp toxins in V. parahaemolyticus. Transcriptomic analysis of bile acid treated AHPND-causing V. parahaemolyticus led to the modulation of several genes involved in metabolic pathways, membrane transport, a bacterial secretion system and RND efflux pumps, Lastly, based on our results, we propose a model to show the involvement of bile acids and LvASBT in the colonization of AHPND-causing V. parahaemolyticus in the shrimp stomach and its subsequent migration to the hepatopancreas.

Alrefai, W. A., and Gill, R. K. Bile acid transporters: structure, function, regulation and pathophysiological implications. Pharmaceutical Research 24, 1803-1823, 2007.

Bachmann, V., Kostiuk, B., Unterweger, D., Diaz-Satizabal, L., Ogg, S., and Pukatzki, S. Bile salts modulate the mucin-activated type VI secretion system of pandemic Vibrio cholerae. PLoS Neglected Tropical Diseases 9, e0004031, 2015.

Begley, M., Gahan, C. G., and Hill, C. The interaction between bacteria and bile. Federation of European Microbiological Societies Microbiology Reviews 29, 625-651, 2005.

Bina, J. E., and Mekalanos, J. J. Vibrio cholerae tolC is required for bile resistance and colonization. Infection and Immunity 69, 4681-4685, 2001.

Bina, X. R., Provenzano, D., Nguyen, N., and Bina, J. E. Vibrio cholerae RND family efflux systems are required for antimicrobial resistance, optimal virulence factor production, and colonization of the infant mouse small intestine. Infection and Immunity 76, 3595-3605, 2008.

Bondad-Reantaso, M. G. Acute Hepatopancreatic Necrosis Disease (AHPND) of Penaeid Shrimps: Global Perspective. Proceedings of the ASEAN Regional Technical Consultation on EMS/AHPND and Other Transboundary Diseases for Improved Aquatic Animal Health in Southeast Asia, Tigbauan, Iloilo, Philippines, Aquaculture Department, Southeast Asian Fisheries Development Center, 16-23, 2016.

Caroline, S., and Aguinaldo, P. Challenges in shrimp aquaculture due to viral diseases: Distribution and biology of the five major penaeid viruses and interventions to avoid viral incidence and dispersion. Brazilian Journal of Microbiology 43, 857-864, 2012.

Claro da Silva, T., Polli, J. E., and Swaan, P. W. The solute carrier family 10 (SLC10): beyond bile acid transport. Molecular Aspects of Medicine 34, 252-269, 2013.

Davis, R. A., and Attie, A. D. Deletion of the ileal basolateral bile acid transporter identifies the cellular sentinels that regulate the bile acid pool. Proceedings of the National Academy of Sciences of the United States of America 105, 4965-4966, 2008.

Dawson, P. A., and Karpen, S. J. Intestinal transport and metabolism of bile acids. Journal of Lipid Research 56,1085-1099, 2015.

de Hoon, M. J. L., Imoto, S., Nolan, J., and Miyano, S. Open source clustering software. Bioinformatics 20, 1453-1454, 2004.

Dhar, A. K., Piamsomboon, P., Caro, L. F. A., Kanrar, S., Adami, Jr. R., Juan, Y-S. First report of acute hepatopancreatic necrosis disease (AHPND) occurring in the USA. Diseases of Aquatic Organisms 132, 241-247, 2019.

Eshik, M. M. E., Abedin, M. M., Punom, N. J., Begum, M. K., Rahman, M. S. Molecular identification of AHPND positive Vibrio parahaemolyticus causing an outbreak in south-west shrimp farming regions of Bangladesh. Journal of Bangladesh Academy of Sciences 41, 127-135, 2017.

Fang, F. C., Frawley, E. R., Tapscott, T., and Vázquez-Torres, A. Bacterial stress responses during host infection. Cell Host and Microbe 20, 133-143, 2016.

Fernando, D. M., and Kumar, A. Resistance-nodulation-division multidrug efflux pumps in gram-negative bacteria: Role in virulence. Antibiotics (Basel) 2, 163-181, 2013.

