Shigella IpaD has a dual role: signal transduction from the type III secretion system needle tip and intracellular secretion regulation

Type III secretion systems (T3SSs) are protein injection devices essential for the interaction of many Gram‐negative bacteria with eukaryotic cells. While Shigella assembles its T3SS when the environmental conditions are appropriate for invasion, secretion is only activated after physical contact with a host cell. First, the translocators are secreted to form a pore in the host cell membrane, followed by effectors which manipulate the host cell. Secretion activation is tightly controlled by conserved T3SS components: the needle tip proteins IpaD and IpaB, the needle itself and the intracellular gatekeeper protein MxiC. To further characterize the role of IpaD during activation, we combined random mutagenesis with a genetic screen to identify ipaD mutant strains unable to respond to host cell contact. Class II mutants have an overall defect in secretion induction. They map to IpaD's C‐terminal helix and likely affect activation signal generation or transmission. The Class I mutant secretes translocators prematurely and is specifically defective in IpaD secretion upon activation. A phenotypically equivalent mutant was found in mxiC. We show that IpaD and MxiC act in the same intracellular pathway. In summary, we demonstrate that IpaD has a dual role and acts at two distinct locations during secretion activation.     

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.     

Single amino acid substitutions on the needle tip protein IpaD increased Shigella virulence

Infection of colonic epithelial cells by Shigella is associated with the type III secretion system, which serves as a molecular syringe to inject effectors into host cells. This system includes an extracellular needle used as a conduit for secreted proteins. Two of these proteins, IpaB and IpaD, dock at the needle tip to control secretion and are also involved in the insertion of a translocation pore into host cell membrane allowing effector delivery. To better understand the function of IpaD, we substituted thirteen residues conserved among homologous proteins in other bacterial species. Generated variants were tested for their ability to surface expose IpaB and IpaD, to control secretion, to insert the translocation pore, and to invade host cells. In addition to a first group of seven ipaD variants that behaved similarly to the wild-type strain, we identified a second group with mutations V314D and I319D that deregulated secretion of all effectors, but remained fully invasive. Moreover, we identified a third group with mutations Y153A, T161D, Q165L and Y276A, that exhibited increased levels of translocators secretion, pore formation, and cell entry. Altogether, our results offer a better understanding of the role of IpaD in the control of Shigella virulence.     

Three‐dimensional electron microscopy reconstruction and cysteine‐mediated crosslinking provide a model of the type III secretion system needle tip complex

Type III secretion systems are found in many Gram‐negative bacteria. They are activated by contact with eukaryotic cells and inject virulence proteins inside them. Host cell detection requires a protein complex located at the tip of the device's external injection needle. The Shigella tip complex (TC) is composed of IpaD, a hydrophilic protein, and IpaB, a hydrophobic protein, which later forms part of the injection pore in the host membrane. Here we used labelling and crosslinking methods to show that TCs from a ΔipaB strain contain five IpaD subunits while the TCs from wild‐type can also contain one IpaB and four IpaD subunits. Electron microscopy followed by single particle and helical image analysis was used to reconstruct three‐dimensional images of TCs at ～20 Å resolution. Docking of an IpaD crystal structure, constrained by the crosslinks observed, reveals that TC organisation is different from that of all previously proposed models. Our findings suggest new mechanisms for TC assembly and function. The TC is the only site within these secretion systems targeted by disease‐protecting antibodies. By suggesting how these act, our work will allow improvement of prophylactic and therapeutic strategies.     

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.     

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein. We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n‐octyl‐oligo‐oxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,N‐dimethyldodecylamine N‐oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size‐dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore.     

