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Polar distribution of type III secretion systems in C. trachomatis LGV2 elementary bodies.A. Gallery of representative immunofluorescence micrographs of LGV2 EBs. Fixed bacteria were immunolabelled for the type III secretion system needle component CdsF (red) and co-stained for Chlamydia (green). Scale bar, 1 µm.B. HeLa cells were infected with C. trachomatis LGV2 by adsorption for 2 h at 37°C (moi 5). Infected cells were fixed by high-pressure freezing and freeze-substitution. Left panel, tomographic slice (0.83 nm thick) from a reconstruction of an EB. Right panel shows indicated area of left panel at higher magnification. Outer membrane (OM), inner membrane (IM) and DNA nucleoid (nu) are labelled. Additional membranes are apparent between the inner membrane and nucleoid (white arrow). Scale bars, 100 nm (left), 50 nm (right).C. Left panel, tomographic slice (0.83 nm thick) from a reconstruction of an EB in close proximity to a HeLa cell filopodium. Additional panels show consecutive tomographic slices from the reconstruction. Needle-like projections on the EB surface terminate at the HeLa cell plasma membrane (white arrows). Scale bar, 80 nm.
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Invasion of host cells is a key early event during bacterial infection, but the underlying pathogen-host interactions are yet to be fully visualised in three-dimensional detail. We have captured snapshots of the early stages of bacterial-mediated endocytosis in situ by exploiting the small size of chlamydial elementary bodies (EBs) for whole cell c...
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... needle of the T3SS. We previously employed this antibody to identify T3SSs in RBs at patho- gen synapses connecting intracellular bacteria, the inclu- sion membrane and the host endoplasmic reticulum (Dumoux et al., 2012). Immunofluorescence micrographs revealed CdsF staining on a defined region of the periph- ery for nearly every EB observed (Fig. 1A). Consistent with early observations of unidentified surface projections by EM (Matsumoto, 1982b), these data confirm that the T3SSs components are restricted to one hemisphere of the EB surface ( Betts et al., 2008). Next, to characterize the ultrastructure of chlamydial EBs and their interaction with host cells, cultured HeLa cells ...
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... (0.83 nm thick) from a reconstruction of an EB in close proximity to a HeLa cell filopodium. Additional panels show consecutive tomographic slices from the reconstruction. Needle-like projections on the EB surface terminate at the HeLa cell plasma membrane (white arrows). Scale bar, 80 nm. consistent with earlier reports (Tamura et al., 1971) (Fig. 1B). A highly condensed, electron-dense nucleoid, typically attributed to an assembly of chromatin and chlamydial histone-like proteins was evident in the bacte- rial cytosol (Barry et al., 1992). Strikingly, additional mem- branous structures were also apparent between the cytosolic face of the inner membrane and the nucleoid, suggesting ...
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... histone-like proteins was evident in the bacte- rial cytosol (Barry et al., 1992). Strikingly, additional mem- branous structures were also apparent between the cytosolic face of the inner membrane and the nucleoid, suggesting an organelle-like structure or other previously unrecognized complexity in the architecture of the inner membrane (Fig. 1B, inset). When EBs were visualized in close proximity to the host cell surface, electron tomograms revealed a widening of the periplasmic space, associated with discrete projections emanating from the bacterial surface and terminating at the host cell plasma membrane (Fig. 1C). Widening of the periplasmic space (from ∼ 15 to ∼ 30 nm) and the ∼ ...
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... unrecognized complexity in the architecture of the inner membrane (Fig. 1B, inset). When EBs were visualized in close proximity to the host cell surface, electron tomograms revealed a widening of the periplasmic space, associated with discrete projections emanating from the bacterial surface and terminating at the host cell plasma membrane (Fig. 1C). Widening of the periplasmic space (from ∼ 15 to ∼ 30 nm) and the ∼ 40 nm projections are consistent with the dimensions of the T3SS basal body and needle respectively (Sani et al., 2007). Indeed, such periplasmic expansion specifically accommodates assembled T3SSs in RBs (Dumoux et al., 2012). These initial findings from ...
