Probiotics, live microbial supplements, are often incorporated into foods and beverages to provide putative health benefits. To ensure their beneficial effects, these organisms must survive processing and storage of food, its passage through the upper gastrointestinal tract (GIT), and subsequent chemical ingestion processes until they reach their target organ. However, there is considerable loss of viability of probiotic bacteria in the acidic conditions of the stomach and the high bile concentration in the small intestine. Bacillus subtilis, a spore-forming non-pathogenic bacterium, recently has gained interest in its probiotic properties; it can effectively maintain a favorable balance of microflora in the GIT. In addition, B. subtilis produces an extracellular matrix that protects it from stressful environments. We suggested that the extracellular matrix produced by B. subtilis could protect other probiotic bacteria and therefore potentially could be used as a vehicle for delivering viable probiotic cells to humans. Therefore, we developed a novel cultivation system that enables co-culturing of B. subtilis along with probiotic lactic acid bacteria (LAB) by increasing production of the extracellular matrix by B. subtilis cells. Moreover, we showed that B. subtilis improved survivability of LAB during food preparation, storage and ingestion. Therefore, we believe that the results of our study will provide a novel technique of using a natural system for preservation and delivery of probiotics to humans.

Apart from addressing humanity’s growing demand for fuels, pharmaceuticals, plastics and other value added chemicals, metabolic engineering of microbes can serve as a powerful tool to address questions concerning the characteristics of cellular metabolism. Along these lines, we developed an in vivo metabolic strategy that conclusively identifies the product specificity of glycerate kinase. By deleting E. coli’s phosphoglycerate mutases, we divide its central metabolism into an ‘upper’ and ’lower’ metabolism, each requiring its own carbon source for the bacterium to grow. Glycerate can serve to replace the upper or lower carbon source depending on the product of glycerate kinase. Using this strategy we show that while glycerate kinase from Arabidopsis thaliana produces 3-phosphoglycerate, both E. coli’s enzymes generate 2-phosphoglycerate. This strategy represents a general approach to decipher enzyme specificity under physiological conditions.

Introduction: Recent studies demonstrate that bacterial species are present within the tumor microenvironment (Geller et al., 2017). The presence of F. nucleatum in tumors has been described to increase cancer cell proliferation, promote chemoresistance, and protect tumors against immune cell attack (Zhang et al., 2018). A higher abundance and prevalence of F. nucleatum has been associated with advanced tumor stage and poor prognosis in human colorectal carcinoma patients (Mima et al., 2016; Yan et al., 2017). Bacteriophages (“phages”) are viruses that specifically infect bacteria and play a critical role in regulating bacterial populations. Phages can be engineered to deliver therapeutic payloads (Schmidt, 2019). Reduction of intratumor F. nucleatum and targeted delivery of anticancer payloads to tumors using phages may offer novel approaches for localized cancer treatment. To date there are only a few reports identifying phages that target F. nucleatum (Zheng et al., 2019). Using an initially isolated phage, we have shown that these phages, administered intravenously, can reach tumor-associated F. nucleatum in vivo (Kahan-Hanum et al., 2019). The aim of the current study was to discover novel F. nucleatum-infecting phages that may serve to decrease intratumor F. nucleatum burden and/or deliver a localized payload for anticancer treatment. Methods: For phage isolation, human saliva samples were screened on lawns of F. nucleatum strains, and natural phages were isolated from plaques. Phage sequences were analyzed following sequencing in Illumina using Nextera kits and genome assembly using SPAde genome assembler. To characterize infectivity, double layer plaque assays on different hosts were carried out. Phage editing was carried out by new molecular tools for F. nucleatum that were developed internally. Results: Twelve novel phages were discovered, including phages against all 4 subspecies of F. nucleatum: animalis (n=2), vincentii (n=7), polymorphum (n=2), and nucleatum (n=1). Sequence analysis revealed that the phages differ greatly from one another and possess many unique features. Both temperate (lysogenic, n=4) and lytic (n=8) phages were isolated as determined by the presence of integrases required for lysogenic integration. Of the eight lytic phages, six share very little similarity with known phages or with each other. Moreover, two of the lytic phages are double-stranded lytic RNA phages, of which only eight have previously been reported. Editing of F. nucleatum-targeting phage by synthetic biology tools to introduce new sequences has already been carried out. Conclusions: The newly discovered unique phages against F. nucleatum offer the first step in development of a novel therapy targeting intratumor bacteria. Reduction of intratumoral F. nucleatum bacterial burden and/or delivery of anticancer or immune-stimulating payloads to colonic tumors associated with F. nucleatum may offer novel treatment approaches for patients with colorectal cancer. This abstract is also being presented as Poster B20. Citation Format: Lior Zelcbuch, Sagit Yahav, Nufar Buchshtab, Maya Kahan-Hanum, Ilya Vainberg-Slutskin, Iddo Weiner, Myriam Golembo, Sharon Kredo-Russo, Naomi Zak, Inbar Gahali-Sass, Sailaja Puttagunta, Merav Bassan. Novel phages targeting the intratumor-associated bacteria Fusobacterium nucleatum [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr PR07.

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