Background

Intestinal parasitosis is among the most prevalent infectious diseases in the world. In Brazil, they are neglected diseases that mainly affect children and adolescents from vulnerable social groups, such as indigenous, rural, and low socio-economic communities [1]. In the world, there is a prevalence of about 1,5 billion infected people [2], and the parasites with the highest incidence are Ascaris lumbricoides, Trichuris trichiura, and Ancylostoma duodenale [3], and in Brazil, the average prevalence of parasitic intestinal infection is 46% of the population; in other words, almost half of the population is directly impacted [4]. Infection occurs through the ingestion of food and water contaminated with eggs or larvae eliminated in the feces of infected people or through contact with soil contaminated with embryonated eggs or larvae of the parasites [5].

Helminthic transmission occurs when infected individuals releases eggs or viable vermin into the environment, mostly in places where there is no basic sanitation, like sewage services and water treatment, such as peripheries of cities and rural areas. The common symptoms of helminthic infections are abdominal pain, diarrhea, intestinal gas, loss of appetite, weight loss, and fatigue. Furthermore, helminthiasis can generate deficits in the physical and mental development of patients, which is worrisome, since the most affected group are children and adolescents between 5 and 14 years [6].

As an aggravating factor, the COVID-19 pandemic interrupted numerous interventions and programs to eliminate and control these diseases, such as research and treatment of cases. The most vulnerable groups became even more exposed to infectious diseases due to a lack of access to health, education, quality food, drinking water, basic sanitation, and housing [7].

Therefore, we observed that a high load of helminthic infections causes serious problems related to malnutrition in children through reduced food intake, malabsorption, and endogenous nutrient loss [8]. In addition, there is a high prevalence of nutritional problems related to anemia due to the excessive destruction of blood cells and microvilli of the intestinal mucosa [9]. Such factors compromise the immune system and generate an imbalance of absorption of macronutrients and micronutrients, leading to the deficiency of vitamins and minerals important for health maintenance. Moreover, they are associated with worsening cognitive and body development of preschool and school children [10]. 

The current global strategy to control infections is preventive chemotherapy, that is, the repeated large-scale administration of anthelmintic drugs to at-risk populations, especially school-age children [11]. Although this recommendation has been shown to be efficient in reducing cases, preventive chemotherapy does not interrupt the cycle of infection and reinfection. Additionally, the use of long-term medications causes increased resistance of parasites to current therapeutic options and does not offer adequate and targeted treatment for cases of malnutrition [12].

From this, our team sought a biotechnological solution for helminth parasitosis.

Inspiration

This year, the iGEM UFMG_UFV team seeks to solve a local, endemic problem of great nutritional and public health relevance. Currently, the alternative for treatment is preventive chemotherapy, and, over time, we have noticed that drug resistance has become a frequent problem and infections are increasingly persistent. On top of that, malnutrition has increased in underdeveloped countries, often associated with high contamination by helminths.

With the use of synthetic biology, we are able to look directly at the problem, proposing more effective solutions to control helminth infections, in addition to assisting in the recovery of intestinal health and nutritional status.

But why did we choose to work with the Lactobacillus genus? The answer is simple: some studies show that bacterial species of this genus, such as Lactobacillus acidophilus, have significant anthelmintic potential [13]. Furthermore, Lactobacillus are capable of inducing changes in the expression of T lymphocytes, such as stimulating the production of Th1 cytokines and inhibiting cytokines of Th2 cells, which leads to an increase in the body's immune response. In this way, Lactobacillus present themselves as a major player in the regulation of the immune system [14].

Our project

Unlike conventional treatments to eliminate parasites and the major problem related to resistance, the iGEM UFMG_UFV 2022 Team seeks to eliminate the problem early on. With this, the ProChi project proposes a solution to the problem of helminth infection, egg elimination, and malnutrition through synthetic biology with a new combination therapy option of Probiotic + Chitinase.

We have developed a new therapeutic option based on L. acidophilus acting as a probiotic genetically modified to produce chitinase enzymes that degrades chitin, which is an essential component of the egg walls and pharynx of worms (helminth nematodes, specifically) and its use has been associated with the fights against these parasites [15]. In this way, the chitin layer of the parasite would be destroyed, the helminth eliminated and intestinal health would be rescued by the action of the probiotic.

