Team:Costa Rica/Description
This year's team is integrated by students from three different Costa Rican universities.
Although none of us has taken a specific course on Synthetic Biology as part of our curriculae, some of us
have assisted research projects related to this field. Therefore, we were really excited to
put that knowledge into action and propose our own project. Antibiotics resistance is one of the greatest threats to human health, food security and
development worldwide (WHO, 2018). The increasing and inadequate use of this antimicrobials have
promoted the emergence of multidrug-resistant bacteria, each time more difficult to treat (Aslam
et al., 2018). As our team is also concerned about this issue, we decided to survey Costa Rican
people about this topic as part of our Human Practice. Surprised of the lack of information
among the interviewed population, we decided to relate our project to antibiotics resistance. Among the highly resistant bacteria there is Clostridium difficile, also classificated as
Clostridiodes difficile. This is a Gram-positive, anaerobic, spore forming bacillus
(Petrosillo, Granata & Cataldo, 2018). It is commonly present in small concentrations in some
mammals and is easily found on surfaces of human hospitals centers. This bacteria is an
opportunistic pathogen, which infection is attributed to its broad antibiotic resistance and
increased fitness (Jiménez et al., 2018). Around 15-25% of all cases of antibiotic-associated
diarrhea is due to C.difficile infection (CDI). It is the main cause of nosocomial
diarrhea worldwide and can also cause pseudomembranous colitis and toxic megacolon (Petrosillo
et al., 2018). In the United States this disease causes 15,000 deaths per year and is
responsible of around $5.4 billion health care costs annually (Balsells et al., 2019). In Costa Rica, C. difficile is considered an endemic disease in our hospitalarian system.
Eight new cases are diagnosed weekly in our country (current population of approximately 5
million people) and there has been an important outbreak. Among the 29 public hospitals of Costa
Rica 21 reported intrahospital cases between 2011 and 2014 (Mairena, 2014). Actually, this
problem is so close to us that even one of our team members have suffered from C.
difficile. The current treatments to eradicate this bacteria are based on the use of more antibiotics or
faecal transplants. The last one can be disgusting for the patients and requires a healthy donor
willing to donate the sample. As for the antibiotics, only few of the currently available are
effective (metronidazole and vancomycin) (Peng et al., 2018). In addition, after treatment,
around 20-30% of the patients present recurrent infection, which is harder and more expensive to
treat than the first time (Balsells et al., 2019). Therefore, there is an urge to find
alternative treatments to CDI (Ramírez-Vargas et al., 2017).
As we interviewed Dr. Manuel Antonio Villalobos, who is in charge of fecal transplants in San
Juan de Dios Hospital, we validated our project. He helped us emphasize with the problem of
C.difficile in our country. As he explained, it is such an issue that the hospital
conditioned a room specifically for patients with this illness and people still dies. Dr.
Villalobos supported the facts we had about C.difficile in Costa Rica and gave us
feedback about our idea. Our project aims to produce a bioengineered Lactobacillus casei with the capacity to sense
when C. difficile is present and as a result produce a lysis protein specific for this
pathogen. Our goal is that this approach could be adapted to treat other antibiotic resistant
bacteria and used for animals as well. DiffEASY is a project that seeks for the creation of an innovative method to treat
Clostridioides difficile infection (previously and frequently named as Clostridium
difficile) by implementing the use of synthetic biology to bioengineer a
Lactobacillus casei, acting as a probiotic against the infection. We elaborated a system using the chasis Lactobacillus casei. It was engineered to sense
the virulence of C. difficile and attack them as a response. In order to do so, we based
our project in two modules: the detection of C. difficile virulence peptides and the
inhibition of C. difficile growth. For the detection of C. difficile virulence, we took advantage of the quorum sensing used
by the bacteria, specifically, the accessory gene regulator system that had been discovered and
previously described (Darkoh et al., 2017). The molecular mechanism is shown in figure 1.
Figure 1. Molecular mechanism of the quorum system accessory gene regulator of Clostridiodes difficile (Agr). Basically, the bacterium produces an autoinducing peptide (AIP) that activates the transcription
of genes promoting the virulence of C.difficile. Although, the promoter activated by the transcription factor (AgrA) in C. difficile isn’t
known yet, there is an homolog system in S. aureus that has been well studied. Considering
that AgrA is based on two independent domains joined by a linker, we created a chimera protein
with the recognition domain of C. difficile and the DNA Binding domain of S.
aureus. Therefore, when the AIP binds to AgrA, the transcriptional factor will bind to
the known DNA promoter of S. aureus. Also, it’s important to note that this homologue
system from S. aureus had already been tested in Lactobacillus, showing that instead of promoting transcription
in the presence of the inducer, the transcription is inhibited (Martin et al., 2013 & Lubkowicz et al., 2018) Figure 2. Integration of both modules. When the AIP is present the
expression of the lysin starts and the treatment against the infection begins to work.