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... (SPG) buffer ( Scidmore, 2005). While EBs purified and stored under these harsh conditions remain invasion com- petent, they may not fully retain native structural features. To minimize structural artefacts, we infected adherent cells cultured directly on EM sample grids with EBs released naturally from co-cultured infected cells (Fig. S1). Following a brief incubation with the released EBs, the grids containing the freshly infected cells were rapidly vitrified by plunge freezing, and whole-cell cryo- electron tomography was performed. This new method largely eliminates potential artefacts caused by centrifugation-assisted inoculation, the mechanical stress of the ...
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... situ T3SS structure from intact bacterial membranes of Yersinia enterocolitica shows overall agreement in the characteristic size and shape (∼ 30 nm basal body plus ∼ 35 nm needle) ( Kudryashev et al., 2013) (Fig. 3B, left and centre panels). These structural similarities, along with the polarized labelling seen by immunofluorescence microscopy (Fig. 1A) and immunogold EM (Fig. S3), indi- cate the complexes are chlamydial ...
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... notion of pre-established EB polarity is further sup- ported by the presence of complex membranous struc- tures located between the inner membrane surface and the nucleoid (Fig. 1B). In cryo-electron tomograms of EBs released from infected cells, these structures are evident as a previously unidentified tubular invagination of the inner membrane originating from the hemisphere generally opposite the T3SS array ( Figs 4A and S4A). Membranes forming this tubule appear to have the same thickness as the inner membrane ...
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... were carried out by diluting the stored LGV2 serovar in infection medium (DMEM, 10% FCS, 25 g ml −1 gentamicin) so that the multiplicity of infection was ∼ 1. After centrifugation-assisted inoculation (160 g 10 min), HeLa or U2OS cells seeded 24 h previously were incubated in the infec- tion medium for 80 min before exchange into fresh medium. At an appropriate time-point, cells were fixed with paraformaldehyde for immunostaining or by high-pressure freezing and freeze- substitution for thin-section EM. ...
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... and rinsed in Hank's buffered salt solution (HBSS). Four microlitres of BSA-coated 10 nm colloidal gold (Sigma) was added to the grid before it was plunge-frozen into liquid ethane (Vitrobot Mark III, FEI). Frozen grids were stored in liquid nitrogen until they were loaded into the electron microscope for cryo-electron tomography (see also Fig. ...
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... Supporting Information may be found in the online version of this article at the publisher's web-site: Fig. S1. Sample preparation for cryo-electron tomography. To preserve early stage Chlamydia EBs in association with cells, cultured HeLa or U2OS cells were infected using frozen aliquots of C. trachomatis LGV2 (moi 5, centrifugation-assisted inocula- tion). At ∼ 48 hpi when infected cells begin to release new EB progeny, gold EM grids (seeded ...
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... Actin remodeling downstream of N-WASP and WAVE2 signaling contributes to invasion by forming various actin-rich structures (e.g., hypertrophic microvilli, cap-like pockets, and pedestal-like structures) at sites of Chlamydia adhesion, indicating the presence of multiple pathways for pathogen entry [11][12][13] . Although multiple uptake mechanisms have been implicated as potential pathways for C. trachomatis invasion, the role of host dynamins during this process has been controversial. ...
... 2g, h, 5a, S6B, D), implying the utilization of dynamininsensitive modes of entry by this strain. Although our study demonstrates that Dyn2 and actin dynamics are functionally linked, a comprehensive model of Dyn2 involvement in several invasion mechanisms reported for Chlamydia requires further characterization [11][12][13] . ...
