Project
Description









Abstract

 

Aeromonas hydrophila is a major pathogen threatening China's aquaculture industry. Traditional treatments, like water quality management, balanced diets, vaccines, and antimicrobials, are often costly, environmentally harmful, or labor-intensive. Our project aims to reduce the virulence of A. hydrophila by targeting its quorum sensing system, which relies on AHL molecules to activate virulence factor expression. We introduced six AHL lactonases from various bacteria into Bacillus subtilis. By degrading AHL, our engineered B. subtilis causes quorum quenching and reduces A. hydrophila's pathogenicity. This solution provides a more affordable, eco-friendly, and sustainable approach to protect aquatic animals from A. hydrophila by applying engineered B. subtilis in aquaculture ponds.

Part 1. Aquaculture


Fish, shrimp, and crabs are delicious. Our appetite has led to the giant yet fast-growing industry of aquatic products. According to the Food and Agriculture Organization (FAO), total world fisheries and aquaculture production grew by 45% from 2000 to 2021, reaching a record high of 182 million tons in 2021, valued at over US$400 billion (FAO, 2023). Asia plays a major role in aquaculture production, and among all the countries, China is by far the main producer of aquaculture, with an astonishing 57% share. However, the thriving aquaculture industry in China faces significant threats from various pathogens.

 

 

Figure 1. World map of aquaculture production (FAO,2023).

 

Pathogens such as Aeromonas hydrophila, Vibrio parahaemolyticus, and Streptococcus iniae are commonly found in Chinese fish farms. These bacteria can cause severe diseases, leading to high mortality rates and economic losses (Irshath et al., 2023). Among them, A. hydrophila is the most prevalent pathogen in local areas (Zhejiang Province, China), which can cause motile aeromonad septicemia (MAS) in aquatic animals (Nielsen et al., 2001; Stratev & Odeyemi, 2016). It can also cause human gastroenteritis when people ingest contaminated food (Fleckenstein et al., 2021).

 

The aquaculture industry has adopted various mitigation strategies to combat A. hydrophila. The first method is to maintain optimal water quality. Providing a well-balanced diet also lowers the risk of A. hydrophila infection. Moreover, vaccines can protect against specific strains of A. hydrophila. Other than these, antibiotics and other antimicrobial agents can also be used to treat Aeromonas infections (Harikrishnan & Balasundaram, 2005; Semwal et al., 2023). These strategies show significant results in small-scale tests, but many are not regularly adopted in the real world due to high labor and financial costs. Also, antibiotic treatment can result in antibiotic resistance and therefore must be carefully managed.

 

The use of probiotics in aquaculture to control A. hydrophila is an area of active research and practical application (Harikrishnan & Balasundaram, 2005). Applying probiotics does not require additional facilities and is not labor intensive. Moreover, since living microorganisms can reproduce in ponds, leading to a long-term effect, fish farmers can save money from repeat purchases. Bacillus subtilis is one of the most commonly used probiotics in aquaculture (Nayak et al., 2023; Neissi et al., 2024; Olmos & Paniagua-Michel, 2014). It is safe for both aquatic animals and humans and easy to grow. Therefore, we modified B. subtilis to enhance its inhibitory effect on A. hydrophila.

Part 2. Quorum Quenching

 

Quorum sensing is a sophisticated bacterial communication mechanism that allows bacteria to detect and respond to changes in their population density through the production and sensing of small signaling molecules called autoinducers. The autoinducer of A. hydrophila is N-butanoyl-L-homoserine lactone (C4-HSL), an acyl-homoserine lactone (AHL). When the concentration of C4-HSL reaches a certain threshold, it activates genes responsible for producing virulence factors (Coquant et al., 2020; Garde et al., 2010). These factors include toxins like aerolysin and hemolysin, extracellular proteases such as serine proteases, and biofilm formation (Khajanchi et al., 2009; Swift et al., 1997). These virulence factors facilitate infection by damaging host cells and forming protective biofilms.

 

 

Figure 2. Quorum sensing and quenching in A. hydrophila. A. C4-HSL freely diffuses through the membrane and binds to receptors. The receptors dimerize and function as a transcription factor to trigger target gene expression (Coquant et al., 2020); B. C4-HSL is degraded by strains producing AHL lactonases, leading to quorum quenching.

