Introduction
Fish has continued to be a vital part of our diet since ancient times. The annual fish consumption has increased not only in India but around the globe as well. This has led to the development of an extensive pisciculture industry. Unfortunately, excessive industrialisation procedures, over-intensive exploitation, and poor management have resulted in significant bacterial disease epidemics in pisciculture. These bacteria can be transmitted by consuming raw or undercooked fish that are infected and can lead to severe symptoms in humans, including but not limited to abdominal pain, vomiting and diarrhoea. To mitigate this issue, it is vital to target the infection cycle in its early stages to prevent the bacteria from infecting the fish.
The problem
One of the most prevalent bacterial infections affecting various marine fish and shellfish is vibriosis. Vibriosis is a serious disease in fish, crustaceans and shellfish as recognised by the FAO, United Nations. It leads to significant economic losses and affects multiple sectors, thus hindering the development of the country.In a study of Epinephelus spp. approximately 66.7% of diseases reported were vibriosis. Several Vibrionaceae species have been linked to vibriosis in marine animals. According to a recent study, the most prevalent species triggering vibriosis in aquaculture farms are Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, Vibrio owensii, and Vibrio campbelli. The pathogenicity of these species is aided by a wide range of virulence factors, which allows them to infect a wide range of hosts [1].
In humans, vibriosis results in acute gastroenteritis after consuming raw, undercooked, or improperly handled marine food. Occasionally, cases of septicaemia, ear infections, or wound infections in people who already have medical issues are seen [2]. In fish, vibriosis exhibits symptoms like fatigue and necrosis of the skin and appendages, which causes body malformations, delayed development, liquefaction of internal organs, blindness, opacity of the muscles, and eventual death. Vibriosis affects all stages of growth causing upto 50% mortality [1].
Our bacteria of interest to tackle this infection in fish is V.parahaemolyticus. It is a gram-negative halophile that is found in estuarine and marine environments [2]. V. parahaemolyticus consists of a novel adhesion factor called Multivalent Adhesion Molecule 7 (MAM7)—a surface protein present on the bacteria. It is responsible for the initial host-pathogen adhesion and can trigger the upregulation of additional adhesins and virulence factors unique to the pathogen and the host cell. MAM7 forms a tripartite complex with the protein—Fibronectin (Fn), and the ligand—Phosphatidic acid (PA). It has been extensively studied in V. parahaemolyticus, and hence, it was deemed fit for our project's research [3].
Why MAM7?
Several virulence factors including hemolysin, type III secretion system, type VI secretion system, adhesion factor, iron uptake system, lipopolysaccharide, protease and outer membrane proteins[4] have been identified in V. parahaemolyticus that lead to the onset of vibriosis, consequently increasing the potential targets for inhibition. Most of these factors require direct contact between the pathogen and the host cell to induce the infection cycle. Studies have shown that the expression of MAM7 leads to rapid contact when it is first exposed to the bacteria and can cause increased expression of additional, pathogen and host cell-specific, adhesion molecules and virulents.
Elimination of this protein from V. parahaemolyticus causes the general reduction in host cell cytotoxicity and a lag in its outset [3]. The host cell, which in our case is fish, consist of Phosphatidic acid(PA) and Fibronectin(Fn) as its interacting ligands, present in the extracellular matrix. When the bacteria V. parahaemolyticus is in the vicinity of the host cell, MAM7 present on its surface interacts with the ligands. Binding to PA and Fn is not mutually exclusive; rather, it forms a tripartite complex of MAM7, PA, and Fn[3]. The adhesion of the bacteria, which is the primary step involved in infection, triggers the process of biofilm formation, further leading to cytotoxicity and haemolysis. In addition to its role in adhesion, MAM7 is involved in host cell signalling pathways that eventually lead to breaching of the epithelial cell barrier [5].
Current solutions
Currently, there are multiple solutions to this infection; however, antibiotics still remain to be the most popular strategy for treating and preventing the disease in fish. Antibiotics are frequently used with fish diets or baths to treat bacterial illnesses in real life. Antibiotic overuse has led to the emergence of multidrug resistance in several pathogenic bacteria, including the Vibrio spp. [6]. Every year, millions of fish are vaccinated to combat the problem of antibiotic resistance. Albeit compared to antibiotics, vaccines are a better prevention strategy, they have their drawbacks. Commercial vaccines are uneconomical and labour-intensive. Furthermore, there are challenges when it comes to the inoculation of these vacccines. The efficacy of oral vaccines is low due to the degradation of the antigens present in the vaccines. Immersion vaccines require a large quantity of the vaccine. Furthermore, they are not efficient and provide immunity for a short period. As for the injection vaccines, they are not suitable for fish that are small in size. Moreover, the fish also need to be starved and anaesthetised for their protection which is detrimental to them[7].
