A Multifunctional Region of the Shigella Type 3 Effector IpgB1 Is Important for Secretion from Bacteria and Membrane Targeting in Eukaryotic Cells

Type 3 secretion systems are complex nanomachines used by many Gram–negative bacteria to deliver tens of proteins (effectors) directly into host cells. Once delivered into host cells, effectors often target to specific cellular loci where they usurp host cell processes to their advantage. Here, using the yeast model system, we identify the membrane localization domain (MLD) of IpgB1, a stretch of 20 amino acids enriched for hydrophobic residues essential for the targeting of this effector to the plasma membrane. Embedded within these residues are ten that define the IpgB1 chaperone-binding domain for Spa15. As observed with dedicated class IA chaperones that mask hydrophobic MLDs, Spa15, a class IB chaperone, promotes IpgB1 stability by binding this hydrophobic region. However, despite being stable, an IpgB1 allele that lacks the MLD is not recognized as a secreted substrate. Similarly, deletion of the chaperone binding domains of IpgB1 and three additional Spa15-dependent effectors result in alleles that are no longer recognized as secreted substrates despite the presence of intact N-terminal secretion signal sequences. This is in contrast with MLD-containing effectors that bind class IA dedicated chaperones, as deletion of the MLD of these effectors alleviates the chaperone requirement for secretion. These observations indicate that at least for substrates of class IB chaperones, the chaperone-effector complex plays a major role in defining type 3 secreted proteins and highlight how a single region of an effector can play important roles both within prokaryotic and eukaryotic cells.     

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.     

Characterization of the interaction partners of secreted proteins and chaperones of Shigella flexneri

The type III secretion (TTS) system of Gram-negative pathogenic bacteria is composed of proteins that assemble into the TTS machinery, proteins that are secreted by this machinery and specific chaperones that are required for storage and sometimes secretion of these proteins. Many sequential protein interactions are involved in the TTS pathway to deliver effector proteins to host cells. We used the yeast two-hybrid system to investigate the interaction partners of the Shigella flexneri effectors and chaperones. Libraries of preys containing random fusions with fragments of the TTS proteins were screened using effectors and chaperones as baits. Interactions between the effectors IpaB and IpaC and their chaperone IpgC were detected by this method, and interaction domains were identified. Using a His-tagged IpgC protein to co-purify truncated IpaB and IpaC proteins, we showed that the chaperone-binding domain was unique and located in the N-terminus of these proteins. This domain was not required for the secretion of recombinant proteins but was involved in the stability of IpaC and instability of IpaB. Homotypic interactions were identified with the baits IpaA, IpaB and IpaC. Interactions between effectors and components of the TTS machinery were also selected that might give insights into regulation of the TTS process.     

MxiK and MxiN interact with the Spa47 ATPase and are required for transit of the needle components MxiH and MxiI,but not of Ipa proteins,through the type III secretion apparatus of Shigella flexneri

The type III secretion (TTS) pathway is used by numerous Gram-negative pathogens to inject virulence factors into eukaryotic cells. The Shigella flexneri TTS apparatus (TTSA) spans the bacterial envelope and its assembly requires the products of approximately 20 mxi and spa genes. We present a functional analysis of the mxiK, mxiN and mxiL genes. Inactivation of mxiK and mxiN, but not mxiL, resulted in the assembly of a non-functional TTSA that lacked the outer needle. The amounts of needle components MxiH and MxiI were drastically reduced in mxiK and mxiN mutants and in the secretion defective spa47 mutant, indicating that MxiH and MxiI are degraded if they do not transit through the TTSA. Remarkably, expression of MxiH-His in the mxiN mutant and MxiI-His in the mxiK mutant restored assembly of a functional TTSA, as shown by the ability of these strains to enter into epithelial cells and to secrete Ipa proteins in response to activation by Congo red. Using a two-hybrid screen in yeast and immunoprecipitation assays from S. flexneri extracts, we identified interactions between MxiK and Spa33 and Spa47 and between MxiN and Spa33 and Spa47. These results suggest that transit of the needle components MxiH and MxiI through the TTSA involves the concerted action of the cytoplasmic proteins Spa47, Spa33, MxiK and MxiN. They also show that neither MxiK nor MxiN are absolutely required for secretion of Ipa proteins, provided that the TTSA is correctly assembled.     

