An Inv/Mxi-Spa-like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen

Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals that is endemic in subtropical areas. B. pseudomallei is a facultative intracellular pathogen that may invade and survive within eukaryotic cells for prolonged periods. After internalization, the bacteria escape from endocytic vacuoles into the cytoplasm of infected cells and form membrane protrusions by inducing actin polymerization at one pole. It is believed that survival within phagocytic cells and cell-to-cell spread via actin protrusions is required for full virulence. We have studied the role of a putative type III protein secretion apparatus (Bsa) in the interaction between B. pseudomallei and host cells. The Bsa system is very similar to the Inv/Mxi-Spa type III secretion systems of Salmonella and Shigella. Moreover, B. pseudomallei encodes proteins that are very similar to Salmonella and Shigella Inv/Mxi-Spa secreted proteins required for invasion, escape from endocytic vacuoles, intercellular spread and pathogenesis. Antibodies to putative Bsa-secreted proteins were detected in convalescent serum from a melioidosis patient, suggesting that the system is functionally expressed in vivo. B. pseudomallei mutant strains lacking components of the Bsa secretion and translocation apparatus were constructed. The mutant strains exhibited reduced replication in J774.2 murine macrophage-like cells, an inability to escape from endocytic vacuoles and a complete absence of formation of membrane protrusions and actin tails. These findings indicate that the Bsa type III secretion system plays an essential role in modulating the intracellular behaviour of B. pseudomallei.     

Crystal structure of Spa40, the specificity switch for the Shigella flexneri type III secretion system

The pathogenic bacterium Shigella flexneri uses a type III secretion system to inject virulence factors from the bacterial cytosol directly into host cells. The machinery that identifies secretion substrates and controls the export of extracellular components and effector proteins consists of several inner-membrane and cytoplasmic proteins. One of the inner membrane components, Spa40, belongs to a family of proteins proposed to regulate the switching of substrate specificity of the export apparatus. We show that Spa40 is cleaved within the strictly conserved amino acid sequence NPTH and substitution of the proposed autocatalytic residue abolishes cleavage. Here we also report the crystal structure of the cytoplasmic complex Spa40_C and compare it with the recent structures of the homologues from Escherichia coli and Salmonella typhimurium . These structures reveal the tight association of the cleaved fragments and show that the conserved NPTH sequence lies on a loop which, when cleaved, swings away from the catalytic N257 residue, resulting in different surface features in this region. This structural rearrangement suggests a mechanism by which non-cleaving forms of these proteins interfere with correct substrate switching of the apparatus.     

Spa15 of Shigella flexneri, a third type of chaperone in the type III secretion pathway

The type III secretion (TTS) pathway is used by numerous Gram-negative pathogens to inject virulence factors into eukaryotic cells. In addition to a functional TTS apparatus, secretion of effector proteins depends upon specific chaperones. Using a two-hybrid screen in yeast and a co-purification assay in Shigella flexneri, we demonstrated that Spa15, which is encoded by an operon for components of the TTS apparatus, is associated in the cytoplasm with three proteins that are secreted by the TTS pathway, IpaA, IpgB1 and OspC3. Spa15 was found to be necessary for stability of IpgB1 but not IpaA, and for secretion of IpaA molecules that were stored in the cytoplasm but not those that were synthesized while the secretion apparatus was active. The ability of Spa15 to associate with several non-homologous secreted proteins, the presence of Spa15 homologues in other TTS systems and the location of the corresponding genes within operons for components of the TTS apparatus suggest that Spa15 belongs to a new class of TTS chaperones.     

Shigella Spa33 is an essential C-ring component of type III secretion machinery

Type III secretion machinery (TTSM), composed of a needle, a basal body, and a C-ring compartment, delivers a subset of effectors into host cells. Here, we show that Shigella Spa33 is an essential component of the C-ring compartment involved in mediating the transit of various TTSM-associated translocated proteins. Electron microscopic analysis and pull-down assay revealed Spa33 to be localized beneath the TTSM via interaction with MxiG and MxiJ (basal body components). Spa33 is also capable of interacting with Spa47 (TTSM ATPase), MxiK, MxiN (required for the transit of MxiH, the needle component), Spa32 (required for determining needle length), and several effectors. Genetic and functional analyses of the Spa33 C-terminal region, which is highly conserved in the SpaO-YscQ-HrcQ(B)-FliN family, indicate that some of the conserved residues are crucial for needle formation via interactions with MxiN. Thus, Spa33 plays a central role as the C-ring component in recruiting/exporting TTSM-associated proteins.     

