Conformational stability and differential structural analysis of LcrV, PcrV, BipD, and SipD from type III secretion systems

Diverse Gram-negative bacteria use type III secretion systems (T3SS) to translocate effector proteins into the cytoplasm of eukaryotic cells. The type III secretion apparatus (T3SA) consists of a basal body spanning both bacterial membranes and an external needle. A sensor protein lies at the needle tip to detect environmental signals that trigger type III secretion. The Shigella flexneri T3SA needle tip protein, invasion plasmid antigen D (IpaD), possesses two independently folding domains in vitro. In this study, the solution behavior and thermal unfolding properties of IpaD's functional homologs SipD (Salmonella spp.), BipD (Burkholderia pseudomallei), LcrV (Yersinia spp.), and PcrV (Pseudomonas aeruginosa) were examined to identify common features within this protein family. CD and FTIR data indicate that all members within this group are alpha-helical with properties consistent with an intramolecular coiled-coil. SipD showed the most complex unfolding profile consisting of two thermal transitions, suggesting the presence of two independently folding domains. No evidence of multiple folding domains was seen, however, for BipD, LcrV, or PcrV. Thermal studies, including DSC, revealed significant destabilization of LcrV, PcrV, and BipD after N-terminal deletions. This contrasted with SipD and IpaD, which behaved like two-domain proteins. The results suggest that needle tip proteins share significant core structural similarity and thermal stability that may be the basis for their common function. Moreover, IpaD and SipD possess properties that distinguish them from the other tip proteins.     

YspD: A Potential Therapeutic Target for Drug Design to Combat Yersinia enterocolitica Infection

YspD is an annotated hydrophilic translocator of Ysa–Ysp type III secretion system of Yersinia enterocolitica. YspD has sequence, secondary structure and three-dimensional structure similar to other hydrophilic translocators. All hydrophilic translocators lack transmembrane region and possess intramolecular coiled-coil region. Disordered regions are mostly clustered at the N-terminal. Large loops provide flexibility, allowing conformational changes during oligomerization and protein–protein interaction. LcrV and PcrV have globular N-terminal and C-terminal domains, connected by intramolecular coiled-coil region. YspD, IpaD, SipD and BipD lack globular N-terminal and C-terminal domains. Their N-terminal and C-terminal domain have a bundle like structure connected by the intramolecular coiled-coil. The intramolecular coiled-coil regions (helix-5&9) of YspD showed maximum conservation, followed by helices at N-terminal. Polar interactions are mainly involved during dimerization of YspD, involving polar residues from helix-9 of both the YspD molecules. A methionine forms the boundary of interaction between the two YspD molecules. The two YspD molecules are arranged in antiparallel fashion to form the dimer. N-terminal of YspB interacted with C-terminal of YspD molecule to form a pentameric complex, consisting four YspD molecules and one YspB molecule. Sequence, structural similarity and presence of specific motifs in YspD (like chaperone protein) indicate the ability of N-terminal domain to show self-chaperoning activity and regulate folding and conformational state of YspD during its journey from the bacterial cytoplasm to the needle tip. Structural analysis of YspD and its mechanism of interaction with other proteins would enable us to design drugs against this hydrophilic protein to combat Yersinia infection.

     

Conformational changes in IpaD from Shigella flexneri upon binding bile salts provide insight into the second step of type III secretion

Shigella flexneri uses its type III secretion apparatus (TTSA) to inject host-altering proteins into targeted eukaryotic cells. The TTSA is composed of a basal body and an exposed needle with invasion plasmid antigen D (IpaD) forming a tip complex that controls secretion. The bile salt deoxycholate (DOC) stimulates recruitment of the translocator protein IpaB into the maturing TTSA needle tip complex. This process appears to be triggered by a direct interaction between DOC and IpaD. Fluorescence spectroscopy and NMR spectroscopy are used here to confirm the DOC-IpaD interaction and to reveal that IpaD conformational changes upon DOC binding trigger the appearance of IpaB at the needle tip. Fo?rster resonance energy transfer between specific sites on IpaD was used here to identify changes in distances between IpaD domains as a result of DOC binding. To further explore the effects of DOC binding on IpaD structure, NMR chemical shift mapping was employed. The environments of residues within the proposed DOC binding site and additional residues within the "distal" globular domain were perturbed upon DOC binding, further indicating that conformational changes occur within IpaD upon DOC binding. These events are proposed to be responsible for the recruitment of IpaB at the TTSA needle tip. Mutation analyses combined with additional spectroscopic analyses confirm that conformational changes in IpaD induced by DOC binding contribute to the recruitment of IpaB to the S. flexneri TTSA needle tip. These findings lay the foundation for determining how environmental factors promote TTSA needle tip maturation prior to host cell contact.     

