Bilateral NSF/BIO-BBSRC The roles of contact-dependent inhibition in building mixed bacterial communities

Bacteria are essential to human, animal health, and plant health, but can also cause disease. Bacteria are usually found working together as well-ordered communities. Understanding how communities develop and are maintained is therefore very important. One of the applicants discovered a system used by a range of bacterial species to inhibit the growth of others: contact dependent inhibition (CDI). In this elegant system one bacterial cell, the inhibitor, injects a toxin into a susceptible bacterium (target) when they touch, which inhibits target cell growth. There is evidence of many different types of these toxins in nature, but we do not yet know how they all work, and why they are so prevalent.
In this project we will examine how CDI affects the development of mixed strain bacterial communities. We will use and integrate three different approaches. Firstly, we will study how these toxins work in detail to gain a clear understanding of the changes the target cell undergoes when it is growth inhibited. This will be achieved by a combination of microbial genetics, molecular biology and biochemistry approaches. Second, using advanced microscopy techniques and strains with well-characterized CDI systems, we will document growth and inhibition in real time. We will measure different aspects, including how fast the toxin acts, and whether target cells can recuperate. Finally, based on the knowledge of CDI gained from these molecular and microscopic studies, we will use mathematical approaches to generate, and then also test, predictive computer models of the effect of CDI on bacterial communities.
CDI toxins are present in many different bacteria and based on similarities, the project can contribute new insight into a wide range of microbiological systems, and thus the understanding we generate may be exploited to address different societal challenges. In health care, probiotics build and maintain beneficial bacterial communities, and the understanding of CDI effects on populations may be exploited to improve probiotic strains or probiotic-based strategies. Similarly, approaches to improve plant health may devised where we encourage and help beneficial bacterial communities to grow. Both academics and pharmaceutical companies may be inspired by the understanding of CDI toxin activity to pursue new approaches to antimicrobial drug development. Finally, we envision that CDI systems may be introduced in industrial processes or in synthetic biology systems, where it is important to control mixed strain bacterial populations, for example in live biosensors. We will organize a workshop with academics and industrial representatives to stimulate discussion on applications of CDI. We also will share our enthusiasm and knowledge of this research with talks and activities for the public and at schools, colleges and universities.

Contact Dependent Inhibition systems (CDI) are ubiquitous, bacterial toxin delivery systems that require direct interaction between the toxin delivery CdiAB apparatus of the CDI+ cell and a defined receptor protein on the target, susceptible cell. CDI is active mostly but not exclusively between strains of a species, largely due to receptor specificity, but CdiI immunity proteins protect self and siblings. CDI toxins include tRNAse, DNase, and proton motive force inhibitor activity but also unknowns. Many fundamental questions about CDI remain; there is a significant gap in our understanding of CDI's role in bacterial ecology and evolution. This proposal seeks to close this gap with the overarching hypothesis that CDI systems shape the development and dynamics of microbial communities. Furthermore, we anticipate that each specific CDI system may have unique effects on population dynamics. Our objectives are to generate the knowledge and biological tools to further our understanding of CDI activity with a focus on Escherichia coli and Enterobacter cloacae CDI. This will be achieved using a combination of molecular biology and biochemical approaches, and will generate strains with well-characterized CDI systems. Subsequently, the effect of these CDI systems on population development will be determined based on a set of qualitative and quantitative imaging analyses at the single cell level, microcolony and biofilm level. This data will be used to parameterize a suite of computational models with stochastic implementation that will be constructed to capture important transient interactions leading to population structure. An integrated model that allows in silico predictions of CDI effect on mixed strain population structure will be validated in several rounds by combining all the tools and expertise: generate defined mutant strains with known CDI variables, determine the effect on population structure, and parameterize the model.

Impact on Knowledge environment and a skilled workforce.
This project on the effect of contact dependent inhibition (CDI) on mixed bacterial populations, will train young scientists in discipline specific skills and knowledge, with an emphasis on biochemistry, molecular biology, microbiology or computational biology. The project ensures that these skills and knowledge are applied in a broad, interdisciplinary context. Working in the project team will provide training in disseminating specialist knowledge to academics from related disciplines, and all project participants will gain experience in applying their specialist knowledge to other fields. Experience in professional teamwork and interdisciplinary skills are valued in many societal and professional settings, including industry, and thus will enhance the PDRA's and PhD student's employability. The international and interdisciplinary nature of the work will further enrich the professional training and transferable skills. The management plan and impact activities have been developed to maximize the potential of this project in this regard.

Undergraduate students will benefit from the research environment that the project will create, as the nature of the science lends itself well to training student in experimental skills (design, techniques) in context of summer research projects (at no cost to the grant), and in introducing and fostering interdisciplinary team work. Many aspects of CDI are suitable to apply to projects in the "The International Genetically Engineered Machine (iGEM)" competition in synthetic biology (both UofY and UCSB support iGem teams), and the applicants will continue to actively support their teams.

Benefits to society and commercial sector.
This is a basic science project, and ideas to exploit this knowledge for industrial or related purposes are at an early stage. Initially, the output of our work will mainly be of interest to scientists in academic settings as outlined in the "Academic Beneficiaries" section, to further the understanding of CDI mechanisms and effects. The putative future applications of this knowledge are exciting and broad, with significant potential to benefit the public. Companies working in drug discovery may be able to build upon our understanding of CDI to develop novel antimicrobials to target pathogens. Designer probiotic cocktails may become possible using CDI+ strains to target specific pathogens. Plant health may be enhanced if healthy plant microbiomes can be actively supported. Synthetic biology is addressing a wide range of societal challenges. In systems that rely on mixed microbial populations, there may be a need to delineate boundaries between populations (living biosensors; synthetic microbiomes) and CDI may inspire new strategies to achieve this. This field is now in the pre-translational phase, and therefore SMEs working on developing new products may be the initial non-academic beneficiaries, as well as pharmaceutical companies with active R&D for health care related applications.

Benefits to the wider public
The project lend itself well to engage schools and the general public. The concepts of "battling" and "cooperating" microbes, of building communities and maintaining them lend themselves to vivid illustrations and activities. Discussing with the public the potential benefits for society and the interdisciplinary nature will illustrate and generate excitement about the value of basic research, STEM sciences and scientific team work to the general public of all ages. The nature of long term applications can engage the public in discussions about synthetic biology and new insights in, and approaches to achieve human health.

Our Pathways to Impact identifies the activities we will undertake to deliver these impacts.