Bacterial virulence mediated by orthogonal post-translational modification.

Many bacterial pathogens secrete virulence factors, also known as effector proteins, directly into host cells. These effectors suppress pro-inflammatory host signaling while promoting bacterial infection. A particularly interesting subset of effectors post-translationally modify host proteins using novel chemistry that is not otherwise found in the mammalian proteome, which we refer to as 'orthogonal post-translational modification' (oPTM). In this Review, we profile oPTM chemistry for effectors that catalyze serine/threonine acetylation, phosphate ß-elimination, phosphoribosyl-linked ubiquitination, glutamine deamidation, phosphocholination, cysteine methylation, arginine N-acetylglucosaminylation, and glutamine ADP-ribosylation on host proteins. AMPylation, a PTM that could be considered orthogonal until only recently, is also discussed. We further highlight known cellular targets of oPTMs and their resulting biological consequences. Developing a complete understanding of oPTMs and the host cell processes they hijack will illuminate critical steps in the infection process, which can be harnessed for a variety of therapeutic, diagnostic, and synthetic applications.

A Chemical Probe for Dehydrobutyrine.

Bacterial phosphothreonine lyases, or phospholyases, catalyze a unique post-translational modification that introduces dehydrobutyrine (Dhb) or dehydroalanine (Dha) in place of phosphothreonine or phosphoserine residues, respectively. We report the use of a phospha-Michael reaction to label proteins and peptides modified with Dha or Dhb. We demonstrate that a nucleophilic phosphine probe is able to modify Dhb-containing proteins and peptides that were recalcitrant to reaction with thiol or amine nucleophiles under mild aqueous conditions. Furthermore, we used this reaction to detect multiple Dhb-modified proteins in mammalian cell lysates, including histone H3, a previously unknown target of phospholyases. This method should prove useful for identifying new phospholyase targets, profiling the biomarkers of bacterial infection, and developing enzyme-mediated strategies for bioorthogonal labeling in living cells.

Selectivity within a Family of Bacterial Phosphothreonine Lyases.

Phosphothreonine lyases are bacterial effector proteins secreted into host cells to facilitate the infection process. This enzyme family catalyzes an irreversible elimination reaction that converts phosphothreonine or phosphoserine to dehydrobutyrine or dehydroalanine, respectively. Herein, we report a study of substrate selectivity for each of the four known phosphothreonine lyases. This was accomplished using a combination of mass spectrometry and enzyme kinetics assays for a series of phosphorylated peptides derived from the mitogen-activated protein kinase (MAPK) activation loop. These studies provide the first experimental evidence that VirA, a putative phosphothreonine lyase identified through homology, is indeed capable of catalyzing phosphate elimination. These studies further demonstrate that OspF is the most promiscuous phosphothreonine lyase, whereas SpvC is the most specific for the MAPK activation loop. Our studies reveal that phospholyases are dramatically more efficient at catalyzing elimination from phosphothreonine than from phosphoserine. Together, our data suggest that each enzyme likely has preferred substrates, either within the MAPK family or beyond. Fully understanding the extent of selectivity is key to understanding the impact of phosphothreonine lyases during bacterial infection and to exploiting their unique chemistry for a range of applications.