Thus, fungi chitinases were chosen as objects of study, since it was shown that nematode-predating fungi promote the cleavage of the chitin layer of these worms and their eggs through the expression of proteases and chitinases [16-20]. The plant fungus Pochonia chlamydosporia has been studied for its potential as a biological agent in the control of nematodes. Likewise, our team chose PCCHI44 endochitinase from P. chlamydosporia to characterize its activity, with the ultimate goal of degrading human parasitic nematodes [21]. Also, the exochitinase CfcI from the fungus Aspergillus niger was chosen for the same purpose [22]. We performed protein-ligand docking simulations, in order to confirm that our chitinases would bind correctly to a chitinase polymer.

Our project will evaluate the quantitative and qualitative effects of both chitinases, separately and together, considering that they have different mechanisms of action: endochitinases cleave glycosidic bonds randomly at internal sites in chitin polymer, while exochitinases cleave the ends of the chitin polymer [23]. The criteria for choosing chitinases were their characterization in literature as exo- or endochitinase and also the knowledge of action against helmints [17-21].

Our chassis, L. acidophilus, is a lactic acid bacteria naturally present in the intestine and is also widely used in probiotics related to the treatment of gastrointestinal diseases [14]. Among the immunological benefits to human health attributed to probiotic bacteria, we can highlight a modulation of the intestinal microbiota, interaction with the immune system, infection prevention, glycemic control, cancer prevention, improvement in lipid metabolism, and stimulation of micronutrient adsorption [13,26].

Therefore, we are developing a technology capable of eliminating the worm while helping to nourish the individual through the action of the probiotic. In addition, as it is a new therapeutic option, our project acts by delaying the increase in helminthic resistance. The development of the project also combines other goals, such as the development of a low-cost bioreactor and the description of new biological parts for later use by other scientists.

References

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[2] Guideline: preventive chemotherapy to control soil-borne helminth infections in at-risk population groups. Geneva: World Health Organization; 2017. Avaiable in: https://apps.who.int/iris/handle/10665/258983

[3] Lustigman, Sara, et al. “A Research Agenda for Helminth Diseases of Humans: The Problem of Helminthiases”. PLoS Neglected Tropical Diseases, organizado por Charles D. Mackenzie, vol. 6, n. 4, 2012, p. e1582, https://doi.org/10.1371/journal.pntd.0001582.

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[5] World Health Organization. Guideline: preventive chemotherapy to control soil-borne helminth infections in at-risk population groups. World Health Organization, 2017. https://apps.who.int/iris/handle/10665/258983.

[6] Brazil. Ministry of Health. Health Surveillance Secretariat. Department of Communicable Diseases Surveillance. Practical Guide to the Control of Geohelminties. Brasilia, p. 33, 2018. Available in: http://bvsms.saude.gov.br/bvs/publicacoes/guia_pratico_controle_geohelmintiases.pdf.

[7] PAHO - Pan American Health Organization. COVID-19 threatens plans to eliminate and control infectious diseases, PAHO Director says. Washington (DC); 2020. Avaliable in: www.paho.org/en/news/11-8-2020-covid-19-threatens-plans-eliminate-and-control-infectious-diseases-paho-director.

[8] Hailegebriel, Tamirat. “Undernutrition, Intestinal Parasitic Infection and Associated Risk Factors among Selected Primary School Children in Bahir Dar, Ethiopia”. *BMC Infectious Diseases*, vol. 18, n. 1, 2018, p. 394, https://doi.org/10.1186/s12879-018-3306-3.

[9] Mrimi, Emmanuel C., et al. “Malnutrition, Anemia, Micronutrient Deficiency and Parasitic Infections among Schoolchildren in Rural Tanzania”. *PLOS Neglected Tropical Diseases*, vol. 16, n. 3, 2022, p. e0010261, https://doi.org/10.1371/journal.pntd.0010261.

