Efficient dark-fermentation biohydrogen production by Shigella flexneri SPD1 from biowaste-derived sugars through green solvents approach.

This study evaluated the dark-fermentative hydrogen (H2) production potential of isolated and identified Shigella flexneri SPD1 from various pure (glucose, fructose, sucrose, lactose, and galactose) and biowastes (coconut coir, cotton fiber, groundnut shells, rice-, and wheat-straws)-derived sugars. Among sugars, S. flexneri SPD1 exhibited high H2 production of up to 3.20 mol/mole of hexose using glucose (5.0 g/L). The pre-treatment of various biowastes using green solvents (choline chloride and lactic acid mixture) and enzymatic hydrolysis resulted in the generation of up to 36.0 g/L of sugars. The maximum H2 production is achieved up to 2.92 mol/mol of hexose using cotton-hydrolysate. Further, the upscaling of bioprocess up to 5 L of capacity resulted in a maximum yield of up to 3.06 mol/mol of hexose. These findings suggested that S. flexneri SPD1, a novel H2-producer, can be employed to develop a circular economy-based approach to produce clean energy.

An internal linker and pH biosensing by phosphatidylinositol 5-phosphate regulate the function of the ESCRT-0 component TOM1.

Target of Myb1 (TOM1) facilitates the transport of endosomal ubiquitinated proteins destined for lysosomal degradation; however, the mechanisms regulating TOM1 during this process remain unknown. Here, we identified an adjacent DXXLL motif-containing region to the TOM1 VHS domain, which enhances its affinity for ubiquitin and can be modulated by phosphorylation. TOM1 is an endosomal phosphatidylinositol 5-phosphate (PtdIns5P) effector under Shigella flexneri infection. We pinpointed a consensus PtdIns5P-binding motif in the VHS domain. We show that PtdIns5P binding by TOM1 is pH-dependent, similarly observed in its binding partner TOLLIP. Under acidic conditions, TOM1 retained its complex formation with TOLLIP, but was unable to bind ubiquitin. S. flexneri infection inhibits pH-dependent endosomal maturation, leading to reduced protein degradation. We propose a model wherein pumping of H+ to the cytosolic side of endosomes contributes to the accumulation of TOM1, and possibly TOLLIP, at these sites, thereby promoting PtdIns5P- and pH-dependent signaling, facilitating bacterial survival.

Intact and mutated Shigella diguanylate cyclases increase c-di-GMP.

The intracellular human pathogen Shigella invades the colonic epithelium to cause disease. Prior to invasion, this bacterium navigates through different environments within the human body, including the stomach and the small intestine. To adapt to changing environments, Shigella uses the bacterial second messenger cyclic di-GMP (c di-GMP) signaling system, synthesized by diguanylate cyclases (DGCs) encoding GGDEF domains. Shigella flexneri encodes a total of 9 GGDEF or GGDEF-EAL domain enzymes in its genome, but five of these genes have acquired mutations that presumably inactivated the c-di-GMP synthesis activity of these enzymes. In this study, we examined individual S. flexneri DGCs for their role in c-di-GMP synthesis and pathogenesis. We individually expressed each of the four intact DGCs in a S. flexneri strain, where these four DGCs had been deleted (Δ4DGC). We found that the 4 S. flexneri intact DGCs synthesize c-di-GMP at different levels in vitro and during infection of tissue-cultured cells. We also found that dgcF and dgcI expression significantly reduces invasion and plaque formation, and dgcF expression increases acid sensitivity, and that these phenotypes did not correspond with measured c-di-GMP levels. However, deletion of these four DGCs did not eliminate S. flexneri c-di-GMP, and we found that dgcE, dgcQ, and dgcN, which all have nonsense mutations prior to the GGDEF domain, still produce c-di-GMP. These S. flexneri degenerate DGC pseudogenes are expressed as multiple proteins, consistent with multiple start codons within the gene. We propose that both intact and degenerate DGCs contribute to S. flexneri c-di-GMP signaling.

Structural and functional characterization of the IpaD π-helix reveals critical roles in DOC interaction, T3SS apparatus maturation, and Shigella virulence.

