Contact-dependent growth inhibition (CDI) is a mechanism of inter-cellular competition found in many Gram-negative pathogenic bacteria. CDI+ inhibitor cells make contact with target bacteria using large filamentous CdiA proteins. Upon cell contact, CdiA delivers a toxin derived from its C-terminus into the target bacterium to inhibit cell growth. All CDI+ bacteria protect themselves from intoxication by producing CdiI immunity proteins that bind and inactivate the toxin. Remarkably, there are several different types of CDI toxins, many of which have distinct nuclease activities. The sequence variations in CDI toxins are mirrored by the CdiI immunity protein, which only protect cells from their cognate toxin. Thus, CDI systems encode a complex network of toxin/immunity protein pairs that are used for inter-strain competition. Recent studies have shown that the variable CdiA-CT region is actually composed of two variable domains. The extreme C-terminal domain contains the growth inhibition activity and is often a nuclease. The N-terminal domain is poorly characterized, but our preliminary data suggest that these domains bind to specific membrane proteins to mediate toxin translocation into the cytoplasm of target bacteria. We hypothesize that these N-terminal specificity domains function as modular translocation domains that can transport nuclease cargos through several independent cell-entry pathways. Herein, we propose a combination of complementary structural, biochemical, and genetic approaches to critically test this model.
Aim 1 will use modern solution nuclear magnetic resonance techniques to obtain structural and dynamic information on several different specificity domains and to probe predicted binding sites of the membrane- protein receptors.
Aim2 uses molecular genetic and biochemical approaches to test whether the specificity domains do indeed mediate translocation across the inner membrane target bacteria as our models suggest.
Many pathogenic bacteria use contact dependent inhibition (CDI) to compete with related organisms. During CDI, donor bacteria deliver toxic enzymes to recipient cells. Toxin delivery requires the participation of specificity domains of the protein CdA that recognize particular inner membrane proteins of the recipient cells. This recognition results in uptake of the toxin by recipient cells. This proposal will investigate the mechanism of action o specificity domains using a combination of structural, biochemical and genetic approaches.
