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  <h2 class="reveal-on-scroll">Introduction</h2>

  <p class="reveal-on-scroll">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, <b>excessive industrialisation procedures</b>, <b>over-intensive
      exploitation</b>, 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 <b>severe
      symptoms</b> 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.
  </p>

  <div class="row reveal-on-scroll">
    <div class="col-4"> <img src="https://static.igem.wiki/teams/4200/wiki/description/manstomach.png"
        class="figure-img img-fluid rounded" alt="Fish graphic with vibriosis"> </div>
    <div class="col-8"> <img src="https://static.igem.wiki/teams/4200/wiki/description/exploitaion-description.png"
        style="width: 100%; margin:auto;" alt="Aquaculture farms being exploited "> </div>
  </div>

</div>

<br>

<h2 class="reveal-on-scroll">The problem</h2>

<p class="reveal-on-scroll">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 <i>Epinephelus</i> 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 <i>Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio
    harveyi, Vibrio owensii, and Vibrio campbelli</i>. The pathogenicity of these species is aided by a wide range of
  <b>virulence factors</b>, which allows them to infect a wide range of hosts [1].
</p>

<p class="reveal-on-scroll">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].
</p>

<div>
  <img src="https://static.igem.wiki/teams/4200/wiki/description/fishwithvibriosis.png"
    alt="Image of fish having vibriosis" id="fishBody">

  <img src="https://static.igem.wiki/teams/4200/wiki/description/fishwithvibriosis-eye-no-bg.png"
    alt="Image of fish having vibriosis" id="fishEye">
</div>

<p class="reveal-on-scroll">Our bacteria of interest to tackle this infection in fish is <em>V.parahaemolyticus</em>. It
  is a gram-negative halophile that is found in estuarine and marine environments [2]. <i>V. parahaemolyticus</i>
  consists of a novel adhesion factor called <b>Multivalent Adhesion Molecule 7 (MAM7)</b>—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 <b>tripartite complex</b> with
  the protein—<b>Fibronectin (Fn)</b>, and the ligand—<b>Phosphatidic acid (PA)</b>. It has been extensively studied in
  <i>V. parahaemolyticus</i>, and hence, it was deemed fit for our project's research [3].
</p>

<img src="https://static.igem.wiki/teams/4200/wiki/description/interaction.png"
  alt="*icon of mam7, fibronectin, pa interaction(less priority)*" class="reveal-on-scroll"
  style="width: 40%; margin:auto;">

<h2 class="reveal-on-scroll">Why MAM7?</h2>

<p class="reveal-on-scroll">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 <i>V. parahaemolyticus</i> 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.
</p>

<p class="reveal-on-scroll">Elimination of this protein from <i>V. parahaemolyticus</i> 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 <i>V.
    parahaemolyticus</i> 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 <b>cytotoxicity</b> and <b>haemolysis</b>. In addition to its role in adhesion, MAM7 is involved in
  <b>host cell signalling pathways</b> that eventually lead to breaching of the epithelial cell barrier [5].
</p>

<figure class="figure reveal-on-scroll"> <img src="https://static.igem.wiki/teams/4200/wiki/loose-mam7structure.png"
    class="figure-img img-fluid rounded" alt="Image of MAM7 protein">
  <figcaption class="figure-caption text-center">Fig: Image of MAM7 (viewed on: PyMOL [9])</figcaption>
</figure>

<h2 class="reveal-on-scroll">Current solutions</h2>

<p class="reveal-on-scroll">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
  <b>multidrug resistance</b> in several pathogenic bacteria, including the <i>Vibrio</i> 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 <b>uneconomical</b> and
  <b>labour-intensive</b>. 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].
</p>

<div class="row reveal-on-scroll">
  <div class="col-7">
    <figure class="figure"> <img src="https://static.igem.wiki/teams/4200/wiki/description/graph-1.png"
        class="figure-img img-fluid rounded" alt="Graphs of antibiotic resistance">
      <figcaption class="figure-caption text-center"> Graph depicting relative abundance of strains of the Vibrio
        species.(I- Intermediate, S- Sensitive, R- Resistant)[9] </figcaption>
    </figure>
  </div>
  <div class="col-5">
    <figure class="figure"> <img src="https://static.igem.wiki/teams/4200/wiki/description/graph-2.png"
        class="figure-img img-fluid rounded" alt="Graphs of antibiotic resistance">
      <figcaption class="figure-caption text-center"> Graph depicting Vibrio strains resistant to antibiotics[9].
      </figcaption>
    </figure>
  </div>

  <h2 class="reveal-on-scroll">Our solution</h2>

  <p class="reveal-on-scroll">Our solution involves the synthesis of a <b>novel antimicrobial peptide</b> 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 <b>mimicking</b> 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 <b>chitosan nanoparticles</b>. To know more about the <a
      href="{{ 'pages' | link_url(page='design') }}">Design</a>
    of our project and the <a href="{{ 'pages' | link_url(page='experiments') }}">experimentation timeline</a>. 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 <a href="{{ 'pages' | link_url(page='design') }}">Design</a> 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 <b>biodegradable</b>. Since, MAM7 is present in several pathogenic
    gram negative bacteria, AMPifin can be a <b>potential broad-spectrum solution</b> against bacteria that use MAM7 as
    their adhesion protein. </p>

  <b class="reveal-on-scroll">References</b>

  <ol class="reveal-on-scroll">
    <li class="reveal-on-scroll">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.</li>
    <li class="reveal-on-scroll">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.</li>
    <li class="reveal-on-scroll">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. </li>
    <li class="reveal-on-scroll">L. Li, H. Meng, D. Gu, Y. Li, and M. Jia, “Molecular mechanisms of <i>Vibrio
        parahaemolyticus </i>pathogenesis,”
      Microbiol. Res., vol. 222, pp. 43–51, May 2019, doi: 10.1016/j.micres.2019.03.003. </li>
    <li class="reveal-on-scroll">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. </li>
    <li class="reveal-on-scroll"> PyMOL The PyMOL Molecular Graphics System, Version 2.5.2 Schrödinger, LLC. </li>
    <li class="reveal-on-scroll">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. </li>
    <li class="reveal-on-scroll">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.</li>
    <li class="reveal-on-scroll">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. </li>
    <li class="reveal-on-scroll">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. </li>
    <li class="reveal-on-scroll"> 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. </li>
  </ol>

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