AUTHOR=Marcelino Isabel , Colomé-Calls Núria , Holzmuller Philippe , Lisacek Frédérique , Reynaud Yann , Canals Francesc , Vachiéry Nathalie 

TITLE=Sweet and Sour Ehrlichia: Glycoproteomics and Phosphoproteomics Reveal New Players in Ehrlichia ruminantium Physiology and Pathogenesis

JOURNAL=Frontiers in Microbiology

VOLUME=Volume 10 - 2019

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.00450

DOI=10.3389/fmicb.2019.00450

ISSN=1664-302X

ABSTRACT=Unraveling which proteins and PTMs affect bacterial pathogenesis and physiology in diverse environments is a tough challenge. Here, using mass spectrometry-based assays, we focused on protein phosphorylation and glycosylation in Ehrlichia ruminantium Gardel virulent (ERGvir) and attenuated (ERGatt) variants and how they can modulate Ehrlichia biological processes.
Phosphoproteome characterization revealed that both strains share the same set of phosphoproteins (n=58), 36% being overexpressed in ERGvir. The percentage of tyrosine phosphorylation is high (23%) and 66% of the identified peptides are multi-phosphorylated. Glycoproteomics revealed a high percentage of glycoproteins (67% in ERGvir) with a subset of glycoproteins being specific to ERGvir (n=64/371) and ERGatt (n=36/343). These glycoproteins are involved in several key biological processes such as protein, amino-acid and purine biosynthesis, translation, virulence, DNA repair and replication. Label-free quantitative analysis revealed up-expression in 31 proteins in ERGvir and 8 in ERGatt.  While further PNGase digestion confidently localized 2 and 5 N-glycoproteins in ERGvir and ERGatt, respectively, western blotting suggests that many glycoproteins are O-GlcNAcylated. Twenty three-proteins were found to be simultaneously phosphorylated and glycosylated in both variants.
This work represents the first comprehensive assessment of PTMs on Ehrlichia biology, rising interesting questions regarding ER-host interactions. Phosphoproteome characterization demonstrates an increased versatility of ER phosphoproteins to participate in different mechanisms. The high number of glycoproteins and the lack of glycosyltransferases-coding genes highlight ER dependence on the host and/or vector cellular machinery for its own protein glycosylation. Moreover, these glycoproteins could be crucial to interact and respond to changes in ER environment. PTM crosstalk between of O-GlcNAcylation and phosphorylation could be used as a major cellular signaling mechanism in ER. As little is known about the Ehrlichia proteins/proteome and its signaling biology, the results presented herein provide a useful resource for future hypothesis-driven exploration of Ehrlichia-specific variations in phosphorylation and especially glycosylation-mediated regulation of proteins.