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Introduction

Project

Treating infectious diseases of the gastrointestinal (GI) tract with antibiotics disrupts a patient's gut microbiota and can increase the prevalence of antibiotic resistant strains. The increasing population of multi-drug resistant bacterial strains, both within and outside of health centers, is a growing health concern that is becoming progressively difficult to treat. Additionally, it is a well-recognized fact within the global health community that traditional antibiotics do not represent a sustainable method of treatment for bacterial infections. There is a clear drive towards minimally invasive, prophylactic therapies for such ailments, but is a demand that so far, has not been adequately met. Our project will aim at replacing broad and narrow spectrum antibiotics with “precision therapies” that have etiology targeting capacity at the species level as well as contain minimal cross-talk among healthy tissues, organs, and symbiotic organisms.

We envision a new paradigm for treating infections of the human gastrointestinal tract through exploitation of engineered probiotics that produce anti-microbials with high specificity for pathogens. The anti-microbials we are exploring do not utilize a one-fit all therapy mold, but target unique features specific to organisms at the genetic level. Towards this aim, we have utilized a general-purpose system that will be delivered to pathogenic bacteria from an engineered bacterial species found in the GI tract (Escherichia coli), which will cleave pathogenic genes with single nucleotide resolution. To achieve specific genome targeting, we will utilize the CRISPR/Cas9 system with gRNA engineered to recognize genes from infectious bacteria that contribute to pathogenicity in humans. Our CRISPR/Cas9 system will be delivered from the engineered E. coli to infectious bacteria using bacterial specific phages, minimizing any side-effects to native microbiota and human-host cells. As a proof-of-principle for our engineered probiotic, we are starting by targeting Vibrio cholerae, however we hope to expand the system to other pathogens of the GI tract.

Probiotics:
We used E. coli as our chassis for our experiments in the lab, but we envision that our system would be used in a common probiotic strain of bacteria, such as Lactobacillus acidophilus. Our probiotic system can be used prophylactically to persons in a region experiencing a cholera outbreak, or it can be administered to someone already exposed to V. cholerae to quickly deliver therapeutic phage directly to the site of the infection. We believe our system would be particularly useful to military personnel or aid workers stationed in a cholera outbreak zone where they would be frequently exposed to V. cholerae. Although an oral cholera vaccine exists, its efficacy is relatively low: 52-62% in healthy adults and as low as 38% for the highest risk age group - children under 5 years of age (1).

Once complete, our system will be able to remain as a stable population in a person’s gut until the event of a V. cholerae infection. Detection of V. cholerae will then activate production of the phage delivery system, which will package the gRNA and Cas9 targeting system into a phage coat, exit the probiotic and transfer the system into V. cholerae present in the gut (transmission of a heterologous DNA message via phage was demonstrated by Ortiz and Endy in 2012 and featured in Waterloo’s 2013 iGEM project (2)). Once inside V. cholerae, the targeting system will bind and cleave sites complementary to the gRNA that correspond to selected pathogenicity genes. We proposed using Cas9/gRNA to target and kill pathogens as opposed to traditional phage therapy using a targeted lytic phage because it allowed us to differentiate and kill harmful pathogens of a strain that has both harmful and harmless serotypes. The gRNA in our system, while specific to V. cholerae, can easily be altered with PCR to target unique regions in other gastrointestinal pathogens.

References

Use these links to navigate our project!

• History of Cholera
• Project Details
• Materials and Methods
• Results
• Conclusions

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