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<h2>PROJECT DESCRIPTION</h2>
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<h3><b>Background</b></h2>
<br>
<p class="pl-20 pr-20">
The core motivation behind our project is the ongoing battle against Invasive Fungal Infections (IFIs) in India. The risks posed by opportunistic fungal infections, especially to immunocompromised patients, have been brought into light following the COVID-19 pandemic in the country. Opportunistic fungal infections like mucormycosis, aspergillosis, and candidiasis have ravaged immunocompromised patients battling multiple comorbidities. Tackling these infections has proven difficult due to a limited repertoire of antifungal drugs, their costs, drug interactions, the routes of administration, and side effects like nephrotoxicity. Furthermore, the rampant use of these limited drugs raises the alarming possibility of developing drug-resistant fungal strains.
</p>
<p class="pl-20 pr-20">
Annually, over 150 million cases of fungal infections are reported globally, with up to 1.7 million deaths per year. IFIs also carry a 60-90% mortality rate among all ICU patients. Although fungal infections have been on the rise, it wasn't until the wake of Covid-19 that this issue was brought to the limelight. Hence, fungal infections are sometimes also quoted as a silent crisis.
</p>
<p class="pl-20 pr-20">
Fungi, being eukaryotes, share a greater degree of molecular similarity to humans than prokaryotic pathogens such as bacteria. Thus, finding unique molecular targets cannot selectively affect the fungal pathogen and not the host isn’t easy. In the limited instances where such targets are available, there is always the looming threat of the emergence of drug-resistant fungal strains. Hence, we thought of developing an antifungal based on the natural defense mechanisms of plants and animals.
</p>
<p class="pl-20 pr-20">
We chose to target chitin (a long-chain polymer of N-acetylglucosamine), an essential component of the cell walls of all fungal species, notably absent in eukaryotic cells such as our own. Our molecular weapons- ‘chitinases’ are naturally occurring enzymes known to break down the glycosidic bonds in chitin. These are often found in bacteria, yeasts, plants, actinomycetes, arthropods, and humans, which use them as defensive mechanisms against fungal infections. However, given the diversity in fungal species and chitinase enzymes, selecting the appropriate chitinase on a case-to-case basis would be quite difficult. Our goal is to harness the power of these naturally occurring chitinase enzymes by combining their substrate specificities and potentially even activity through active pairings of their functional domains. As a result, we suggest the production of a specified set of recombinant chitinase enzymes that combine the best characteristics of different wild-type enzymes and are thus more effective.
</p>
<p class="pl-20 pr-20">
Importantly, the chitin biosynthesis pathway in fungi is rather complex and involves the concerted action of several enzymes in a well-regulated fashion. Hence, we hypothesize that the probability of alteration of chitin structure by the target organisms to avoid lysis by our chitinases is rather low. The recombinant proteins are supported by a thorough literature review that demonstrates the project's viability. Therefore, we are developing a novel, eco-friendly and novel therapeutic ‘MOLDEMORT’ to tackle various invasive fungal infections. This solution has potential wide-ranging applications in healthcare, agriculture, and society alike.
</p>
<h3><b>Inspiration</b></h2>
<br>
<p class="pl-20 pr-20">
The project idea surfaced when we discovered fungal contamination in a number of sites both on and off-campus. More research into pathogenic fungus species found that <i>Fusarium sp.</i> (causes eye infections),<i> Rhizopus sp.</i> (causes mucormycosis), and<i> Aspergillus sp.</i> (causes aspergillosis) are common in many public areas and frequently cause problems in immunocompromised individuals. Armed with this information on the pathogenic nature of certain fungal species, we decided to isolate fungal samples within the campus and sequenced their genomes to know which species of fungi usually thrive on walls of the building. We found <i>Aspergillus versicolor</i>, <i>Aspergillus niger</i>, <i>Rhizopus oryzae</i>, <i>Fusarium solani</i>, <i>Nodulisporium indicum</i>, <i>Phanerochaete sordida</i>, and unidentified <i>Trichoderma</i> species.
