Src-Dependent NM2A Tyrosine Phosphorylation Regulates Actomyosin Remodeling.

Non-muscle myosin 2A (NM2A) is a key cytoskeletal enzyme that, along with actin, assembles into actomyosin filaments inside cells. NM2A is fundamental for cell adhesion and motility, playing important functions in different stages of development and during the progression of viral and bacterial infections. Phosphorylation events regulate the activity and the cellular localization of NM2A. We previously identified the tyrosine phosphorylation of residue 158 (pTyr158) in the motor domain of the NM2A heavy chain. This phosphorylation can be promoted by Listeria monocytogenes infection of epithelial cells and is dependent on Src kinase; however, its molecular role is unknown. Here, we show that the status of pTyr158 defines cytoskeletal organization, affects the assembly/disassembly of focal adhesions, and interferes with cell migration. Cells overexpressing a non-phosphorylatable NM2A variant or expressing reduced levels of Src kinase display increased stress fibers and larger focal adhesions, suggesting an altered contraction status consistent with the increased NM2A activity that we also observed. We propose NM2A pTyr158 as a novel layer of regulation of actomyosin cytoskeleton organization.

LAMP2 regulates autophagy in the thymic epithelium and thymic stroma-dependent CD4 T cell development.

Within the thymus, thymic epithelial cells (TECs) provide dedicated thymic stroma microenvironments for T cell development. Because TEC functionality is sensitive to aging and cytoablative therapies, unraveling the molecular elements that coordinate their thymopoietic role has fundamental and clinical implications. Particularly, the selection of CD4 T cells depends on interactions between TCRs expressed on T cell precursors and self-peptides:MHC II complexes presented by cortical TECs (cTECs). Although the macroautophagy/autophagy-lysosomal protein degradation pathway is implicated in CD4 T cell selection, the molecular mechanism that controls the generation of selecting MHC II ligands remains elusive. LAMP2 (lysosomal-associated membrane protein 2) is a well-recognized mediator of autolysosome (AL) maturation. We showed that LAMP2 is highly expressed in cTECs. Notably, genetic inactivation of Lamp2 in thymic stromal cells specifically impaired the development of CD4 T cells that completed positive selection, without misdirecting MHC II-restricted cells into the CD8 lineage. Mechanistically, defects in autophagy in lamp2-deficient cTECs were linked to alterations in MHC II processing, which was associated with a marked reduction in CD4 TCR repertoire diversity selected within the lamp2-deficient thymic stroma. Together, our findings suggest that LAMP2 interconnects the autophagy-lysosomal axis and the processing of selecting self-peptides:MHC II complexes in cTECs, underling its implications for the generation of a broad CD4 TCR repertoire.Abbreviations: AIRE: autoimmune regulator (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy); AL: autolysosome; AP: autophagosome; Baf-A1: bafilomycin A1; B2M: beta-2 microglobulin; CTSL: cathepsin L; CD74/Ii: CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated); CFSE: carboxyfluorescein succinimidyl ester; CFU: colony-forming unit; CLIP: class II-associated invariant chain peptides; cTECs: cortical TECs dKO: double knockout; DN: double negative; DP: double positive; ENPEP/LY51: glutamyl aminopeptidase; FOXP3: forkhead box; P3 IFNG/IFNγ: interferon gamma; IKZF2/HELIOS: IKAROS family zinc finger 2; IL2RA/CD25: interleukin 2 receptor, alpha chain; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; LIP: lymphopenia-induced proliferation; Lm: Listeria monocytogenes; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MHC: major histocompatibility complex; mTECs: medullary TECs; PRSS16/TSSP: protease, serine 16 (thymus); SELL/CD62L: selectin, lymphocyte; SP: single positive; TCR: T cell receptor; TCRB: T cell receptor beta chain; TECs: thymic epithelial cells; UEA-1: Ulex europaeus agglutinin-1; WT: wild-type.

Detection of SARS-CoV-2 infection in thyroid follicular cells from a COVID-19 autopsy series.

