Biology subjects

Michard, C.

Publications and source records attributed to Michard, C..

5 recordsLinked to original sources

bioRxiv2026

PDIM drives the transition from repairable to catastrophic EsxA-mediated vacuole membrane damage during Mycobacterium marinum infection

During infection, pathogenic mycobacteria damage the membrane of the Mycobacterium-containing vacuole (MCV), triggering host repair responses that preserve an intracellular permissive niche before bacteria escape to the cytosol at later stages. How the MCV transitions from repairable injury to catastrophic rupture remains poorly understood. Here, we dissected the respective contributions of the ESX-1 effectors EsxA/EsxB and the cell-envelope lipid phthiocerol dimycocerosate (PDIM) during Mycobacterium marinum infection of the amoeba Dictyostelium discoideum. Combining host reporters for membrane damage and repair with single and double bacterial mutants, we show that EsxA/EsxB and PDIM are both required for full intracellular virulence but act at distinct stages of MCV damage progression. Loss of EsxA/EsxB strongly reduced recruitment of ESCRT and autophagy reporters to the MCV, demonstrating that EsxA initiates repairable membrane lesions. In contrast, PDIM-deficient bacteria retained the ability to recruit early damage reporters and ESCRT machinery but showed reduced autophagy-associated repair, failed to efficiently acquire cytosolic perilipin coating, and remained largely confined within the MCV. Genetic disruption of host autophagy restored cytosolic access and intracellular growth of PDIM-deficient bacteria, indicating that PDIM is specifically required to overcome host repair capacity rather than to initiate damage. Importantly, the sequential requirement for EsxA and PDIM was conserved during infection of murine microglial BV-2 cells. Remarkably, PDIM-defective mutants induced lysenin recruitment, a reporter of sphingomyelin exposure, but progression to autophagy engagement was strongly decreased. Together, our results support a two-step model in which EsxA initiates MCV membrane damage, while PDIM amplifies these lesions towards catastrophic rupture, enabling escape to the cytosol and dissemination.

microbiology↗

bioRxiv2026

PefA, an ALG-2-like Ca2+ sensor, regulates ESCRT and autophagy responses to mycobacterial vacuole damage

Calcium influx is a universal early signal triggering membrane repair pathways, yet how Ca2+ sensors coordinate the balance between ESCRT-mediated sealing and autophagy-based responses at damaged endolysosomes and pathogen-containing vacuoles remains unclear. Here we use the Dictyostelium discoideum-Mycobacterium marinum infection model, a surrogate to study intracellular pathogenesis of Mycobacterium tuberculosis, combined with genetic, imaging, and proteomic analyses to identify the penta-EF-hand protein PefA, an ALG-2-like Ca2+ sensor, as a Ca2+-responsive regulator that orchestrates recruitment of the E3 ubiquitin ligase TrafE, ESCRT components, and the autophagy machinery to damaged membranes. PefA is transcriptionally upregulated and accumulates at the mycobacterial vacuole, promoting timely repair that preserves vacuolar integrity and supports bacterial replication. Loss of PefA impairs ESCRT and autophagy engagement, leading to premature bacterial escape into the cytosol and altered infection outcomes. These findings uncover a conserved Ca2+-dependent mechanism linking membrane damage sensing to coordinated repair pathways and shapes host-pathogen interactions, with direct relevance to tuberculosis pathogenesis and host resilience to infection. TeaserThe PefA calcium sensor times membrane repair and autophagy to control vacuole integrity during mycobacterial infection.

microbiology↗

bioRxiv2024

An evolutionary-conserved VPS34-PIKfyve-TRPML1-Myosin II axis regulates the speed of amoeboid cell migration

Amoeboid cell migration is key to efficient T cell immunity. Spatial polarization of organelles within cells, including endo-lysosomes, is a prerequisite of migration. However, how ultrastructural polarization is linked to the signaling requirements governing T cell migration, remains unknown. Here we show that signaling molecules generated by endo-lysosome-localized kinases regulate velocity of amoeboid migration. Specifically, imaging of T cells identified accumulation of endo-lysosomes decorated with the lipid kinases VPS34-PIKfyve at the uropod of polarized cells. Activity of VPS34 and PIKfyve regulated speed, but not directedness, of migrating T cells. Mechanistically, PI(3,5)P2 generated by the sequential action of VPS34 and PIKfyve mediated Ca2+ efflux from lysosomes via the mucolipin TRP cation channel 1 (TRPML1), thus controlling activity of myosin IIA and hence the generation of propulsive force through retrograde actin flow. The VPS34-PIKfyve kinases also regulated velocity of myeloid cells, as well as of the amoeba Dictyostelium discoideum - establishing the axis as an evolutionary conserved speed control system of amoeboid cell migration. Graphical AbstractO_LIThe VPS34-PIKfyve axis is active on endo-lysosomes at the uropod of migrating T cells. C_LIO_LIVPS34 and PIKfyve promote myosin IIA activation and retrograde action flow. C_LIO_LIAmoeboid cell migration speed is controlled by VPS34 and PIKfyve via TRPML1. C_LIO_LIRegulation of amoeboid migration speed is a conserved function of the VPS34-PIKfyve axis. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/575998v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1b70e64org.highwire.dtl.DTLVardef@111d052org.highwire.dtl.DTLVardef@83bc24org.highwire.dtl.DTLVardef@b51931_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Dual control of host actin polymerization by a Legionella effector pair

Host actin cytoskeleton is often targeted by pathogenic bacteria through the secretion of effectors. Legionella pneumophila virulence relies on the injection of the largest known arsenal of bacterial proteins, over 300 Dot/Icm Type 4 Secretion System effectors, into the host cytosol. Here we define the functional interactions between VipA and LegK2, two effectors with antagonistic activities towards actin polymerization that have been proposed to interfere with the endosomal pathway. We confirmed the prominent role of LegK2 effector in Legionella infection, as the deletion of legK2 results in defects in the inhibition of actin polymerization at the Legionella Containing Vacuole, as well as in endosomal escape of bacteria and subsequent intracellular replication. More importantly, we observed the restoration of the{Delta} legK2 mutant defects, upon deletion of vipA gene, making LegK2/VipA the first example of effector-effector suppression pair that targets the actin cytoskeleton and whose functional interaction impacts L. pneumophila virulence. We demonstrated that LegK2 and VipA do not modulate each others activity in a metaeffector relationship. Instead, the antagonistic activities of the LegK2/VipA effector pair would target different substrates, Arp2/3 for LegK2 and G-actin for VipA, to temporally control actin polymerization at the LCV and interfere with phagosome maturation and endosome recycling, thus contributing to the intracellular life cycle of the bacterium. Strikingly, the functional interaction between LegK2 and VipA is consolidated by an evolutionary history that has refined the best effector repertoire for the benefit of L. pneumophila virulence.

microbiology↗

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↗