Shigella infection causes destruction of the human colonic epithelial barrier. The Golgi network and recycling endosomes are essential for maintaining epithelial barrier function. Here we show that ...Shigella epithelial invasion induces fragmentation of the Golgi complex with consequent inhibition of both secretion and retrograde transport in the infected host cell. Shigella induces tubulation of the Rab11-positive compartment, thereby affecting cell surface receptor recycling. The molecular process underlying the observed damage to the Golgi complex and receptor recycling is a massive redistribution of plasma membrane cholesterol to the sites of Shigella entry. IpaB, a virulence factor of Shigella that is known to bind cholesterol, is necessary and sufficient to induce Golgi fragmentation and reorganization of the recycling compartment. Shigella infection-induced Golgi disorganization was also observed in vivo, suggesting that this mechanism affecting the sorting of cell surface molecules likely contributes to host epithelial barrier disruption associated with Shigella pathogenesis.
► Shigella inhibits cell surface receptor recycling ► Shigella disrupts the Golgi network and its secretory function ► Cholesterol delocalization by Shigella entry underlies Golgi disruption ► The cholesterol-binding Shigella effector IpaB disrupts the Golgi network
There are several endocytic pathways, which are either dependent on or independent of clathrin. This study focuses on a poorly characterized mechanism—clathrin‐ and caveolae‐independent ...endocytosis—used by the interleukin‐2 receptor β (IL‐2Rβ). We address the question of its regulation in comparison with the clathrin‐dependent pathway. First, we show that Ras‐related C3 botulinum toxin substrate 1 (Rac1) is specifically required for IL‐2Rβ entry, and we identify p21‐activated kinases (Paks) as downstream targets. By RNA interference, we show that Pak1 and Pak2 are both necessary for IL‐2Rβ uptake, in contrast to the clathrin‐dependent route. We observe that cortactin, a partner of actin and dynamin—two essential endocytic factors—is required for IL‐2Rβ uptake. Furthermore, we find that cortactin acts downstream from Paks, suggesting control of its function by these kinases. Thus, we describe a cascade composed of Rac1, Paks and cortactin specifically regulating IL‐2Rβ internalization. This study indicates Paks as the first specific regulators of the clathrin‐independent endocytosis pathway.
Determination of protein stoichiometry in living cells is key to understanding basic biological processes. This is particularly important for receptor-mediated endocytosis, a highly regulated ...mechanism that requires the sequential assembly of numerous factors. Here, we describe a quantitative approach to analyze receptor clustering dynamics at the plasma membrane. Our workflow combines TIRF live imaging of a CRISPR-Cas9-edited cell line expressing a GFP-tagged receptor in a physiological relevant environment, a calibration technique for single-molecule analysis of GFP, and detection and tracking with an open-source software. This method allows to determine the number of receptor molecules at the plasma membrane in real time.
Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the ...cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44(+) melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins.
Growing evidence indicates that kinases are central to the regulation of endocytic pathways. Previously, we identified p21-activated kinase 1 (Pak1) as the first specific regulator of clathrin- and ...caveolae-independent endocytosis used by the interleukin 2 receptor subunit (IL-2R). Here, we address the mechanism by which Pak1 regulates IL-2Rβ endocytosis. First, we show that Pak1 phosphorylates an activator of actin polymerization, cortactin, on its serine residues 405 and 418. Consistently, we observe a specific inhibition of IL-2Rβ endocytosis when cells overexpress a cortactin, wherein these serine residues have been mutated. In addition, we show that the actin polymerization enhancer, neuronal Wiskott-Aldrich syndrome protein (N-WASP), is involved in IL-2Rβ endocytosis. Strikingly, we find that Pak1 phosphorylation of cortactin on serine residues 405 and 418 increases its association with N-WASP. Thus, Pak1, by controlling the interaction between cortactin and N-WASP, could regulate the polymerization of actin during clathrin-independent endocytosis.
The secreton (type II secretion) and type IV pilus biogenesis branches of the general secretory pathway in Gram‐negative bacteria share many features that suggest a common evolutionary origin. Five ...components of the secreton, the pseudopilins, are similar to subunits of type IV pili. Here, we report that when the 15 genes encoding the pullulanase secreton of Klebsiella oxytoca were expressed on a high copy number plasmid in Escherichia coli, one pseudopilin, PulG, was assembled into pilus‐like bundles. Assembly of the ‘secreton pilus’ required most but not all of the secreton components that are essential for pullulanase secretion, including some with no known homologues in type IV piliation machineries. Two other pseudopilins, pullulanase and two outer membrane‐associated secreton components were not associated with pili. Thus, PulG is probably the major component of the pilus. Expression of a type IV pilin gene, the E.coli K‐12 gene ppdD, led to secreton‐dependent incorporation of PpdD pilin into pili without diminishing pullulanase secretion. This is the first demonstration that pseudopilins can be assembled into pilus‐like structures.