Spa32 interaction with the inner‐membrane Spa40 component of the type III secretion system of Shigella flexneri is required for the control of the needle length by a molecular tape measure mechanism

The effectors of enterocyte invasion by Shigella are dependent on a type III secretion system that contains a needle whose length average does not exceed 50 nm. Previously, we reported that Spa32 is required for needle length control as well as to switch substrate specificity from MxiH to Ipa proteins secretion. To identify functional domains of Spa32, 11 truncated variants were constructed and analysed for their capacity (i) to control the needle's length; (ii) to secrete the Ipa proteins; and (iii) to invade HeLa cells. Deletion at either the N‐terminus or C‐terminus affect Spa32 function in all cases, but Spa32 variants lacking internal residues 37–94 or 130–159 retained full Spa32 function. Similarly, a Spa32 variant obtained by inserting of the YscP's ruler domain retained Spa32 function although it programmed slightly elongated needles. Using the GST pull‐down assay, we show that residues 206–246 are required for Spa32 binding to the C‐terminus of Spa40, an inner membrane protein required for Ipa proteins secretion. Our data clearly demonstrate that shortening Spa32 affects the length of the needle in a comparable manner to the spa32 mutant, indicating that the control of needle length does not require a molecular ruler mechanism.     

Spa47 is an oligomerization‐activated type three secretion system (T3SS) ATPase from Shigella flexneri

Gram‐negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti‐infective agents.     

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.     

Structural analysis of ligand‐bound states of the Salmonella type III secretion system ATPase InvC

Translocation of virulence effector proteins through the type III secretion system (T3SS) is essential for the virulence of many medically relevant Gram‐negative bacteria. The T3SS ATPases are conserved components that specifically recognize chaperone–effector complexes and energize effector secretion through the system. It is thought that functional T3SS ATPases assemble into a cylindrical structure maintained by their N‐terminal domains. Using size‐exclusion chromatography coupled to multi‐angle light scattering and native mass spectrometry, we show that in the absence of the N‐terminal oligomerization domain the Salmonella T3SS ATPase InvC can form monomers and dimers in solution. We also present for the first time a 2.05 å resolution crystal structure of InvC lacking the oligomerization domain (InvCΔ79) and map the amino acids suggested for ATPase intersubunit interaction, binding to other T3SS proteins and chaperone–effector recognition. Furthermore, we validate the InvC ATP‐binding site by co‐crystallization of InvCΔ79 with ATPγS (2.65 å) and ADP (2.80 å). Upon ATP‐analogue recognition, these structures reveal remodeling of the ATP‐binding site and conformational changes of two loops located outside of the catalytic site. Both loops face the central pore of the predicted InvC cylinder and are essential for the function of the T3SS ATPase. Our results present a fine functional and structural correlation of InvC and provide further details of the homo‐oligomerization process and ATP‐dependent conformational changes underlying the T3SS ATPase activity.     

Functional analysis of the type III secretion system in enteropathogenic Escherichia coli O157:H45

A mass outbreak of Escherichia coli O157:H45 was first reported in Japan in 1998. This pathogen was classified as an enteropathogenic E. coli (EPEC) O157 because it was characterized by the Shiga toxin gene (stx)-negative and bundle-forming pilus (bfp) gene-positive genotypes. In this study, we investigated the type III secretion system in EPEC O157. Although no type III secreted proteins, Esps (E. colisecreted proteins), in EPEC O157:H45 were detectable in culture supernatant, secreted proteins were induced by the introduction of an EPEC plasmid-encoded regulator, per. In further contrast to EHEC O157:H7, EPEC O157:H45 triggered the accumulation of tyrosine phosphorylated proteins beneath the adherent bacteria. These results suggest that regulation of the type III secretion apparatus and host signal transduction events between E. coli O157:H45 and O157:H7 are completely different.     

Structure of Spa15, a type III secretion chaperone from Shigella flexneri with broad specificity

Type III secretion (TTS) systems are used by many Gram-negative pathogens to inject virulence proteins into the cells of their hosts. Several of these virulence effectors require TTS chaperones that maintain them in a secretion-competent state. Whereas most chaperones bind only one effector, Spa15 from the human pathogen Shigella flexneri and homologous chaperones bind several seemingly unrelated effectors, and were proposed to form a special subgroup. Its 1.8 A crystal structure confirms this specific classification, showing that Spa15 has the same fold as other TTS effector chaperones, but forms a different dimer. The presence of hydrophobic sites on the Spa15 surface suggests that the different Spa15 effectors all possess similar structural elements that can bind these sites. Furthermore, the Spa15 structure reveals larger structural differences between class I chaperones than previously anticipated, which does not support the hypothesis that chaperone-effector complexes are structurally conserved and function as three-dimensional secretion signals.     

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.     

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.     