High resolution structure of BipD: an invasion protein associated with the type III secretion system of Burkholderia pseudomallei

Burkoldheria pseudomallei is a Gram-negative bacterium that possesses a protein secretion system similar to those found in Salmonella and Shigella. Recent work has indicated that the protein encoded by the BipD gene of B. pseudomallei is an important secreted virulence factor. BipD is similar in sequence to IpaD from Shigella and SipD from Salmonella and is therefore likely to be a translocator protein in the type-III secretion system of B. pseudomallei. The crystal structure of BipD has been solved at a resolution of 2.1 A revealing the detailed tertiary fold of the molecule. The overall structure is appreciably extended and consists of a bundle of antiparallel alpha-helical segments with two small beta-sheet regions. The longest helices of the molecule form a four-helix bundle and most of the remaining secondary structure elements (three helices and two three-stranded beta-sheets) are formed by the region linking the last two helices of the four-helix bundle. The structure suggests that the biologically active form of the molecule may be a dimer formed by contacts involving the C-terminal alpha-helix, which is the most strongly conserved part of the protein. Comparison of the structure of BipD with immunological and other data for IpaD indicates that the C-terminal alpha-helix is also involved in contacts with other proteins that form the translocon.     

The crystal structures of the Salmonella type III secretion system tip protein SipD in complex with deoxycholate and chenodeoxycholate

The type III secretion system (T3SS) is a protein injection nanomachinery required for virulence by many human pathogenic bacteria including Salmonella and Shigella. An essential component of the T3SS is the tip protein and the Salmonella SipD and the Shigella IpaD tip proteins interact with bile salts, which serve as environmental sensors for these enteric pathogens. SipD and IpaD have long central coiled coils and their N-terminal regions form α-helical hairpins and a short helix α3 that pack against the coiled coil. Using AutoDock, others have predicted that the bile salt deoxycholate binds IpaD in a cleft formed by the α-helical hairpin and its long central coiled coil. NMR chemical shift mapping, however, indicated that the SipD residues most affected by bile salts are located in a disordered region near helix α3. Thus, how bile salts interact with SipD and IpaD is unclear. Here, we report the crystal structures of SipD in complex with the bile salts deoxycholate and chenodeoxycholate. Bile salts bind SipD in a region different from what was predicted for IpaD. In SipD, bile salts bind part of helix α3 and the C-terminus of the long central coiled coil, towards the C-terminus of the protein. We discuss the biological implication of the differences in how bile salts interact with SipD and IpaD.     

Self-chaperoning of the type III secretion system needle tip proteins IpaD and BipD

Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence: IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intramolecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure.     

Crystal structure of PrgI-SipD: insight into a secretion competent state of the type three secretion system needle tip and its interaction with host ligands

Many infectious gram-negative bacteria, including Salmonella typhimurium, require a Type Three Secretion System (T3SS) to translocate virulence factors into host cells. The T3SS consists of a membrane protein complex and an extracellular needle together that form a continuous channel. Regulated secretion of virulence factors requires the presence of SipD at the T3SS needle tip in S. typhimurium. Here we report three-dimensional structures of individual SipD, SipD in fusion with the needle subunit PrgI, and of SipD:PrgI in complex with the bile salt, deoxycholate. Assembly of the complex involves major conformational changes in both SipD and PrgI. This rearrangement is mediated via a π bulge in the central SipD helix and is stabilized by conserved amino acids that may allow for specificity in the assembly and composition of the tip proteins. Five copies each of the needle subunit PrgI and SipD form the T3SS needle tip complex. Using surface plasmon resonance spectroscopy and crystal structure analysis we found that the T3SS needle tip complex binds deoxycholate with micromolar affinity via a cleft formed at the SipD:PrgI interface. In the structure-based three-dimensional model of the T3SS needle tip, the bound deoxycholate faces the host membrane. Recently, binding of SipD with bile salts present in the gut was shown to impede bacterial infection. Binding of bile salts to the SipD:PrgI interface in this particular arrangement may thus inhibit the T3SS function. The structures presented in this study provide insight into the open state of the T3SS needle tip. Our findings present the atomic details of the T3SS arrangement occurring at the pathogen-host interface.     