Influence of oligomerization state on the structural properties of invasion plasmid antigen B from Shigella flexneri in the presence and absence of phospholipid membranes

Shigella flexneri causes bacillary dysentery, an important cause of mortality among children in the developing world. Shigella secretes effector proteins via its type III secretion system (T3SS) to promote bacterial uptake into human colonic epithelial cells. The T3SS basal body spans the bacterial cell envelope anchoring a surface‐exposed needle. A pentamer of invasion plasmid antigen D lies at the nascent needle tip and invasion plasmid antigen B (IpaB) is recruited into the needle tip complex on exposure to bile salts. From here, IpaB forms a translocon pore in the host cell membrane. Although the mechanism by which IpaB inserts into the membrane is unknown, it was recently shown that recombinant IpaB can exist as either a monomer or tetramer. Both of these forms of IpaB associate with membranes, however, only the tetramer forms pores in liposomes. To reveal differences between these membrane‐binding events, Cys mutations were introduced throughout IpaB, allowing site‐specific fluorescence labeling. Fluorescence quenching was used to determine the influence of oligomerization and/or membrane association on the accessibility of different IpaB regions to small solutes. The data show that the hydrophobic region of tetrameric IpaB is more accessible to solvent relative to the monomer. The hydrophobic region appears to promote membrane interaction for both forms of IpaB, however, more of the hydrophobic region is protected from solvent for the tetramer after membrane association. Limited proteolysis demonstrated that changes in IpaB's oligomeric state may determine the manner by which it associates with phospholipid membranes and the subsequent outcome of this association. Proteins 2014; 82:3013–3022. © 2014 Wiley Periodicals, Inc.     

Structures of the Shigella flexneri type 3 secretion system protein MxiC reveal conformational variability amongst homologues

Many Gram-negative pathogenic bacteria use a complex macromolecular machine, known as the type 3 secretion system (T3SS), to transfer virulence proteins into host cells. The T3SS is composed of a cytoplasmic bulb, a basal body spanning the inner and outer bacterial membranes, and an extracellular needle. Secretion is regulated by both cytoplasmic and inner membrane proteins that must respond to specific signals in order to ensure that virulence proteins are not secreted before contact with a eukaryotic cell. This negative regulation is mediated, in part, by a family of proteins that are thought to physically block the entrance to the secretion apparatus until an appropriate signal is received following host cell contact. Despite weak sequence homology between proteins of this family, the crystal structures of Shigella flexneri MxiC we present here confirm the conservation of domain topology with the homologue from Yersinia sp. Interestingly, comparison of the Shigella and Yersinia structures reveals a significant structural change that results in substantial domain re-arrangement and opening of one face of the molecule. The conservation of a negatively charged patch on this face suggests it may have a role in binding other components of the T3SS.     

A Gatekeeper Chaperone Complex Directs Translocator Secretion during Type Three Secretion

Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ∼20 individual protein components that form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.     

Using disruptive insertional mutagenesis to identify the in situ structure‐function landscape of the Shigella translocator protein IpaB

Bacterial type III secretion systems (T3SS) are used to inject proteins into mammalian cells to subvert cellular functions. The Shigella T3SS apparatus (T3SA) is comprised of a basal body, cytoplasmic sorting platform and exposed needle with needle “tip complex” (TC). TC maturation occurs when the translocator protein IpaB is recruited to the needle tip where both IpaD and IpaB control secretion induction. IpaB insertion into the host membrane is the first step of translocon pore formation and secretion induction. We employed disruptive insertional mutagenesis, using bacteriophage T4 lysozyme (T4L), within predicted IpaB loops to show how topological features affect TC functions (secretion control, translocon formation and effector secretion). Insertions within the N‐terminal half of IpaB were most likely to result in a loss of steady‐state secretion control, however, all but the two that were not recognized by the T3SA retained nearly wild‐type hemolysis (translocon formation) and invasiveness levels (effector secretion). In contrast, all but one insertion in the C‐terminal half of IpaB maintained secretion control but were impaired for hemolysis and invasion. These nature of the data suggest the latter mutants are defective in a post‐secretion event, most likely due to impaired interactions with the second translocator protein IpaC. Intriguingly, only two insertion mutants displayed readily detectable T4L on the bacterial surface. The data create a picture in which the makeup and structure of a functional T3SA TC is highly amenable to physical perturbation, indicating that the tertiary structure of IpaB within the TC is more plastic than previously realized.     