Our solution
Our solution involves the synthesis of a novel antimicrobial peptide that will bind to MAM7 and prevent the bacteria from adhering to the cell surface, thereby inhibiting the progression of vibriosis. The antimicrobial peptide will be created by mimicking the structure of fibronectin. The peptide will be produced using the pET-22b(+) vector system and BL21 as our chassis. To deliver our peptide, it will be encapsulated in chitosan nanoparticles. To know more about the Design of our project and the experimentation timeline. Using chitosan nanoparticles for the delivery will be an asset to AMPifin since they are proven to have immunoregulatory properties in fish. For more information on the design and the methods of peptide production and delivery, refer to our Design page. Our peptide is preferable when compared to current solutions because it is unlikely to induce antibiotic resistance and is sustainable. Furthermore it does not disrupt the microbiota of the ecosystem and is biodegradable. Since, MAM7 is present in several pathogenic gram negative bacteria, AMPifin can be a potential broad-spectrum solution against bacteria that use MAM7 as their adhesion protein.
References- M. Y. Ina-Salwany et al., “Vibriosis in Fish: A Review on Disease Development and Prevention,” J. Aquat. Anim. Health, vol. 31, no. 1, pp. 3–22, Mar. 2019, doi: 10.1002/AAH.10045.
- V. Letchumanan, K. G. Chan, and L. H. Lee, “Vibrio parahaemolyticus: a review on the pathogenesis, prevalence, and advance molecular identification techniques,” Front. Microbiol., vol. 5, no. DEC, 2014, doi: 10.3389/FMICB.2014.00705.
- A. M. Krachler and K. Orth, “Functional characterization of the interaction between bacterial adhesin Multivalent Adhesion Molecule 7 (MAM7) protein and its host cell ligands,” J. Biol. Chem., vol. 286, no. 45, pp. 38939–38947, Nov. 2011, doi: 10.1074/JBC.M111.291377.
- L. Li, H. Meng, D. Gu, Y. Li, and M. Jia, “Molecular mechanisms of Vibrio parahaemolyticus pathogenesis,” Microbiol. Res., vol. 222, pp. 43–51, May 2019, doi: 10.1016/j.micres.2019.03.003.
- J. Lim, D. H. Stones, C. A. Hawley, C. A. Watson, and A. M. Krachler, “Multivalent Adhesion Molecule 7 Clusters Act as Signaling Platform for Host Cellular GTPase Activation and Facilitate Epithelial Barrier Dysfunction,” PLoS Pathog., vol. 10, no. 9, p. e1004421, Sep. 2014, doi: 10.1371/journal.ppat.1004421.
- PyMOL The PyMOL Molecular Graphics System, Version 2.5.2 Schrödinger, LLC.
- S. Elmahdi, L. V. DaSilva, and S. Parveen, “Antibiotic resistance of Vibrio parahaemolyticus and Vibrio vulnificus in various countries: A review,” Food Microbiol., vol. 57, pp. 128–134, Aug. 2016, doi: 10.1016/J.FM.2016.02.008.
- S. Ben Hamed, S. T. Tapia-Paniagua, M. Á. Moriñigo, and M. J. T. Ranzani-Paiva, “Advances in vaccines developed for bacterial fish diseases, performance and limits,” Aquac. Res., vol. 52, no. 6, pp. 2377–2390, Jun. 2021, doi: 10.1111/ARE.15114.
- S. Elmahdi, L. V. DaSilva, and S. Parveen, “Antibiotic resistance of Vibrio parahaemolyticus and Vibrio vulnificus in various countries: A review,” Food Microbiol., vol. 57, pp. 128–134, Aug. 2016, doi: 10.1016/J.FM.2016.02.008.
- S. Ben Hamed, S. T. Tapia-Paniagua, M. Á. Moriñigo, and M. J. T. Ranzani-Paiva, “Advances in vaccines developed for bacterial fish diseases, performance and limits,” Aquac. Res., vol. 52, no. 6, pp. 2377–2390, Jun. 2021, doi: 10.1111/ARE.15114.
- Y. Deng et al., “Prevalence, virulence genes, and antimicrobial resistance of Vibrio species isolated from diseased marine fish in South China,” Sci. Rep., vol. 10, no. 1, p. 14329, Dec. 2020, doi: 10.1038/s41598-020-71288-0.