Our system stops when the presence of AIP decreases. In order to inhibit the growth of C. difficile we will use a lysin specific to
Clostrioides taxid (Mayer M, 2011). This lysin has a N-acetylmuramoyl-L-alanine amidase
catalytic domain that had been shown to inhibit the growth of C. difficil in vitro,
without killing Lactobacillus spp. At the end, we will merge the 2 modules (Figure 2). Therefore, when the input AIP is present, a
transduction signal will activate and release the lysin, stopping when there is no AIP left in
the medium. The following video summarizes our project.
Aslam, B., Wang, W., Arshad, M. I., Khurshid, M., Muzammil, S., Rasool, M. H. &
Baloch, Z. (2018). Antibiotic resistance: a rundown of a global crisis. Infection
and Drug Resistance, Volume 11, 1645–1658.doi:10.2147/idr.s173867
Balsells, E., Shi, T., Leese, C., Lyell, I., Burrows, J., Wiuff, C., … & Nair, H.
(2018). Global burden of Clostridium difficile infections: a systematic
review and meta-analysis. Journal of Global Health, 9(1).
doi:10.7189/jogh.09.010407
Darkoh, C. & DuPont, H. L. (2017). The accessory gene regulator-1 as a therapeutic target for C. difficile infections. Expert opinion on therapeutic targets, 21(5), 451–453. doi:10.1080/14728222.2017.1311863
Jiménez, A., Araya, R., Paniagua, D., Camacho, Z., Du, T., Golding, G. R. & Quesada,
C. (2018). Molecular epidemiology and antimicrobial resistance of Clostridium
difficile in a national geriatric hospital in Costa Rica. Journal of
Hospital Infection, 99(4), 475–480. doi:10.1016/j.jhin.2018.03.027
Mairena, J. (2014). Infección hospitalaria por Clostridium está dentro del comportamiento habitual. Retrieved from: https://www.ccss.sa.cr/noticia?infeccion-hospitalaria-por-clostridium-esta-dentro-del-comportamiento-habitual
Martin, M. J., Clare, S., Goulding, D., Faulds-Pain, A., Barquist, L., Browne, H. P. & Wren, B. W. (2013). The agr locus regulates virulence and colonization genes in Clostridium difficile 027. Journal of bacteriology , 195(16), 3672–3681. doi:10.1128/JB.00473-13
Mayer, M. J., Garefalaki, V., Spoerl, R., Narbad, A. & Meijers, R. (2011). Structure-based modification of a Clostridium difficile-targeting endolysin affects activity and host range. Journal of bacteriology, 193(19), 5477–5486. doi:10.1128/JB.00439-11
Lubkowicz D., Chun Loong Ho, In Young Hwang, Wen Shan Yew, Yung Seng Lee & Chang, W. (2018). Reprogramming Probiotic Lactobacillus reuteri as a Biosensor for Staphylococcus aureus Derived AIP-I Detection. ACS Synthetic Biology, 7 (5), 1229-1237. doi: 10.1021/acssynbio.8b00063
Peng, Z., Ling, L., Stratton, C. W., Li, C., Polage, C. R., Wu, B. & Tang, Y. W. (2018). Advances in the diagnosis and treatment of Clostridium difficile infections. Emerging microbes & infections, 7(1), 15. doi:10.1038/s41426-017-0019-4
Petrosillo, N., Granata, G., & Cataldo, M. (2018). Novel Antimicrobials for the
Treatment of Clostridium difficile Infection. Frontiers in Medicine,
5.doi:10.3389/fmed.2018.00096
Ramírez, G., Quesada, C., Acuña, L., López, D., Murillo, T., del Mar Gamboa, M.,
Rodríguez, C. (2017). A Clostridium difficile Lineage Endemic to Costa Rican
Hospitals Is Multidrug Resistant by Acquisition of Chromosomal Mutations and Novel
Mobile Genetic Elements. Antimicrobial Agents and Chemotherapy,
61(4).doi:10.1128/aac.02054-16
World Health Organization. (2018). Antibiotic Resistance. Retrieved from https://www.who.int/news-room/fact-sheets/detail/antibiotic-resistance
Inspiration
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References