Fong, J. C., Syed, K. A., Klose, K. E., and Yildiz, F. H. Role of Vibrio polysaccharide (vps) genes in VPS production, biofilm formation and Vibrio cholerae pathogenesis. Microbiology 156, 2757-2769, 2010.

Goldstein, J., and Levy, C. Novel and emerging therapies for cholestatic liver diseases. Liver International 38, 1520-1535, 2018.

Gotoh, K., Kodama, T., Hiyoshi, H., Izutsu, K., Park, K. S., Dryselius, R., Akeda, Y., Honda, T., and Iida, T. Bile acid-induced virulence gene expression of Vibrio parahaemolyticus reveals a novel therapeutic potential for bile acid sequestrants. PLoS One 5, e13365, 2010.

Greene, N. P., Kaplan, E., Crow, A., and Koronakis, V. Antibiotic resistance mediated by the MacB ABC transporter family: A structural and functional perspective. Frontiers in Microbiology 9, 950, 2018.

Han, J. E., Tang, K. F., Tran, L. H., and Lightner, D. V. (2015). Photorhabdus insect-related (Pir) toxin-like genes in a plasmid of Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND) of shrimp Diseases of Aquatic Organisms 113, 33-40, 2015.

Hay, A. J., and Zhu, J. Host intestinal signal-promoted biofilm dispersal induces Vibrio cholerae colonization. Infection and Immunity 83, 317-323, 2015.

Hegade, V. S., Kendrick, S. F., Dobbins, R. L., Miller, S. R., Richards, D., Storey, J., Dukes, G., Gilchrist, K., Vallow, S., Alexander, G. J., Corrigan, M., Hirschfield, G. M., and Jones, D. E. BAT117213: Ileal bile acid transporter (IBAT) inhibition as a treatment for pruritus in primary biliary cirrhosis: study protocol for a randomised controlled trial. BioMed Central Gastroenterology 16, 71, 2016.

Hofmann, A. F., and Eckmann, L. How bile acids confer gut mucosal protection against bacteria. Proceedings of the National Academy of Sciences of the United States of America 103, 4333-4334, 2006.

Hong, X., Lu, L., and Xu, D. Progress in research on acute hepatopancreatic necrosis disease (AHPND). Aquaculture International 24, 577-593, 2016.

Hung, D. T., and Mekalanos, J. J. Bile acids induce cholera toxin expression in Vibrio cholerae in a ToxT-independent manner. Proceedings of the National Academy of Sciences of the United States of America 102, 3028-3033, 2005.

Hung, D. T., Zhu, J., Sturtevant, D., and Mekalanos, J. J. Bile acids stimulate biofilm formation in Vibrio cholerae. Molecular Microbiology 59, 193-201, 2006.

Jamal, M. T., Abdulrahman, I. A., Harbi, M. A., and Chithambaran, S. Probiotics as alternative control measures in shrimp aquaculture: A review. Journal of Applied Biology and Biotechnology 7, 69-77, 2019.

Karunasagar, I., and Karunasagar, I. Ecology, virulence factors and global spread of Vibrio parahaemolyticus. Asian Fisheries Science 31S, 15-28, 2018.

Koestler, B. J., and Waters, C. M. Bile acids and bicarbonate inversely regulate intracellular cyclic di-GMP in Vibrio cholerae. Infection and Immunity 82, 3002-3014, 2014.

Kugita, H. OIE initiatives on acute hepatopancreatic necrosis disease (AHPND) and other aquatic animal diseases in Asia. Proceedings of the ASEAN Regional Technical Consultation on EMS/AHPND and Other Transboundary Diseases for Improved Aquatic Animal Health in Southeast Asia, Tigbauan, Iloilo, Philippines, Aquaculture Department, Southeast Asian Fisheries Development Center, 11-15, 2016.

Kumar, R., Chang, C. H., Ng, T. H., Ding, J. Y., Tseng, T. C., Lo, C. F., Wang, H. C. Draft genome sequence of Vibrio parahaemolyticus strain M1-1, which causes Acute Hepatopancreatic Necrosis Disease (AHPND) in shrimp in Vietnam. Genome Announcements 6, e01468-17, 2018.

