Mass Spectrometry Offers Insight into the Role of Ser/Thr/Tyr Phosphorylation in the Mycobacteria

Abstract

Phosphorylation is a post translational modification which can rapidly regulate biochemical pathways by altering protein function, and has been associated with pathogenicity in bacteria. Once engulfed by host macrophages, pathogenic bacteria are exposed to harsh conditions and must respond rapidly in order to survive. The causative agent of TB, Mycobacterium tuberculosis, is unusual amongst the bacteria because it can survive within the host macrophage for decades in a latent state, demonstrating a remarkable capacity to successfully evade the host immune response. This ability may be mediated in part by regulatory mechanisms such as ser/thr/tyr phosphorylation. Mass spectrometry-based proteomics has afforded us the capacity to identify hundreds of phosphorylation sites in the bacterial proteome, allowing for comparative phosphoproteomic studies in the mycobacteria. There remains an urgent need to validate the reported phosphosites, and to elucidate their biological function in the context of pathogenicity. However, given the sheer number of putative phosphorylation events in the mycobacterial proteome, and the technical difficulty of assigning biological function to a phosphorylation event, it will not be trivial to do so. There are currently six published phosphoproteomic investigations of a member of mycobacteria. Here, we combine the datasets from these studies in order to identify commonly detected phosphopeptides and phosphosites in order to present high confidence candidates for further validation. By applying modern mass spectrometry-based techniques to improve our understanding of phosphorylation and other PTMs in pathogenic bacteria, we may identify candidates for therapeutic intervention.

Multisystem Analysis of Mycobacterium tuberculosis Reveals Kinase-Dependent Remodeling of the Pathogen-Environment Interface

“…2A ; see also Table S3A in the supplemental material). Although these results partially overlap those from prior studies ( 21 , 26 – 28 ) ( Fig. 2B ), many new phosphoproteins were identified, substantially expanding the M. tuberculosis phosphoproteome.…”

“…Decreases in the level of phosphorylation were maximal or nearly maximal at the earliest time point (12 h) for many of these proteins, which is consistent with their being direct in vivo substrates of PknA and/or PknB ( Table S3F ). Notably, Thr was the phosphorylated residue in 88% of the phosphopeptides that were significantly decreased in level versus 48% overall, consistent with the preference of PknB and PknA for Thr phosphorylation ( 21 , 27 ). The Glu-X-X-Thr(P) and Thr(P)-X-Pro motifs were significantly associated with decreased Thr phosphorylation ( Fig.…”

Serum proteomics of active tuberculosis patients and contacts reveals unique processes activated during Mycobacterium tuberculosis infection

“…TB burden is especially dramatic in sub-Saharan countries 52 ; Mozambique, as the country of origin in the second cohort is from this region and, therefore, verification of the initial quantitative proteomics study in this second group of participants from a distant geographical region increases the strength of our findings and suggests a common host-acquired immune response to TB. Recently, mass spectrometry has been used to study the effect of phosphorylation in mycobacterial proteins known to be involved in MTB virulence 53 . Although, MTB strain identification was not performed during patient diagnosis, it would be of interest to investigate whether or not different MTB strains possessing distinct pathogenicity traits promote similar defense mechanisms in the host.…”

Proteomic Investigation Uncovers Potential Targets and Target Sites of Pneumococcal Serine-Threonine Kinase StkP and Phosphatase PhpP

“…Nevertheless, the presented highly confident phosphorylation sites can hint to yet unknown dephosphorylation targets of the pneumococcal PhpP and thus provides the basis for a deeper insight into the regulatory mechanisms of this phosphatase. However, phosphorylation is dynamic and can change rapidly (Macek et al, 2009;Calder et al, 2016). For sure, not all phosphorylation sites could be detected within this MS based approach focused on an extension of available phosphoproteomic data for S. pneumoniae with an emphasis on the StkP/PhpP couple.…”