NMR Model of PrgI–SipD Interaction and Its Implications in the Needle-Tip Assembly of the Salmonella Type III Secretion System

Salmonella and other pathogenic bacteria use the type III secretion system (T3SS) to inject virulence proteins into human cells to initiate infections. The structural component of the T3SS contains a needle and a needle tip. The needle is assembled from PrgI needle protomers and the needle tip is capped with several copies of the SipD tip protein. How a tip protein docks on the needle is unclear. A crystal structure of a PrgI–SipD fusion protein docked on the PrgI needle results in steric clash of SipD at the needle tip when modeled on the recent atomic structure of the needle. Thus, there is currently no good model of how SipD is docked on the PrgI needle tip. Previously, we showed by NMR paramagnetic relaxation enhancement (PRE) methods that a specific region in the SipD coiled coil is the binding site for PrgI. Others have hypothesized that a domain of the tip protein—the N-terminal α-helical hairpin—has to swing away during the assembly of the needle apparatus. Here, we show by PRE methods that a truncated form of SipD lacking the α-helical hairpin domain binds more tightly to PrgI. Further, PRE-based structure calculations revealed multiple PrgI binding sites on the SipD coiled coil. Our PRE results together with the recent NMR-derived atomic structure of the Salmonella needle suggest a possible model of how SipD might dock at the PrgI needle tip. SipD and PrgI are conserved in other bacterial T3SSs; thus, our results have wider implication in understanding other needle-tip complexes.     

Control of effector export by the Pseudomonas aeruginosa type III secretion proteins PcrG and PcrV

Pseudomonas aeruginosa uses a type III secretion system to inject protein effectors into a targeted host cell. Effector secretion is triggered by host cell contact. How effector secretion is prevented prior to cell contact is not well understood. In all secretion systems studied to date, the needle tip protein is required for controlling effector secretion, but the mechanism by which needle tip proteins control effector secretion is unclear. Here we present data that the P. aeruginosa needle tip protein, PcrV, controls effector secretion by assembling into a functional needle tip complex. PcrV likely does not simply obstruct the secretion channel because the pore‐forming translocator proteins can still be secreted while effector secretion is repressed. This finding suggests that PcrV controls effector secretion by affecting the conformation of the apparatus, shifting it from the default, effector secretion ‘on’ conformation, to the effector secretion ‘off’ conformation. We also present evidence that PcrG, which can bind to PcrV and is also involved in controlling effector export, is cytoplasmic and that the interaction between PcrG and PcrV is not required for effector secretion control by either protein. Taken together, these data allow us to propose a working model for control of effector secretion by PcrG and PcrV.     

Oligomerization of PcrV and LcrV, protective antigens of Pseudomonas aeruginosa and Yersinia pestis

Protective antigens of Pseudomonas aeruginosa (PcrV) and Yersinia pestis (LcrV) are key elements of specialized machinery, the type III secretion system (T3SS), which enables the injection of effector molecules into eukaryotic cells. Being positioned at the injectisome extremity, V proteins participate in the translocation process across the host cell plasma membrane. In this study, we demonstrate the assembly of V proteins into oligomeric doughnut-like complexes upon controlled refolding of the proteins in vitro. The oligomeric nature of refolded PcrV was revealed by size exclusion chromatography, native gel electrophoresis, and native mass spectrometry, which ascertain the capacity of the protein to multimerize into higher-order species. Furthermore, transmission electron microscopy performed on oligomers of both PcrV and LcrV revealed the presence of distinct structures with approximate internal and external diameters of 3-4 and 8-10 nm, respectively. The C-terminal helix, alpha12, of PcrV and notably the hydrophobic residues Val(255), Leu(262), and Leu(276) located within this helix, were shown to be crucial for oligomerization. Moreover, the corresponding mutant proteins produced in P. aeruginosa were found to be non-functional in in vivo type III-dependent cytotoxicity assays by directly affecting the correct assembly of PopB/D translocon within the host cell membranes. The detailed understanding of structure-function relationships of T3SS needle tip proteins will be of value in further developments of new vaccines and antimicrobials.     

