Port of entry--the type III secretion translocon

Many Gram-negative plant and animal pathogenic bacteria use a specialized type III secretion system (TTSS) as a molecular syringe to inject effector proteins directly into the host cell. Protein translocation across the eukaryotic host cell membrane is presumably mediated by a bacterial translocon. The structure of this predicted transmembrane complex and the mechanism of transport are far from being understood. In bacterial pathogens of animals, several putative type III secretion translocon proteins (TTPs) have been identified. Interestingly, TTP sequences are not conserved among different bacterial species, however, there are structural similarities such as transmembrane segments and coiled-coil regions. Accumulating evidence suggests that TTPs are components of oligomeric protein channels that are inserted into the host cell membrane by the TTSS.     

Biogenesis, regulation, and targeting of the type III secretion system

The type III secretion system (T3SS) is employed by a number of Gram-negative bacterial pathogens to inject toxins into eukaryotic cells. The biogenesis of this complex machinery requires the regulated interaction between over 20 cytosolic, periplasmic, and membrane-imbedded proteins, many of which undergo processes such as polymerization, partner recognition, and partial unfolding. Elements of this intricate macromolecular system have been characterized through electron microscopy, crystallography, and NMR techniques, allowing for an initial understanding of the spatiotemporal regulation of T3SS-related events. Here, we report recent advances in the structural characterization of T3SS proteins from a number of bacteria, and provide an overview of recently identified small molecule T3SS inhibitors that could potentially be explored for novel antibacterial development.     

A bacterial type III secretion system inhibits actin polymerization to prevent pore formation in host cell membranes

The bacterial pathogen Yersinia pseudotuberculosis uses type III secretion machinery to translocate Yop effector proteins through host cell plasma membranes. A current model suggests that a type III translocation channel is inserted into the plasma membrane, and if Yops are not present to fill the channel, the channel will form a pore. We examined the possibility that Yops act within the host cell to prevent pore formation. Yop- mutants of Y.pseudotuberculosis were assayed for pore-forming activity in HeLa cells. A YopE- mutant exhibited high levels of pore-forming activity. The GTPase-downregulating function of YopE was required to prevent pore formation. YopE+ bacteria had increased pore-forming activity when HeLa cells expressed activated Rho GTPases. Pore formation by YopE- bacteria required actin polymerization. F-actin was concentrated at sites of contact between HeLa cells and YopE- bacteria. The data suggest that localized actin polymerization, triggered by the type III machinery, results in pore formation in cells infected with YopE- bacteria. Thus, translocated YopE inhibits actin polymerization to prevent membane damage to cells infected with wild-type bacteria.     

Oligomerization of type III secretion proteins PopB and PopD precedes pore formation in Pseudomonas

Pseudomonas aeruginosa is the agent of opportunistic infections in immunocompromised individuals and chronic respiratory illnesses in cystic fibrosis patients. Pseudomonas aeruginosa utilizes a type III secretion system for injection of toxins into the host cell cytoplasm through a channel on the target membrane (the 'translocon'). Here, we have functionally and structurally characterized PopB and PopD, membrane proteins implicated in the formation of the P.aeruginosa translocon. PopB and PopD form soluble complexes with their common chaperone, PcrH, either as stable heterodimers or as metastable heterooligomers. Only oligomeric forms are able to bind to and disrupt cholesterol-rich membranes, which occurs within a pH range of 5-7 in the case of PopB/PcrH, and only at acidic pH for PcrH-free PopD. Electron microscopy reveals that upon membrane association PopB and PopD form 80 A wide rings which encircle 40 A wide cavities. Thus, formation of metastable oligomers precedes membrane association and ring generation in the formation of the Pseudomonas translocon, a mechanism which may be similar for other pathogens that employ type III secretion systems.     

Synergistic pore formation by type III toxin translocators of Pseudomonas 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.     

Hierarchical protein targeting and secretion is controlled by an affinity switch in the type III secretion system of enteropathogenic Escherichia coli

Type III secretion (T3S), a protein export pathway common to Gram‐negative pathogens, comprises a trans‐envelope syringe, the injectisome, with a cytoplasm‐facing translocase channel. Exported substrates are chaperone‐delivered to the translocase, EscV in enteropathogenic Escherichia coli, and cross it in strict hierarchical manner, for example, first “translocators”, then “effectors”. We dissected T3S substrate targeting and hierarchical switching by reconstituting them in vitro using inverted inner membrane vesicles. EscV recruits and conformationally activates the tightly membrane‐associated pseudo‐effector SepL and its chaperone SepD. This renders SepL a high‐affinity receptor for translocator/chaperone pairs, recognizing specific chaperone signals. In a second, SepD‐coupled step, translocators docked on SepL become secreted. During translocator secretion, SepL/SepD suppress effector/chaperone binding to EscV and prevent premature effector secretion. Disengagement of the SepL/SepD switch directs EscV to dedicated effector export. These findings advance molecular understanding of T3S and reveal a novel mechanism for hierarchical trafficking regulation in protein secretion channels.     