Studies of the conformational stability of invasion plasmid antigen B from Shigella

Shigella spp. are the causative agent of shigellosis, the second leading cause of diarrhea in children of ages 2–5. Despite many years of research, a protective vaccine has been elusive. We recently demonstrated that invasion plasmid antigens B and D (IpaB and IpaD) provide protection against S. flexneri and S. sonnei. These proteins, however, have very different properties which must be recognized and then managed during vaccine formulation. Herein, we employ spectroscopy to assess the stability of IpaB as well as IpgC (invasion protein gene), IpaB's cognate chaperone, and the IpaB/IpgC complex. The resulting data are mathematically summarized into a visual map illustrating the stability of the proteins and their complex as a function of pH and temperature. The IpaB/IpgC complex exhibits thermal stability at higher pH values but, though initially stable, quickly unfolds with increasing temperature when maintained at lower pH. In contrast, IpaB is a much more complex protein exhibiting increased stability at higher pH, but shows initial instability at lower pH values with pH 5 showing a distinct transition. IpgC precipitates at and below pH 5 and is stable above pH 7. Most strikingly, it is clear that complex formation results in stabilization of the two components. This work serves as a basis for the further development of IpaB as a vaccine candidate as well as extends our understanding of the structural stability of the Shigella type III secretion system.     

The type III secretion system needle tip complex mediates host cell sensing and translocon insertion

Type III secretion systems (T3SSs) are essential virulence determinants of many Gram-negative bacterial pathogens. The Shigella T3SS consists of a cytoplasmic bulb, a transmembrane region and a hollow 'needle' protruding from the bacterial surface. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which proteins that facilitate host cell invasion are translocated. As the needle is implicated in host cell sensing and secretion regulation, its tip should contain components that initiate host cell contact. Through biochemical and immunological studies of wild-type and mutant Shigella T3SS needles, we reveal tip complexes of differing compositions and functional states, which appear to represent the molecular events surrounding host cell sensing and pore formation. Our studies indicate that the interaction between IpaB and IpaD at needle tips is key to host cell sensing, orchestration of IpaC secretion and its subsequent assembly at needle tips. This allows insertion into the host cell membrane of a translocation pore that is continuous with the needle.     

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.     

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.     

The Structure of the Salmonella typhimurium Type III Secretion System Needle Shows Divergence from the Flagellar System

The type III secretion system (T3SS) is essential for the infectivity of many pathogenic Gram-negative bacteria. The T3SS contains proteins that form a channel in the inner and outer bacterial membranes, as well as an extracellular needle that is used for transporting and injecting effector proteins into a host cell. The homology between the T3SS and the bacterial flagellar system has been firmly established, based upon both sequence similarities between respective proteins in the two systems and the structural homology of higher-order assemblies. It has previously been shown that the Shigella flexneri needle has a helical symmetry of ∼ 5.6 subunits/turn, which is quite similar to that of the most intensively studied flagellar filament (from Salmonella typhimurium), which has ∼ 5.5 subunits/turn. We now show that the Sa. typhimurium needle, expected by homology arguments to be more similar to the Sa. typhimurium flagellar filament than is the needle from Shigella, actually has ∼ 6.3 subunits/turn. It is not currently understood how host cell contact, made at the tip of the needle, is communicated to the secretory system at the base. In contrast to the Sa. typhimurium flagellar filament, which shows a nearly crystalline order, the Sa. typhimurium needle has a highly variable symmetry, which could be used to transmit information about host cell contact.     