Ultrastructural analysis of IpaD at the tip of the nascent MxiH type III secretion apparatus of Shigella flexneri

Shigella flexneri is a Gram-negative enteric pathogen that is the predominant cause of bacillary dysentery. Shigella uses a type III secretion system to deliver effector proteins that alter normal target cell functions to promote pathogen invasion. The type III secretion apparatus (T3SA) consists of a basal body, an extracellular needle, and a tip complex that is responsible for delivering effectors into the host cell cytoplasm. IpaD [Ipa (invasion plasmid antigen)] is the first protein to localize to the T3SA needle tip, where it prevents premature effector secretion and serves as an environmental sensor for triggering recruitment of the translocator protein IpaB to the needle tip. Thus, IpaD would be expected to form a stable structure whose overall architecture supports its functions. It is not immediately obvious from the published IpaD crystal structure (Protein Data Bank ID 2j0o) how a multimer of IpaD would be incorporated at the tip of the first static T3SA intermediate, nor what its functional role would be in building a mature T3SA. Here, we produce three-dimensional reconstructions from transmission electron microscopy images of IpaD localized at the Shigella T3SA needle tip for comparison to needle tips from a Shigella ipaD-null mutant. The results demonstrate that IpaD resides as a homopentamer at the needle tip of the T3SA. Furthermore, comparison to tips assembled from the distal domain IpaD(Δ192-267) mutation shows that IpaD adopts an elongated conformation that facilitates its ability to control type III secretion and stepwise assembly of the T3SA needle tip complex.     

Characterization of the Interaction between the Salmonella Type III Secretion System Tip Protein SipD and the Needle Protein PrgI by Paramagnetic Relaxation Enhancement

Many Gram-negative bacteria that cause major diseases and mortality worldwide require the type III secretion system (T3SS) to inject virulence proteins into their hosts and cause infections. A structural component of the T3SS is the needle apparatus, which consists of a base, an external needle, and a tip complex. In Salmonella typhimurium, the external needle is assembled by the polymerization of the needle protein PrgI. On top of this needle sits a tip complex, which is partly formed by the tip protein SipD. How SipD interacts with PrgI during the assembly of the T3SS needle apparatus remains unknown. The central region of PrgI forms an α-helical hairpin, whereas SipD has a long central coiled-coil, which is a defining structural feature of other T3SS tip proteins as well. Using NMR paramagnetic relaxation enhancement, we have identified a specific region on the SipD coiled-coil that interacts directly with PrgI. We present a model of how SipD might dock at the tip of the needle based on our paramagnetic relaxation enhancement results, thus offering new insight about the mechanism of assembly of the T3SS needle apparatus.     

Identification of the bile salt binding site on IpaD from Shigella flexneri and the influence of ligand binding on IpaD structure

Type III secretion (TTS) is an essential virulence factor for Shigella flexneri, the causative agent of shigellosis. The Shigella TTS apparatus (TTSA) is an elegant nanomachine that is composed of a basal body, an external needle to deliver effectors into human cells, and a needle tip complex that controls secretion activation. IpaD is at the tip of the nascent TTSA needle where it controls the first step of TTS activation. The bile salt deoxycholate (DOC) binds to IpaD to induce recruitment of the translocator protein IpaB into the maturing tip complex. We recently used spectroscopic analyses to show that IpaD undergoes a structural rearrangement that accompanies binding to DOC. Here, we report a crystal structure of IpaD with DOC bound and test the importance of the residues that make up the DOC binding pocket on IpaD function. IpaD binds DOC at the interface between helices α3 and α7, with concomitant movement in the orientation of helix α7 relative to its position in unbound IpaD. When the IpaD residues involved in DOC binding are mutated, some are found to lead to altered invasion and secretion phenotypes. These findings suggest that adoption of a DOC bound structural state for IpaD primes the Shigella TTSA for contact with host cells. The data presented here and in the studies leading up to this work provide the foundation for developing a model of the first step in Shigella TTS activation.     