Lai, H. C., Ng, T. H., Ando, M., Lee, C. T., Chen, I. T., Chuang, J. C, Mavichak, R., Chang, S. H., Yeh, M. D., Chiang, Y. A., Takeyama, H., Hamaguchi, H. O., Lo, C. F., Aoki, T., and Wang, H. C. Pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in shrimp. Fish and Shellfish Immunology 47, 1006-1014, 2015.

Leaño, E. M. Regional Response on AHPND and Other Emerging Shrimp Diseases in the Asia-Pacific. Proceedings of the ASEAN Regional Technical Consultation on EMS/AHPND and Other Transboundary Diseases for Improved Aquatic Animal Health in Southeast Asia, Tigbauan, Iloilo, Philippines, Aquaculture Department, Southeast Asian Fisheries Development Center, 24-32, 2016.

Lee, C. T., Chen, I. T., Yang, Y. T., Ko, T. P., Huang, Y. T., Huang, J. Y., Huang, M. F., Lin, S. J., Chen, C. Y., Lin, S. S., Lightner, D. V., Wang, H. C., Wang, A. H., Wang, H. C., Hor, L., and Lo, C. F. The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin. Proceedings of the National Academy of Sciences of the United States of America 112, 10798-10803. 2015.

León-Sicairos, N., Angulo-Zamudio, U. A., de la Garza, M., Velázquez-Román, J., Flores-Villaseñor, H. M., and Canizalez-Román, A. Strategies of Vibrio parahaemolyticus to acquire nutritional iron during host colonization. Frontiers in Microbiology 6, 702, 2015.

Letchumanan, V., Chan, K. G., Khan, T. M., Bukhari, S. I., Ab Mutalib, N. S., Goh, B. H., and Lee, L. H. Bile sensing: The activation of Vibrio parahaemolyticus virulence. Frontiers in Microbiology 8, 728, 2017.

Li, P., Rivera-Cancel, G., Kinch, L. N., Salomon, D., Tomchick, D. R., Grishin, N. V., and Orth, K. Bile salt receptor complex activates a pathogenic type III secretion system. Elife 5, 5718, 2016.

Li, P., Kinch, L. N., Ray, A., Dalia, A. B., Cong, Q., Nunan, L. M., Camilli, A., Grishin, N. V., Salomon, D., and Orth, K. Acute hepatopancreatic necrosis disease-causing Vibrio parahaemolyticus strains maintain an antibacterial type VI secretion system with versatile effector repertoires. Applied and Environmental Microbiology 83, e00737-17, 2017.

Lightner, D. V. The penaeid shrimp viruses TSV, IHHNV, WSSV, and YHV. Journal of Applied Aquaculture 9, 27-52, 1999.

Liu, M., Yan, M., Liu, L., and Chen, S. Characterization of a novel zinc transporter ZnuA acquired by Vibrio parahaemolyticus through horizontal gene transfer. Frontiers in Cellular and Infection Microbiology 3, 61, 2013.

Lustri, B. C., Sperandio, V., and Moreira, C. G. Bacterial chat: Intestinal metabolites and signals in host-microbiota-pathogen interactions. Infection and Immunity 85, e00476-17, 2017.

Matsuo, T., Nakamura, K., Kodama, T., Mikami, T., Hiyoshi, H., Tsuchiya, T., Ogawa, W., and Kuroda, T. Characterization of all RND-type multidrug efflux transporters in Vibrio parahaemolyticus. MicrobiologyOpen 2, 725-742, 2013.

Matsuo, T., Ogawa, W., Tsuchiya, T., and Kuroda, T. Overexpression of vmeTUV encoding a multidrug efflux transporter of Vibrio parahaemolyticus causes bile acid resistance. Gene 541, 19-25, 2014.

Merritt, M. E., and Donaldson, J. R. Effect of bile salts on the DNA and membrane integrity of enteric bacteria. Journal of Medical Microbiology 58, 1533-1541, 2009.

Ng, T. H., Lu, C. W., Lin, S. S., Chang, C. C., Tran, L. H., Chang, W. C., Lo, C.F., and Wang, H. C. The Rho signaling pathway mediates the pathogenicity of AHPND-causing V. parahaemolyticus in shrimp. Cellular Microbiology 20, e12849, 2018.

