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

Abstract

Unraveling which proteins and post-translational modifications (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. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD012589.

GroEL is an immunodominant surface-exposed antigen of Rickettsia typhi

“…This is also true for the GroEL proteins from these bacteria [ 77 ] as well as the GroEL protein from E . ruminantium that was found to be O -GlcNAcylated [ 78 ]. According to the Glycopp prediction server ( https://webs.iiitd.edu.in/raghava/glycopp/ [ 79 ]), four O - and 5 N -glycosylation sites exist in the R .…”

“…In this context it is interesting that several phosphorylated proteins were found in E . rumantium although corresponding enzymes that could be responsible for these phosphorylations were not detectable in the bacterial proteome [ 78 ]. Therefore, it may be finally discussed whether surface-exposed molecules of intracellular bacteria are accessible for eukaryotic cytosolic enzymes that could add protein modifications.…”

Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922

“…Selected protein spots were excised from the gels digested with trypsin and analysed by liquid chromatography coupled to mass spectrometry (Orbitrap Velos, Thermo, Waltham, MA, USA), as described previously [ 32 ]. Proteins were identified through searching against E. coli proteins found in the UniProt database, using Mascot (Matrix Science, London, UK) to search the SwissProt database (2018_11, taxonomy restricted to E. coli proteins).…”

Importance of protein Ser/Thr/Tyr phosphorylation for bacterial pathogenesis

“…The recent acquisition of large bacterial phosphoproteomes has allowed the assessment of phosphosite sequence features, representing putative kinase Phosphorylation consensus sequences have been identified in datasets obtained from M. tuberculosis [51,125], Helicobacter pylori [145], S. flexneri [64], and E. coli [63,146,147], including bacteria-specific N-and C-terminal phosphorylation preferences [139,147]. Interestingly, a phosphoproteomic dataset from the tick-transmitted Ehrlichia ruminantium pathogen was enriched in recognition motifs for several eukaryal kinases, suggesting significant host cell interaction [148]. Another general theme of bacterial phosphoproteomes is that they, similar to the eukaryal ones, are dominated by pSer sites, followed by pThr and, to a lesser extent, pTyr modifications.…”

“…[125], for example, used a TMT-based quantitative approach to identify phosphorylation events specific to M. tuberculosis virulence by comparing virulent and attenuated substrains. Similarly, Marcelino et al [148] used labelfree quantification to compare the phosphorylation patterns of virulent versus attenuated variants of E. ruminantium that had been grown together with bovine aortic endothelial cells. Misra et al used a hypervirulent strain of Listeria monocytogenes, harboring an activating mutation in the virulence gene activator PrfA, to identify virulence-associated phosphorylation events.…”