Chlamydia invasion of epithelial cells is a pathogen-driven process involving two functionally distinct effectors – TarP and TmeA. They collaborate to promote robust actin dynamics at sites of entry. Here, we extend studies on the molecular mechanism of invasion by implicating the host GTPase dynamin 2 (Dyn2) in the completion of pathogen uptake. Importantly, Dyn2 function is modulated by TarP and TmeA at the levels of recruitment and activation through oligomerization, respectively. TarP-dependent recruitment requires phosphatidylinositol 3-kinase and the small GTPase Rac1, while TmeA has a post-recruitment role related to Dyn2 oligomerization. This is based on the rescue of invasion duration and efficiency in the absence of TmeA by the Dyn2 oligomer-stabilizing small molecule activator Ryngo 1-23. Notably, Dyn2 also regulated turnover of TarP- and TmeA-associated actin networks, with disrupted Dyn2 function resulting in aberrant turnover dynamics, thus establishing the interdependent functional relationship between Dyn2 and the effectors TarP and TmeA.
... Overall, the data support a developmental cycle that includes an asymmetrically dividing RB population. Asymmetry has been documented for both the EB and RB cells (28)(29)(30)(31). In addition, it has been shown that the division plane can form asymmetrically during RB division (28,32). ...
Chlamydia trachomatis is an obligate intracellular bacterial pathogen responsible for both ocular and sexually transmitted infections. All Chlamydiae are reliant on a complex developmental cycle, consisting of both infectious and noninfectious cell forms.
... C. trachomatis entry into the host is preceded by activation of either PDGFRβ or Abl kinases, which leads to phosphorylation of TarP, stimulation of Vav2 and several actin nucleators or actin-binding proteins including cortactin (Elwell et al., 2008;Patel et al., 2014). As a result, EBs undergo envelopment by macropinosomes and actinrich filopodia, resulting in encapsulation into early vacuoles (Nans et al., 2014). The exact role of cortactin in this scenario is largely unclear. ...
Pathogenic bacteria possess a great potential of causing infectious diseases and represent a serious threat for human and animal health. Understanding the molecular basis of infection development can provide new valuable strategies for disease prevention and better control. In host-pathogen interactions, actin-cytoskeletal dynamics plays a crucial role for successful adherence, invasion and intracellular motility of many intruding microbial pathogens. Cortactin, a major cellular factor that promotes actin polymerization and other functions, appears as a central regulator of host-pathogen interactions and different human diseases including cancer development. Various important microbes have been reported to hijack cortactin signaling during infection. The primary regulation of cortactin appears to proceed via serine and/or tyrosine phosphorylation events by upstream kinases, acetylation and interaction with various other host proteins, including the Arp2/3 complex, filamentous actin, the actin nucleation promoting factor N-WASP, focal adhesion kinase FAK, the large GTPase dynamin-2, the guanine nucleotide exchange factor Vav2 and the actin-stabilizing protein CD2AP. Given that many signaling factors can affect cortactin activities, several microbes target certain unique pathways, while also sharing some common features. Here we review our current knowledge on the hallmarks of cortactin as a major target for eminent Gram-negative and Gram-positive bacterial pathogens in humans.
... Three environmental strains of Chlamydiae and their amoebae host were high-pressure frozen, followed by the CEMOVIS method (Pilhofer et al. 2014). Results from their work and another group's work (Nans et al. 2014) show localization of bacteria within host cells, the surrounding membrane morphology, and T3SS needle structures engaging with host cell membranes (Figs. 2a-2d). ...
Animals, plants, and fungi live in a microbe-dominated world. Investigating the interactions and processes at the host-microbe interface offers insight to how bacteria influence the development, health, and disease of the host. Optimization of existing imaging technologies and development of novel instrumentation will provide the tools needed to fully understand the dynamic relationship that occurs at the host-microbe interface throughout the lifetime of the host. In this review, we describe the current methods used in cryo-electron microscopy (cryo-EM) including cryo-fixation, sample processing, FIB-SEM, and cryotomography. Further, we highlight the new advances associated with these methods that open the cryo-EM discipline to large, complex multicellular samples, like symbiotic tissues. We describe the advantages and challenges associated with correlative imaging techniques and sample thinning methods like lift-out. By highlighting recent pioneering studies in the large-volume or symbiotic sample workflows, we provide insight into how symbiotic model systems will benefit from cryo-EM methods to provide artefact-free, near-native, macromolecular-scale resolution imaging at the host-microbe interface throughout the development and maintenance of symbiosis. Cryo-EM methods have brought a deep fundamental understanding of prokaryotic biology since its conception. We propose the application of existing and novel cryo-EM techniques to symbiotic systems is the logical next step that will bring an even greater understanding how microbes interact with their host tissues.
