Checking the possible antifungal activity of chimeric chitinase along with glucanases
As fungal cell walls are made up of chitin-glucan complexes (CGCs), it is logical to assume that chitinases acting along with glucanases will potentially elicit an antifungal activity greater than that when either of these components is acting alone. This idea was further backed up by Dr. Robin Allshire who had worked on epigenetic mechanisms in yeast strains showcasing antifungal resistance. Many plant species have been shown to express glucanases in combination with chitinases against pathogenic infestations . 1,3-β-glucanase, a protein belonging to the family of glucanases, seems to be an optimal glucanase showing antifungal activity since the most abundant component in the fungal cell wall is 1,3-β-glucan . Hence, a protein combo that could enzymatically cleave chitin, as well as 1,3-β-glucan (major components of the fungal cell walls), is likely to act as a potent antifungal agent. There have also been reports suggesting a synergistic activity between 1,3-β-glucanases and chitinases .
So as an extension of our project, we seek to express and purify 1,3-β-glucanase and assay its antifungal activity. To move forth in this direction, we would have to standardise the expression conditions and the methodology of purification for our glucanase and characterise the optimum pH and temperature conditions required for the maximal synergistic activity with our chitinase. Previously used methods for the purification of 1,3-β-glucanase from different sources can guide our efforts etc .
Chimeric chitinase application in agriculture
Biocontrol, the process of inhibiting the growth of unfavourable insects or pests using biological agents, has been gaining considerable traction due to its simplicity and eco-friendliness. The agriculture sector is facing huge losses due to fungal infestations . This necessitates the creation of novel biocontrol agents that can prevent or inhibit fungal growth.The emergence of fungal strains resistant to commonly used antifungals, environmental hazards caused by such chemicals, their huge cost, and unavailability make it imperative to develop new biocontrol agents. Chitinases which lyse fungal cell walls could act as a potent biocontrol agent in the field of agriculture. Chitin being present in the exoskeleton of arthropods makes them vulnerable to enzymes like chitinases. Hence, chitinases could also play an essential role as an eco-friendly biopesticide, decreasing cases of pest infestations in the agriculture sector .
However, using enzymes as a biocontrol agent does have its limitations due to the narrow range of pH and temperature where it shows maximal activity. This restricts its usage to a limited geographical area and even seasons. To tackle such problems, formulations containing stabilizing agents.
Engineering genetically modified plants that could express our broad-spectrum chitinase under stimulation by fungal pathogens could greatly enhance agricultural productivity. Plants lack chitin in their cell walls, whereas most pathogens and pests that affect them possess chitin, making the expression of our chitinase under such challenging conditions highly favourable to the survival of plants.
Production of chimeric chitinase from human chitinases
Homo sapiens possess two functional chitinases: acidic mammalian chitinase (AMcase or CHIA) and chitotriosidase 1(CHIT1). Both of these proteins are true chitinases (Endo-type) and belong to the GH18 family . Several chitinase-like proteins (CLPs) or chitinase-like-lectins (chi-lectins) are also expressed in several mammals, including human beings. Chitinase domain–containing 1 (CHID1), stabilin-1- interacting chitinase-like protein (SI-CLP), oviductal glycoprotein 1 (OVGP1), Ym1 and Ym2 are examples of such proteins. Although the latter proteins do not exhibit true chitinase activity due to substitutions in their catalytic domain, they still possess carbohydrate-binding activity . Many of these proteins have been reported to be involved in immune responses and inflammatory reactions against a myriad of pathogens, including fungi. The human chitinase CHIT1 has been found to be getting expressed in tissues such as the lung, spleen, liver, thymus, and lacrimal gland. In contrast, AMcase has been found to be expressed in immune cells, and both of these chitinases are involved in immune reactions .
Using the same logic of domain fusion and protein engineering employed in our project, we can engineer chimeric human chitinases that possess the chitin-binding domain and catalytic domain of different human chitinases chitinase-like proteins, which could have the potential to elicit an enhanced antifungal response. Taking the protein’s size to be considered, appropriate culture systems like bacterial or insect cell culture will have to be sought out to express and purify such chimeric proteins. Since creating such proteins is a novel endeavour, appropriate linker sequences that could link the different domains of interest without affecting their domain architecture or functional properties have to be developed.
Engineering such chimeric human chitinases will open new avenues in the field of medicine and biology. Invasive fungal infections are one of the leading causes of mortality among immunocompromised patients, and the available therapeutic measures have often been proved to be inadequate under such situations. The availability of chimeric human chitinases to be administered to such patients seem to be a safe, efficient and cost-effective way of saving their lives. To render such chimeric human chitinases as a therapeutic measure, we need to create a delivery system that could precisely deliver the protein of interest into the site of infection where it can neutralize the fungal pathogens. The possible broad-spectrum antifungal activity which is hypothesized to be possessed by such chimeric chitinases will enable such a treatment method to neutralize multiple fungal species which are invasive in nature. The high degree of conservation of these chitinases and chitinase-like proteins across mammalian species makes it less likely for the chimeric chitinase to elicit an immune response when administered as a therapeutic agent.
Chitinases as a potential remedy against wall moulds
Moulds or mildew are fungi that occur in damp places and areas where decaying organic matter is abundant. These growths can be found in both indoor and outdoor locations and have the potential to spread rapidly. These moulds produce spores invisible to the naked eye but can cause the formation of a new mould in any place favourable to its growth.
They are also culprits of several fungal infections in human beings, especially among immunocompromised patients, for whom these infections can be life-threatening. Moulds inhabit many indoor and damp outdoor areas. Exposure to such conditions has shown a direct correlation to an increased incidence of upper respiratory tract symptoms, cough, wheeze, and asthma exacerbation . Since these medical conditions are some of the most common morbidities affecting vast sections of the population, finding a solution to this menace caused by fungal moulds seems to be of utmost importance.
Engineering chimeric chitinases with broad-spectrum activity will potentially keep the growth and spread of such fungal moulds under control. Its superior chitinolytic activity would neutralize the fungal components of the moulds and significantly reduce the menace caused by moulds. To enable such activity for our chitinase, it is necessary to find a delivery system whereby our chitinase can act on the moulds present on hard surfaces like walls. The delivery system should also enable the chitinase to show its chitinolytic activity for a prolonged period enabling it to clear the moulds effectively. The development of such a delivery system could make our chitinase a potent solution to the long-existing problem of mould growth.
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