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Allombert, J.

Publications and source records attributed to Allombert, J..

2 recordsLinked to original sources
bioRxiv2019

Orchestrated delivery of Legionella effectors by the Icm/Dot secretion system

Secretion of bacterial effector proteins into host cells plays a key role in bacterial virulence. Yet, the dynamics of the secretion systems activity remains poorly understood, especially when machineries deal with the export of numerous effectors. We address the question of multi-effector secretion by focusing on the Legionella pneumophila Icm/Dot T4SS that translocates a record number of 300 effectors. We set up a kinetic translocation assay, based on the {beta}-lactamase translocation reporter system combined with the effect of the protonophore CCCP. When used for translocation analysis of Icm/Dot substrates constitutively produced by L. pneumophila, this assay allows a fine monitoring of the secretion activity of the T4SS, independently of the expression control of the effectors. We observed that effectors are translocated with a specific timing, suggesting a control of their docking/translocation by the T4SS. Their delivery is accurately organized to allow effective manipulation of the host cell, as exemplified by the sequential translocation of effectors targeting Rab1, namely SidM/DrrA, LidA, LepB. Remarkably, the timed delivery of effectors does not depend only on their interaction with chaperone proteins but implies cyclic-di-GMP signaling, as the diguanylate cyclase Lpl0780/Lpp0809, contributes to the timing of translocation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/754762v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@17b7245org.highwire.dtl.DTLVardef@18b2443org.highwire.dtl.DTLVardef@1f9b7ceorg.highwire.dtl.DTLVardef@19275ff_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIThe dynamics of multi-effector secretion is addressed in the paradigm Legionella Icm/Dot T4SS C_LIO_LIA kinetic translocation assay allowing a fine monitoring of the T4SS activity is set up C_LIO_LISpecific secretion patterns consistent with sequential functions in the host are reported C_LIO_LIT4SS-dependent translocation is promoted by a diguanylate cyclase C_LIO_LIUnreported control of Type 4 secretion by the second messenger c-di-GMP signaling is revealed C_LI

microbiology↗
bioRxiv2019

The Coxiella burnetii secreted protein kinase CstK influences vacuole development and interacts with the GTPase-activating protein TBC1D5

Coxiella burnetii is the etiological agent of the emerging zoonosis Q fever. Crucial to the pathogenesis of this intracellular pathogen is the secretion of bacterial effectors into host cells by a Type 4b Secretion System (T4SS), to subvert host cell membrane trafficking, leading to the biogenesis of a parasitophorous vacuole allowing intracellular replication. The characterization of prokaryotic Serine/Threonine Protein Kinases (STPKs) in bacterial pathogens is emerging as an important strategy to better understand host-pathogen interactions. In this study, we investigated CstK (for Coxiella Ser/Thr kinase), a bacterial protein kinase identified in C. burnetii by in silico analysis. Here, we demonstrated that this putative protein kinase undergoes autophosphorylation on Ser, Thr, and Tyr residues, and phosphorylates a classical eukaryotic protein kinase substrate in vitro. This dual Ser/Thr and Tyr kinase activity is similarly observed for eukaryotic dual specificity Tyr phosphorylation-regulated kinase class. CstK is translocated during infections and localizes at Coxiella-containing vacuoles (CCVs). Moreover, a C. burnetii mutant strain overexpressing CstK displays a severe CCVs development phenotype, suggesting a finely tuned regulation by the bacterial kinase during infection. Protein-protein interaction studies identified the Rab7-GTPase activating protein (GAP) TBC1D5 as a candidate CstK-specific host target, suggesting a role for this eukaryotic GAP in Coxiella infections. Indeed, CstK colocalizes with TBC1D5 in non-infected cells, and TBC1D5 is recruited at CCVs during infection. Accordingly, depletion of TBC1D5 from infected cells significantly affects CCVs development. Our results indicate that CstK has a critical role during infection as a bacterial effector protein that interacts with host proteins to facilitate vacuole biogenesis and intracellular replication.

microbiology↗