Biology subjects

Gueho, A.

Publications and source records attributed to Gueho, A..

7 recordsLinked to original sources

bioRxiv2024

Membrane microdomains are crucial for Mycobacterium marinum EsxA-dependent membrane damage, escape to the cytosol and infection

Infection by pathogenic mycobacteria such as Mycobacterium tuberculosis disrupts the membrane of the Mycobacterium-Containing Vacuole (MCV). The key effector EsxA, secreted via the ESX-1 type-VII system, is pivotal in this process, yet its membranolytic activity is not fully elucidated. Infecting the amoeba Dictyostelium discoideum with Mycobacterium marinum, we demonstrate that the composition of the MCV membrane, notably its sterol-rich microdomains, significantly influences damage and rupture. Disruption of these microdomains through the knockout of organizing proteins, termed vacuolins, or through sterol depletion, markedly diminishes M. marinum-induced membrane damage and cytosolic escape, thereby increasing cellular resistance to infection. Furthermore, we establish that vacuolins and sterols are essential for the in vitro partitioning of EsxA within membranes. Extending our findings to mammalian cells, we show that the role of microdomain organizers and sterols is evolutionarily conserved; specifically, flotillin knockdown and sterol depletion enhance the resistance of murine microglial cells to M. marinum infection. Our results underscore the critical role of host membrane microdomains in facilitating mycobacterial membranolytic activity and subsequent cytosolic access, pivotal for a successful infection.

cell biology↗

bioRxiv2023

Evolutionarily conserved ovarian fluid proteins are responsible for extending egg viability in salmonid fish

In contrast to most fish species, salmonids exhibit the unique ability to hold their eggs for several days after ovulation without significant loss of viability. During this period, eggs are held in the body cavity in a biological fluid, the coelomic fluid (CF) that is responsible for preserving egg viability. To identify CF proteins responsible for preserving egg viability, a proteomic comparison was performed using 3 salmonid species and 3 non-salmonid species to identify salmonid-specific highly abundant proteins. In parallel, rainbow trout CF fractions were purified and used in a biological test to estimate their egg viability preservation potential. The most biologically active CF fractions were then subjected to mass spectrometry analysis. We identified 50 CF proteins that are overabundant in a salmonid-specific manner and present in analytical fractions exhibiting the highest egg viability preservation potential. Here we show that salmonid CF is a complex biological fluid and that several proteins are responsible for preserving egg viability. Among these key players are proteins related to immunity, calcium binding, lipid metabolism, proteolysis, extracellular matrix and sialic acid metabolic pathway.

evolutionary biology↗

bioRxiv2022

A PI(3,5)P2 probe reveals PIKfyve is required for Rab7 acquisition and the delivery and fusion of early macropinosomes to phagosomes

Phagosome maturation is tightly regulated to ensure efficient delivery of the complex arsenal of antimicrobial activities that kill and digest captured microbes. Like other endocytic pathways, phagosome maturation is regulated by a combination of Rab GTPases and phosphoinositide signalling lipids (PIPs) which define membrane identity and recruit specific effectors. PIKfyve is a PI-5 kinase, which converts PI(3)P to PI(3,5)P2 on endosomes. Disruption of PIKfyve results in severe defects in phagosomal maturation but the underlying mechanism remains unclear. Here, we use the model professional phagocyte, Dictyostelium discoideum to dissect the role of PIKfyve in the crucial first steps of phagosome maturation. We find that, although early Rab5 dynamics are unaffected, loss of PIKfyve prevents phagosomes from acquiring Rab7 by fusion with a pool of Rab7 and V-ATPase positive endosomes. By following PIP dynamics using our recently characterised PI(3,5)P2-probe SnxA, we delineate multiple subpopulations of Rab7-positive endosomes that fuse sequentially with phagosomes. We identify one of these as PI(3,5)P2-positive macropinosomes, which dock and fuse with phagosomes in a PIKfyve-dependent manner. We therefore show that Dictyostelium phagosomes primarily accumulate Rab7 by vesicular fusion rather than from a cytosolic pool, and that this requires PIKfyve. In particular PI(3,5)P2 defines a specific subset of fusogenic macropinosomes, which we propose enables content mixing and the efficient bulk delivery of lysosomal components to phagosomes.

cell biology↗

bioRxiv2021

Disruption of vacuolin microdomains in the host Dictyostelium discoideum increases resistance to Mycobacterium marinum-induced membrane damage and infection