... This interaction leads to the uptake of EBs via actin-dependent or -independent mechanisms. Actin-dependent mechanisms have been extensively studied, including the emergence of macropinosome, actinrich filopodia, or phagocytic cups-based internalization (Dumoux et al., 2015a;Nans et al., 2014) [ Figure 20]. Actin-mediated internalization of EBs is initiated by injecting presynthesized effectors into the cytoplasm of host cells through T3SS, affecting actin filaments and leading to the typically non-phagocytic uptake of EB (Ferrell and Fields, 2016). ...
... One pole is characterized by tubular inner membrane invaginations, while the other pole exhibits asymmetric periplasmic expansion to accommodate a range of T3SS. Once internalized by host cells, these EBs lose their polarity [ Figure 21] (Dumoux et al., 2015a;Nans et al., 2014). An effector protein of Chlamydia, known as the translocated actin recruiting phosphoprotein (Tarp), is responsible for driving changes in the cytoskeleton and cell signaling (Clifton et al., 2004;Ghosh et al., 2020;Keb et al., 2021;Shehat et al., 2021). ...
Chlamydia trachomatis is an obligate intracellular bacterium, which grows mainly in epithelial cells of the mucous membranes of the genital tract. Asymptomatic in most cases, infections with this pathogen can lead to pelvic inflammations. The inflammation itself can lead to fibrosis and tubal infertility in women. To better understand the pathological manifestations in the female genital tract (FGT), it is important to study the response of epithelial cells, which constitute the first line of host defense against C. trachomatis infection. To this end, we developed a simplified protocol to isolate a very pure fraction of primary epithelial cells from the FGT of patients undergoing hysterectomy. We observed that these primary epithelial cells were less permissive to C. trachomatis infection than the cell line classically used in laboratories, i.e. HeLa cells. We have shown that the difference in culture medium and the addition of serum in HeLa cell cultures explain a large part of these differences. However, when tested in an identical culture medium, primary ectocervical epithelial cells were found to be less permissive than HeLa cells towards C. trachomatis infection. Finally, primary epithelial cells expressed overall more pro-inflammatory cytokines, both basally and after C. trachomatis infection, than HeLa cells, suggesting a strong capacity of primary epithelial cells to mount an inflammatory response. We then focused on understanding why type I interferon (IFN-I) acts synergistically with C. trachomatis infection towards the pro-inflammatory response of epithelial cells. We demonstrated that IFN-I, but not C. trachomatis, increased the expression of several bacterial pattern recognition receptors (PRRs). Expression silencing of TLR3 receptor, or deletion of this gene, prevented synergetic effect between IFN-I and C. trachomatis. We also identified the intermediate signaling pathway between IFN-I receptor activation and TLR3 expression, as well as the signaling downstream of TLR3 activation, which results in the expression of the pro-inflammatory cytokines interleukin-6 and 8. Based on these data, we conclude that IFN-I exacerbates the host inflammatory response triggered by C. trachomatis infection via increased TLR3 expression, and that this synergetic effect between IFN-I and bacteria on pro-inflammatory signaling in epithelial cells may play a role in the tissue damage that results from infection in some of the patients.
... Both clathrin-mediated entry and receptor binding constitute features generally proposed to be part of the adhesin-mediated, zipper mechanism (Veiga et al., 2007). Finally, Chlamydia entry happens through filopodial capture, prior to phagocytic cup zippering tightly around the EBs (Nans et al., 2014), suggesting a mixed mechanism of entry. ...
