UC Santa Barbara

Bacteria have evolved numerous strategies to establish their communities, enabling them to compete and survive in diverse environments. Cell density and complex conditions often trigger bacteria to regulate specific gene expressions, providing them with a competitive advantage. One prevalent mechanism observed among Gram-negative bacteria is the Contact-dependent Growth inhibition (CDI) system, which requires close contact with neighboring cells to deliver toxic effectors. The CDI system utilizes the two-partner secretion system to facilitate toxin delivery. The cycle starts with the biogenesis of a beta-barrel protein, CdiB on the CDI+ cell outer membrane. Its periplasmic domain pushes a large filamentous CdiA protein through the lumen of CdiB and allows it to secrete until it reaches a secretion arrest signal. In this conformation, the receptor binding domain (RBD) is located at the distal end of the filament, waiting for the target cell outer membrane receptor recognition. Furthermore, a cognate immunity protein, CdiI is encoded within the CDI+ cell that safeguards cells from self- and kin-intoxications. The CdiA filament features an intricate design with domains designated for different functions. Strikingly, the filament is well conserved across species up until the VENN motif at the C-terminus. Sequence alignment revealed that post-VENN sequences encode the effector proteins targeting different components of the bacterial cells. This thesis reports several aspects of CDI, including the two unique RNase effectors isolated from Escherichia coli STEC_O31 and O32:H37, a distinctive protein translocation to import CdiA-CT, and a novel protease toxin found in Citrobacter rodentium DBS100. Chapter I provides a general introduction to contract-dependent growth inhibition and other inhibitory systems, offering an overview of our current understanding of key features of the CDI. Chapter II presents a detailed characterization of an EndoU toxin from STEC_O31. In Chapter II, we introduce a different class of RNase toxin that utilizes an elongation factor (EF-Tu) as a co-factor for activity from Escherichia coli O32:H37. Chapter IV focuses on a translocation mechanism of CdiA-CTO32:H37 via SbmA in E. coli. Finally, in Chapter V, we expand our search for new toxin effectors, leading to the discovery of a cysteine protease isolated from Citrobacter rodentium DBS100.