New Roles for PagP in the Bacterial Outer Membrane Stress Response

Abstract

The ability of Gram-negative bacteria to modulate outer membrane (OM) composition in response to stressful environments is essential for their survival and replication within host tissues. The OM enzyme PagP catalyzes the transfer of palmitate from a glycerophospholipid to lipid A. Lipid A is the endotoxic portion of LPS responsible for transmembrane signalling to initiate the immune response. Palmitoylation of lipid A can either attenuate or stimulate the immune response depending on where the palmitate chain is attached to a specific lipid A molecule. Here we report that the Escherichia coli PagP homolog is a multifunctional enzyme, which displays two distinct active sites exposed on either side of the bacterial OM. E. coli PagP converts phosphatidylglycerol (PG) to palmitoyl-PG (PPG) using the same cell surface active site involved in the palmitoylation of lipid A. PPG is then serially degraded to bis(monoacylglycero)phosphate (BMP) and either lyso-PG or lyso-BMP by a novel lipase active site located in PagP on the periplasmic side of the OM. The periplasmic lipase active site can be inactivated with the Y87F amino acid substitution. BMP is a novel glycerophosphoglycerol (GPG) that has not previously been reported in bacterial lipid metabolism. Not all PagP homologs have this ability to remodel GPGs. We have identified a divergent lipid A palmitoyltransferase in Pseudomonas aeruginosa that does not palmitoylate PG. The P. aeruginosa homolog also has different lipid A regiospecificity, adding palmitate on the opposite glucosamine at the 3’-position compared to the 2-position of the proximal sugar observed for the E. coli homolog. We have determined that P. aeruginosa PagP is representative of a distinct clade of PagP evolved to fulfill different functions. Although this minor clade is inclusive of homologs that lack obvious sequence similarity with the major clade enterobacterial PagP, we have identified conserved catalytic and structural elements within the minor clade that contribute to our growing understanding of homologous PagP structure/function relationships. A comparative analysis of all available sequences of minor clade PagP homologs has revealed invariant His, Ser, and Tyr residues that are necessary for catalysis. Additionally, a 4-amino acid conserved signature indel or CSI is unique to bacteria clustered phylogenetically within the γ-subclass of Proteobacteria.