Nishiyama, S-i., Takahashi, Y., Yamamoto, K., Suzuki, D., Itoh, Y., Sumita, K., Uchida, Y., Homma, M., Imada, K., and Kawagishi, I. Identification of a Vibrio cholerae chemoreceptor that senses taurine and amino acids as attractants. Scientific Reports 6, 20866, 2016.

Nunan, L., Lightner, D., Pantoja, C., and Gomez-Jimenez, S. Detection of acute hepatopancreatic necrosis disease (AHPND) in Mexico. Diseases of Aquatic Organisms 111, 81-86, 2014.

Pace, J. L., Chai, T. J., Rossi, H. A., and Jiang, X. Effect of bile on Vibrio parahaemolyticus. Applied and Environmental Microbiology 63, 2372-2377, 1997.

Petronella, N., and Ronholm, J. The mechanisms that regulate Vibrio parahaemolyticus virulence gene expression differ between pathotypes. Microbial Genomics 4, e000182, 2018.

Plecha, S. C., and Withey, J. H. Mechanism for inhibition of Vibrio cholerae ToxT activity by the unsaturated fatty acid components of bile. Journal of Bacteriology 197, 1716-1725, 2015.

Pride, A. C., Herrera, C. M., Guan, Z., Giles, D. K., and Trent, M. S. The outer surface lipoprotein VolA mediates utilization of exogenous lipids by Vibrio cholerae. mBio 4, e00305-13, 2013.

Provenzano, D. and Klose, K. E. Altered expression of the ToxR-regulated porins OmpU and OmpT diminishes Vibrio cholerae bile resistance, virulence factor expression, and intestinal colonization. Proceedings of the National Academy of Sciences of the United States of America 97, 10220-10224, 2000.

Rao, A., Kosters, A., Mells, J. E., Zhang, W., Setchell, K. D., Amanso, A. M., Wynn, G. M., Xu, T., Keller, B. T., Yin, H., Banton, S., Jones, D. P., Wu, H., Dawson, P. A., Karpen, S. J. Inhibition of ileal bile acid uptake protects against nonalcoholic fatty liver disease in high-fat diet-fed mice. Science Translational Medicine 8, 357ra122. 2016.

Ribet, D., and Cossart, P. How bacterial pathogens colonize their hosts and invade deeper tissues. Microbes and Infection 17, 173-183, 2015.

Rodrigues, S., Paillard, C., Van Dillen, S., Tahrioui, A., Berjeaud, J. M., Dufour, A., Bazire, A. Relation between biofilm and virulence in Vibrio tapetis: A transcriptomic study. Pathogens 7, 92, 2018.

Salah Ud-Din, A. I., Tikhomirova, A., and Roujeinikova, A. Structure and functional diversity of GCN5-related N-acetyltransferases (GNAT). International Journal of Molecular Sciences 17, 1018, 2016.

Sanchez, L. M., Cheng, A. T., Warner, C. J., Townsley, L., Peach, K. C., Navarro, G., Shikuma, N. J., Bray, W. M., Riener, R. M., Yildiz, F. H., and Linington, R. G. Biofilm formation and detachment in gram-negative pathogens is modulated by select bile acids. PLoS One 11, e0149603, 2016.

Sannasiddappa, T. H., Lund, P. A., and Clarke, S. R. In vitro antibacterial activity of unconjugated and conjugated bile salts on Staphylococcus aureus. Frontiers in Microbiology 8, 1581, 2017.

Seper, A., Pressler, K., Kariisa, A., Haid, A. G., Roier, S., Leitner, D. R., Reidl, J., Tamayo, R., and Schild, S. Identification of genes induced in Vibrio cholerae in a dynamic biofilm system. The International Journal of Medical Microbiology 304, 749-763, 2014.

Shinn, A. P., Pratoomyot, J., Griffiths, D., Trong T. Q., Vu, N. T., Jiravanichpaisal, P., and Briggs, M. Asian shrimp production and economic costs of disease. Asian Fisheries Science 31S, 29-54. 2018.