Invasive pathogenic bacteria exploit the host cell cytoskeleton and intracellular signaling pathways inducing their own uptake into non-phagocytic cells. After entry, some bacteria establish their replicative niche inside a membrane-bound compartment, while others escape to the cytosol where they replicate and disseminate to neighboring cells by means of actin-based motility. The major steps in bacterial invasion, i.e., entry, survival, replication and dissemination, reveal both common and unique mechanisms that drive a successful infection.
... This control shows that Cya detection in the supernatant was not due to 174 bacterial lysis. Finally, the Cya reporter was not recovered in the supernatants of the mxiD 175 strain, demonstrating that its secretion was dependent on the T3S system. 176 177 Evidence for the direct secretion of CT295 in the inclusion lumen 178 C. trachomatis GlgA is detected both in the inclusion lumen and in the cytoplasm (17). ...
Microorganisms with an intracellular development lifestyle exert a strong pressure on the metabolism of their host, since they obtain all their nutriments from its cytoplasm. The obligate intracellular bacteria Chlamydia trachomatis provides an extreme illustration of this: they rely on the host not only for the supply of glucose, their main carbon source, but probably also, at least partially, for the supply of the energy currency generated through glucose catabolism, adenosine triphosphate (ATP). These bacteria undergo a particular biphasic developmental cycle: the infectious bacteria, or elementary bodies (EBs), adhere to the membrane of a host cell, typically of the epithelium of the genital tract, and trigger their internalization. Once inside a membrane-bound compartment, called an inclusion, the bacteria express a new set of genes and convert to reticulate bodies (RBs). This only replicative form of the bacteria has a higher metabolism than EBs. Bacteria multiply in the inclusion several times until RBs convert back to EBs, which, once released, can initiate a new infectious cycle. The metabolic pressure exerted by the bacteria on their host thus evolves with time. However, whether infection modulates the metabolism of its host, and the degree of the reliance of individual steps of the bacterial development cycle on host metabolism, remain largely unknown. In this work, using primary epithelial cells and a cell line of non tumoral origin, we showed that the two main ATP producing pathways of the host, glycolysis and oxidative phosphorylation, remained fairly stable during infection. These results suggest that, against our expectations, there is no significant shift of the host metabolism towards glycolysis during infection. Inhibition of either pathway strongly reduced the capacity of the bacteria to undergo a developmental cycle. While EBs showed some degree of energetic autonomy in the synthesis of the first proteins expressed at the onset of infection, a functional glycolysis was necessary for the establishment of early inclusions, while oxidative phosphorylation is less needed at this early stage of development. The relative importance of the two pathways to sustain the initial steps of infection correlates with their relative contribution in maintaining ATP levels in epithelial cells, glycolysis being the main contributor. Altogether, this work confirms the dependence of the bacteria on the ATP production capacity of the host. However, ATP consumption by the bacteria appears to be fairly balanced with the normal production capacity of the host, and the autonomous production capacity of the bacteria, so that no major shift in host metabolism is required to meet bacterial needs.
... Interestingly, only the metabolically inactive EBs were affected. Whole-cell cryo-electron tomography revealed polarization of the type 3 secretion system on EBs during distinct stages of their development [32]. This may increase their susceptibility to deformation. ...
The family Chlamydiaceae currently comprises a single genus Chlamydia, with 11 validly published species and seven more taxa. It includes the human pathogens Chlamydia (C.) trachomatis, C. pneumoniae and C. psittaci, a zoonotic agent causing avian chlamydiosis and human psittacosis, as well as other proven or potential pathogens in ruminants, birds, snakes, reptiles and turtles. During routine testing of 15 apparently healthy captive flamingos in a zoo in 2011, an atypical strain of Chlamydiaceae was detected by real-time PCR of cloacal swab samples. Sequence analysis of the 16S rRNA gene revealed high similarity to the uncultured Chlamydiales bacterium clone 122, which previously had been found in gulls. As more samples were collected during annual campaigns of the flamingo ringing program in southern France from 2012 to 2015, Chlamydiaceae-specific DNA was detected by PCR in 30.9% of wild birds. From these samples, three strains were successfully grown in cell culture. Ultrastructural analysis, comparison of 16S and 23S rRNA gene sequences, whole-genome analysis based on de novo hybrid-assembled sequences of the new strains as well as subsequent calculation of taxonomic parameters revealed that the relatedness of the flamingo isolates to established members of the family Chlamydiaceae was sufficiently distant to indicate that the three strains belong to two distinct species within a new genus. Based on these data, we propose the introduction of Chlamydiifrater gen. nov., as a new genus, and Chlamydiifrater phoenicopteri sp. nov. and Chlamydiifrater volucris sp. nov., as two new species of the genus.