Shigella spp. are highly pathogenic members of the Enterobacteriaceae family, causing ∼269 million cases of bacillary dysentery and >200,000 deaths each year. Like many Gram-negative pathogens, Shigella rely on their type three secretion system (T3SS) to inject effector proteins into eukaryotic host cells, driving both cellular invasion and evasion of host immune responses. Exposure to the bile salt deoxycholate (DOC) significantly enhances Shigella virulence and is proposed to serve as a critical environmental signal present in the small intestine that prepares Shigella's T3SS for efficient infection of the colonic epithelium. Here, we uncover critical mechanistic details of the Shigella-specific DOC signaling process by describing the role of a π-helix secondary structure element within the T3SS tip protein invasion plasmid antigen D (IpaD). Biophysical characterization and high-resolution structures of IpaD mutants lacking the π-helix show that it is not required for global protein structure, but that it defines the native DOC binding site and prevents off target interactions. Additionally, Shigella strains expressing the π-helix deletion mutants illustrate the pathogenic importance of its role in guiding DOC interaction as flow cytometry and gentamycin protection assays show that the IpaD π-helix is essential for DOC-mediated apparatus maturation and enhanced invasion of eukaryotic cells. Together, these findings add to our understanding of the complex Shigella pathogenesis pathway and its evolution to respond to environmental bile salts by identifying the π-helix in IpaD as a critical structural element required for translating DOC exposure to virulence enhancement.

Determining glycosyltransferase functional order via lethality due to accumulated O-antigen intermediates, exemplified with Shigella flexneri O-antigen biosynthesis.

The O antigen (OAg) polysaccharide is one of the most diverse surface molecules of Gram-negative bacterial pathogens. The structural classification of OAg, based on serological typing and sequence analysis, is important in epidemiology and the surveillance of outbreaks of bacterial infections. Despite the diverse chemical structures of OAg repeating units (RUs), the genetic basis of RU assembly remains poorly understood and represents a major limitation in assigning gene functions in polysaccharide biosynthesis. Here, we describe a genetic approach to interrogate the functional order of glycosyltransferases (GTs). Using Shigella flexneri as a model, we established an initial glycosyltransferase (IT)-controlled system, which allows functional order allocation of the subsequent GT in a 2-fold manner as follows: (i) first, by reporting the growth defects caused by the sequestration of UndP through disruption of late GTs and (ii) second, by comparing the molecular sizes of stalled OAg intermediates when each putative GT is disrupted. Using this approach, we demonstrate that for RfbF and RfbG, the GT involved in the assembly of S. flexneri backbone OAg RU, RfbG, is responsible for both the committed step of OAg synthesis and the third transferase for the second L-Rha. We also show that RfbF functions as the last GT to complete the S. flexneri OAg RU backbone. We propose that this simple and effective genetic approach can be also extended to define the functional order of enzymatic synthesis of other diverse polysaccharides produced both by Gram-negative and Gram-positive bacteria.IMPORTANCEThe genetic basis of enzymatic assembly of structurally diverse O antigen (OAg) repeating units (RUs) in Gram-negative pathogens is poorly understood, representing a major limitation in our understanding of gene functions for the synthesis of bacterial polysaccharides. We present a simple genetic approach to confidently assign glycosyltransferase (GT) functions and the order in which they act during assembly of the OAg RU. We employed this approach to determine the functional order of GTs involved in Shigella flexneri OAg assembly. This approach can be generally applied in interrogating GT functions encoded by other bacterial polysaccharides to advance our understanding of diverse gene functions in the biosynthesis of polysaccharides, key knowledge in advancing biosynthetic polysaccharide production.

The Shigella kinase effector OspG modulates host ubiquitin signaling to escape septin-cage entrapment.

Shigella flexneri is a Gram-negative bacterium causing severe bloody dysentery. Its pathogenesis is largely dictated by a plasmid-encoded type III secretion system (T3SS) and its associated effectors. Among these, the effector OspG has been shown to bind to the ubiquitin conjugation machinery (E2~Ub) to activate its kinase activity. However, the cellular targets of OspG remain elusive despite years of extensive efforts. Here we show by unbiased phosphoproteomics that a major target of OspG is CAND1, a regulatory protein controlling the assembly of cullin-RING ubiquitin ligases (CRLs). CAND1 phosphorylation weakens its interaction with cullins, which is expected to impact a large panel of CRL E3s. Indeed, global ubiquitome profiling reveals marked changes in the ubiquitination landscape when OspG is introduced. Notably, OspG promotes ubiquitination of a class of cytoskeletal proteins called septins, thereby inhibiting formation of cage-like structures encircling cytosolic bacteria. Overall, we demonstrate that pathogens have evolved an elaborate strategy to modulate host ubiquitin signaling to evade septin-cage entrapment.