Objective: To understand whether thyroid cells can be directly infected by the SARS-CoV-2 virus and to establish a putative correlation with the expression of the host entry machinery: ACE-2, TMPRSS2, and furin. Methods: We assessed the presence of SARS-CoV-2 virus at the gene level by RT-PCR, viral RNA transcripts localization by in situ hybridization, and by detecting viral proteins by immunohistochemistry for the nucleocapsid and the spike proteins. Furthermore, we also described the immunoexpression of key host factors for virus entry in the COVID-19 thyroid samples. Results: We performed RT-PCR for SARS-CoV-2 in all autopsy specimens and detected viral genome positivity in 13 of 15 thyroid tissues and in a lung specimen. In 9 of the 14 positive samples, we were also able to confirm SARS-CoV-2 signal by in situ hybridization. Immunohistochemistry for the viral nucleocapsid and spike protein was also positive for ten and nine of the RT-PCR-positive cases, respectively, but revealed a lower sensitivity. We also described, for the first time in a COVID-19 series, the immunohistochemical expression of ACE-2, TMPRSS2, and furin in the thyroid. Conclusions: Our results obtained in thyroid specimens from deceased COVID-19 patients indicate that thyrocytes can be directly infected by SARS-CoV-2 since we detected the presence of SARS-CoV-2 genome in follicular cells. Nevertheless, we did not find a clear correlation between the presence of viral genome and the expression of the host factors for virus entry, namely ACE-2, TMPRSS2, and furin.

Searching for SARS-CoV-2 in Cancer Tissues: Results of an Extensive Methodologic Approach based on ACE2 and Furin Expression.

SARS-CoV-2 pandemics have been massively characterized on a global scale by the rapid generation of in-depth genomic information. The main entry gate of SARS-CoV-2 in human cells is the angiotensin-converting enzyme 2 (ACE2) receptor. The expression of this protein has been reported in several human tissues, suggesting a correlation between SARS-CoV-2 organotropism and ACE2 distribution. In this study, we selected (a series of) 90 patients who were submitted to surgery for tumor removal between the beginning of the SARS-CoV-2 pandemic and the closure of operating rooms (by the end of March 2020) in two different countries-Portugal and Brazil. We evaluated the expressions of ACE2 and furin (another important factor for virus internalization) in colon (n = 60), gastric (n = 19), and thyroid (n = 11) carcinomas. In a subseries of cases with PCR results for SARS-CoV-2 detection in the peri-operatory window (n = 18), we performed different methodological approaches for viral detections in patient tumor samples. Our results show that colon and gastric carcinomas display favorable microenvironments to SARS-CoV-2 tropism, presenting high expression levels of ACE2 and furin. From the subseries of 18 cases, 11 tested positive via PCR detection performed in tumor blocks; however, a direct association between the ACE2 expression and SARS-CoV-2 infection was not demonstrated in cancer cells using histology-based techniques, such as immunohistochemistry or in situ hybridization. This study raises the possibility of ACE2-mediated viral tropism in cancer tissues to be clarified in future studies.

Encapsulation of the septal cell wall protects Streptococcus pneumoniae from its major peptidoglycan hydrolase and host defenses.

Synthesis of the capsular polysaccharide, a major virulence factor for many pathogenic bacteria, is required for bacterial survival within the infected host. In Streptococcus pneumoniae, Wze, an autophosphorylating tyrosine kinase, and Wzd, a membrane protein required for Wze autophosphorylation, co-localize at the division septum and guarantee the presence of capsule at this subcellular location. To determine how bacteria regulate capsule synthesis, we studied pneumococcal proteins that interact with Wzd and Wze using bacterial two hybrid assays and fluorescence microscopy. We found that Wzd interacts with Wzg, the putative ligase that attaches capsule to the bacterial cell wall, and recruits it to the septal area. This interaction required residue V56 of Wzd and both the transmembrane regions and DNA-PPF domain of Wzg. When compared to the wild type, Wzd null pneumococci lack capsule at midcell, bind the peptidoglycan hydrolase LytA better and are more susceptible to LytA-induced lysis, and are less virulent in a zebrafish embryo infection model. In this manuscript, we propose that the Wzd/Wze pair guarantees full encapsulation of pneumococcal bacteria by recruiting Wzg to the division septum, ensuring that capsule attachment is coordinated with peptidoglycan synthesis. Impairing the encapsulation process, at localized subcellular sites, may facilitate elimination of bacteria by strategies that target the pneumococcal peptidoglycan.