 

The process of disrupting quorum sensing is called quorum quenching. By interfering with the signaling pathways, quorum quenching can effectively reduce bacterial virulence, thereby reducing the ability of A. hydrophila to infect aquatic animals (Chen et al., 2013; Chu et al., 2014). Quorum-quenching enzymes, such as AHL lactonases, acylases, and oxidoreductases, play a crucial role in this process. These enzymes degrade or modify the autoinducers, thereby preventing them from binding to their receptors and triggering the quorum-sensing response (Chen et al., 2013).

Part 3. Modification of Bacillus subtilis

 

Part 3.1 Bacillus subtilis

B. subtilis is a safe, commonly used Gram-positive bacterium in labs. Its environmental resilience enables it to thrive in harsh conditions. With its rapid growth and ability to thrive in simple media, B. subtilis emerges as a cost-effective choice for large-scale production. Equipped with a robust protein secretion system, B. subtilis can efficiently export proteins into the extracellular environment (Su et al., 2020; Urdaci et al., 2004).

 

We chose to use B. subtilis WB600, a modified strain lacking six intrinsic proteases. WB600 facilitates the production of recombinant proteins with increased yield and stability (Wu et al., 1991). B. subtilis WB600 holds promise for producing extraneous quorum-quenching enzymes to inhibit quorum sensing of A. hydrophila.

 

Part 3.2 Genes and Plasmids

Our selected extraneous quorum-quenching enzymes are derived from a broad range of microorganisms. Safety is our first priority, after literature research, six AHL lactonases are screened from gene donors that are safe for both humans and aquatic animals.

 

B. subtilis itself is considered first. YtnP from B. subtilis WB600 and AiiA from B. subtilis BS-1 are our targets (Pan et al., 2008; Schneider et al., 2012). Then, we turn to other lab strains. Agrobacterium tumefaciens is a common soil and plant bacterium. The C58 strain is commonly used in plant transformation. We plan to extract AttM from it (Carlier et al., 2003). Finally, we find Microbacterium testaceum StLB037, Solibacillus silvestris StLB214, and Arthrobacter sp. IBN110. M. testaceum is an endophytic bacterium, while Arthrobacter and S. silvestris are soil bacteria (Dsouza et al., 2015; Krishnamurthi et al., 2009; Morohoshi et al., 2012; Morohoshi et al., 2011; Wang et al., 2010), which are all generally considered safe for animals. AiiM derived from M. testaceum StLB037 (Wang et al., 2010), AhlS derived from S. silvestris StLB214 (Morohoshi et al., 2012), and AhlD derived from Arthrobacter sp. IBN110 (Park et al., 2003) are AHL lactonases we desire.

 

Figure 3. A. Map of pHT43-BsAiiA; B. Plasmid map of pHT43-BsYtnP.

 

For B. subtilis WB600 to produce quorum-quenching enzymes to combat quorum sensing of A. hydrophila, we used the pHT43 secretion plasmid vector. pHT43 contains a strong Pgrac promoter for B. subtilis, followed by RBS and a SPamyQ secretion peptide. Connecting the AHL lactonase genes with the secretion peptide enables high-efficiency extracellular expression.

Summary

 

China leads the world in aquaculture production, but the huge industry is threatened by A. hydrophila infection. Existing strategies to combat A. hydrophila, including maintaining water quality, providing balanced diets, vaccination, and antimicrobial treatments are often costly, environmentally harmful, or labor-intensive. However, the use of engineered probiotics, such as B. subtilis, has emerged as a promising approach to control A. hydrophila due to its affordability, eco-friendliness, and long-term effectiveness. Quorum sensing is essential for the pathogenicity of A. hydrophila. Quorum-quenching enzymes, like AHL lactonases, disrupt this system, reducing bacterial virulence. Using the pHT43 vector, we engineered B. subtilis WB600 to produce and secrete six AHL lactonases derived from different bacterial gene donors. In the future, when our engineered B. subtilis is applied to aquaculture ponds, it will inhibit the virulence factors of A. hydrophila, leading to improved aquatic animal health and increased productivity.