Maintenance of bacterial outer membrane lipid asymmetry: insight into MlaA.

The outer membrane (OM) of Gram-negative bacteria acts as an effective barrier to protect against toxic compounds. By nature, the OM is asymmetric with the highly packed lipopolysaccharide (LPS) at the outer leaflet and glycerophospholipids at the inner leaflet. OM asymmetry is maintained by the Mla system, in which is responsible for the retrograde transport of glycerophospholipids from the OM to the inner membrane. This system is comprised of six Mla proteins, including MlaA, an OM lipoprotein involved in the removal of glycerophospholipids that are mis-localized at the outer leaflet of the OM. Interestingly, MlaA was initially identified - and called VacJ - based on its role in the intracellular spreading of Shigella flexneri.Many open questions remain with respect to the Mla system and the mechanism involved in the translocation of mislocated glycerophospholipids at the outer leaflet of the OM, by MlaA. After summarizing the current knowledge on MlaA, we focus on the impact of mlaA deletion on OM lipid composition and biophysical properties of the OM. How changes in OM lipid composition and biophysical properties can impact the generation of membrane vesicles and membrane permeability is discussed. Finally, we explore whether and how MlaA might be a candidate for improving the activity of antibiotics and as a vaccine candidate.Efforts dedicated to understanding the relationship between the OM lipid composition and the mechanical strength of the bacterial envelope and, in turn, how such properties act against external stress, are needed for the design of new targets or drugs for Gram-negative infections.

Microtubules provide force to promote membrane uncoating in vacuolar escape for a cyto-invasive bacterial pathogen.

Intracellular bacterial pathogens gain entry to mammalian cells inside a vacuole derived from the host membrane. Some of them escape the bacteria-containing vacuole (BCV) and colonize the cytosol. Bacteria replicating within BCVs coopt the microtubule network to position it within infected cells, whereas the role of microtubules for cyto-invasive pathogens remains obscure. Here, we show that the microtubule motor cytoplasmic dynein-1 and specific activating adaptors are hijacked by the enterobacterium Shigella flexneri. These host proteins were found on infection-associated macropinosomes (IAMs) formed during Shigella internalization. We identified Rab8 and Rab13 as mediators of dynein recruitment and discovered that the Shigella effector protein IpaH7.8 promotes Rab13 retention on moving BCV membrane remnants, thereby facilitating membrane uncoating of the Shigella-containing vacuole. Moreover, the efficient unpeeling of BCV remnants contributes to a successful intercellular spread. Taken together, our work demonstrates how a bacterial pathogen subverts the intracellular transport machinery to secure a cytosolic niche.

PEtN-Modified O-Antigen Enhances Shigella Pathogenesis by Promoting Epithelial Cell Invasion and Inhibiting Complement Binding.

Shigellosis poses an ongoing global public health threat. The presence and length of the O-antigen in lipopolysaccharide play critical roles in Shigella pathogenesis. The plasmid-mediated opt gene encodes a phosphoethanolamine (PEtN) transferase that catalyzes the addition of PEtN to the O-antigen of Shigella flexneri serotype X and Y strains, converting them into serotype Xv and Yv strains, respectively. Since 2002, these modified strains have become prevalent in China. Here we demonstrate that PEtN-mediated O-antigen modification in S. flexneri increase the severity of corneal infection in guinea pigs without any adaptive cost. This heightened virulence is associated with epithelial cell adhesion and invasion, as well as an enhanced inflammatory response of macrophage. Notably, PEtN addition allow S. flexneri to attenuate the binding of complement C3 and better resist phagocytosis, potentially contributing to the retention of S. flexneri in the host environment.

Transcriptional profiling of zebrafish identifies host factors controlling susceptibility to Shigella flexneri.