</p>
<p class="pl-20 pr-20">
We then grew interested in the question of how fungal infections caused by these species and others could be treated. A survey of literature revealed that the current antifungals used for treatment include various compounds such as amphotericin B (AmB), caspofungin, triazoles, etc., but these antifungal agents have severe limitations due to lack of sufficient fungicidal effect and toxic side effects. Along with this, there have also been several reports of the emergence of drug-resistant fungal strains due to the limited molecular targets of the existing drugs.
</p>
<p class="pl-20 pr-20">
We decided to step in and try to create a new antifungal drug that targets the polymer chitin, a ubiquitous component of fungal cell walls. According to the literature survey, the lysis of chitin contained in fungal cell walls by naturally occurring chitinase enzymes would undermine the integrity of fungal cells, leading to their death. By harnessing the power of synthetic biology and protein engineering, the team IISER_TVM has engineered a chimeric chitinase to tackle the issue of invasive fungal infections.
</p>
<h4><b>Reference:</b></h4>
<ol class="ordered-list">
<li><p>Ambati et. al. "Dectin-1-Targeted Antifungal Liposomes Exhibit Enhanced Efficacy | Msphere". Msphere, 2021, </p>
</li>
<li><p>Kainz, Katharina et al. "Fungal Infections In Humans: The Silent Crisis". Microbial Cell, vol 7, no. 6, 2020, pp. 143-145. Shared Science Publishers OG, doi:10.15698/mic2020.06.718. Accessed 19 Aug 2021.
</li>
<li><p>Bongomin, Felix et al. "Global And Multi-National Prevalence Of Fungal Diseases—Estimate Precision". Journal Of Fungi, vol 3, no. 4, 2017, p. 57. MDPI AG, doi:10.3390/jof3040057. Accessed 19 Aug 2021.
</li></ol>
</section>
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</div>
<!--
<section class="sample-text-area">
<div class="container box_1170">
<h2 class="text-center">Bacterial Chimeric Chitinase Design</h2>
<br>
<p class="pl-20 pr-20">
We designed two chimeric bacteria chitinases from three bacterial sources: <i>Pseudoalteromonas</i> sp DL-6, <i>Amycolatopsis orientalis</i>, and <i>Serratia marcescens</i>.
</p>
<p class="pl-20 pr-20">
<b>First chimeric combination:</b> We replaced the catalytic domain of <i>Serratia marcescens</i> with the catalytic domain of <i>Pseudoalteromonas</i> following the basic structure of <i>Serratia</i> chitinase.
</p>
<h4 class="pl-20 pr-20">Basic Outline</h4>
<p class="pl-20 pr-20">
Amino Acids in the beginning of <i>Serratia</i> ChiB natural sequence — CBD of <i>S. marcescens</i> Chi B — CD of <i>Pseudoalteromonas</i> Chi A — Chi B natural linker between CD and CBD of <i>S. marcescens</i> — CBD of <i>S. marcescens</i>
</p>
</div>
<div class="image-block, text-center">
<img src="assets/img/project/BC1_Domain_Structure.png" alt="sample image" class="img-fluid">
<p><strong>Figure 1.</strong> Unannotated image</p>
</div>
<div class="container box_1170">
<br><p class="pl-20 pr-20">
<b>Second chimeric combination:</b> We chose chitinase from Amycolaptosis orientalis which lacks a chitin-binding domain. So we added the chitin-binding domain of S. marcescens Chi B in the C-terminal of the Amycolatopsis orientalis chitinase.