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[11] WHO. Summary of global update on preventive chemotherapy implementation in 2015: Weekly epidemiological record. 30 September 2016. Available in: https://www.who.int/publications/i/item/who-wer9638-468-475

[12] Mphahlele, Morutse, et al. “Anthelmintic Resistance in Livestock”. *Helminthiasis*, IntechOpen, 2020, https://doi.org/10.5772/intechopen.87124

[13] De Vrese, Michael, e J. Schrezenmeir. “Probiotics, Prebiotics, and Synbiotics”. Food Biotechnology, organizado por Ulf Stahl et al., vol. 111, Springer Berlin Heidelberg, 2008, p. 1–66. DOI.org (Crossref), https://doi.org/10.1007/10_2008_097.

[14] Kye, Yeon‐Jin, et al. “Lactobacillus Acidophilus PIN7 Paraprobiotic Supplementation Ameliorates DSS‐induced Colitis through Anti‐inflammatory and Immune Regulatory Effects”. Journal of Applied Microbiology, vol. 132, n. 4, 2022, p. 3189–200, https://doi.org/10.1111/jam.15406.

[15] Hong, Sin-Hyoung, et al. “Antifungal Activity and Expression Patterns of Extracellular Chitinase and β-1,3-Glucanase in Wickerhamomyces Anomalus EG2 Treated with Chitin and Glucan”. Microbial Pathogenesis, vol. 110, 2017, p. 159–64, https://doi.org/10.1016/j.micpath.2017.06.038.

[16] Yang, Jinkui, et al. “Nematicidal Enzymes from Microorganisms and Their Applications”. Applied Microbiology and Biotechnology, vol. 97, n. 16, 2013, p. 7081–95, https://doi.org/10.1007/s00253-013-5045-0.

[17] Braga, F.R.; et al. Ovicidal action of a crude enzymatic extract of fungus Pochonia chlamydosporia against Ancylostoma sp eggs. Revista da Sociedade Brasileira de Medicina Tropical, v. 44, n. 1, 2011, https://doi.org/10.1590/s0037-86822011000100027.

[18] Khan, A.; et al. Effects of Paecilomyces lilacinus protease and chitinase on the eggshell structures and hatching of Meloidogyne javanica juveniles. Biological control, v. 31, n. 3, p. 346-352, 2004, https://doi.org/10.1016/j.biocontrol.2004.07.011.

[19] Chen, L.; 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.

[20] Jin, N.; et al. Isolation and characterization of Aspergillus niger NBC001 underlying suppression against Heterodera glycines. Scientific Reports, v. 9, n. 1, p. 1-13, 2019, https://doi.org/10.1038/s41598-018-37827-6.

[21] Mi, Q.; et al. “Cloning and Overexpression of Pochonia Chlamydosporia Chitinase Gene Pcchi44, a Potential Virulence Factor in Infection against Nematodes”. Process Biochemistry, vol. 45, n. 5, 2010, p. 810–14, https://doi.org/10.1016/j.procbio.2010.01.022.

[22] Van Munster, Jolanda M., et al. “Biochemical Characterization of Aspergillus Niger CfcI, a Glycoside Hydrolase Family 18 Chitinase That Releases Monomers during Substrate Hydrolysis”. Microbiology, vol. 158, n. 8, 2012, p. 2168–79, https://doi.org/10.1099/mic.0.054650-0.

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[24] Photograph by Catalina Maya Rendón, distributed under a CC-BY 2.0 license. Available on: https://commons.wikimedia.org/wiki/File:Collage_Helminth_eggs.png.

[25] Photograph by Mogana Das Murtey and Patchamuthu Ramasamy, distributed under a CC-BY 2.0 license. Available on: https://en.m.wikipedia.org/wiki/File:Lactobacillus_acidophilus_SEM.jpg.

[26] Lee, Chih-Wei, et al. “Synbiotic Combination of Djulis (Chenopodium Formosanum) and Lactobacillus Acidophilus Inhibits Colon Carcinogenesis in Rats”. Nutrients, vol. 12, n. 1, 2019, p. 103. DOI.org (Crossref), https://doi.org/10.3390/nu12010103.