Mechanisms protecting host cells against bacterial pore-forming toxins

Abstract

Pore-forming toxins (PFTs) are key virulence determinants produced and secreted by a variety of human bacterial pathogens. They disrupt the plasma membrane (PM) by generating stable protein pores, which allow uncontrolled exchanges between the extracellular and intracellular milieus, dramatically disturbing cellular homeostasis. In recent years, many advances were made regarding the characterization of conserved repair mechanisms that allow eukaryotic cells to recover from mechanical disruption of the PM membrane. However, the specificities of the cell recovery pathways that protect host cells against PFT-induced damage remain remarkably elusive. During bacterial infections, the coordinated action of such cell recovery processes defines the outcome of infected cells and is, thus, critical for our understanding of bacterial pathogenesis. Here, we review the cellular pathways reported to be involved in the response to bacterial PFTs and discuss their impact in single-cell recovery and infection.

Keywords: Actomyosin remodeling; Blebbing; Cholesterol-dependent cytolysins; Host signaling; Plasma membrane damage; Plasma membrane repair; Pore-forming toxins; Shedding.

Fig. 1

Proposed overview model depicting calcium-dependent PM repair mechanisms that protect host cells against PFTs. (1) Toxin oligomerization, pore formation, and calcium influx initiate the activation of calcium-dependent protective events. (2) Annexins are recruited to damaged areas according to their differential calcium sensitivity (gray scale) and assemble into 2D protein arrays to clog PM pores. (3) PM blebbing and shedding occur at damaged sites and involve recruitment of ESCRT subunits, which facilitate budding and ATP-dependent release of PM vesicles containing PFT pores, annexins, and ESCRT components. (4) In parallel, calcium influx triggers PM docking and exocytosis of cortical lysosomes. Upon assembly of SNARE complexes, the calcium sensor Synaptotagmin VII (Syt-VII) enables fusion of lysosomes with the PM and release of lysosomal enzymes, in particular ASM. (5) ASM hydrolyses PM sphingomyelin, producing ceramide domains, which facilitate membrane invagination and endocytosis of PFTs’ pores and incomplete pore structures. Ceramide domains may also contribute to annexin recruitment. (6) PFTs traffic to MVBs through ESCRT-dependent sorting and are degraded via MVB–lysosomal fusion. Toxins may also be recycled back to the PM and further secreted

Fig. 2

Proposed model for potassium-dependent host-protective mechanisms against PFT intoxication. (1) Potassium efflux and release of ATP occur across PFT-assembled pores. Extracellular ATP activates the P₂X₇ receptor and cation channel, triggering further potassium efflux and calcium influx. These events activate several recovery processes that include: (2) PM translocation of lysosomal ASM, subsequent remodeling of PM lipid composition, and release of phosphatidylserine (PS)-enriched PM vesicles. This process may enable the secretion of cytokines and occurs downstream activation of p38. (3) Inflammasome activation, caspase-1 processing and activation of IL-1 beta secretion. Caspase-1 activation increases lipid metabolism and membrane biogenesis pathways. (4) Potassium efflux also activates MAPK signaling, in particular p38 and JNK, which further control the UPR and protective transcriptional responses required for survival against PFTs

Fig. 3

Proposed model illustrating the protective mechanisms of actomyosin remodeling in response to PFT intoxication. (1, 2) Pore formation and the subsequent calcium influx induce cortical actomyosin remodeling by: disassembling actomyosin structures; activating the GTPases Rac1 and RhoA, and enhancing calpain activity which breaks cytoskeletal–PM contacts and disrupts interactions between actin and actin-binding proteins. Cortical lysosomal positioning is maintained by interactions between Rab3A and NMIIA. The rise in cytosolic calcium activates Rab3 and promotes actin remodeling contributing to binding, docking, and fusion of cortical lysosomes with the PM. (3) Actomyosin remodeling and lysosome secretion lower PM tension and modify the PM lipid composition causing PM blebbing, ruffling, and shedding or internalization of PFT pores. Actomyosin reorganization is regulated by RhoA and Rac1 and is stabilized by the formation of NMIIA cortical bundles. Concomitant ER expansion and ER–cytoskeletal interactions also contribute to stabilize the cortical actomyosin network. (4) Following shedding or internalization of PFT pores, cells re-establish cytoskeletal organization and recover normal cytosolic calcium levels

Fig. 4

Schematic representation of shedding of large cytoplasm-containing blebs or extrusions, and thinning of epithelia damaged by PFTs. (1) The ion imbalance generated by pore formation promotes apical actomyosin remodeling and vesicle secretion. (2) Both processes lower PM tension contributing to PM blebbing, remodeling, and shedding. The cytosolic ion imbalance alters organelle dynamics and causes organelle damage, including: lipid-droplet formation, mitochondria fission and enlargement, ER expansion and vacuolation, lysosomal secretion and rupture. (3) Damaged organelles are detected in the proximity of the PM and within cytoplasmic extrusions or large cell particles (e.g., blebs and villi) released by intoxicated cells. (4) Epithelial integrity and cellular homeostasis are maintained by transient (~ h) contraction and thinning of the epithelial actomyosin network and removal of damaged organelles via autophagy. Toxin pores are eliminated by PM shedding, endocytosis, and autophagic targeting

Fig. 5

Model illustrating how the main host-protective responses to PFT intoxication influence the outcome of bacterial infections. (1) Lysosome exocytosis and actomyosin remodeling promote the secretion of lysosomal enzymes that alter PM lipid composition and enable activation of endocytic pathways which allow pathogen internalization. (2) The release of hydrolytic enzymes contributes for pathogen killing. (3) Alterations in PM tension caused by PFT-induced damage promote the formation and release of PM protrusions and/or blebs that allow the dissemination of L. monocytogenes in enclosed vesicles or allow the shedding of intracellular bacteria. Released bacteria-containing vesicles can also be subsequently engulfed and killed by recruited phagocytes (efferocytosis). (4) Large PM blebs may sustain the replication of intracellular P. aeruginosa. (5) Autophagy targets PFT-producing bacteria in the host cytosol, upon vacuolar escape, and either promotes pathogen killing or the formation of SLAPs, a niche for L. monocytogenes replication