Threonine eliminylation by bacterial phosphothreonine lyases rapidly causes cross-linking of mitogen-activated protein kinase (MAPK) in live cells.

Old long-lived proteins contain dehydroalanine (Dha) and dehydrobutyrine (Dhb), two amino acids engendered by dehydration of serines and threonines, respectively. Although these residues have a suspected role in protein cross-linking and aggregation, their direct implication has yet to be determined. Here, we have taken advantage of the ability of the enteropathogen Shigella to convert the phosphothreonine residue of the pT-X-pY consensus sequence of ERK and p38 into Dhb and followed the impact of dehydration on the fate of the two MAPKs. To that end, we have generated the first antibodies recognizing Dhb-modified proteins and allowing tracing them as they form. We showed that Dhb modifications accumulate in a long-lasting manner in Shigella-infected cells, causing subsequent formation of covalent cross-links of MAPKs. Moreover, the Dhb signal correlates precisely with the activation of the Shigella type III secretion apparatus, thus evidencing injectisome activity. This observation is the first to document a causal link between Dhb formation and protein cross-linking in live cells. Detection of eliminylation is a new avenue to phosphoproteome regulation in eukaryotes that will be instrumental for the development of type III secretion inhibitors.

Shigella flexneri targets the HP1γ subcode through the phosphothreonine lyase OspF.

HP1 proteins are transcriptional regulators that, like histones, are targets for post-translational modifications defining an HP1-mediated subcode. HP1γ has multiple phosphorylation sites, including serine 83 (S83) that marks it to sites of active transcription. In a guinea pig model for Shigella enterocolitis, we observed that the defective type III secretion mxiD Shigella flexneri strain caused more HP1γ phosphorylation in the colon than the wild-type strain. Shigella interferes with HP1 phosphorylation by injecting the phospholyase OspF. This effector interacts with HP1γ and alters its phosphorylation at S83 by inactivating ERK and consequently MSK1, a downstream kinase. MSK1 that here arises as a novel HP1γ kinase, phosphorylates HP1γ at S83 in the context of an MSK1-HP1γ complex, and thereby favors its accumulation on its target genes. Genome-wide transcriptome analysis reveals that this mechanism is linked to up-regulation of proliferative gene and fine-tuning of immune gene expression. Thus, in addition to histones, bacteria control host transcription by modulating the activity of HP1 proteins, with potential implications in transcriptional reprogramming at the mucosal barrier.

Targeting of chromatin readers: a novel strategy used by the Shigella flexneri virulence effector OspF to reprogram transcription.

Shigella flexneri, a gram-negative bacterium responsible of bacillary dysentery, uses multiple strategies to overcome host immune defense. We have decrypted how this bacterium manipulates host-cell chromatin binders to take control of immune gene expression. We found that OspF, an injected virulence factor previously identified as a repressor of immune gene expression, targets the chromatin reader HP1γ. Heterochromatin Protein 1 family members specifically recognize and bind histone H3 methylated at Lys 9. Although initially identified as chromatin-associated transcriptional silencers in heterochromatin, their location in euchromatin indicates an active role in gene expression. Notably, HP1γ phosphorylation at Serine 83 defines a subpopulation exclusively located to euchromatin, targeted to the site of transcriptional elongation. We showed that OspF directly interacts with HP1γ, and causes HP1 dephosphorylation, suggesting a model in which this virulence effector "uses" HP1 proteins as beacons to target and repress immune gene expression (Harouz, et al. EMBO J (2014)). OspF alters HP1γ phosphorylation mainly by inactivating the Erk-activated kinase MSK1, spotlighting it as a new HP1 kinase. In vivo, infectious stresses trigger HP1γ phosphorylation in the colon, principally in the lamina propria and the intestinal crypts. Several lines of evidence suggest that HP1 proteins are modified as extensively as histones, and decrypting the impact of these HP1 post-translational modifications on their transcriptional activities in vivo will be the next challenges to be taken up.

Bacterial nucleoid structure probed by active drag and resistive pulse sensing.

Recent biophysical approaches have provided key insights into the enthalpic and entropic forces that compact the nucleoid in the cell. Our biophysical approach combines two complementary, non-invasive and label-free techniques: a precisely timed steerable optical trap and a high throughput microcapillary Coulter counter. We demonstrate the ability of the latter technique to probe the physical properties and size of many purified nucleoids, at the individual nucleoid level. The DNA-binding protein H-NS is central to the organization of the bacterial genome. Our results show that nucleoids purified from the Δhns strain in the stationary phase expand approximately five fold more than the form observed in WT bacteria. This compaction is consistent with the role played by H-NS in regulating the nucleoid structure and the significant organizational changes that occur as the cell adapts to the stationary phase. We also study the permeability to the flow of ions and find that in the experiment nucleoids behave as solid colloids.