Shigella flexneri is a human-adapted pathovar of Escherichia coli that can invade the intestinal epithelium, causing inflammation and bacillary dysentery. Although an important human pathogen, the host response to S. flexneri has not been fully described. Zebrafish larvae represent a valuable model for studying human infections in vivo. Here, we use a Shigella-zebrafish infection model to generate mRNA expression profiles of host response to Shigella infection at the whole-animal level. Immune response-related processes dominate the signature of early Shigella infection (6 h post-infection). Consistent with its clearance from the host, the signature of late Shigella infection (24 h post-infection) is significantly changed, and only a small set of immune-related genes remain differentially expressed, including acod1 and gpr84. Using mutant lines generated by ENU, CRISPR mutagenesis and F0 crispants, we show that acod1- and gpr84-deficient larvae are more susceptible to Shigella infection. Together, these results highlight the power of zebrafish to model infection by bacterial pathogens and reveal the mRNA expression of the early (acutely infected) and late (clearing) host response to Shigella infection.

Shigella flexneri remodeling and consumption of host lipids during infection.

IMPORTANCE: Bacterial pathogens have vastly distinct sites that they inhabit during infection. This requires adaptation due to changes in nutrient availability and antimicrobial stress. The bacterial surface is a primary barrier, and here, we show that the bacterial pathogen Shigella flexneri increases its surface decorations when it transitions to an intracellular lifestyle. We also observed changes in bacterial and host cell fatty acid homeostasis. Specifically, intracellular S. flexneri increased the expression of their fatty acid degradation pathway, while the host cell lipid pool was significantly depleted. Importantly, bacterial proliferation could be inhibited by fatty acid supplementation of host cells, thereby providing novel insights into the possible link between human malnutrition and susceptibility to S. flexneri.

Diffusible signal factors (DSFs) bind and repress VirF, the leading virulence activator of Shigella flexneri.

Shigella, the aetiological agent of human bacillary dysentery, controls the expression of its virulence determinants through an environmentally stimulated cascade of transcriptional activators. VirF is the leading activator and is essential for proper virulence expression. In this work, we report on in vitro and in vivo experiments showing that two autoinducers of the DSF family, XcDSF and BDSF interact with the jelly roll module of VirF causing its inhibition and affecting the expression of the entire virulence system of Shigella, including its ability to invade epithelial cells. We propose a molecular model explaining how the binding of XcDSF and BDSF causes inhibition of VirF by preventing its dimerization. Overall, our experimental results suggest that XcDSF and BDSF may contribute to "colonisation resistance" in the human gut or, alternatively, may be exploited for the fine-tuning of Shigella virulence expression as the bacterium migrates from the lumen to approach the intestinal mucosa. Our findings also stress how a detailed understanding of the interaction of DSF ligands with VirF may contribute to the rational development of innovative antivirulence drugs to treat shigellosis.

Shigella flexneri Adapts to Niche-Specific Stresses through Modifications in Cell Envelope Composition and Decoration.

Shigella flexneri is the primary causative agent of worldwide shigellosis. As the pathogen transverses the distinct niches of the gastrointestinal tract it necessitates dynamic adaptation strategies to mitigate host antimicrobials such as dietary fatty acids (FAs) and the bile salt, deoxycholate (DOC). This study investigates the dynamics of the S. flexneri cell envelope, by interrogating adaptations following FA or DOC exposure. We deciphered the effects of FAs and DOC on bacterial membrane fatty acid and lipopolysaccharide (LPS) compositions. We identified novel LPS-based strategies by the pathogen to support resistance to these host compounds. In particular, expression of S. flexneri very-long O antigen (VL-Oag) LPS was found to play a central role in stress mitigation, as VL-Oag protects against antimicrobial FAs, but its presence rendered S. flexneri susceptible to DOC stress. Collectively, this work underpins the importance for S. flexneri to maintain appropriate regulation of cell envelope constituents, in particular VL-Oag LPS, to adequately adapt to diverse stresses during infection.

Exploring the use of Congo red agar in improving the serotyping of non-serotypeable Shigella with In-silico evidence.