Evidence for targeting of Yop effectors by the chromosomally encoded Ysa type III secretion system of Yersinia enterocolitica

Yersinia enterocolitica O:8 has two contact-dependent type III secretion systems (TTSSs). The Ysa TTSS is encoded by a set of genes located on the chromosome and exports Ysp proteins. The Ysc TTSS and the Yop effector proteins it exports are encoded by genes located on plasmid pYVe8081. In this study, secretion of YspG, YspH, and YspJ by the Ysa TTSS was shown to require pYVe8081. Furthermore, mutations that blocked the function of the Ysc TTSS did not affect YspG, YspH, and YspJ production. This indicated that YspG, YspH, and YspJ are encoded by genes located on pYVe8081 and that they may correspond to Yops. A comparison of Ysps with Yop effectors secreted by Y. enterocolitica indicated that YspG, YspH, and YspJ have apparent molecular masses similar to those of YopN, YopP, and YopE, respectively. Immunoblot analysis demonstrated that antibodies directed against YopN, YopP, and YopE recognized YspG, YspH, and YspJ. Furthermore, mutations in yopN, yopP, and yopE specifically blocked YopN, YopP, and YopE secretion by the Ysc TTSS and YspG, YspH, and YspJ secretion by the Ysa TTSS. These results indicate YspG, YspH, and YspJ are actually YopN, YopP, and YopE. Additional analysis demonstrated that YopP and YspH secretion was restored to yopP mutants by complementation in trans with a wild-type copy of the yopP gene. Examination of Y. enterocolitica-infected J774A.1 macrophages revealed that both the Ysc and Ysa TTSSs contribute to YopP-dependent suppression of tumor necrosis factor alpha production. This indicates that both the Ysa and Ysc TTSSs are capable of targeting YopP and that they influence Y. enterocolitica interactions with macrophages. Taken together, these results suggest that the Ysa and Ysc TTSSs contribute to Y. enterocolitica virulence by exporting both unique and common subsets of effectors.     

Process of protein transport by the type III secretion system.

The type III secretion system (TTSS) of gram-negative bacteria is responsible for delivering bacterial proteins, termed effectors, from the bacterial cytosol directly into the interior of host cells. The TTSS is expressed predominantly by pathogenic bacteria and is usually used to introduce deleterious effectors into host cells. While biochemical activities of effectors vary widely, the TTSS apparatus used to deliver these effectors is conserved and shows functional complementarity for secretion and translocation. This review focuses on proteins that constitute the TTSS apparatus and on mechanisms that guide effectors to the TTSS apparatus for transport. The TTSS apparatus includes predicted integral inner membrane proteins that are conserved widely across TTSSs and in the basal body of the bacterial flagellum. It also includes proteins that are specific to the TTSS and contribute to ring-like structures in the inner membrane and includes secretin family members that form ring-like structures in the outer membrane. Most prominently situated on these coaxial, membrane-embedded rings is a needle-like or pilus-like structure that is implicated as a conduit for effector translocation into host cells. A short region of mRNA sequence or protein sequence in effectors acts as a signal sequence, directing proteins for transport through the TTSS. Additionally, a number of effectors require the action of specific TTSS chaperones for efficient and physiologically meaningful translocation into host cells. Numerous models explaining how effectors are transported into host cells have been proposed, but understanding of this process is incomplete and this topic remains an active area of inquiry.     

Revisiting the chlamydial type III protein secretion system: clues to the origin of type III protein secretion

The bacterial type III secretion pathway delivers effector proteins into eukaryotic cells. Analysis of the type III system and flagellar export genes in the obligate parasites of the family Chlamydiales suggests that the type III system arose from the flagellar export system in chlamydiae or related bacteria.     

Pseudomonas syringae type III secretion system targeting signals and novel effectors studied with a Cya translocation reporter