References

Carlier, A., Uroz, S., Smadja, B., Fray, R., Latour, X., Dessaux, Y., & Faure, D. (2003). The Ti plasmid of Agrobacterium tumefaciens harbors an attM-paralogous gene, aiiB, also encoding N-Acyl homoserine lactonase activity. Appl Environ Microbiol, 69(8), 4989-4993. https://doi.org/10.1128/aem.69.8.4989-4993.2003

Chen, F., Gao, Y., Chen, X., Yu, Z., & Li, X. (2013). Quorum quenching enzymes and their application in degrading signal molecules to block quorum sensing-dependent infection. Int J Mol Sci, 14(9), 17477-17500. https://doi.org/10.3390/ijms140917477

Chu, W., Zhou, S., Zhu, W., & Zhuang, X. (2014). Quorum quenching bacteria Bacillus sp. QSI-1 protect zebrafish (Danio rerio) from Aeromonas hydrophila infection. Sci Rep, 4, 5446. https://doi.org/10.1038/srep05446

Coquant, G., Grill, J. P., & Seksik, P. (2020). Impact of N-Acyl-Homoserine Lactones, Quorum Sensing Molecules, on Gut Immunity. Front Immunol, 11, 1827. https://doi.org/10.3389/fimmu.2020.01827

Dsouza, M., Taylor, M. W., Turner, S. J., & Aislabie, J. (2015). Genomic and phenotypic insights into the ecology of Arthrobacter from Antarctic soils. BMC Genomics, 16(1), 36. https://doi.org/10.1186/s12864-015-1220-2

FAO. (2023). World Food and Agriculture – Statistical Yearbook 2023. Rome Retrieved from https://openknowledge.fao.org/handle/20.500.14283/cc8166en

Fleckenstein, J. M., Matthew Kuhlmann, F., & Sheikh, A. (2021). Acute Bacterial Gastroenteritis. Gastroenterol Clin North Am, 50(2), 283-304. https://doi.org/10.1016/j.gtc.2021.02.002

Garde, C., Bjarnsholt, T., Givskov, M., Jakobsen, T. H., Hentzer, M., Claussen, A., Sneppen, K., Ferkinghoff-Borg, J., & Sams, T. (2010). Quorum sensing regulation in Aeromonas hydrophila. J Mol Biol, 396(4), 849-857. https://doi.org/10.1016/j.jmb.2010.01.002

Harikrishnan, R., & Balasundaram, C. (2005). Modern Trends in Aeromonas hydrophila Disease Management with Fish. Reviews in Fisheries Science, 13(4), 281-320. https://doi.org/10.1080/10641260500320845

Irshath, A. A., Rajan, A. P., Vimal, S., Prabhakaran, V. S., & Ganesan, R. (2023). Bacterial Pathogenesis in Various Fish Diseases: Recent Advances and Specific Challenges in Vaccine Development. Vaccines (Basel), 11(2). https://doi.org/10.3390/vaccines11020470

Khajanchi, B. K., Sha, J., Kozlova, E. V., Erova, T. E., Suarez, G., Sierra, J. C., Popov, V. L., Horneman, A. J., & Chopra, A. K. (2009). N-acylhomoserine lactones involved in quorum sensing control the type VI secretion system, biofilm formation, protease production, and in vivo virulence in a clinical isolate of Aeromonas hydrophila. Microbiology (Reading), 155(Pt 11), 3518-3531. https://doi.org/10.1099/mic.0.031575-0

Krishnamurthi, S., Chakrabarti, T., & Stackebrandt, E. (2009). Re-examination of the taxonomic position of Bacillus silvestris Rheims et al. 1999 and proposal to transfer it to Solibacillus gen. nov. as Solibacillus silvestris comb. nov. Int J Syst Evol Microbiol, 59(Pt 5), 1054-1058. https://doi.org/10.1099/ijs.0.65742-0

Morohoshi, T., Tominaga, Y., Someya, N., & Ikeda, T. (2012). Complete genome sequence and characterization of the N-acylhomoserine lactone-degrading gene of the potato leaf-associated Solibacillus silvestris. J Biosci Bioeng, 113(1), 20-25. https://doi.org/10.1016/j.jbiosc.2011.09.006

Morohoshi, T., Wang, W. Z., Someya, N., & Ikeda, T. (2011). Genome sequence of Microbacterium testaceum StLB037, an N-acylhomoserine lactone-degrading bacterium isolated from potato leaves. J Bacteriol, 193(8), 2072-2073. https://doi.org/10.1128/JB.00180-11

