Formation Of Translocon Research Articles

Novel humanized anti-PcrV monoclonal antibody COT-143 protects mice from lethal Pseudomonas aeruginosa infection via inhibition of toxin translocation by the type III secretion system.

Treatment of Pseudomonas aeruginosa infection is challenging due to its intrinsic and acquired antibiotic resistance. As the number of current therapeutic options for P. aeruginosa infections is limited, developing novel treatments against the pathogen is an urgent clinical priority. The suppression of virulence of P. aeruginosa could be a new therapeutic option, and the type III secretion system (T3SS), which enables the bacteria to translocate various kinds of toxins into host cells and inhibits cellular functions, is considered as one possible target. In this report, we examined T3SS inhibition by COT-143/INFEX702, a humanized monoclonal antibody against PcrV, T3SS component, and present the crystal structure of the antibody-PcrV complex. COT-143 inhibited T3SS-dependent cytotoxicity and protected mice from the mortality caused by P. aeruginosa infection. The inhibition of cytotoxicity coincided with inhibition of translocon formation in a host cell membrane, which is necessary for T3SS intoxication. COT-143 protected murine neutrophils and facilitated phagocytosis of P. aeruginosa. These results suggest that COT-143 facilitates P. aeruginosa clearance by protecting neutrophil via inhibition of T3SS-dependent toxin translocation. This is the first report to show that an anti-PcrV antibody directly interferes with translocon formation to inhibit intoxication of host cells.

Tracing the origin of fish immunoglobulins

We have studied the origin of immunoglobulin genes in fish. There are two evolutionary lines of bony fish, Actinopterygii and Sarcopterygii. The former gave rise to most of the current fish and the latter to the animals that went to land. Non-teleost actinopterygians are significant evolutionary, sharing a common ancestor with sarcopterygians. There are three different immunoglob- ulin isotypes in ray-finned fish: IgM, IgD and IgT. We deduce that translocon formation in im- munoglobulins genes occurred already in non-teleost Actinopterygii. We establish a relationship between no teleosts and teleostean fish at the domain level of different immunoglobulins. We found two evolutionary lines of immunoglobulin. A line that starts from Immunoglobulin M and another from an ancestral Immunoglobulin W. The M line is stable, and the W line gives rise to the IgD of the fish. Immunoglobulin T emerges by recombination between both lines.

Live imaging of Yersinia translocon formation and immune recognition in host cells.

Yersinia enterocolitica employs a type three secretion system (T3SS) to translocate immunosuppressive effector proteins into host cells. To this end, the T3SS assembles a translocon/pore complex composed of the translocator proteins YopB and YopD in host cell membranes serving as an entry port for the effectors. The translocon is formed in a Yersinia-containing pre-phagosomal compartment that is connected to the extracellular space. As the phagosome matures, the translocon and the membrane damage it causes are recognized by the cell-autonomous immune system. We infected cells in the presence of fluorophore-labeled ALFA-tag-binding nanobodies with a Y. enterocolitica strain expressing YopD labeled with an ALFA-tag. Thereby we could record the integration of YopD into translocons and its intracellular fate in living host cells. YopD was integrated into translocons around 2 min after uptake of the bacteria into a phosphatidylinositol-4,5-bisphosphate enriched pre-phagosomal compartment and remained there for 27 min on average. Damaging of the phagosomal membrane as visualized with recruitment of GFP-tagged galectin-3 occurred in the mean around 14 min after translocon formation. Shortly after recruitment of galectin-3, guanylate-binding protein 1 (GBP-1) was recruited to phagosomes, which was accompanied by a decrease in the signal intensity of translocons, suggesting their degradation or disassembly. In sum, we were able for the first time to film the spatiotemporal dynamics of Yersinia T3SS translocon formation and degradation and its sensing by components of the cell-autonomous immune system.

The Tip Complex: From Host Cell Sensing to Translocon Formation.

Type III secretion systems are used by some Gram-negative bacteria to inject effector proteins into targeted eukaryotic cells for the benefit of the bacterium. The type III secretion injectisome is a complex nanomachine comprised of four main substructures including a cytoplasmic sorting platform, an envelope-spanning basal body, an extracellular needle and an exposed needle tip complex. Upon contact with a host cell, secretion is induced, resulting in the formation of a translocon pore in the host membrane. Translocon formation completes the conduit needed for effector secretion into the host cell. Control of type III secretion occurs in response to environmental signals, with the final signal being host cell contact. Secretion control occurs primarily at two sites-the cytoplasmic sorting platform, which determines secretion hierarchy, and the needle tip complex, which is critical for sensing and responding to environmental signals. The best-characterized injectisomes are those from Yersinia, Shigella and Salmonella species where there is a wealth of information on the tip complex and the two translocator proteins. Of these systems, the best characterized from a secretion regulation standpoint is Shigella. In the Shigella system, the tip complex and the first secreted translocon both contribute to secretion control and, thus, both are considered components of the tip complex. In this review, all three of these type III secretion systems are described with discussion focused on the structure and formation of the injectisome tip complex and what is known of the transition from nascent tip complex to assembled translocon pore.