Cells Responding to Closely Related Cholesterol-Dependent Cytolysins Release Extracellular Vesicles with a Common Proteomic Content Including Membrane Repair Proteins.

The plasma membrane (PM) protects cells from extracellular threats and supports cellular homeostasis. Some pathogens produce pore-forming toxins (PFTs) that disrupt PM integrity by forming transmembrane pores. High PFT concentrations cause massive damage leading to cell death and facilitating infection. Sub-lytic PFT doses activate repair mechanisms to restore PM integrity, support cell survival and limit disease. Shedding of extracellular vesicles (EVs) has been proposed as a key mechanism to eliminate PFT pores and restore PM integrity. We show here that cholesterol-dependent cytolysins (CDCs), a specific family of PFTs, are at least partially eliminated through EVs release, and we hypothesize that proteins important for PM repair might be included in EVs shed by cells during repair. To identify new PM repair proteins, we collected EVs released by cells challenged with sub-lytic doses of two different bacterial CDCs, listeriolysin O and pneumolysin, and determined the EV proteomic repertoire by LC-MS/MS. Intoxicated cells release similar EVs irrespectively of the CDC used. Also, they release more and larger EVs than non-intoxicated cells. A cluster of 70 proteins including calcium-binding proteins, molecular chaperones, cytoskeletal, scaffold and membrane trafficking proteins, was detected enriched in EVs collected from intoxicated cells. While some of these proteins have well-characterized roles in repair, the involvement of others requires further study. As proof of concept, we show here that Copine-1 and Copine-3, proteins abundantly detected in EVs released by intoxicated cells, are required for efficient repair of CDC-induced PM damage. Additionally, we reveal here new proteins potentially involved in PM repair and give new insights into common mechanisms and machinery engaged by cells in response to PM damage.

Stabilin-1 plays a protective role against Listeria monocytogenes infection through the regulation of cytokine and chemokine production and immune cell recruitment.

Scavenger receptors are part of a complex surveillance system expressed by host cells to efficiently orchestrate innate immune response against bacterial infections. Stabilin-1 (STAB-1) is a scavenger receptor involved in cell trafficking, inflammation, and cancer; however, its role in infection remains to be elucidated. Listeria monocytogenes (Lm) is a major intracellular human food-borne pathogen causing severe infections in susceptible hosts. Using a mouse model of infection, we demonstrate here that STAB-1 controls Lm-induced cytokine and chemokine production and immune cell accumulation in Lm-infected organs. We show that STAB-1 also regulates the recruitment of myeloid cells in response to Lm infection and contributes to clear circulating bacteria. In addition, whereas STAB-1 appears to promote bacterial uptake by macrophages, infection by pathogenic Listeria induces the down regulation of STAB-1 expression and its delocalization from the host cell membrane.We propose STAB-1 as a new SR involved in the control of Lm infection through the regulation of host defense mechanisms, a process that would be targeted by bacterial virulence factors to promote infection.

Listeria monocytogenes Interferes with Host Cell Mitosis through Its Virulence Factors InlC and ActA.

Listeria monocytogenes is among the best-characterized intracellular pathogens. Its virulence factors, and the way they interfere with host cells to hijack host functions and promote the establishment and dissemination of the infection, have been the focus of multiple studies over the last 30 years. During cellular infection, L. monocytogenes was shown to induce host DNA damage and delay the host cell cycle to its own benefit. However, whether the cell cycle stage would interfere with the capacity of Listeria to infect human cultured cell lines was never assessed. We found here that L. monocytogenes preferentially infects cultured cells in G2/M phases. Inside G2/M cells, the bacteria lead to an increase in the overall mitosis duration by delaying the mitotic exit. We showed that L. monocytogenes infection causes a sustained activation of the spindle assembly checkpoint, which we correlated with the increase in the percentage of misaligned chromosomes detected in infected cells. Moreover, we demonstrated that chromosome misalignment in Listeria-infected cells required the function of two Listeria virulence factors, ActA and InlC. Our findings show the pleiotropic role of Listeria virulence factors and their cooperative action in successfully establishing the cellular infection.