Shigella flexneri Spa15 crystal structure verified in solution by double electron electron resonance

Shigella flexneri Spa15 is a chaperone of the type 3 secretion system, which binds a number of effectors to ensure their stabilization prior to secretion. One of these effectors is IpgB1, a mimic of the human Ras-like Rho guanosine triphosphatase RhoG. In this study, Spa15 alone and in complex with IpgB1 has been studied by double electron electron resonance, an experiment that gives distance information showing the spacial separation of attached spin labels. This distance is explained by determining the crystal structure of the spin-labeled Spa15 where labels are seen to be buried in hydrophobic pockets. The double electron electron resonance experiment on the Spa15 complex with IpgB1 shows that IpgB1 does not bind Spa15 in the same way as is seen in the homologous Salmonella sp. chaperone:effector complex InvB:SipA.     

Characterization of the Yersinia enterocolitica type III secretion ATPase YscN and its regulator, YscL

Type III secretion is a mechanism used by a broad range of gram-negative bacteria to neutralize eukaryotic defenses by enabling translocation of bacterial proteins directly into the cytoplasm of host cells. The bacterial energy source for secretion is ATP, which is consumed by an ATPase that couples ATP hydrolysis to the unfolding of secreted proteins and the dissociation of their chaperones just prior to secretion. By studying the biochemical properties of YscN and YscL of Yersinia enterocolitica, we have characterized them as the ATPase and ATPase regulator, respectively, of the type III secretion system of this organism. In vivo, YscL and YscN interact with each other, and the overexpression of glutathione S-transferase-YscL abolishes secretion and down-regulates the expression of secretion apparatus components.     

IpgB1 and IpgB2, two homologous effectors secreted via the Mxi-Spa type III secretion apparatus, cooperate to mediate polarized cell invasion and inflammatory potential of Shigella flexenri

Type III secretion systems (T3SS) are present in many pathogenic gram-negative bacteria and mediate the translocation of bacterial effector proteins into host cells. Here, we report the phenotypic characterization of S. flexneri ipgB1 and ipgB2 mutants, in which the genes encoding the IpgB1 and IpgB2 effectors have been inactivated, either independently or simultaneously. Like IpgB1, we found that IpgB2 is secreted by the T3SS and its secretion requires the Spa15 chaperone. Upon infection of semi-confluent HeLa cells, the ipgB2 mutant exhibited the same invasive capacity as the wild-type strain and the ipgB1 mutant was 50% less invasive. Upon infection of polarised Caco2-cells, the ipgB2 mutant did not show a significant defect in invasion and the ipgB1 mutant was slightly more invasive than the wild-type strain. Entry of the ipgB1 ipgB2 mutant in polarized cells was reduced by 70% compared to the wild-type strain. Upon infection of the cornea in Guinea pigs, the ipgB2 mutant exhibited a wild-type phenotype, the ipgB1 mutant was hypervirulent and elicited a more pronounced proinflammatory response, while the ipgB1 ipgB2 mutant was highly attenuated. The attenuated phenotype of the ipgB1 ipgB2 mutant was confirmed using a murine pulmonary model of infection and histopathology and immunochemistry studies.     

Enhanced secretion through the Shigella flexneri Mxi-Spa translocon leads to assembly of extracellular proteins into macromolecular structures

Genes required for entry of Shigella flexneri into epithelial cells in vitro are clustered in two adjacent loci, one of which encodes secretory proteins, the IpaA–D proteins, and the other their dedicated secretion apparatus, the Mxi–Spa translocon. Ipa secretion, which is induced upon contact of bacteria with epithelial cells, is prevented during growth in vitro. Here, we show that ipaB and ipaD mutations lead to enhanced secretion of a set of about 15 proteins. These extracellular proteins and some Ipas associate in organized structures consisting of extended sheets. Growth of the wild-type strain in the presence of Congo red is shown to induce protein secretion through the Mxi–Spa translocon. Cultures grown to stationary phase in the presence of Congo red contain extracellular filaments whose composition and morphology are similar to those produced by the hyper-secreting ipaB and ipaD mutants.     