Visualization of translocons in Yersinia type III protein secretion machines during host cell infection.

Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery—also named injectisome—assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their formation have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology, we provide novel information about the spatiotemporal organization of T3SS translocons and their regulation by host cell factors.

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.

SipB-SipC Complex Is Essential for Translocon Formation

The delivery of effector proteins by Salmonella across the host cell membrane requires a subset of effectors secreted by the type III secretion system (TTSS) known as translocators. SipC and SipB are translocator proteins that are inserted into host membranes and presumably form a channel that translocates type III effectors into the host cell. The molecular events of how these translocators insert into the host cell membrane remain unknown. We have previously shown that the SipC C-terminal amino acid region (321–409) is required for the translocation of effectors into host cells. In this study, we demonstrate that the ability to form SipC-SipB complex is essential for their insertion into the host membrane. The SipB-interacting domain of SipC is near its C-terminal amino acid region (340–409). In the absence of SipB, SipC was not detected in the membrane fraction. Furthermore, SipC mutants that no longer interact with SipB are defective in inserting into the host cell membrane. We propose a mechanism whereby SipC binds SipB through its C-terminal region to facilitate membrane-insertion and subsequent translocon formation in the host cell membrane.

A Mutant with Aberrant Extracellular LcrV-YscF Interactions Fails To Form Pores and Translocate Yop Effector Proteins but Retains the Ability To Trigger Yop Secretion in Response to Host Cell Contact

The plasmid-encoded type three secretion system (TTSS) of Yersinia spp. is responsible for the delivery of effector proteins into cells of the innate immune system, where these effectors disrupt the target cells' activity. Successful translocation of effectors into mammalian cells requires Yersinia to both insert a translocon into the host cell membrane and sense contact with host cells. To probe the events necessary for translocation, we investigated protein-protein interactions among TTSS components of the needle-translocon complex using a chemical cross-linking-based approach. We detected extracellular protein complexes containing YscF, LcrV, and YopD that were dependent upon needle formation. The formation of these complexes was evaluated in a secretion-competent but translocation-defective mutant, the YscFD28AD46A strain (expressing YscF with the mutations D28A and D46A). We found that one of the YscF and most of the LcrV and YopD cross-linked complexes were nearly absent in this mutant. Furthermore, the YscFD28AD46A strain did not support YopB insertion into mammalian membranes, supporting the idea that the LcrV tip complex is required for YopB insertion and translocon formation. However, the YscFD28AD46A strain did secrete Yops in the presence of host cells, indicating that a translocation-competent tip complex is not required to sense contact with host cells to trigger Yop secretion. In conclusion, in the absence of cross-linkable LcrV-YscF interactions, translocon insertion is abolished, but Yersinia still retains the ability to sense cell contact.

Association of a Protective Monoclonal IgA with the O Antigen of Salmonella enterica Serovar Typhimurium Impacts Type 3 Secretion and Outer Membrane Integrity

Invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium is an energetically demanding process, involving the transfer of effector proteins from invading bacteria into host cells via a specialized organelle known as the Salmonella pathogenicity island 1 (SPI-1) type 3 secretion system (T3SS). By a mechanism that remains poorly understood, entry of S. Typhimurium into epithelial cells is inhibited by Sal4, a monoclonal, polymeric IgA antibody that binds an immunodominant epitope within the O-antigen (O-Ag) component of lipopolysaccharide. In this study, we investigated how the binding of Sal4 to the surface of S. Typhimurium influences T3SS activity, bacterial energetics, and outer membrane integrity. We found that Sal4 treatment impaired T3SS-mediated translocon formation and attenuated the delivery of tagged effector proteins into epithelial cells. Sal4 treatment coincided with a partial reduction in membrane energetics and intracellular ATP levels, possibly explaining the impairment in T3SS activity. Sal4's effects on bacterial secretion and energetics occurred concurrently with an increase in O-Ag levels in culture supernatants, alterations in outer membrane permeability, and changes in surface ultrastructure, as revealed by transmission electron microscopy and cryo-electron microscopy. We propose that Sal4, by virtue of its ability to bind and cross-link the O-Ag, induces a form of outer membrane stress that compromises the integrity of the S. Typhimurium cell envelope and temporarily renders the bacterium avirulent.

