The Mycobacterium tuberculosis protein O-phosphorylation landscape

Abstract

Protein phosphorylation is a main mechanism for translating extracellular signals into cellular adaptations. In bacteria, the two-component system has been the paradigm of protein phosphorylation. Increasingly, however, protein serine/threonine and tyrosine phosphorylation (O-phosphorylation) mediated by serine/threonine protein kinases (STPKs) is identified. Mycobacterium tuberculosis (Mtb) in particular has a larger repertoire of both STPKs and O-phosphoproteins than most bacteria, suggesting a more prevalent role of STPKs in Mtb. Many studies have identified individual STPK functions and substrates, but a systems-level understanding and the full scope of O-phosphorylation in bacteria in general and Mtb in particular remains unknown. In this dissertation, we aimed to establish a systems-wide understanding of Mtb O-phosphorylation. To this end, we combined kinase loss-of-function (LoF) and gain-of-function (GoF) mutants with mass spectrometry (MS)-based phosphoproteomics. We found that the Mtb phosphoproteome is 5x larger than was previously reported. By identifying hypo-phosphorylated proteins in the LoF mutants and hyper-phosphorylated protein in the GoF mutants, we comprehensively identified over 1,400 STPK-substrates in the bacterium, assigning putative STPK substrates and functions. We showed that the O-phosphoproteome is organized as a higher-order network of a complexity that has previously only been associated with eukaryotes. Transcription factors (TFs) were highly phosphorylated, suggesting a large functional interface between O-phosphorylation and transcription. We further characterized this interface by using transcriptomics and discovered large transcriptional effects of individual STPKs. We used these data to predict functional phosphorylation events and validated these predictions for the putative zinc regulator Zur. These data provide the deepest bacterial O-phosphoproteome to date, identify a complex O-phosphorylation network, and provide a resource to assign thousands of specific phosphorylation events to individual STPKs and their transcriptional effects. Together, these data challenge the paradigm of TCSs as the main bacterial phosphosignaling mechanism and suggest that Mtb regulates a large swath of physiology through O-phosphorylation.