Absolute Proteome Composition and Dynamics during Dormancy and Resuscitation of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis remains a health concern due to its ability to enter a non-replicative dormant state linked to drug resistance. Understanding transitions into and out of dormancy will inform therapeutic strategies. We implemented a universally applicable, label-free approach to estimate absolute cellular protein concentrations on a proteome-wide scale based on SWATH mass spectrometry. We applied this approach to examine proteomic reorganization of M. tuberculosis during exponential growth, hypoxia-induced dormancy, and resuscitation. The resulting data set covering >2,000 proteins reveals how protein biomass is distributed among cellular functions during these states. The stress-induced DosR regulon contributes 20% to cellular protein content during dormancy, whereas ribosomal proteins remain largely unchanged at 5%-7%. Absolute protein concentrations furthermore allow protein alterations to be translated into changes in maximal enzymatic reaction velocities, enhancing understanding of metabolic adaptations. Thus, global absolute protein measurements provide a quantitative description of microbial states, which can support the development of therapeutic interventions.

Systems-level Proteomics of Two Ubiquitous Leaf Commensals Reveals Complementary Adaptive Traits for Phyllosphere Colonization

“…Beforehand, the OpenSWATH output was filtered to contain only features below an mScore of 0.01 (1% FDR). The sum of the five most intense transitions per peptide averaged over the three most intense peptides per protein was used as the protein intensity for all proteins identified by SWATH MS (29,45). Also proteins with only one or two peptides were included.…”

“…Such near-com-plete SWATH assay libraries covering over 70% proteome coverage are to date only available for few other organisms, i.e. Saccharomyces cerevisiae, Mycobacterium tuberculosis, and Streptococcus pyogenes (28,29,31). The two SWATH assay libraries are available for download through the SWATHAtlas database (www.SWATHAtlas.org) as a public resource for future proteomic studies of these two model strains of the plant microbiota.…”

“…However, because proteome coverage per species was low in studies on complex natural communities, complementary approaches are required for an in-depth understanding of bacterial fitness traits on leaves. The recently developed Sequential Windowed Aquisition of all THeoretical fragment ions Mass Spectrometry (SWATH MS) 1 technique allows for the reproducible quantification of thousands of proteins in a single run, overcoming technical limitations of previous studies and proteomic strategies and enabling systems level insights into each strain's physiology (27)(28)(29). In short, the SWATH MS approach generates a complete map of all detectable fragment ions derived from peptide precursors in a given sample using data independent acquisition (DIA).…”

Systems proteomics approaches to study bacterial pathogens: application to Mycobacterium tuberculosis

“…The results showed that whereas ribosomal proteins remain largely unchanged, products of DosR regulon genes were strongly induced to constitute 20% of the cellular protein content during dormancy. A quarter of 631 differentially expressed proteins had metabolic functions and 80% of them constituted connected metabolic pathways with at least four enzymes [44]. …”

A Proteomic Signature of Dormancy in the Actinobacterium Micrococcus luteus

“…Targeting resuscitation has been a primary focus by the World Health Organization (WHO) as an avenue for treatment of widespread tuberculosis infection (17). A link between latency and dormancy was suggested by Wayne from results showing initiation of dormancy in oxygen-poor (hypoxic) tissue environments (18), an observation leading to an in vitro hypoxia M. tuberculosis model of dormancy (19)(20)(21)(22)(23). The actual state and underlying mechanisms of tuberculosis latency are not well understood, and diverse definitions exist for latent and dormant phenotypic states (24).…”