Intestinal epithelial cells are constantly exposed to pathogens and mechanical forces. However, the impact of mechanical forces on infections leading to diarrheal diseases remains largely unknown. ...Here, we addressed whether flow and peristalsis impact the infectivity of the human pathogen Shigella within a 3D colonic epithelium using Intestine-Chip technology. Strikingly, infection is significantly increased and minimal bacterial loads are sufficient to invade enterocytes from the apical side and trigger loss of barrier integrity, thereby shifting the paradigm about early stage Shigella invasion. Shigella quickly colonizes epithelial crypt-like invaginations and demonstrates the essential role of the microenvironment. Furthermore, by modulating the mechanical forces of the microenvironment, we find that peristalsis impacts Shigella invasion. Collectively, our results reveal that Shigella leverages the intestinal microenvironment by taking advantage of the microarchitecture and mechanical forces to efficiently invade the intestine. This approach will enable molecular and mechanistic interrogation of human-restricted enteric pathogens.
Display omitted
•Intestine-Chip reveals that Shigella infects enterocytes efficiently from the gut lumen•The crypt-like structure of the intestine is critical for Shigella adhesion•Peristaltic motion (mechanical forces) enhances Shigella invasion•Shigella exploits intestine architecture and mechanical forces to maximize infectivity
Grassart et al. employ Organ-on-Chip to interrogate the impact of intestinal mechanical forces on Shigella infection. In the 3D microenvironment, Shigella infects enterocytes from the apical side, exploiting the crypt-like invaginations for early colonization, while intestinal peristalsis enhances invasion. Collectively, Organ-on-Chip reveals that Shigella leverages the intestinal microarchitecture and mechanical forces.
Recent advances in organoid and organ-on-chip (OoC) technologies offer an unprecedented level of tissue mimicry. These models can recapitulate the diversity of cellular composition, 3D organization, ...and mechanical stimulation. These approaches are intensively used to understand complex diseases. This review focuses on the latest advances in this field to study host-microorganism interactions.
SARS‐CoV‐2 infection results in impaired interferon response in patients with severe COVID‐19. However, how SARS‐CoV‐2 interferes with host immune responses is incompletely understood. Here, we ...sequence small RNAs from SARS‐CoV‐2‐infected human cells and identify a microRNA (miRNA) derived from a recently evolved region of the viral genome. We show that the virus‐derived miRNA produces two miRNA isoforms in infected cells by the enzyme Dicer, which are loaded into Argonaute proteins. Moreover, the predominant miRNA isoform targets the 3′UTR of interferon‐stimulated genes and represses their expression in a miRNA‐like fashion. Finally, the two viral miRNA isoforms were detected in nasopharyngeal swabs from COVID‐19 patients. We propose that SARS‐CoV‐2 can potentially employ a virus‐derived miRNA to hijack the host miRNA machinery, which could help to evade the interferon‐mediated immune response.
Synopsis
SARS‐CoV‐2 produces two miRNAs derived from a conserved stem‐loop structure of the ORF‐7a transcript in a Dicer‐dependent manner. These virus‐derived miRNAs interact with host Argonaute and can repress host innate immune response genes in cultured cells.
CoV2‐miR‐O7a.1 and CoV2‐miR‐O7a.2 are generated from the SARS‐CoV‐2 genome by a Dicer‐dependent cleavage of a stem‐loop structure of ORF7a.
The stem‐loop structure is under selective pressure and is conserved in various SARS‐CoV‐2 variants but not in SARS‐CoV.
CoV2‐miR‐O7a.2 can target 3′UTRs of interferon‐stimulated genes and represses their expression in a miRNA‐like fashion.
CoV2‐miR‐O7a.1 and CoV2‐miR‐O7a.2 are both detected in nasopharyngeal swabs from COVID‐19 patients.
SARS‐CoV‐2 produces two miRNAs derived from a conserved stem‐loop structure of the ORF‐7a transcript in a Dicer‐dependent manner. These virus‐derived miRNAs interact with host Argonaute and can repress host innate immune response genes in cultured cells.
PDF
