Structural Basis for Rcs Phosphorelay Cascade

Project Summary: One of the key factors that contributes to Klebsiella pneumoniae's pathogenicity is its ability to produce extracellular polysaccharide capsule in response to environmental stress. Capsule production is regulated by the Rcs regulatory phosphorelay cascade. Rcs cascade is inactive by default but becomes significantly activated when the bacteria experience environmental changes such as the presence of antimicrobials. It is a multi-step process that involves several proteins and protein complexes to activate. Mechanisms underlying the activation/inhibition process are incompletely understood. A key player of the Rcs cascade is the integral membrane protein, IgaA, that negatively regulates the system. The current working model suggests that a signal is first received by the lipoprotein RcsF (outer membrane protein), followed by the transduction of the signal to the essential protein IgaA, triggering autophosphorylation of RcsC where phosphate is sequentially transferred from RcsC to RcsD, then ending in the phosphorylation of the transcriptional regulator RcsB. Upon phosphorylation, RcsB positively and negatively regulates the production of capsules and stress-related genes in response to environmental changes. This proposal aims to answer fundamental questions regarding how IgaA interacts with RcsF and sends a stress signal to RcsC/RcsD/RcsB leading to activation/inhibition of Rcs phosphorelay system. Specific aim 1 will use a combination of biophysical methods that measure protein-protein interactions to interrogate the binding affinities of IgaA for RcsF, RcsD, and RcsC. Specific aim 2 will use cryo-electron microscopy and/or x- ray crystallography to determine the structures of IgaA bound to RcsF, RcsD, and RcsC. Specific aim 3 will use biochemical and genetic experiments to establish how mutations in the binding interfaces of IgaA/RcsF/RcsD/RcsC affect Rcs signaling and capsule production. The structural, genetic, and biochemical experiments proposed here will shed light on the molecular mechanism of the Rcs phosphorelay system. In addition, this study will lay the foundation for how capsule synthesis is regulated in Klebsiella and thus provide avenues for drug target studies.