Conserved tandem arginines for PbgA/YejM allow Salmonella to regulate LpxC and control lipopolysaccharide biogenesis during infection

Abstract

Salmonella enterica serovar Typhimurium uses PbgA/YejM, a conserved multi-pass transmembrane protein with a soluble periplasmic domain (PD), to balance the glycerophospholipid (GPL) and lipopolysaccharide (LPS) concentrations within the outer membrane (OM). The lipid homeostasis and virulence defects of pbgA{Delta}191-586 mutants, which are deleted for the PD, can be suppressed by substitutions in three LPS regulators, LapB/YciM, FtsH, and LpxC. We reasoned that S. Typhimurium uses the PbgA PD to regulate LpxC through functional interactions with LapB and FtsH. In the stationary phase of growth, pbgA{Delta}191-586 mutants accumulated LpxC and overproduced LPS precursors, known as lipid A-core molecules. Trans-complementation fully decreased the LpxC and lipid A-core levels for the mutants, while substitutions in LapB, FtsH, and LpxC variably reduced the concentrations. PbgA binds lipid A-core, in part, using dual arginines, R215 and R216, which are located near the plasma membrane. Neutral, conservative, and non-conservative substitutions were engineered at these positions to test whether the side-chain charges for residues 215 and 216 influenced LpxC regulation. Salmonellae that expressed PbgA with dual alanines or aspartic acids overproduced LpxC, accumulated lipid A-core and short-LPS molecules, and were severely attenuated in mice. Bacteria that expressed PbgA with tandem lysines were fully virulent in mice and yielded LpxC and lipid A-core levels that were similar to the wild type. Thus, S. Typhimurium uses the cationic charge of PbgA R215 and R216 to down-regulate LpxC and decrease lipid A-core biosynthesis in response to host stress and this regulatory mechanism enhances their virulence during bacteremia. IMPORTANCESalmonella enterica serovar Typhimurium causes self-limiting gastroenteritis in healthy individuals and severe systemic disease in immunocompromised humans. The pathogen manipulates the immune system of its host by regulating the lipid, protein, and polysaccharide content of the outer membrane (OM) bilayer. Lipopolysaccharides (LPS) comprise the external leaflet of the OM, and are essential for establishing the OM barrier and providing gram-negative microbes with intrinsic antimicrobial resistance. LPS molecules are potent endotoxins and immunomodulatory ligands that bind host-pattern receptors, which control host resistance and adaptation during infection. Salmonellae use the cationic charge of dual arginines for PbgA/YejM to negatively regulate LPS biosynthesis. The mechanism involves PbgA binding to an LPS precursor and activating a conserved multi-protein signal transduction network that cues LpxC proteolysis, the rate-limiting enzyme. The cationic charge of the tandem arginines is critical for the ability of salmonellae to survive intracellularly and to cause systemic disease in mice.