Pseudomonas syringae pv. tomato strain DC3000 is a pathogen of tomato and Arabidopsis: The hrp-hrc-encoded type III secretion system (TTSS), which injects bacterial effector proteins (primarily called Hop or Avr proteins) into plant cells, is required for pathogenicity. In addition to being regulated by the HrpL alternative sigma factor, most avr or hop genes encode proteins with N termini that have several characteristic features, including (i) a high percentage of Ser residues, (ii) an aliphatic amino acid (Ile, Leu, or Val) or Pro at the third or fourth position, and (iii) a lack of negatively charged amino acids within the first 12 residues. Here, the well-studied effector AvrPto was used to optimize a calmodulin-dependent adenylate cyclase (Cya) reporter system for Hrp-mediated translocation of P. syringae TTSS effectors into plant cells. This system includes a cloned P. syringae hrp gene cluster and the model plant Nicotiana benthamiana. Analyses of truncated AvrPto proteins fused to Cya revealed that the N-terminal 16 amino acids and/or codons of AvrPto are sufficient to direct weak translocation into plant cells and that longer N-terminal fragments direct progressively stronger translocation. AvrB, tested because it is poorly secreted in cultures by the P. syringae Hrp system, was translocated into plant cells as effectively as AvrPto. The translocation of several DC3000 candidate Hop proteins was also examined by using Cya as a reporter, which led to identification of three new intact Hop proteins, designated HopPtoQ, HopPtoT1, and HopPtoV, as well as two truncated Hop proteins encoded by the naturally disrupted genes hopPtoS4::tnpA and hopPtoAG::tnpA. We also confirmed that HopPtoK, HopPtoC, and AvrPphE(Pto) are translocated into plant cells. These results increased the number of Hrp system-secreted proteins in DC3000 to 40. Although most of the newly identified Hop proteins possess N termini that have the same features as the N termini of previously described Hop proteins, HopPtoV has none of these characteristics. Our results indicate that Cya should be a useful reporter for exploring multiple aspects of the Hrp system in P. syringae.     

Small-molecule type III secretion system inhibitors block assembly of the Shigella type III secreton

Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds.     

The Salmonella type III secretion translocon protein SspC is inserted into the epithelial cell plasma membrane upon infection

Salmonella species translocate effector proteins into the host cell cytoplasm using a type III secretion system (TTSS). The translocation machinery probably contacts the eukaryotic cell plasma membrane to effect protein transfer. Data presented here demonstrate that both SspB and SspC, components of the translocation apparatus, are inserted into the epithelial cell plasma membrane 15 min after Salmonella typhimurium infection. In addition, a yeast two-hybrid interaction between SspC and an eukaryotic intermediate filament protein was identified. Three individual carboxyl-terminal point mutations within SspC that disrupt the yeast two-hybrid interaction were isolated. Strains expressing the mutant SspC alleles were defective for invasion, translocation of effector molecules and membrane localization of SspC. These data indicate that insertion of SspC into the plasma membrane of target cells is required for invasion and effector molecule translocation and that the carboxyl terminus of SspC is essential for these functions.     

Key steps in type III secretion system (T3SS) towards translocon assembly with potential sensor at plant plasma membrane

Many plant‐ and animal‐pathogenic Gram‐negative bacteria employ the type III secretion system (T3SS) to translocate effector proteins from bacterial cells into the cytosol of eukaryotic host cells. The effector translocation occurs through an integral component of T3SS, the channel‐like translocon, assembled by hydrophilic and hydrophobic proteinaceous translocators in a two‐step process. In the first, hydrophilic translocators localize to the tip of a proteinaceous needle in animal pathogens, or a proteinaceous pilus in plant pathogens, and associate with hydrophobic translocators, which insert into host plasma membranes in the second step. However, the pilus needs to penetrate plant cell walls in advance. All hydrophilic translocators so far identified in plant pathogens are characteristic of harpins: T3SS accessory proteins containing a unitary hydrophilic domain or an additional enzymatic domain. Two‐domain harpins carrying a pectate lyase domain potentially target plant cell walls and facilitate the penetration of the pectin‐rich middle lamella by the bacterial pilus. One‐domain harpins target plant plasma membranes and may play a crucial role in translocon assembly, which may also involve contrapuntal associations of hydrophobic translocators. In all cases, sensory components in the target plasma membrane are indispensable for the membrane recognition of translocators and the functionality of the translocon. The conjectural sensors point to membrane lipids and proteins, and a phosphatidic acid and an aquaporin are able to interact with selected harpin‐type translocators. Interactions between translocators and their sensors at the target plasma membrane are assumed to be critical for translocon assembly.     

Suppression of bacterial cell–cell signalling,biofilm formation and type III secretion system by citrus flavonoids