Spontaneous formation of IpaB ion channels in host cell membranes reveals how Shigella induces pyroptosis in macrophages

The Gram-negative bacterium Shigella flexneri invades the colonic epithelium and causes bacillary dysentery. S. flexneri requires the virulence factor invasion plasmid antigen B (IpaB) to invade host cells, escape from the phagosome and induce macrophage cell death. The mechanism by which IpaB functions remains unclear. Here, we show that purified IpaB spontaneously oligomerizes and inserts into the plasma membrane of target cells forming cation selective ion channels. After internalization, IpaB channels permit potassium influx within endolysosomal compartments inducing vacuolar destabilization. Endolysosomal leakage is followed by an ICE protease-activating factor-dependent activation of Caspase-1 in macrophages and cell death. Our results provide a mechanism for how the effector protein IpaB with its ion channel activity causes phagosomal destabilization and induces macrophage death. These data may explain how S. flexneri uses secreted IpaB to escape phagosome and kill the host cells during infection and, may be extended to homologs from other medically important enteropathogenic bacteria.     

The Common Structural Architecture of Shigella flexneri and Salmonella typhimurium Type Three Secretion Needles

The Type Three Secretion System (T3SS), or injectisome, is a macromolecular infection machinery present in many pathogenic Gram-negative bacteria. It consists of a basal body, anchored in both bacterial membranes, and a hollow needle through which effector proteins are delivered into the target host cell. Two different architectures of the T3SS needle have been previously proposed. First, an atomic model of the Salmonella typhimurium needle was generated from solid-state NMR data. The needle subunit protein, PrgI, comprises a rigid-extended N-terminal segment and a helix-loop-helix motif with the N-terminus located on the outside face of the needle. Second, a model of the Shigella flexneri needle was generated from a high-resolution 7.7-Å cryo-electron microscopy density map. The subunit protein, MxiH, contains an N-terminal α-helix, a loop, another α-helix, a 14-residue-long β-hairpin (Q51–Q64) and a C-terminal α-helix, with the N-terminus facing inward to the lumen of the needle. In the current study, we carried out solid-state NMR measurements of wild-type Shigella flexneri needles polymerized in vitro and identified the following secondary structure elements for MxiH: a rigid-extended N-terminal segment (S2-T11), an α-helix (L12-A38), a loop (E39-P44) and a C-terminal α-helix (Q45-R83). Using immunogold labeling in vitro and in vivo on functional needles, we located the N-terminus of MxiH subunits on the exterior of the assembly, consistent with evolutionary sequence conservation patterns and mutagenesis data. We generated a homology model of Shigella flexneri needles compatible with both experimental data: the MxiH solid-state NMR chemical shifts and the state-of-the-art cryoEM density map. These results corroborate the solid-state NMR structure previously solved for Salmonella typhimurium PrgI needles and establish that Shigella flexneri and Salmonella typhimurium subunit proteins adopt a conserved structure and orientation in their assembled state. Our study reveals a common structural architecture of T3SS needles, essential to understand T3SS-mediated infection and develop treatments.     

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.     

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.     

The Type III Secretion System-Related CPn0809 from Chlamydia pneumoniae

Chlamydia pneumoniae is an intracellular Gram-negative bacterium that possesses a type III secretion system (T3SS), which enables the pathogen to deliver, in a single step, effector proteins for modulation of host-cell functions into the human host cell cytosol to establish a unique intracellular niche for replication. The translocon proteins located at the top of the T3SS needle filament are essential for its function, as they form pores in the host-cell membrane. Interestingly, unlike other Gram-negative bacteria, C. pneumoniae has two putative translocon operons, named LcrH_1 and LcrH_2. However, little is known about chlamydial translocon proteins. In this study, we analyzed CPn0809, one of the putative hydrophobic translocators encoded by the LcrH_1 operon, and identified an ‘SseC-like family’ domain characteristic of T3S translocators. Using bright-field and confocal microscopy, we found that CPn0809 is associated with EBs during early and very late phases of a C. pneumoniae infection. Furthermore, CPn0809 forms oligomers, and interacts with the T3SS chaperone LcrH_1, via its N-terminal segment. Moreover, expression of full-length CPn0809 in the heterologous host Escherichia coli causes a grave cytotoxic effect that leads to cell death. Taken together, our data indicate that CPn0809 likely represents one of the translocon proteins of the C. pneumoniae T3SS, and possibly plays a role in the translocation of effector proteins in the early stages of infection.