Listeria monocytogenes Wall Teichoic Acid Glycosylation Promotes Surface Anchoring of Virulence Factors, Resistance to Antimicrobial Peptides, and Decreased Susceptibility to Antibiotics.

The cell wall of Listeria monocytogenes (Lm), a major intracellular foodborne bacterial pathogen, comprises a thick peptidoglycan layer that serves as a scaffold for glycopolymers such as wall teichoic acids (WTAs). WTAs contain non-essential sugar substituents whose absence prevents bacteriophage binding and impacts antigenicity, sensitivity to antimicrobials, and virulence. Here, we demonstrated, for the first time, the triple function of Lm WTA glycosylations in the following: (1) supporting the correct anchoring of major Lm virulence factors at the bacterial surface, namely Ami and InlB; (2) promoting Lm resistance to antimicrobial peptides (AMPs); and (3) decreasing Lm sensitivity to some antibiotics. We showed that while the decoration of WTAs by rhamnose in Lm serovar 1/2a and by galactose in serovar 4b are important for the surface anchoring of Ami and InlB, N-acetylglucosamine in serovar 1/2a and glucose in serovar 4b are dispensable for the surface association of InlB or InlB/Ami. We found that the absence of a single glycosylation only had a slight impact on the sensibility of Lm to AMPs and antibiotics, however the concomitant deficiency of both glycosylations (rhamnose and N-acetylglucosamine in serovar 1/2a, and galactose and glucose in serovar 4b) significantly impaired the Lm capacity to overcome the action of antimicrobials. We propose WTA glycosylation as a broad mechanism used by Lm, not only to properly anchor surface virulence factors, but also to resist AMPs and antibiotics. WTA glycosyltransferases thus emerge as promising drug targets to attenuate the virulence of bacterial pathogens, while increasing their susceptibility to host immune defenses and potentiating the action of antibiotics.

Virulence gene repression promotes Listeria monocytogenes systemic infection.

The capacity of bacterial pathogens to infect their hosts depends on the tight spatiotemporal regulation of virulence genes. The Listeria monocytogenes (Lm) metal efflux pump repressor CadC is highly expressed during late infection stages, modulating lipoprotein processing and host immune response. Here we investigate the potential of CadC as broad repressor of virulence genes. We show that CadC represses the expression of the bile salt hydrolase impairing Lm resistance to bile. During late infection, in absence of CadC-dependent repression, the constitutive bile salt hydrolase expression induces the overexpression of the cholic acid efflux pump MdrT that is unfavorable to Lm virulence. We establish the CadC regulon and show that CadC represses additional virulence factors activated by σB during colonization of the intestinal lumen. CadC is thus a general repressor that promotes Lm virulence by down-regulating, at late infection stages, genes required for survival in the gastrointestinal tract. This demonstrates for the first time how bacterial pathogens can repurpose regulators to spatiotemporally repress virulence genes and optimize their infectious capacity.

Phage resistance at the cost of virulence: Listeria monocytogenes serovar 4b requires galactosylated teichoic acids for InlB-mediated invasion.

The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen.

Perfringolysin O-Induced Plasma Membrane Pores Trigger Actomyosin Remodeling and Endoplasmic Reticulum Redistribution.