Aim: This study investigated the quorum sensing, biofilm and type three secretion system (TTSS) inhibitory properties of citrus flavonoids. Methods and Results: Flavonoids were tested for their ability to inhibit quorum sensing using Vibrio harveyi reporter assay. Biofilm assays were carried out in 96‐well plates. Inhibition of biofilm formation in Escherichia coli O157:H7 and V. harveyi by citrus flavonoids was measured. Furthermore, effect of naringenin on expression of V. harveyi TTSS was investigated by semi‐quantitative PCR. Differential responses for different flavonoids were observed for different cell–cell signalling systems. Among the tested flavonoids, naringenin, kaempferol, quercetin and apigenin were effective antagonists of cell–cell signalling. Furthermore, these flavonoids suppressed the biofilm formation in V. harveyi and E. coli O157:H7. In addition, naringenin altered the expression of genes encoding TTSS in V. harveyi. Conclusion: The results of the study indicate a potential modulation of bacterial cell–cell communication, E. coli O157:H7 biofilm and V. harveyi virulence, by flavonoids especially naringenin, quercetin, sinensetin and apigenin. Among the tested flavonoids, naringenin emerged as potent and possibly a nonspecific inhibitor of autoinducer‐mediated cell–cell signalling. Naringenin and other flavonoids are prominent secondary metabolites present in citrus species. Therefore, citrus, being a major source of some of these flavonoids and by virtue of widely consumed fruit, may modulate the intestinal microflora. Significance and Impact of the Study: Currently, a limited number of naturally occurring compounds have demonstrated their potential in inhibition of cell–cell communications; therefore, citrus flavonoids may be useful as lead compounds for the development of antipathogenic agents.     

Regulation of the type III secretion system in phytopathogenic bacteria

The type III secretion system (TTSS) is a specialized protein secretion machinery used by numerous gram-negative bacterial pathogens of animals and plants to deliver effector proteins directly into the host cells. In plant-pathogenic bacteria, genes encoding the TTSS were discovered as hypersensitive response and pathogenicity (hrp) genes, because mutation of these genes typically disrupts the bacterial ability to cause diseases on host plants and to elicit hypersensitive response on nonhost plants. The hrp genes and the type III effector genes (collectively called TTSS genes hereafter) are repressed in nutrient-rich media but induced when bacteria are infiltrated into plants or incubated in nutrient-deficient inducing media. Multiple regulatory components have been identified in the plant-pathogenic bacteria regulating TTSS genes under various conditions. In Ralstonia solanacearum, several signal transduction components essential for the induction of TTSS genes in plants are dispensable for the induction in inducing medium. In addition to the inducing signals, recent studies indicated the presence of negative signals in the plant regulating the Pseudomonas syringae TTSS genes. Thus, the levels of TTSS gene expression in plants likely are determined by the interactions of multiple signal transduction pathways. Studies of the hrp regulons indicated that TTSS genes are coordinately regulated with a number of non-TTSS genes.     

Endofungal bacterium controls its host by an hrp type III secretion system

Burkholderia rhizoxinica and Rhizopus microsporus form a unique symbiosis in which intracellular bacteria produce the virulence factor of the phytopathogenic fungus. Notably, the host strictly requires endobacteria to sporulate. In this study, we show that the endofungal bacteria possess a type III secretion system (T3SS), which has a crucial role in the maintenance of the alliance. Mutants defective in type III secretion show reduced intracellular survival and fail to elicit sporulation of the host. Furthermore, genes coding for T3SS components are upregulated during cocultivation of the bacterial symbiont with their host. This is the first report on a T3SS involved in bacterial–fungal symbiosis. Phylogenetic analysis revealed that the T3SS represents a prototype of a clade of yet uncharacterized T3SSs within the hrp superfamily of T3SSs from plant pathogenic microorganisms. In a control experiment, we demonstrate that under laboratory conditions, rhizoxin production was not required for establishment of the symbiotic interaction.     

High-yield production of secreted active proteins by the Pseudomonas aeruginosa type III secretion system

The Escherichia coli system is the system of choice for recombinant protein production because it is possible to obtain a high protein yield in inexpensive media. The accumulation of protein in an insoluble form in inclusion bodies remains a major disadvantage. Use of the Pseudomonas aeruginosa type III secretion system can avoid this problem, allowing the production of soluble secreted proteins.     

Hierarchical effector protein transport by the Salmonella Typhimurium SPI-1 type III secretion system

Background

Type III secretion systems (TTSS) are employed by numerous pathogenic and symbiotic bacteria to inject a cocktail of different “effector proteins” into host cells. These effectors subvert host cell signaling to establish symbiosis or disease.

Methodology/Principal Findings

We have studied the injection of SipA and SptP, two effector proteins of the invasion-associated Salmonella type III secretion system (TTSS-1). SipA and SptP trigger different host cell responses. SipA contributes to triggering actin rearrangements and invasion while SptP reverses the actin rearrangements after the invasion has been completed. Nevertheless, SipA and SptP were both pre-formed and stored in the bacterial cytosol before host cell encounter. By time lapse microscopy, we observed that SipA was injected earlier than SptP. Computer modeling revealed that two assumptions were sufficient to explain this injection hierarchy: a large number of SipA and SptP molecules compete for transport via a limiting number of TTSS; and the TTSS recognize SipA more efficiently than SptP.

Conclusions/Significance

This novel mechanism of hierarchical effector protein injection may serve to avoid functional interference between SipA and SptP. An injection hierarchy of this type may be of general importance, allowing bacteria to precisely time the host cell manipulation by type III effectors.