How do bacterial extracellular Contractile Injection Systems bind target cells? A remarkable diversity of receptor binding domains - post revision analysis

How do bacterial extracellular Contractile Injection Systems bind target cells? A remarkable diversity of receptor binding domains Nimrod Nachmias1,, Zhiren Wang2,, Feng Jiang2,#, and Asaf Levy1,# 1 Department of Plant Pathology and Microbiology, Institute of Environmental Science, The Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Rehovot, Israel 2 NHC Key Laboratory of Systems Biology of Pathogens, Key Laboratory of Pathogen Infection Prevention and Control (Ministry of Education), State Key Laboratory of Respiratory Health and Multimorbidity, National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Abstract: Extracellular contractile injection systems (eCIS) represent a diverse family of bacteriophage tail-derived protein toxin delivery complexes in prokaryotes. eCIS are vital for microbial interaction with hosts and they use tail fiber proteins for target cell binding. However, the identity of the tail fiber proteins and their target cells are unknown. Here, we conducted a comprehensive exploration of eCIS tail fiber genes, providing insights into their remarkable diversity, target cells, functional adaptations, and intriguing evolutionary dynamics. We identified 3,445 eCIS tail fiber proteins found in 2,585 eCIS loci from 1,069 microbes. These fibers can be categorized by five new N-terminal domains responsible for tail fiber attachment to eCIS baseplates. Importantly, we leveraged structure prediction to divide the fibers into 276 structural clusters and dissected over a thousand domain fold families which seem to facilitate glycan and protein binding on the surface of potential eukaryotes or bacterial targets. These domains are likely acquired from diverse genomes of eukaryotes, bacteria, and viruses. Finally, we experimentally showed that the tail fibers from Paenibacillus eCIS can bind and direct effector injection into THP1 murine monocytes, and may bind D-mannose. This study reveals the exceptional diversity of eCIS target specificity determinants, suggests new eCIS target cells in Nature, and provides thousands of proteins that adhere to different cell types.