Clostridium perfringens produces an arsenal of toxins that act together to cause severe infections in humans and livestock animals. Perfringolysin O (PFO) is a cholesterol-dependent pore-forming toxin encoded in the chromosome of virtually all C. perfringens strains and acts in synergy with other toxins to determine the outcome of the infection. However, its individual contribution to the disease is poorly understood. Here, we intoxicated human epithelial and endothelial cells with purified PFO to evaluate the host cytoskeletal responses to PFO-induced damage. We found that, at sub-lytic concentrations, PFO induces a profound reorganization of the actomyosin cytoskeleton culminating into the assembly of well-defined cortical actomyosin structures at sites of plasma membrane (PM) remodeling. The assembly of such structures occurs concomitantly with the loss of the PM integrity and requires pore-formation, calcium influx, and myosin II activity. The recovery from the PM damage occurs simultaneously with the disassembly of cortical structures. PFO also targets the endoplasmic reticulum (ER) by inducing its disruption and vacuolation. ER-enriched vacuoles were detected at the cell cortex within the PFO-induced actomyosin structures. These cellular events suggest the targeting of the endothelium integrity at early stages of C. perfringens infection, in which secreted PFO is at sub-lytic concentrations.

Stathmin recruits tubulin to Listeria monocytogenes-induced actin comets and promotes bacterial dissemination.

The tubulin cytoskeleton is one of the main components of the cytoarchitecture and is involved in several cellular functions. Here, we examine the interplay between Listeria monocytogenes (Lm) and the tubulin cytoskeleton upon cellular infection. We show that non-polymeric tubulin is present throughout Lm actin comet tails and, to a less extent, in actin clouds. Moreover, we demonstrate that stathmin, a regulator of microtubule dynamics, is also found in these Lm-associated actin structures and is required for tubulin recruitment. Depletion of host stathmin results in longer comets containing less F-actin, which may be correlated with higher levels of inactive cofilin in the comet, thus suggesting a defect on local F-actin dynamics. In addition, intracellular bacterial speed is significantly reduced in stathmin-depleted cells, revealing the importance of stathmin/tubulin in intracellular Lm motility. In agreement, the area of infection foci and the total bacterial loads are also significantly reduced in stathmin-depleted cells. Collectively, our results demonstrate that stathmin promotes efficient cellular infection, possibly through tubulin recruitment and control of actin dynamics at Lm-polymerized actin structures.

Mechanisms protecting host cells against bacterial pore-forming toxins.

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.

Scavenger Receptors: Promiscuous Players during Microbial Pathogenesis.

Innate immunity is the most broadly effective host defense, being essential to clear the majority of microbial infections. Scavenger Receptors comprise a family of sensors expressed in a multitude of host cells, whose dual role during microbial pathogenesis gained importance over recent years. SRs regulate the recruitment of immune cells and control both host inflammatory response and bacterial load. In turn, pathogens have evolved different strategies to overcome immune response, avoid recognition by SRs and exploit them to favor infection. Here, we discuss the most relevant findings regarding the interplay between SRs and pathogens, discussing how these multifunctional proteins recognize a panoply of ligands and act as bacterial phagocytic receptors.

MouR controls the expression of the Listeria monocytogenes Agr system and mediates virulence.

The foodborne pathogen Listeria monocytogenes (Lm) causes invasive infection in susceptible animals and humans. To survive and proliferate within hosts, this facultative intracellular pathogen tightly coordinates the expression of a complex regulatory network that controls the expression of virulence factors. Here, we identified and characterized MouR, a novel virulence regulator of Lm. Through RNA-seq transcriptomic analysis, we determined the MouR regulon and demonstrated how MouR positively controls the expression of the Agr quorum sensing system (agrBDCA) of Lm. The MouR three-dimensional structure revealed a dimeric DNA-binding transcription factor belonging to the VanR class of the GntR superfamily of regulatory proteins. We also showed that by directly binding to the agr promoter region, MouR ultimately modulates chitinase activity and biofilm formation. Importantly, we demonstrated by in vitro cell invasion assays and in vivo mice infections the role of MouR in Lm virulence.

l-Rhamnosylation of wall teichoic acids promotes efficient surface association of Listeria monocytogenes virulence factors InlB and Ami through interaction with GW domains.

