Overview

Once we achieve the expression of chitinases in our chassis, we will work to ensure the effectiveness of this enzyme action in parasite control. For that, we aim to test the activity of expressed and purified chitinases from our culture on the nematode Caenorhabditis elegans. This experiment will be able to demonstrate the capacity of chitin degradation on the egg walls and cuticles of C. elegans by chitinases expressed by L. acidophilus.

Experimental Design

Caenorhabditis elegans is a model nematode that we selected to work with since it demonstrated advantages of well-known anatomy, besides the body and eggshell transparency, which could allow us to easily visualize its cells using standard microscopy[1]. In addition, this organism isn't capable to establish a human disease in contrast to the agents that cause helminthiases, which ensures security for the project's involved people.

After contacting Professor Dr. Viviane Alves, one of the biggest references in the field of microbiology in Brazil, we were presented with a protocol for C. elegans culture based on its embryonic cell growth [2]. Our next steps involve performing this protocol to continue our proof of concept.

To obtain the chitinases expressed by the cultivated L. acidophilus, we began to perform a protocol of purification of these enzymes. Because we introduced a signal peptide to our constructs that will induce the secretion of chitinases expressed, we can purify these enzymes just by performing nickel column affinity chromatography. This procedure is possible because of the histidine-tail added to the protein. Then, we can use the proteins in the anthelmintic test in C. elegans plates. We can also directly add the engineered bacteria we grew to C. elegans plates to observe if the chitinases would disrupt the nematode's chitin layer.

We already started to design the guidelines to implement this proof of concept, but, unfortunately, due to lack of time, rushed schedule and failure to obtain purified chitinase we will work to do this in the future.

Future Perspectives

To finish our proof of concept we are going to add different concentrations of our chitinases to our plates of C. elegans [3,4]. Thus, we will be able to analyze how the morphology of eggs and adults was affected by different concentrations of our chitinases comparing their anatomy with the negative control that will not be challenged and also comparing it with a positive control that will be challenged with commercialized chitinases.

For this comparison, we will photograph the different life stages of C. elegans before and after exposure to chitinase using a microscope. Then, we will observe some characteristics that indicate the action of chitinases against the parasite. After that, we are going to perform some quantitative analyses of nematicidal activity, like quantitative mortality tests, litter size assays, and growth analyses [3,4].

We hope to visualize digested C. elegans eggshells after exposure to purified chitinases, with a barrier no longer visible around the embryos, making them more vulnerable[2]. These effects of immobilization of the embryo’s development and death, consequently result in a reduction of the nematode population density and could control human infectious diseases [5]. Regarding the action against the adult nematodes, we expect to visualize degraded nematode cuticles and injuries on the pharynx of C. elegans, making the worm unfeasible [3]. This will prove that the chitinases expressed by Lactobacillus acidophilus are capable of combating a nematode and will indicate their role in the control of parasitic diseases.

References

[1] Carretero, Maria, et al. “C. Elegans as Model for Drug Discovery”. Current Topics in Medicinal Chemistry, vol. 17, no 18, 2017, p. 2067–76. PubMed, https://doi.org/10.2174/1568026617666170131114401.

[2] Sangaletti, Rachele, e Laura Bianchi. “A Method for Culturing Embryonic C. Elegans Cells”. Journal of Visualized Experiments: JoVE, no 79, setembro de 2013, p. e50649. PubMed, https://doi.org/10.3791/50649.

[3] Chen, Lin et al. Enhanced nematicidal potential of the chitinase pachi from Pseudomonas aeruginosa in association with Cry21Aa. Scientific Reports, v. 5, n. 1, p. 1-11, 2015, https://doi.org/10.1038/srep14395.

[4] Bischof, Larry J., et al. “Assays for Toxicity Studies in C. Elegans with Bt Crystal Proteins”. Methods in Molecular Biology (Clifton, N.J.), vol. 351, 2006, p. 139–54. PubMed, https://doi.org/10.1385/1-59745-151-7:139.

[5] Gortari, Maria Cecilia, e Roque Alberto Hours. “Fungal Chitinases and Their Biological Role in the Antagonism onto Nematode Eggs. A Review”. Mycological Progress, 2008. agris.fao.org, https://doi.org/10.1007/s11557-008-0571-3.