An antibody targeting type III secretion system induces broad protection against Salmonella and Shigella infections

Salmonella and Shigella bacteria are food- and waterborne pathogens that are responsible for enteric infections in humans and are still the major cause of morbidity and mortality in the emerging countries. The existence of multiple Salmonella and Shigella serotypes as well as the emergence of strains resistant to antibiotics requires the development of broadly protective therapies. Recently, the needle tip proteins of the type III secretion system of these bacteria were successfully utilized (SipD for Salmonella and IpaD for Shigella) as vaccine immunogens to provide good prophylactic cross-protection in murine models of infections. From these experiments, we have isolated a cross-protective monoclonal antibody directed against a conserved region of both proteins. Its conformational epitope determined by Deep Mutational Scanning is conserved among needle tip proteins of all pathogenic Shigella species and Salmonella serovars, and are well recognized by this antibody. Our study provides the first in vivo experimental evidence of the importance of this common region in the mechanism of virulence of Salmonella and Shigella and opens the way to the development of cross-protective therapeutic agents.     

NMR identification of the binding surfaces involved in the Salmonella and Shigella Type III secretion tip‐translocon protein–protein interactions

The type III secretion system (T3SS) is essential for the pathogenesis of many bacteria including Salmonella and Shigella, which together are responsible for millions of deaths worldwide each year. The structural component of the T3SS consists of the needle apparatus, which is assembled in part by the protein–protein interaction between the tip and the translocon. The atomic detail of the interaction between the tip and the translocon proteins is currently unknown. Here, we used NMR methods to identify that the N‐terminal domain of the Salmonella SipB translocon protein interacts with the SipD tip protein at a surface at the distal region of the tip formed by the mixed α/β domain and a portion of its coiled‐coil domain. Likewise, the Shigella IpaB translocon protein and the IpaD tip protein interact with each other using similar surfaces identified for the Salmonella homologs. Furthermore, removal of the extreme N‐terminal residues of the translocon protein, previously thought to be important for the interaction, had little change on the binding surface. Finally, mutations at the binding surface of SipD reduced invasion of Salmonella into human intestinal epithelial cells. Together, these results reveal the binding surfaces involved in the tip‐translocon protein–protein interaction and advance our understanding of the assembly of the T3SS needle apparatus. Proteins 2016; 84:1097–1107. © 2016 Wiley Periodicals, Inc.     

Identification of the MxiH needle protein residues responsible for anchoring invasion plasmid antigen D to the type III secretion needle tip

The pathogenesis of Shigella flexneri requires a functional type III secretion apparatus to serve as a conduit for injecting host-altering effector proteins into the membrane and cytoplasm of the targeted cell. The type III secretion apparatus is composed of a basal body and an exposed needle that is an extended polymer of MxiH with a 2.0-nm inner channel. Invasion plasmid antigen D (IpaD) resides at the tip of the needle to control type III secretion. The atomic structures of MxiH and IpaD have been solved. MxiH (8.3 kDa) is a helix-turn-helix, whereas IpaD (36.6 kDa) has a dumbbell shape with two globular domains flanking a central coiled-coil that stabilizes the protein. These structures alone, however, have not been sufficient to produce a workable in silico model by which IpaD docks at the needle tip. Thus, the work presented here provides an initial step in understanding this important protein-protein interaction. We have identified key MxiH residues located in its PSNP loop and the contiguous surface that uniquely contribute to the formation of the IpaD-needle interface as determined by NMR chemical shift mapping. Mutation of Asn-43, Leu-47, and Tyr-50 residues severely affects the stable maintenance of IpaD at the Shigella surface and thus compromises the invasive phenotype of S. flexneri. Other residues could be mutated to give rise to intermediate phenotypes, suggesting they have a role in tip complex stabilization while not being essential for tip complex formation. Initial in vitro fluorescence polarization studies confirmed that specific amino acid changes adversely affect the MxiH-IpaD interaction. Meanwhile, none of the mutations appeared to have a negative effect on the MxiH-MxiH interactions required for efficient needle assembly.     