OBJECTIVES: To assess if congo red could make non-serotypeable Shigella strains serotypeable and to employ molecular docking to determine the basis of the same phenomenon. METHODS: We used 42 bacterial strains of non-serotypeable Shigella collected from 2012 to 2019 for this study. Each bacterial strain was freshly inoculated onto congo red agar and incubated at 37° C for 18-24 h. Bacterial colonies obtained were re-subjected to biochemical tests followed by serotyping and serogrouping. In-silico studies to investigate the interaction between MxiC protein of T3SS and O-antigen LPS with congo red were performed. RESULTS: Of the total 42 non-serotypeable Shigella studied, (26/42)62% were capable of being serotyped following the use of congo red agar, 65% were Shigella flexneri, 19% were Shigella dysenteriae, while 2 strains (7%) each of Shigella boydii and Shigella sonnei were detected. We observed no change in their biochemical properties. The in-silico molecular docking studies revealed high binding affinity between congo red and the B-Chain of Mxi C. Out of the 5 chains of the O-Antigen, congo red showed robust binding with the B-chain with the involvement of a cluster of hydrophobic interactions between them. This may have a crucial role in the conversion of non-serotypeable strains to serotypeable strains on exposure to congo red as observed in our study. CONCLUSION: Congo red agar as a medium converts a sizeable percentage of non-serotypeable Shigella strains to serotypeable Shigella strains.

Role of the MDR Efflux Pump AcrAB in Epithelial Cell Invasion by Shigella flexneri.

The tripartite complex AcrAB-TolC is the major RND pump in Escherichia coli and other Enterobacteriaceae, including Shigella, the etiological agent of bacillary dysentery. In addition to conferring resistance to many classes of antibiotics, AcrAB plays a role in the pathogenesis and virulence of several bacterial pathogens. Here, we report data demonstrating that AcrAB specifically contributes to Shigella flexneri invasion of epithelial cells. We found that deletion of both acrA and acrB genes causes reduced survival of S. flexneri M90T strain within Caco-2 epithelial cells and prevents cell-to-cell spread of the bacteria. Infections with single deletion mutant strains indicate that both AcrA and AcrB favor the viability of the intracellular bacteria. Finally, we were able to further confirm the requirement of the AcrB transporter activity for intraepithelial survival by using a specific EP inhibitor. Overall, the data from the present study expand the role of the AcrAB pump to an important human pathogen, such as Shigella, and add insights into the mechanism governing the Shigella infection process.

Comparative study of GBP recruitment on two cytosol-dwelling pathogens, Francisella novicida and Shigella flexneri highlights differences in GBP repertoire and in GBP1 motif requirements.

Guanylate-Binding Proteins are interferon-inducible GTPases that play a key role in cell autonomous responses against intracellular pathogens. Despite sharing high sequence similarity, subtle differences among GBPs translate into functional divergences that are still largely not understood. A key GBP feature is the formation of supramolecular GBP complexes on the bacterial surface. Such complexes are observed when GBP1 binds lipopolysaccharide (LPS) from Shigella and Salmonella and further recruits GBP2-4. Here, we compared GBP recruitment on two cytosol-dwelling pathogens, Francisella novicida and S. flexneri. Francisella novicida was coated by GBP1 and GBP2 and to a lower extent by GBP4 in human macrophages. Contrary to S. flexneri, F. novicida was not targeted by GBP3, a feature independent of T6SS effectors. Multiple GBP1 features were required to promote targeting to F. novicida while GBP1 targeting to S. flexneri was much more permissive to GBP1 mutagenesis suggesting that GBP1 has multiple domains that cooperate to recognize F. novicida atypical LPS. Altogether our results indicate that the repertoire of GBPs recruited onto specific bacteria is dictated by GBP-specific features and by specific bacterial factors that remain to be identified.

Integrative structural analysis of the type III secretion system needle complex from Shigella flexneri.

The type III secretion system (T3SS) is a large, transmembrane protein machinery used by various pathogenic gram-negative bacteria to transport virulence factors into the host cell during infection. Understanding the structure of T3SSs is crucial for future developments of therapeutics that could target this system. However, much of the knowledge about the structure of T3SS is available only for Salmonella, and it is unclear how this large assembly is conserved across species. Here, we combined cryo-electron microscopy, cross-linking mass spectrometry, and integrative modeling to determine the structure of the T3SS needle complex from Shigella flexneri. We show that the Shigella T3SS exhibits unique features distinguishing it from other structurally characterized T3SSs. The secretin pore complex adopts a new fold of its C-terminal S domain and the pilotin MxiM[SctG] locates around the outer surface of the pore. The export apparatus structure exhibits a conserved pseudohelical arrangement but includes the N-terminal domain of the SpaS[SctU] subunit, which was not present in any of the previously published virulence-related T3SS structures. Similar to other T3SSs, however, the apparatus is anchored within the needle complex by a network of flexible linkers that either adjust conformation to connect to equivalent patches on the secretin oligomer or bind distinct surface patches at the same height of the export apparatus. The conserved and unique features delineated by our analysis highlight the necessity to analyze T3SS in a species-specific manner, in order to fully understand the underlying molecular mechanisms of these systems. The structure of the type III secretion system from Shigella flexneri delineates conserved and unique features, which could be used for the development of broad-range therapeutics.