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, manipulates the host phagosome maturation pathway to replicate intracellularly. Mycobacterium marinum, a closely-related species, and Dictyostelium discoideum, a social amoeba and alternative phagocytic host, have been used as models to study host-pathogen interactions occurring during mycobacterial infections. Vacuolins, functional homologues of the mammalian flotillins, organize membrane microdomains and play a role in vesicular trafficking. Various pathogens have been reported to manipulate their membrane association and function. During infection of D. discoideum with M. marinum, Vacuolin C was specifically and highly induced and all three vacuolin isoforms were enriched at the mycobacteria-containing-vacuole (MCV). In addition, absence of vacuolins reduced escape from the MCV and conferred resistance to M. marinum infection. Moreover, ESAT-6, the membrane-disrupting virulence factor of M. marinum, was less associated with membranes when vacuolins were absent. Together, these results suggest that vacuolins are important host factors that are manipulated by mycobacteria to inflict membrane damage and escape from their compartment.

microbiology↗

bioRxiv2019

Co-ordinated Ras and Rac activity shapes macropinocytic cups and enables phagocytosis of geometrically diverse bacteria

Engulfment of extracellular material by phagocytosis or macropinocytosis depends on the ability of cells to generate specialised cup shaped protrusions. To effectively capture and internalise their targets, these cups are organised into a ring or ruffle of actin-driven protrusion encircling a non-protrusive interior domain. These functional domains depend on the combined activities of multiple Ras and Rho family small GTPases, but how their activities are integrated and differentially regulated over space and time is unknown. Here, we show that the amoeba Dictyostelium discoideum coordinates Ras and Rac activity using the multidomain protein RGBARG (RCC1, RhoGEF, BAR and RasGAP-containing protein). We find RGBARG uses a tripartite mechanism of Ras, Rac and phospholipid interactions to localise at the protruding edge and interface with the interior of both macropinocytic and phagocytic cups. There, RGBARG shapes the protrusion by driving Rac activation at the rim whilst suppressing expansion of the active Ras interior domain. Consequently, cells lacking RGBARG form enlarged, flat interior domains unable to generate large macropinosomes. During phagocytosis, we find that disruption of RGBARG causes a geometry-specific defect in engulfing rod-shaped bacteria and ellipsoidal beads. This demonstrates the importance of co-ordinating small GTPase activities during engulfment of more complex shapes and thus the full physiological range of microbes, and how this is achieved in a model professional phagocyte.

cell biology↗

bioRxiv2019

Proteomic characterization of the Mycobacterium marinum-containing vacuole in Dictyostelium discoideum

Mycobacterium tuberculosis, the causative agent of tuberculosis, is able to manipulate the phagosome compartment in which it resides in order to establish a permissive replicative compartment called the Mycobacterium-containing vacuole (MCV). Mycobacterium marinum, a fish pathogen and a close relative of the tuberculosis group, is able to infect the free-living amoeba and professional phagocyte Dictyostelium discoideum and to manipulate its phagosome maturation. By using this host-pathogen model system, we have established an innovative process to isolate MCVs. This procedure allowed us to isolate the M. marinum-MCV at 1, 3 and 6 hours post infection to study the early M. marinum-MCV proteome. By using isobaric labelling and mass spectrometry, we quantitatively compared the proteomic composition of those MCVs isolated at different stages of the early infection phase to understand how M. marinum impacts on this compartment to divert it from the normal phagosomal pathway. Furthermore, we also compared the manipulated compartment M. marinum-MCV to non- or less manipulated compartments containing different mycobacteria strains: the non-pathogenic M. smegmatis, the avirulent M. marinum-L1D or the attenuated M. marinum-RD1.

systems biology↗

bioRxiv2018

PIKfyve/Fab1 is required for efficient V-ATPase delivery to phagosomes, phagosomal killing, and restriction of Legionella infection

By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum.\n\nPIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5)2) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation in Dictyostelium. Disruption of PIKfyve inhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently, PIKfyve- cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lacking PIKfyve are more susceptible to infection by the intracellular pathogen Legionella pneumophila. We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes.\n\nImportanceCells that capture or eat bacteria must swiftly kill them to prevent pathogens from surviving long enough to escape the bactericidal pathway and establish an infection. This is achieved by the rapid delivery of components that produce an antimicrobial environment in the phagosome, the compartment containing the captured microbe. This is essential both for the function of immune cells and for amoebae that feed on bacteria in their environment. Here we identify a central component of the pathway used by cells to deliver antimicrobial components to the phagosome and show that bacteria survive over three times as long within the host if this pathway is disabled. We show that this is of general importance for killing a wide range of pathogenic and non-pathogenic bacteria, and that it is physiologically important if cells are to avoid infection by the opportunistic human pathogen Legionella.

cell biology↗