Rodolphe Barrangou

The m-CAFEs SFA (Microbial Community Analysis and Functional Evaluation in Soils Science Focus Area) is a multi-institutional project that will advance our understanding of the mechanisms and interactions or rhizosphere microbiomes governing nutrient cycling. This will be achieved by developing powerful new in situ community manipulation capabilities to test model predictions and establish causal mechanisms. Our work builds upon a two-year pilot effort that has developed fabricated ecosystems (EcoFABs) enabling reproducible plant growth and colonization within these controlled environments designed for systems biology, imaging, and community manipulation technologies. We have used genetic manipulation approaches within the EcoFABs to understand the molecular mechanisms underpinning these processes and provide much needed insights into the ~50% of microbial genes of unknown function. In this three-year SFA period, we will rapidly develop novel CRISPR-Cas and phage-based rhizosphere microbiome editing technologies to investigate uncultivated microbial activities and interactions within controlled lab environments. We will use both defined microbial assemblies and native soil-derived enriched microbial communities to further develop and apply this powerful approach to dissecting functions of plant-rhizosphere microbiomes for eventual extension to fully-complex communities. The defined microbial assemblies will enable detailed characterization of constituent isolates whereas the enrichments of native microbial communities will enable investigation of community functions of uncultivated microbes. Given the vast number of unknown microbial interactions and gene functions within these communities, we will use powerful modeling approaches combined with our EcoFAB data to simulate interventions to prioritize targeted manipulations that provide new insights into activities and interactions of microbes including uncultivated species. Ultimately, the new experimental tools and detailed understanding organism interactions obtained by the m-CAFEs team will enable the predictable control of rhizosphere microbiomes, with important implications for sustainable energy production and environmental health.

This study proposes to continue to investigate Lactobacillus species for probiotic properties using microbiological and genetic approaches. The genome sequence information of two significant probiotic species, Lactobacillus acidophilus and Lactobacillus gasseri, will be the primary model organisms for this study. Other lactobacilli will also be investigated when appropriate. The impact of environmental signals encountered in foods and the gastrointestinal tract (food-dairy components, acid, bile, heat, cold, oxidative stresses, etc) will be evaluated using whole genome microarrays, for these model organisms and for other lactobacilli for which genome information is available. Discovery and confirmation of genetic traits that direct beneficial activities and outcomes of probiotic Lactobacillus species is expected to promote the use of effective cultures in a variety of functional food, and dairy food scenarios and, thereby, sustain future expansion of this category.

Diarrheal disease is the second leading cause of death in children under the age of 5 worldwide with rotavirus responsible for 40 percent of hospitalizations due to diarrheal illness1. It is estimated that rotavirus killed approximately 215,000 children in 2013. The World Health Organization recommends including a rotavirus vaccine in all global vaccination protocols and there are currently two vaccines licensed worldwide2. The global implementation is ongoing but in countries where data is available, vaccination has resulted in a 33% reduction in hospitalization due to rotavirus morbidities. Unfortunately, both vaccines have limited efficacy (50-60%) in developing countries and are associated with a low level risk of intussusception3. Next generation vaccines are under development and will need to address several key concerns and limitations inherent with the current modified-live vaccines. IgA has been shown to be important in protection against rotaviral infection in humans and in animal models4-9. An orally delivered vaccine that induces protective mucosal and systemic antibody responses would be ideal for use in the campaign against rotavirus. Induction of IgA through systemic immunization has proven to be difficult. The mucosal immune system is, in many respects, independent of the systemic immune system. For example, ninety percent of intestinal IgA is produced locally and induction of mucosal immunity is best achieved via mucosal infection or vaccination10-12. Commensal organisms of the intestinal tract have evolved to cope with the hostile environment presented by the gastrointestinal tract and some commensals, now considered probiotics, have been shown to enhance health via beneficial interaction with the mucosal immune system13,14. Appropriately selected commensal organisms could be powerful vectors for the delivery of therapeutics and vaccines. We have developed an orally-delivered mucosal vaccine platform that employs the commensal bacteria Lactobacillus acidophilus (LA). We have brought together several adjuvant and antigen-expression strategies in unique constructs that show great potential to induce mucosal and systemic antibody responses. This platform offers several important feasibility advantages as it employs a commensal designated as GRAS (generally regarded as safe) by the FDA, is inexpensive to produce, does not require cold-chain, and is needleless. Investigation of immunodominant epitopes from the rotavirus envelope have identified two regions, a 4 amino acid fragment of the VP8 trypsin cleavage fragment of VP4 and a 14 amino acid fragment corresponding to AA242-259 from VP6, that induce protective IgA15-17. In the proposed studies, we will utilize CRISPR-Cas to engineer a state-of-the-art vaccine construct and then determine the immunogenicity and efficacy of this novel vaccine in a Balb/c mouse model. We will explore correlates of protection and determine the influence of the interaction between vaccine, host, and intestinal microbiome on immunization and challenge outcomes.

The m-CAFEs SFA (Microbial Community Analysis and Functional Evaluation in Soils Science Focus Area) is a multi-institutional project that will advance our understanding of the mechanisms and interactions or rhizosphere microbiomes governing nutrient cycling. This will be achieved by developing powerful new in situ community manipulation capabilities to test model predictions and establish causal mechanisms. Our work builds upon a two-year pilot effort that has developed fabricated ecosystems (EcoFABs) enabling reproducible plant growth and colonization within these controlled environments designed for systems biology, imaging, and community manipulation technologies. We have used genetic manipulation approaches within the EcoFABs to understand the molecular mechanisms underpinning these processes and provide much needed insights into the ~50% of microbial genes of unknown function. In this three-year SFA period, we will rapidly develop novel CRISPR-Cas and phage-based rhizosphere microbiome editing technologies to investigate uncultivated microbial activities and interactions within controlled lab environments. We will use both defined microbial assemblies and native soil-derived enriched microbial communities to further develop and apply this powerful approach to dissecting functions of plant-rhizosphere microbiomes for eventual extension to fully-complex communities. The defined microbial assemblies will enable detailed characterization of constituent isolates whereas the enrichments of native microbial communities will enable investigation of community functions of uncultivated microbes. Given the vast number of unknown microbial interactions and gene functions within these communities, we will use powerful modeling approaches combined with our EcoFAB data to simulate interventions to prioritize targeted manipulations that provide new insights into activities and interactions of microbes including uncultivated species. Ultimately, the new experimental tools and detailed understanding organism interactions obtained by the m-CAFEs team will enable the predictable control of rhizosphere microbiomes, with important implications for sustainable energy production and environmental health.

CRISPR-Cas systems are revolutionary technologies that enable precise genome editing in a broad variety of organisms, encompassing humans, animals, plants and bacteria. Here, we focus on developing novel enzymes and proteins for use in altering the human genome to treat diseases via gene therapies.