Comparative Ser/Thr/Tyr phosphoproteomics between two mycobacterial species: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG.

Kehilwe C Nakedi,Jonathan M Blackburn,Andrew J M Nel,Shaun Garnett,Nelson C Soares

doi:10.3389/fmicb.2015.00237

Abstract

Ser/Thr/Tyr protein phosphorylation plays a critical role in regulating mycobacterial growth and development. Understanding the mechanistic link between protein phosphorylation signaling network and mycobacterial growth rate requires a global view of the phosphorylation events taking place at a given time under defined conditions. In the present study we employed a phosphopeptide enrichment and high throughput mass spectrometry-based strategy to investigate and qualitatively compare the phosphoproteome of two mycobacterial model organisms: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG. Cells were harvested during exponential phase and our analysis detected a total of 185 phospho-sites in M. smegmatis, of which 106 were confidently localized [localization probability (LP) = 0.75; PEP = 0.01]. By contrast, in M. bovis BCG the phosphoproteome comprised 442 phospho-sites, of which 289 were confidently localized. The percentage distribution of Ser/Thr/Tyr phosphorylation was 39.47, 57.02, and 3.51% for M. smegmatis and 35, 61.6, and 3.1% for M. bovis BCG. Moreover, our study identified a number of conserved Ser/Thr phosphorylated sites and conserved Tyr phosphorylated sites across different mycobacterial species. Overall a qualitative comparison of the fast and slow growing mycobacteria suggests that the phosphoproteome of M. smegmatis is a simpler version of that of M. bovis BCG. In particular, M. bovis BCG exponential cells exhibited a much more complex and sophisticated protein phosphorylation network regulating important cellular cycle events such as cell wall biosynthesis, elongation, cell division including immediately response to stress. The differences in the two phosphoproteomes are discussed in light of different mycobacterial growth rates.

Highlights

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), a major health concern worldwide
There is increasing evidence indicating that protein phosphorylation plays an essential role during mycobacterial cell division and environmental adaptation
Our analysis included three rounds of TiO2 chromatography to enrich phosphorylated peptides, followed by subsequent analysis of phosphorylation-events using liquid chromatography coupled with state-of-the-art high resolution tandem mass spectrometry (LC/MS/MS) (Supplementary Figure S2a and S2b), in order to compare the phosphoproteomes of these two model mycobacterium species during exponential growth

Summary

Introduction

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), a major health concern worldwide. The majority of latently infected individuals will remain asymptomatic throughout their lives, with the risk of developing active TB disease from a latent infection being ∼10% per lifetime; in HIV positive individuals though, this risk increases to 10% per year (WHO, 2014). This latent infected population is a substantial reservoir of potential new TB cases and is a significant public health concern. Current thinking suggests that there is most likely a continuum of states between latent TB infection (LTBI), subclinical TB and active TB disease, but to date no M. tuberculosis bacilli have been observed in LTBI individuals, so the exact physiological state of M. tuberculosis during a latent infection remains unknown

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Conclusion