</p>
<h4 class="pl-20 pr-20">Basic Outline</h4>
<p class="pl-20 pr-20">
Chitinase seq. of Amycolatopsis orientalis — CBD of S. marcescens Chi B
</p>
</div>
<div class="image-block, text-center">
<img src="assets/img/project/BC2_Domain_Structure.png" alt="sample image" class="img-fluid">
<p><strong>Figure 1.</strong> Unannotated image</p>
</div>
<br>
<br>
<br>
<div class="container box_1170">
<h2 class="text-center">Plant Chimeric Chitinase Design</h2>
<br>
<p class="pl-20 pr-20">
Apart from bacterial chitinases, we chose plant chitinases based on suggestions from structural biology experts since these chitinases would have evolved with more efficient chitinolytic activity. We designed two chimeric plant chitinase combinations using chitinases from two sources: <i>Triticum aestivum</i> (Wheat) and <i>Hordeum vulgare</i> (Barley). The cloning and the succeeding downstream assay experiments could not be performed due to lack of time. Both of these plant chitinases show efficient chitinolytic activity and retain activity at body temperature and pH range.
</p>
<p class="pl-20 pr-20">
<b>First chimeric combination:</b> We joined the sequences of both barley and wheat chitinases using common (GGGS)\(_3\) linkers. Wheat chitinase doesn’t have a chitin-binding domain, so we added barley CBD in its C-terminal in the combination.
</p>
<h4 class="pl-20 pr-20">Basic Outline</h4>
<p class="pl-20 pr-20">
AA seq before Barley CBD — Barley CBD — Barley CD — AA after CD barley — (GGGS)\(_{3}\) — AA seq before wheat CD — Wheat CD — natural linker from barley — Barley CBD
</p>
</div>
<div class="image-block, text-center">
<img src="assets/img/project/PC1_Domain_Structure.png" alt="sample image" class="img-fluid">
<p><strong>Figure 1.</strong> Unannotated image</p>
</div>
<div class="container box_1170">
<br><p class="pl-20 pr-20">
<b>Second chimeric combination:</b> We created a chimeric chitinase with a wheat catalytic domain flanked by two barley chitin-binding domains.
</p>
<h4 class="pl-20 pr-20">Basic Outline</h4>
<p class="pl-20 pr-20">
AA before barley chitinase — Barley CBD — natural linker from barley — Wheat CD — natural linker from Barley — Barley CBD
</p>
</div>
<div class="image-block, text-center">
<img src="assets/img/project/PC2_Domain_Structure.png" alt="sample image" class="img-fluid">
<p><strong>Figure 1.</strong> Unannotated image</p>
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<script src="./assets/js/jquery.slicknav.min.js"></script>
<!-- Jquery Slick , Owl-Carousel Plugins -->
<script src="./assets/js/owl.carousel.min.js"></script>
<script src="./assets/js/slick.min.js"></script>
<!-- One Page, Animated-HeadLin -->
<script src="./assets/js/wow.min.js"></script>
<script src="./assets/js/animated.headline.js"></script>
<script src="./assets/js/jquery.magnific-popup.js"></script>
<!-- Date Picker -->
<script src="./assets/js/gijgo.min.js"></script>
<!-- Nice-select, sticky -->
<script src="./assets/js/jquery.nice-select.min.js"></script>
<script src="./assets/js/jquery.sticky.js"></script>
<!-- Progress -->
<script src="./assets/js/jquery.barfiller.js"></script>
<!-- counter , waypoint,Hover Direction -->
<script src="./assets/js/jquery.counterup.min.js"></script>
<script src="./assets/js/waypoints.min.js"></script>
<script src="./assets/js/jquery.countdown.min.js"></script>
<script src="./assets/js/hover-direction-snake.min.js"></script>
<!-- contact js -->
<script src="./assets/js/contact.js"></script>
<script src="./assets/js/jquery.form.js"></script>
<script src="./assets/js/jquery.validate.min.js"></script>
<script src="./assets/js/mail-script.js"></script>
<script src="./assets/js/jquery.ajaxchimp.min.js"></script>
<!-- Jquery Plugins, main Jquery -->
<script src="./assets/js/plugins.js"></script>
<script src="./assets/js/main.js"></script>
</body>
</html>