... Here, we elucidate how in vivo cross-linking of human plasma to the surface of live bacteria can provide detailed information of protein-protein complexes in their native environment, with possible implications for the studies of conformational differences between in vitro crystallized and electron cryo-microscopically imaged and reconstructed macromolecular complexes and in vivo cross-linked ones. The approach detailed here should also open up new avenues for the future integration of XL-MS data with in vivo prokaryotic electron cryo-tomographic imaging and model reconstruction of host-pathogen complexes (Nans et al., 2014(Nans et al., , 2015Chowdhury et al., 2020). ...
Protein–protein interactions are central in many biological processes, but they are challenging to characterize, especially in complex samples. Protein cross-linking combined with mass spectrometry (MS) and computational modeling is gaining increased recognition as a viable tool in protein interaction studies. Here, we provide insights into the structure of the multicomponent human complement system membrane attack complex (MAC) using in vivo cross-linking MS combined with computational macromolecular modeling. We developed an affinity procedure followed by chemical cross-linking on human blood plasma using live Streptococcus pyogenes to enrich for native MAC associated with the bacterial surface. In this highly complex sample, we identified over 100 cross-linked lysine–lysine pairs between different MAC components that enabled us to present a quaternary model of the assembled MAC in its native environment. Demonstrating the validity of our approach, this MAC model is supported by existing X-ray crystallographic and electron cryo-microscopic models. This approach allows the study of protein–protein interactions in native environment mimicking their natural milieu. Its high potential in assisting and refining data interpretation in electron cryo-tomographic experiments will be discussed.
... The use of cryo-electron tomography has recently been used to capture snapshots of how chlamydia EBs are taken up by nonphagocytic cells. These approaches have revealed that C. trachomatis orients its polarized T3SS to make direct contact with the host cell membrane [37]. Contact between the T3SS and the host cell membrane induces the formation of macropinosomes, phagocytic cups, and actin-rich filopodia [26]. ...
As an obligate intracellular pathogen, host cell invasion is paramount to Chlamydia trachomatis proliferation. While the mechanistic underpinnings of this essential process remain ill-defined, it is predicted to involve delivery of prepackaged effector proteins into the host cell that trigger plasma membrane remodeling and cytoskeletal reorganization. The secreted effector proteins TmeA and TarP, have risen to prominence as putative key regulators of cellular invasion and bacterial pathogenesis. Although several studies have begun to unravel molecular details underlying the putative function of TarP, the physiological function of TmeA during host cell invasion is unknown. Here, we show that TmeA employs molecular mimicry to bind to the GTPase binding domain of N-WASP, which results in recruitment of the actin branching ARP2/3 complex to the site of chlamydial entry. Electron microscopy revealed that TmeA mutants are deficient in filopodia capture, suggesting that TmeA/N-WASP interactions ultimately modulate host cell plasma membrane remodeling events necessary for chlamydial entry. Importantly, while both TmeA and TarP are necessary for effective host cell invasion, we show that these effectors target distinct pathways that ultimately converge on activation of the ARP2/3 complex. In line with this observation, we show that a double mutant suffers from a severe entry defect nearly identical to that observed when ARP3 is chemically inhibited or knocked down. Collectively, our study highlights both TmeA and TarP as essential regulators of chlamydial invasion that modulate the ARP2/3 complex through distinct signaling platforms, resulting in plasma membrane remodeling events that are essential for pathogen uptake.