Wall teichoic acids (WTAs) are important surface glycopolymers involved in various physiological processes occurring in the Gram-positive cell envelope. We previously showed that the decoration of Listeria monocytogenes (Lm) WTAs with l-rhamnose conferred resistance against antimicrobial peptides. Here, we show that WTA l-rhamnosylation also contributes to physiological levels of autolysis in Lm through a mechanism that requires efficient association of Ami, a virulence-promoting autolysin belonging to the GW protein family, to the bacterial cell surface. Importantly, WTA l-rhamnosylation also controls the surface association of another GW protein, the invasin internalin B (InlB), that promotes Lm invasion of host cells. Whereas WTA N-acetylglucosaminylation is not a prerequisite for GW protein surface association, lipoteichoic acids appear to also play a role in the surface anchoring of InlB. Strikingly, while the GW domains of Ami, InlB and Auto (another autolysin contributing to cell invasion and virulence) are sufficient to mediate surface association, this is not the case for the GW domains of the remaining six uncharacterized Lm GW proteins. Overall, we reveal WTA l-rhamnosylation as a bacterial surface modification mechanism that contributes to Lm physiology and pathogenesis by controlling the surface association of GW proteins involved in autolysis and infection.

Genome Sequence of Listeria monocytogenes 2542, a Serotype 4b Strain from a Cheese-Related Outbreak in Portugal.

We report here the draft genome sequence of Listeria monocytogenes 2542, a serotype 4b clinical strain recovered from a placental sample during a cheese-related listeriosis outbreak in Portugal.

Epithelial Keratins Modulate cMet Expression and Signaling and Promote InlB-Mediated Listeria monocytogenes Infection of HeLa Cells.

The host cytoskeleton is a major target for bacterial pathogens during infection. In particular, pathogens usurp the actin cytoskeleton function to strongly adhere to the host cell surface, to induce plasma membrane remodeling allowing invasion and to spread from cell to cell and disseminate to the whole organism. Keratins are cytoskeletal proteins that are the major components of intermediate filaments in epithelial cells however, their role in bacterial infection has been disregarded. Here we investigate the role of the major epithelial keratins, keratins 8 and 18 (K8 and K18), in the cellular infection by Listeria monocytogenes. We found that K8 and K18 are required for successful InlB/cMet-dependent L. monocytogenes infection, but are dispensable for InlA/E-cadherin-mediated invasion. Both K8 and K18 accumulate at InlB-mediated internalization sites following actin recruitment and modulate actin dynamics at those sites. We also reveal the key role of K8 and K18 in HGF-induced signaling which occurs downstream the activation of cMet. Strikingly, we show here that K18, and at a less extent K8, controls the expression of cMet and other surface receptors such TfR and integrin ß1, by promoting the stability of their corresponding transcripts. Together, our results reveal novel functions for major epithelial keratins in the modulation of actin dynamics at the bacterial entry sites and in the control of surface receptors mRNA stability and expression.

Control of cytoskeletal dynamics during cellular responses to pore forming toxins.

Following damage by pore forming toxins (PFTs) host cells engage repair processes and display profound cytoskeletal remodeling and concomitant plasma membrane (PM) blebbing. We have recently demonstrated that host cells utilize similar mechanisms to control cytoskeletal dynamics in response to PFTs and during cell migration. This involves assembly of cortical actomyosin bundles, reorganisation of the endoplasmic reticulum (ER) network, and the interaction between the ER chaperone Gp96 and the molecular motor Non-muscle Myosin Heavy Chain IIA (NMHCIIA). Consequently, Gp96 regulates actomyosin activity, PM blebbing and cell migration, and protects PM integrity against PFTs. In addition, we observed that PFTs increase association of Gp96 and ER vacuoles with the cell surface or within PM blebs loosely attached to the cell body. Similarly, gut epithelial cells damaged by PFTs in vivo were shown to release microvilli structures or directly purge cytoplasmic content. Cytoplasmic purging involves profound cytoskeletal remodeling and ER vacuolation, suggesting that our observations recapitulate recovery processes in vivo. Here, we discuss our findings in light of the current understanding of PM repair mechanisms and in vivo recovery responses to PFTs.