The role of bacteriocins on S. pneumoniae diversity

While mankind has only recently added chemical weapons to it's arsenal, toxic agents have been the weapons of choice for bacteria and fungi for millions of years. Humans have exploited microbial products, such as penicillin and streptomycin, to cure bacterial diseases. Since Fleming's discovery of penicillin, produced by the Penicillium mold, these drugs have worked with remarkable success, transforming the medical landscape and dramatically improving human health. The microbial use of antimicrobials, however, is considerably less friendly. Instead, microbes are believed to use these hugely diverse toxins to kill each other, thereby enabling producing strains to gain ground in the struggle for resources. This struggle is a key aspect of microbial ecology and evolution, equally important for microbes living in the soil and those living as pathogens within the human body. Recent theoretical studies have shown that competition between strains of bacteria producing different toxins, called bacteriocins, can cause the evolution of a nearly limitless variety of bacterial types that produce diverse bacteriocins and mechanisms of bacteriocin resistance. Thus, it appears, at least in theory, that the bacterial arms-race of one strain against another closely resembles our own arms-race against them; in both cases new drugs are developed (evolve) while others lose efficacy due to the evolution of resistance. The aims of this proposal are therefore to understand the evolutionary causes and consequences of this widespread chemical warfare between bacteria.

To address these aims we will employ a strategy combining the development of new theory with an approach called experimental evolution, which examines the evolution of bacteria in the lab over thousands of generations. Our study system will be based upon bacteriocins produced by the gram-positive pathogen S. pneumoniae. S. pneumoniae remains one of the leading causes of bacterial diseases in children and the elderly worldwide. The bacteriocins in this species are highly diverse as are the signaling molecules that induce them. Hundreds of distinct combinations of signal and toxin can exist, the causes and consequences of which are unknown.

We will use the following plan to answer these questions. First, we will characterize the diversity of S. pneumoniae toxins and resistance by competing strains against one another in pair-wise tournaments. We will next seek to understand the genetics of toxicity and resistance by sequencing the relevant genes from natural bacterial strains. Using the information gained in these analyses, we will build theoretical models to simulate the coevolution of multi-toxin communities of bacteria. We will ask how well these models predict the actual diversity of toxicity profiles in natural populations. In addition, we will use the model to generate novel predictions about evolution of synthetic bacterial communities. Finally, we will test these predictions by allowing mixtures of bacterial strains to evolve in the lab for 1,000s of generations. After this period of evolution we will study phenotypic and genetic changes that competing populations have accumulated.

Our results will have several fundamental and applied implications. Scientists are working to specifically assemble microbial communites to perform heath and environment related services. It is thus clearly of considerable importance to develop a focused and predictive understanding of rules governing populations of bacterial pathogens. Furthermore, bacteriocins are increasingly being explored as alternative antimicrobials and have been developed as food preservatives. Antibiotic resistance in S. pneumoniae is increasing and the need for alternative chemotherapeutic agents is real. The successful deployment of bacteriocins relies on an understanding of their diversity and the responses cells evolve to resist them.

The last decade has seen a renaissance in our understanding of bacterial diversity and the key roles bacteria play in regulating human health. Lacking from this work is an appreciation of the factors leading to the origin and maintenance of bacterial communities. While many models seeking to understand the forces underlying bacterial diversity focus on resources, these have failed to explain the origin and the astounding diversity of bacterial populations. In addition to competing for resources, bacterial also compete directly with one another using antimicrobial toxins, called bacteriocins. These toxins are secreted by nearly all bacterial species and are used to kill competitors, thereby providing a direct fitness advantage to toxin producing cells. Recent theoretical work has shown that "interference" competition using bacteriocins can cause the evolution of a nearly limitless variety of coexisting toxin types and strains resistant to these novel compounds. Toxin production, therefore, may be a potent mechanism for the generation and maintenance of bacterial diversity. The aims of this proposal are to understand the evolutionary causes and consequences of bacteriocin diversity, focusing on the bacteriocins produced by the gram-positive pathogen Streptococcus pneumoniae. To address these aims we will use a multi-disciplinary approach combining spatially explicit cellular automaton simulations of S. pneumoniae bacteriocins and experimental evolution to track bacterial evolution over 1000s of generations. Because of increasing rates of antibiotic resistance, bacteriocins are increasingly being explored as alternative therapeutic agents. The successful deployment of bacteriocins relies on an understanding of their diversity and the responses cells evolve to resist them. Our results may identify novel therapies for S. pneumoniae and establish the framework for similar studies in other pathogens.

The most immediate impact of this work will be in the academic research community. Because of the interdisciplinary nature of this project, the data generated will benefit researchers from several fields, including evolutionary biology, microbiology, and microbial ecology, as well as researchers interested in antimicrobial peptide activity and drug discovery. Our findings will have the most direct impact on the widening community of ecologists and evolutionary biologists studying the causes of bacterial diversity in natural and human associated environments, and in particular the role of interference competition in driving this diversity. The benefit to these researchers will be the generation and analysis of an empirical and theoretical dataset that will facilitate advances and drive new ideas in this field. Chemical and biomedical industries are a second group of beneficiaries, owing to the considerable commercial interest in exploiting bacteriocins as antibacterial drugs and natural food preservatives. The bacteriocin Nisin, for example, produced by Lactococcus lactis, is used commercially to preserve meats and cheese. In addition, bacteriocins in S. pneumoniae, like those from other bacterial species, may have commercial potential because they are likely to be narrow-spectrum and thus less associated with evolved peptide resistance in non-target bacterial species. The PDRA and technician employed on the grant will directly benefit through training and techniques learned in microbiology, bioinformatics, and theoretical model development. A final group of beneficiaries is the general public, as this research will improve awareness and knowledge of mechanisms underlying biodiversity and evolutionary change, and also inform the general public of the development and potential of novel forms of antimicrobial therapy. The mechanisms to ensure this research impacts the work of the academic community will be the development and dissemination of community resources (e.g. bacterial clones and constructs, sequence data), publication of our findings in high-impact international peer-reviewed journals, and attendance and presentation at international meetings. Industrial and commercial opportunities will be pursued through the University of Manchester database of industrial partners. Key measures of success will be the publication of our work and invitations to present our finding at major meetings (ESEB, GRC, ISPPD) and seminar series worldwide. Given the potential commercial impact of our work, another measure of success will be if our findings lead to novel commercializable research directions, either by independent groups or through collaborative ventures. The realization of the impact of our work within the academic community and general public is expected to be rapid (1-3 years) while any commercial applications of our findings are anticipated to occur on a longer time frame (5-10 years). A final measure of success is the career development of key staff. It is hoped that through skills learned during this project the PDRA and technician will continue with careers in science, either as independent researchers or employed within the public or private sector.