Nayak, A., Harshitha, M., Dubey, S., Munang'andu, H. M., Chakraborty, A., Karunasagar, I., & Maiti, B. (2023). Evaluation of Probiotic Efficacy of Bacillus subtilis RODK28110C3 Against Pathogenic Aeromonas hydrophila and Edwardsiella tarda Using In Vitro Studies and In Vivo Gnotobiotic Zebrafish Gut Model System. Probiotics Antimicrob Proteins. https://doi.org/10.1007/s12602-023-10127-w

Neissi, A., Majidi Zahed, H., & Roshan, R. (2024). Probiotic performance of B. subtilis MS. 45 improves aquaculture of rainbow trout Oncorhynchus mykiss during acute hypoxia stress. Sci Rep, 14(1), 3720. https://doi.org/10.1038/s41598-024-54380-7

Nielsen, M. E., Hoi, L., Schmidt, A. S., Qian, D., Shimada, T., Shen, J. Y., & Larsen, J. L. (2001). Is Aeromonas hydrophila the dominant motile Aeromonas species that causes disease outbreaks in aquaculture production in the Zhejiang Province of China? Dis Aquat Organ, 46(1), 23-29. https://doi.org/10.3354/dao046023

Olmos, J., & Paniagua-Michel, J. (2014). Bacillus subtilis A Potential Probiotic Bacterium to Formulate FunctionalFeeds for Aquaculture. Journal of Microbial & Biochemical Technology, 6, 1-5.

Pan, J., Huang, T., Yao, F., Huang, Z., Powell, C. A., Qiu, S., & Guan, X. (2008). Expression and characterization of aiiA gene from Bacillus subtilis BS-1. Microbiol Res, 163(6), 711-716. https://doi.org/10.1016/j.micres.2007.12.002

Park, S. Y., Lee, S. J., Oh, T. K., Oh, J. W., Koo, B. T., Yum, D. Y., & Lee, J. K. (2003). AhlD, an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria. Microbiology (Reading), 149(Pt 6), 1541-1550. https://doi.org/10.1099/mic.0.26269-0

Schneider, J., Yepes, A., Garcia-Betancur, J. C., Westedt, I., Mielich, B., & López, D. (2012). Streptomycin-induced expression in Bacillus subtilis of YtnP, a lactonase-homologous protein that inhibits development and streptomycin production in Streptomyces griseus. Appl Environ Microbiol, 78(2), 599-603. https://doi.org/10.1128/aem.06992-11

Semwal, A., Kumar, A., & Kumar, N. (2023). A review on pathogenicity of Aeromonas hydrophila and their mitigation through medicinal herbs in aquaculture. Heliyon, 9(3), e14088. https://doi.org/10.1016/j.heliyon.2023.e14088

Stratev, D., & Odeyemi, O. A. (2016). Antimicrobial resistance of Aeromonas hydrophila isolated from different food sources: A mini-review. J Infect Public Health, 9(5), 535-544. https://doi.org/10.1016/j.jiph.2015.10.006

Su, Y., Liu, C., Fang, H., & Zhang, D. (2020). Bacillus subtilis: a universal cell factory for industry, agriculture, biomaterials and medicine. Microb Cell Fact, 19(1), 173. https://doi.org/10.1186/s12934-020-01436-8

Swift, S., Karlyshev, A. V., Fish, L., Durant, E. L., Winson, M. K., Chhabra, S. R., Williams, P., Macintyre, S., & Stewart, G. S. (1997). Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules. J Bacteriol, 179(17), 5271-5281. https://doi.org/10.1128/jb.179.17.5271-5281.1997

Urdaci, M. C., Bressollier, P., & Pinchuk, I. (2004). Bacillus clausii probiotic strains: antimicrobial and immunomodulatory activities. J Clin Gastroenterol, 38(6 Suppl), S86-90. https://doi.org/10.1097/01.mcg.0000128925.06662.69

Wang, W. Z., Morohoshi, T., Ikenoya, M., Someya, N., & Ikeda, T. (2010). AiiM, a novel class of N-acylhomoserine lactonase from the leaf-associated bacterium Microbacterium testaceum. Appl Environ Microbiol, 76(8), 2524-2530. https://doi.org/10.1128/AEM.02738-09

Wu, X. C., Lee, W., Tran, L., & Wong, S. L. (1991). Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. J Bacteriol, 173(16), 4952-4958. https://doi.org/10.1128/jb.173.16.4952-4958.1991