Sweet and sour Ehrlichia: glycoproteomics and phosphoproteomics reveal new players in Ehrlichia ruminantium physiology and pathogenesis

ABSTRACT: Unraveling which proteins and PTMs affect bacterial pathogenesis and physiology in diverse environments is a tough challenge. Herein, we used mass spectrometry-based assays to study protein phosphorylation and glycosylation in Ehrlichia ruminantium Gardel virulent (ERGvir) and attenuated (ERGatt) variants and, how they can modulate Ehrlichia biological processes. The characterization of the S/T/Y phosphoproteome 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 key biological processes such as protein, amino-acid and purine biosynthesis, translation, virulence, DNA repair and replication. Label-free quantitative analysis revealed over-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 detected in both the phospho- and glycoproteome, for the two 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. PTMs 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 further hypothesis-driven exploration of Ehrlichia protein regulation by phosphorylation and glycosylation events.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Ehrlichia Ruminantium Str. Gardel

SUBMITTER: Isabel Marcelino  

LAB HEAD: Nathalie Vachiery

PROVIDER: PXD012589 | Pride | 2019-03-01

REPOSITORIES: Pride