Last year the world faced a pandemic that has already caused more than 4.4 million deaths, according to the World Health Organization (WHO). However, it is not the only disease that is encroaching on human lives. Over the last 30 years, the world has faced at least 30 new infectious diseases, including Swine flu, well-known Ebola, SARS, etc. There are beneficial conditions for that — favorable climate and weather, ecosystem changes, human susceptibility to infections, international trade and travel, or even lack of public health services . All factors sum up and contribute to the excellent environment for the new emerging or reemerging infectious diseases.
One of the examples is amebiasis, an infectious disease caused mostly by Entamoeba histolytica .
E. histolytica cysts enter the human body orally through contaminated food, water or human to human contact . For this reason, this infection mainly affects the developing world in tropical and subtropical regions where there is poor sanitation, lack of publicly available health care, favorable climate for pathogens propagation, lack of knowledge about food processing and keeping conditions, defecation into water sources such as rivers .
After exposure to E. histolytica cysts, there are two possible pathogenesis pathways. First, trophozoites stay in the large intestine, proliferate asymptomatically, and leave the intestine as newly formed cysts. In this case, an infected person becomes an infection carrier. Second, trophozoites proliferate and adhere to the colonic epithelium by Gal/GalNac lectin, and cause cell lysis. It leads to the destruction of the protective mucous barrier, surrounding cells fagocytation [4, 5]. Symptoms of intestinal amebiasis include abdominal pain, ulcerative colitis with mucus and blood, bloody diarrhea which later on progresses to raspberry-jelly-like stool, appendicitis, and ulcers. However, in more progressed inflammation — extraintestinal amebiasis, trophozoites can enter blood circulation, travel to the liver and cause an amebic liver abscess, or less frequently lung, brain abscess, or skin infection .
About 50 million people have a symptomatic form of amebiasis, and 100 000 of them die annually . The real numbers of the infection should be higher because only 10 percent of infected people feel symptoms and some cases do not get identified because of poor data collection in regions where this infection is endemic . However, this infection occurs in developed countries as travelers and immigrants disseminate infection. As we saw from the SARS-CoV-2 situation, a local problem can easily transform into a global pandemic.
The annual rates, lack of publicly available diagnostic tests, inexistent prevention tools, and deadly consequences of pathogenesis progression have inspired us to create effective prevention and sensitive diagnostic tools to improve the control of this infection. Probiotics, a preventive measure, are going to lower the rates of annual amebiasis cases by abolishing E. histolytica trophozoites in their earliest development stages, and the diagnostic test is going to improve the monitoring of the infection progression and accelerate the detection of an invasive form. In this way, the global spread of the infection would be prevented and many lives would be saved.
Although this infection is known for at least 160 years, there are no designed vaccines or other active prevention tools . For this reason, there is a high need for prevention of the infection for a successful control of its spredness. Moreover, currently used methods designed to diagnose amebiasis are not efficient or unavailable in the regions where this infection has spread the most [9, 10].
When creating our project, we looked at amebiasis holistically and comprehensively. Our team decided to target E. histolytica from two angles: prevention and diagnostics.
As a tool to prevent amebiasis, our team created probiotics capable of naringenin biosynthesis. Naringenin is a flavonoid naturally present in plants, and it was chosen as an active substance as experimental data showed that it reduced Entamoeba histolytica virulence . To reach the intestine where the infection may start, we decided to use probiotic bacteria (namely, Escherichia coli Nissle 1917 and Lactobacillus casei BL23), which occur naturally in the intestine, and are adapted to that environment . What is more, E. coli Nissle 1917 have been used to treat several gastrointestinal disorders and some studies have revealed that E. coli Nissle 1917 secreted outer membrane vesicles help to maintain intestinal barrier  preventing intestinal infections by induction of antimicrobial peptides secretion and regulation of cytokines or chemokines responses of immune or intestinal epithelial cells . As E. coli Nissle 1917 is facultative anaerobe colonizing mostly the large intestine  and L. casei BL23 is colonizing the small intestine , our approach is to create the naringenin synthesis of moderate and non toxic amounts dispersed through the all over intestine. This prevents E. histolytica cysts development into infective trophozoites and further infection progression.
We understood that synthesis efficiency in the intestine conditions would not be the same as the highest reached efficiency for industry use in recent publications [16, 17]. For this purpose, our team decided to use as much as possible synthesis efficiency-enhancing mechanisms to achieve the higher concentrations of naringenin. While keeping this in mind, we looked for the most effective promoters for both chassis organisms and included an mRNA cyclisation mechanism  to enhance translation in bacteria. Thus, we chose to connect enzymes of naringenin biosynthesis with linkers to make metabolic flux used for naringenin synthesis more efficient.
For safety purpose, we used a novel VapXD kill-switch  dependent on bile concentration and temperature so that GMO probiotics that left the human body would not cause any harm to the environment. Finally, by CRISPR-Cas9 combined with Lambda Red recombineering  or by CRISPR-Cas9D10A nickase-assisted genome editing , the whole naringenin construct was inserted into E. coli Nissle 1917 and L. casei BL23 genomes, respectively. This creates a more stable system and prevents plasmids transfer to other microbiome bacteria.
For the diagnostic part, we have chosen to create a rapid, point-of-care, user-friendly diagnostic test identifying extraintestinal amebiasis. The main components of this test are aptamers, specific to the E. histolytica secreted proteins. These single stranded DNA sequences fold into tertiary structures for particular fit with target proteins . We have chosen aptamers instead of antibodies or DNA interaction based detection tests because of robust detection, enhanced stability of test components under various environment conditions, overcomed cross-reactions possibility  and simpler test design enabling to scale this test. In our test design, aptamers conjugated with polydiacetylene (PDA) polymers bind to particular biomarkers and induce conformational changes. Those changes show up as transitions from blue to red . We also thought about uncertainties of our diagnostic test and precisely chose protein excreted by E. histolytica for much clearer determination of illness condition. Pyruvate phosphate dikinase are particularly unique to E. histolytica  and will be detected by aptamers based blood tests. On this biomarker we chose to do SELEX - systematic evolution of ligands by exponential enrichment . Final product consists of 3 detection fields and on each of them just a single drop of blood is needed for amebiasis detection.
As mentioned earlier, new infections emerge almost every year. Combating those threats is almost impossible without certain tools such as preventive substances, specific, sensitive diagnostic methods, and effective treatment. With deeper analysis of other infectious diseases agents and pathogenesis, we would be able to create probiotics for a broader range of deadly pathogens. In addition, relying on intestinal production of pharmacoactive products can diminish drug overdose problems if certain genetic circuits controlling the synthesis of those compounds are included in applied probiotics.
Furthermore, it drags our attention and determination towards scaling our diagnostic test to identify intestinal amebiasis or even other infectious diseases. Our project builds up the basis for developing such tests because we have framed the workflow for creating diagnostic tests for specific antigens.