Silva, A. J., and Benitez, J. A. Vibrio cholerae biofilms and cholera pathogenesis. PLoS Neglected Tropical Diseases 10, e0004330, 2016.

Soonthornchai, W., Chaiyapechara, S., Jarayabhand, P., Söderhäll, K., and Jiravanichpaisal, P. Interaction of vibrio spp. with the inner surface of the digestive tract of Penaeus monodon. PLoS One 10, e0135783, 2015.

Soto-Rodriguez, S. A., Gomez-Gil, B., Lozano-Olvera, R., Betancourt-Lozano, M., and Morales-Covarrubias, M. S. Field and experimental evidence of Vibrio parahaemolyticus as the causative agent of acute hepatopancreatic necrosis disease of cultured shrimp (Litopenaeus vannamei) in Northwestern Mexico. Applied and Environmental Microbiology 81, 1689-1699, 2015.

Teschler, J. K., Zamorano-Sánchez, D., Utada, A. S., Warner, C. J., Wong, G. C., Linington, R. G., and Yildiz, F. H. Living in the matrix: assembly and control of Vibrio cholerae biofilms. Nature Reviews Microbiology 13, 255-268, 2015.

Tran, L., Nunan, L., Redman, R. M., Mohney, L. L., Pantoja, C. R., Fitzsimmons, K., and Lightner, D. V. Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp. Diseases of Aquatic Organisms 105, 45–55, 2013.

Tseng, Y. T., Kumar, R., and Wang, H. C. LvRas and LvRap are both important for WSSV replication in Litopenaeus vannamei. Fish and Shellfish Immunology 88, 150-160, 2019.

Tung, T. C. Stomach metabolic alteration in response to AHPND infection in shrimp. Master thesis. Retrieved from National Cheng Kung University of Department of Biotechnology and Bioindustry Sciences, 2018.

Urdaneta, V. and Casadesús, J. Interactions between bacteria and bile salts in the gastrointestinal and hepatobiliary tracts. Frontiers in Medicine 4, 163, 2017.

Vonk, H. J. Emulgators in the digestive fluids of invertebrates. Archives Internationales de Physiologie et de Biochimie 70, 67-85, 1962.

Wang, H. C., Tseng, C. W., Lin, H. Y., Chen, I. T., Chen, Y. H., Chen, Y. M., Chen, T. Y., and Yang, H. L. RNAi knockdown of the Litopenaeus vannamei Toll gene (LvToll) significantly increases mortality and reduces bacterial clearance after challenge with Vibrio harveyi. Developmental and Comparative Immunology 34, 49-58, 2010.

Wibbenmeyer, J. A., Provenzano, D., Landry, C. F., Klose, K. E., and Delcour, A. H. Vibrio cholerae OmpU and OmpT porins are differentially affected by bile. Infection and Immunity 70, 121-126, 2002.

Yang, M., Liu, Z., Hughes, C., Stern, A. M., Wang, H., Zhong, Z., Kan, B., Fenical, W., and Zhu, J. Bile salt-induced intermolecular disulfide bond formation activates Vibrio cholerae virulence. Proceedings of the National Academy of Sciences of the United States of America 110, 2348-2353, 2013.

Zhu, J., and Mekalanos, J. J. Quorum sensing-dependent biofilms enhance colonization in Vibrio cholerae. Developmental Cell 5, 647-656, 2003.

Zhang, X., Yuan, J, Sun, Y, Li, S, Gao, Y, Yu, Y, Liu, C., Wang, Q., Lv, X., Zhang, X., Ma, K. Y., Wang, X., Lin, W., Wang, L., Zhu, X., Zhang, C., Zhang J., Jin, S., Yu, K., Kong, J., Xu, P., Chen, J., Zhang, H., Sorgeloos, P., Sagi, A., Alcivar-Warren, A., Liu, Z., Wang, L., Ruan, J., Chu, K. H., Liu, B., Li, F., and Xiang, J. Penaeid shrimp genome provides insights into benthic adaptation and frequent molting. Nature Communications 10, 356, 2019.
Zhou, X., and Cai, J. The state of world fisheries and aquaculture 2018 - Meeting the sustainable development goals. Fisheries and Agriculture Organization of United Nations 17-34, 2018