Membrane targeting and pore formation by the type III secretion system translocon

The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative species to initiate infection. Toxins secreted through the system are synthesized in the bacterial cytoplasm and utilize the T3SS to pass through both bacterial membranes and the periplasm, thus being introduced directly into the eukaryotic cytoplasm. A key element of the T3SS of all bacterial pathogens is the translocon, which comprises a pore that is inserted into the membrane of the target cell, allowing toxin injection. Three macromolecular partners associate to form the translocon: two are hydrophobic and one is hydrophilic, and the latter also associates with the T3SS needle. In this review, we discuss recent advances on the biochemical and structural characterization of the proteins involved in translocon formation, as well as their participation in the modification of intracellular signalling pathways upon infection. Models of translocon assembly and regulation are also discussed.

Mobility of BtuB and OmpF in the Escherichia coli Outer Membrane: Implications for Dynamic Formation of a Translocon Complex

Diffusion of two Escherichia coli outer membrane proteins—the cobalamin (vitamin B12) receptor (BtuB) and the OmpF porin, which are implicated in the cellular import pathways of colicins and phages—was measured in vivo. The lateral mobility of these proteins is relevant to the mechanism of formation of the translocon for cellular import of colicins such as the rRNase colicin E3. The diffusion coefficient ( D) of BtuB, the primary colicin receptor, complexed to fluorescent antibody or colicin, is 0.05 ± 0.01 μm 2/s and 0.10 ± 0.02 μm 2/s, respectively, over a timescale of 25–150 ms. Mutagenesis of the BtuB TonB box, which eliminates or significantly weakens the interaction between BtuB and the TonB energy-transducing protein that is anchored in the cytoplasmic membrane, resulted in a fivefold larger value of D, 0.27 ± 0.06 μm 2/s for antibody-labeled BtuB, indicating a cytoskeletal-like interaction of TonB with BtuB. OmpF has a diffusion coefficient of 0.006 ± 0.002 μm 2/s, ∼10-fold smaller than that of BtuB, and is restricted within a domain of diameter 100 nm, showing it to be relatively immobile compared to BtuB. Thus, formation of the outer membrane translocon for cellular import of the nuclease colicins is a demonstrably dynamic process, because it depends on lateral diffusion of BtuB and collisional interaction with relatively immobile OmpF.

Structural Basis of Chaperone Recognition of Type III Secretion System Minor Translocator Proteins

The type III secretion system (T3SS) is a complex nanomachine employed by many Gram-negative pathogens, including the nosocomial agent Pseudomonas aeruginosa, to inject toxins directly into the cytoplasm of eukaryotic cells. A key component of all T3SS is the translocon, a proteinaceous channel that is inserted into the target membrane, which allows passage of toxins into target cells. In most bacterial species, two distinct membrane proteins (the "translocators") are involved in translocon formation, whereas in the bacterial cytoplasm, however, they remain associated to a common chaperone. To date, the strategy employed by a single chaperone to recognize two distinct translocators is unknown. Here, we report the crystal structure of a complex between the Pseudomonas translocator chaperone PcrH and a short region from the minor translocator PopD. PcrH displays a 7-helical tetratricopeptide repeat fold that harbors the PopD peptide within its concave region, originally believed to be involved in recognition of the major translocator, PopB. Point mutations introduced into the PcrH-interacting region of PopD impede translocator-chaperone recognition in vitro and lead to impairment of bacterial cytotoxicity toward macrophages in vivo. These results indicate that T3SS translocator chaperones form binary complexes with their partner molecules, and the stability of their interaction regions must be strictly maintained to guarantee bacterial infectivity. The PcrH-PopD complex displays homologs among a number of pathogenic strains and could represent a novel, potential target for antibiotic development.