Interplay between predicted inner‐rod and gatekeeper in controlling substrate specificity of the type III secretion system

The type III secretion apparatus (T3SA) is a multi‐protein complex central to the virulence of many Gram‐negative pathogens. Currently, the mechanisms controlling the hierarchical addressing of needle subunits, translocators and effectors to the T3SA are still poorly understood. In Shigella, MxiC is known to sequester effectors within the cytoplasm prior to receiving the activation signal from the needle. However, molecules involved in linking the needle and MxiC are unknown. Here, we demonstrate a molecular interaction between MxiC and the predicted inner‐rod component MxiI suggesting that this complex plugs the T3SA entry gate. Our results suggest that MxiI–MxiC complex dissociation facilitates the switch in secretion from translocators to effectors. We identified MxiC^F206S variant, unable to interact with MxiI, which exhibits a constitutive secretion phenotype although it remains responsive to induction. Moreover, we identified the mxiI^Q67A mutant that only secretes translocators, a phenotype that was suppressed by coexpression of the MxiC^F206S variant. We demonstrated the interaction between MxiI and MxiC homologues in Yersinia and Salmonella. Lastly, we identified an interaction between MxiC and chaperone IpgC which contributes to understanding how translocators secretion is regulated. In summary, this study suggests the existence of a widely conserved T3S mechanism that regulates effectors secretion.     

Regulation of type III secretion system in Pseudomonas syringae

Pseudomonas syringae is a model phytopathogenic bacterium that uses the type III secretion system (T3SS) to cause lethal diseases in staple crops and thus presents a threat to food security worldwide. Great progress has been made in delineating the biochemical mechanisms and cellular targets of T3SS effectors, but less is known about the signalling pathways and molecular mechanisms of T3SS regulators. In recent years, thanks to the popularity and power of genome-wide mutant screening and high-throughput sequencing, new regulatory proteins (such as RhpR, AefR, AlgU and CvsR) and proteases (such as Lon and RhpP) have been identified as T3SS regulators in P. syringae pathovars. The detailed mechanisms of previously illustrated regulators (such as HrpRS, HrpL and HrpGV) have also been further studied. Notably, the two-component system RhpRS has been determined to play key roles in the modulation of T3SS via direct regulation of hrpRS and other virulence-related pathways by sensing changes in environmental signals. In addition, secondary messengers (such as c-di-GMP and ppGpp) have been shown to fine-tune the activity of T3SS. Overall, these studies have suggested the existence of a highly intricate regulatory network for T3SS, which controls the pathogenicity of P. syringae. This short review summarizes studies of P. syringae T3SS regulation and the known mechanisms of key regulators.     

Interfacial amino acids support Spa47 oligomerization and shigella type three secretion system activation

Like many Gram-negative pathogens, Shigella rely on a type three secretion system (T3SS) for injection of effector proteins directly into eukaryotic host cells to initiate and sustain infection. Protein secretion through the needle-like type three secretion apparatus (T3SA) requires ATP hydrolysis by the T3SS ATPase Spa47, making it a likely target for in vivo regulation of T3SS activity and an attractive target for small molecule therapeutics against shigellosis. Here, we developed a model of an activated Spa47 homo-hexamer, identifying two distinct regions at each protomer interface that we hypothesized to provide intermolecular interactions supporting Spa47 oligomerization and enzymatic activation. Mutational analysis and a series of high-resolution crystal structures confirm the importance of these residues, as many of the engineered mutants are unable to form oligomers and efficiently hydrolyze ATP in vitro. Furthermore, in vivo evaluation of Shigella virulence phenotype uncovered a strong correlation between T3SS effector protein secretion, host cell membrane disruption, and cellular invasion by the tested mutant strains, suggesting that perturbation of the identified interfacial residues/interactions influences Spa47 activity through preventing oligomer formation, which in turn regulates Shigella virulence. The most impactful mutations are observed within the conserved Site 2 interface where the native residues support oligomerization and likely contribute to a complex hydrogen bonding network that organizes the active site and supports catalysis. The critical reliance on these conserved residues suggests that aspects of T3SS regulation may also be conserved, providing promise for the development of a cross-species therapeutic that broadly targets T3SS ATPase oligomerization and activation.     

A translocator‐specific export signal establishes the translocator–effector secretion hierarchy that is important for type III secretion system function

Type III secretion systems are used by many Gram‐negative pathogens to directly deliver effector proteins into the cytoplasm of host cells. To accomplish this, bacteria secrete translocator proteins that form a pore in the host‐cell membrane through which the effector proteins are then introduced into the host cell. Evidence from multiple systems indicates that the pore‐forming translocator proteins are exported before effectors, but how this secretion hierarchy is established is unclear. Here we used the Pseudomonas aeruginosa translocator protein PopD as a model to identify its export signals. The N‐terminal secretion signal and chaperone, PcrH, are required for export under all conditions. Two novel signals in PopD, one proximal to the chaperone binding site and one at the very C‐terminus of the protein, are required for export of PopD before effector proteins. These novel export signals establish the translocator–effector secretion hierarchy, which in turn, is critical for the delivery of effectors into host cells.