Insights into the GSDMB-mediated cellular lysis and its targeting by IpaH7.8.

The multifunctional GSDMB protein is an important molecule in human immunity. The pyroptotic and bactericidal activity of GSDMB is a host response to infection by the bacterial pathogen Shigella flexneri, which employs the virulence effector IpaH7.8 to ubiquitinate and target GSDMB for proteasome-dependent degradation. Furthermore, IpaH7.8 selectively targets human but not mouse GSDMD, suggesting a non-canonical mechanism of substrate selection. Here, we report the crystal structure of GSDMB in complex with IpaH7.8. Together with biochemical and functional studies, we identify the potential membrane engagement sites of GSDMB, revealing general and unique features of gasdermin proteins in membrane recognition. We further illuminate how IpaH7.8 interacts with GSDMB, and delineate the mechanism by which IpaH7.8 ubiquitinates and suppresses GSDMB. Notably, guided by our structural model, we demonstrate that two residues in the α1-α2 loop make the mouse GSDMD invulnerable to IpaH7.8-mediated degradation. These findings provide insights into the versatile functions of GSDMB, which could open new avenues for therapeutic interventions for diseases, including cancers and bacterial infections.

A hierarchy of cell death pathways confers layered resistance to shigellosis in mice.

Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease driven by bacterial colonization of colonic intestinal epithelial cells. Vertebrates have evolved programmed cell death pathways that sense invasive enteric pathogens and eliminate their intracellular niche. Previously we reported that genetic removal of one such pathway, the NAIP-NLRC4 inflammasome, is sufficient to convert mice from resistant to susceptible to oral Shigella flexneri challenge (Mitchell et al., 2020). Here, we investigate the protective role of additional cell death pathways during oral mouse Shigella infection. We find that the Caspase-11 inflammasome, which senses Shigella LPS, restricts Shigella colonization of the intestinal epithelium in the absence of NAIP-NLRC4. However, this protection is limited when Shigella expresses OspC3, an effector that antagonizes Caspase-11 activity. TNFα, a cytokine that activates Caspase-8-dependent apoptosis, also provides potent protection from Shigella colonization of the intestinal epithelium when mice lack both NAIP-NLRC4 and Caspase-11. The combined genetic removal of Caspases-1, -11, and -8 renders mice hyper-susceptible to oral Shigella infection. Our findings uncover a layered hierarchy of cell death pathways that limit the ability of an invasive gastrointestinal pathogen to cause disease.

Localization of host endocytic and actin-associated proteins during Shigella flexneri intracellular motility and intercellular spreading.

Shigella flexneri (S. flexneri), the causative agent of bacillary dysentery, uses an effector-mediated strategy to hijack host cells and cause disease. To propagate and spread within human tissues, S. flexneri bacteria commandeer the host actin cytoskeleton to generate slender actin-rich comet tails to move intracellularly, and later, plasma membrane actin-based protrusions to move directly between adjacent host cells. To facilitate intercellular bacterial spreading, large micron-sized endocytic-like membrane invaginations form at the periphery of neighboring host cells that come into contact with S. flexneri-containing membrane protrusions. While S. flexneri comet tails and membrane protrusions consist primarily of host actin cytoskeletal proteins, S. flexneri membrane invaginations remain poorly understood with only clathrin and the clathrin adapter epsin-1 localized to the structures. Tangentially, we recently reported that Listeria monocytogenes, another actin-hijacking pathogen, exploits an assortment of caveolar and actin-bundling proteins at their micron-sized membrane invaginations formed during their cell-to-cell movement. Thus, to further characterize the S. flexneri disease process, we set out to catalog the distribution of a variety of actin-associated and caveolar proteins during S. flexneri actin-based motility and cell-to-cell spreading. Here we show that actin-associated proteins found at L. monocytogenes comet tails and membrane protrusions mimic those present at S. flexneri comet tails with the exception of α-actinins 1 and 4, which were shed from S. flexneri membrane protrusions. We also demonstrate that all known host endocytic components found at L. monocytogenes membrane invaginations are also present at those formed during S. flexneri infections.