Mapping of the chaperone AcrH binding regions of translocators AopB and AopD and characterization of oligomeric and metastable AcrH‐AopB‐AopD complexes in the type III secretion system of Aeromonas hydrophila

In the type III secretion system (T3SS) of Aeromonas hydrophila, AcrH acts as a chaperone for translocators AopB and AopD. AcrH forms a stable 1:1 monomeric complex with AopD, whereas the 1:1 AcrH-AopB complex exists mainly as a metastable oligomeric form and only in minor amounts as a stable monomeric form. Limited protease digestion shows that these complexes contain highly exposed regions, thus allowing mapping of intact functional chaperone binding regions of AopB and AopD. AopD uses the transmembrane domain (DF1, residues 16-147) and the C-terminal amphipathic helical domain (DF2, residues 242-296) whereas AopB uses a discrete region containing the transmembrane domain and the putative N-terminal coiled coil domain (BF1, residues 33-264). Oligomerization of the AcrH-AopB complex is mainly through the C-terminal coiled coil domain of AopB, which is dispensable for chaperone binding. The three proteins, AcrH, AopB, and AopD, can be coexpressed to form an oligomeric and metastable complex. These three proteins are also oligomerized mainly through the C-terminal domain of AopB. Formation of such an oligomeric and metastable complex may be important for the proper formation of translocon of correct topology and stoichiometry on the host membrane.

Twin Arginine Translocation (Tat)-dependent Export in the Apparent Absence of TatABC or TatA Complexes Using Modified Escherichia coli TatA Subunits That Substitute for TatB

The twin arginine translocation pathway exports folded proteins across the cytoplasmic membrane of many bacteria. In Escherichia coli and other Gram-negative bacteria, TatA, TatB, and TatC are all essential for efficient translocation, and current models suggest that separate TatABC and TatA complexes coalesce at the point of translocation. However, other microbes appear only to possess tatA and tatC genes. In Escherichia coli, virtually no translocation is observed when only TatA and TatC are present, but several mutations at the extreme N terminus of TatA were shown to support translocation. Here we show that these apparently bifunctional mutant TatA variants can function as typical TatA components because translocation is observed when they are co-expressed with TatBC, and they assemble into large, heterogeneous complexes that resemble wild type TatA complexes. However, cells expressing TatC plus the mutant TatA variants do not contain complexes that resemble the expected 370-kDa TatABC complex, clearly indicating that the mutant TatA forms cannot assemble efficiently, or stably, into this complex. The simultaneous expression of wild type TatA furthermore blocks translocation activity, suggesting that the mutant TatA forms preferentially bind to other TatA molecules rather than TatC. Surprisingly, we observe translocation in the absence of detectable free TatA, when translational fusions of the mutant TatAs with TatC are expressed. Transport can thus proceed in the simultaneous absence of TatABC and TatA complexes at detectable levels, and we conclude that the active translocon may be formed from dynamic twin arginine translocation complexes, one or more of which may await characterization.

The Plug Domain of Yeast Sec61p Is Important for Efficient Protein Translocation, but Is Not Essential for Cell Viability

The Sec61/SecY translocon mediates translocation of proteins across the membrane and integration of membrane proteins into the lipid bilayer. The structure of the translocon revealed a plug domain blocking the pore on the lumenal side. It was proposed to be important for gating the protein conducting channel and for maintaining the permeability barrier in its unoccupied state. Here, we analyzed in yeast the effect of introducing destabilizing point mutations in the plug domain or of its partial or complete deletion. Unexpectedly, even when the entire plug domain was deleted, cells were viable without growth phenotype. They showed an effect on signal sequence orientation of diagnostic signal-anchor proteins, a minor defect in cotranslational and a significant deficiency in posttranslational translocation. Steady-state levels of the mutant protein were reduced, and when coexpressed with wild-type Sec61p, the mutant lacking the plug competed poorly for complex partners. The results suggest that the plug is unlikely to be important for sealing the translocation pore in yeast but that it plays a role in stabilizing Sec61p during translocon formation.

Synergistic Pore Formation by Type III Toxin Translocators ofPseudomonas aeruginosa

Type III secretion/translocation systems are essential actors in the pathogenicity of Gram-negative bacteria. The injection of bacterial toxins across the host cell plasma membranes is presumably accomplished by a proteinaceous structure, the translocon. In vitro, Pseudomonas aeruginosa translocators PopB and PopD form ringlike structures observed by electron microscopy. We demonstrate here that PopB and PopD are functionally active and sufficient to form pores in lipid vesicles. Furthermore, the two translocators act in synergy to promote membrane permeabilization. The size-based selectivity observed for the passage of solutes indicates that the membrane permeabilization is due to the formation of size-defined pores. Our results provide also new insights into the mechanism of translocon pore formation that may occur during the passage of toxins from the bacterium into the cell. While proteins bind to lipid vesicles equally at any pH, the kinetics of membrane permeabilization accelerate progressively with decreasing pH values. Electrostatic interactions and the presence of anionic lipids were found to be crucial for pore formation whereas cholesterol did not appear to play a significant role in functional translocon formation.