IpaD is localized at the tip of the Shigella flexneri type III secretion apparatus

Type III secretion (T3S) systems are used by numerous Gram-negative pathogenic bacteria to inject virulence proteins into animal and plant host cells. The core of the T3S apparatus, known as the needle complex, is composed of a basal body transversing both bacterial membranes and a needle protruding above the bacterial surface. In Shigella flexneri, IpaD is required to inhibit the activity of the T3S apparatus prior to contact of bacteria with host and has been proposed to assist translocation of bacterial proteins into host cells. We investigated the localization of IpaD by electron microscopy analysis of cross-linked bacteria and mildly purified needle complexes. This analysis revealed the presence of a distinct density at the needle tip. A combination of single particle analysis, immuno-labeling and biochemical analysis, demonstrated that IpaD forms part of the structure at the needle tip. Anti-IpaD antibodies were shown to block entry of bacteria into epithelial cells.     

IpaD is localized at the tip of the Shigella flexneri type III secretion apparatus

Type III secretion (T3S) systems are used by numerous Gram-negative pathogenic bacteria to inject virulence proteins into animal and plant host cells. The core of the T3S apparatus, known as the needle complex, is composed of a basal body transversing both bacterial membranes and a needle protruding above the bacterial surface. In Shigella flexneri, IpaD is required to inhibit the activity of the T3S apparatus prior to contact of bacteria with host and has been proposed to assist translocation of bacterial proteins into host cells. We investigated the localization of IpaD by electron microscopy analysis of cross-linked bacteria and mildly purified needle complexes. This analysis revealed the presence of a distinct density at the needle tip. A combination of single particle analysis, immuno-labeling and biochemical analysis, demonstrated that IpaD forms part of the structure at the needle tip. Anti-IpaD antibodies were shown to block entry of bacteria into epithelial cells.     

Deoxycholate interacts with IpaD of Shigella flexneri in inducing the recruitment of IpaB to the type III secretion apparatus needle tip

Type III secretion (TTS) is an essential virulence function for Shigella flexneri that delivers effector proteins that are responsible for bacterial invasion of intestinal epithelial cells. The Shigella TTS apparatus (TTSA) consists of a basal body that spans the bacterial inner and outer membranes and a needle exposed at the pathogen surface. At the distal end of the needle is a "tip complex" composed of invasion plasmid antigen D (IpaD). IpaD not only regulates TTS, but is required for the recruitment and stable association of the translocator protein IpaB at the TTSA needle tip in the presence of deoxycholate or other bile salts. This phenomenon is not accompanied by induction of TTS or the recruitment of IpaC to the Shigella surface. We now show that IpaD specifically binds fluorescein-labeled deoxycholate and, based on energy transfer measurements and docking simulations, this interaction appears to occur where the N-terminal domain of IpaD meets its central coiled-coil, a region that may also be involved in needle-tip interactions. TTS is initiated as a series of distinct steps and that small molecules present in the bacterial milieu are capable of inducing the first step of TSS through interactions with the needle tip protein IpaD. Furthermore, the amino acids proposed to be important for deoxycholate binding by IpaD appear to have significant roles in regulating tip complex composition and pathogen entry into host cells.     

Functional insights into the Shigella type III needle tip IpaD in secretion control and cell contact

Type III secretion apparatus (T3SA) are complex nanomachines that insert a translocation pore into the host cell membrane through which effector proteins are injected into the cytosol. In Shigella, the pore is inserted by a needle tip complex that also controls secretion. IpaD is the key protein that rules the composition of the tip complex before and upon cell contact or Congo red (CR) induction. However, how IpaD is involved in secretion control and translocon insertion remains not fully understood. Here, we report the phenotypic analysis of 20 10‐amino acids deletion variants all along the coiled‐coil and the central domains of IpaD (residues 131–332). Our results highlight three classes of T3S phenotype; (i) wild‐type secretion, (ii) constitutive secretion of all classes of effectors, and (iii) constitutive secretion of translocators and early effectors, but not of late effectors. Our data also suggest that the composition of the tip complex defines both the T3SA inducibility state and late effectors secretion. Finally, we shed light on a new aspect regarding the contact of the needle tip with cell membrane by uncoupling the Shigella abilities to escape macrophage vacuole, and to insert the translocation pore or to invade non‐phagocytic cells.