Crystal structure of Yersinia enterocolitica type III secretion chaperone SycT

Pathogenic Yersinia species use a type III secretion (TTS) system to deliver a number of cytotoxic effector proteins directly into the mammalian host cell. To ensure effective translocation, several such effector proteins transiently bind to specific chaperones in the bacterial cytoplasm. Correspondingly, SycT is the chaperone of YopT, a cysteine protease that cleaves the membrane-anchor of Rho-GTPases in the host. We have analyzed the complex between YopT and SycT and determined the structure of SycT in three crystal forms. Biochemical studies indicate a stoichometric effector/chaperone ratio of 1:2 and the chaperone-binding site contains at least residues 52-103 of YopT. The crystal structures reveal a SycT homodimer with an overall fold similar to that of other TTS effector chaperones. In contrast to the canonical five-stranded anti-parallel beta-sheet flanked by three alpha-helices, SycT lacks the dimerization alpha-helix and has an additional beta-strand capable of undergoing a conformational change. The dimer interface consists of two beta-strands and the connecting loops. Two hydrophobic patches involved in effector binding in other TTS effector chaperones are also found in SycT. The structural similarity of SycT to other chaperones and the spatial conservation of effector-binding sites support the idea that TTS effector chaperones form a single functional and structural group.     

Salmonella type III secretion-associated chaperones confer secretion-pathway specificity

Type III protein secretion systems (TTSSs) are ancestrally related to the flagellar export system and are essential for the virulence of many bacteria pathogenic for humans, animals and plants. Most proteins destined to travel the TTSS pathway possess at least two domains that specifically target them to the secretion apparatus. One of the domains is located within the amino terminal first approximately 20 amino acids and the second domain, located within the first approximately 140 amino acids, serves as a binding site for specific chaperones. It has been previously proposed that these two secretion signals are capable of operating independently of one another to facilitate secretion into the extracellular environment. We have found that in the absence of their chaperone-binding domains, the Salmonella typhimurium TTSS-secreted proteins SptP and SopE are no longer targeted for secretion through their cognate TTSS and, instead, are secreted through the flagellar export pathway. These results indicate the existence of an 'ancestral' flagellar secretion signal within TTSS-exported proteins that is revealed in the absence of the chaperone-binding domain. Furthermore, we found that secretion into culture supernatants as well as translocation into host cells by the cognate TTSS require both, the amino terminal and chaperone-binding domains. We conclude from these studies that a critical function for the TTSS-associated chaperones is to confer secretion-pathway specificity to their cognate secreted proteins.     

A directed screen for chlamydial proteins secreted by a type III mechanism identifies a translocated protein and numerous other new candidates

Chlamydiae are strict intracellular parasites that induce their internalization upon contact with the host cell and grow inside an intracellular compartment called an inclusion. They possess a type III secretion (TTS) apparatus, which allows for the translocation of specific proteins in the host cell cytosol. In particular, chlamydial proteins of the Inc family are secreted to the inclusion membrane by a TTS mechanism; other TTS substrates are mostly unknown. Using a secretion assay based on the recognition of TTS signals in Shigella flexneri, we searched for TTS signals in the proteins of unknown function, conserved between three different chlamydial species, Chlamydia pneumoniae, C. trachomatis and C. caviae. We identified 24 new candidate proteins which did not belong to the Inc family. Four of these proteins were also secreted as full-length proteins by a TTS mechanism in S. flexneri, indicating that their translocation does not require other chlamydial proteins. One of these proteins was detected in the cytosol of infected cells using specific antibodies, directly demonstrating that it is translocated in the host cell during bacterial proliferation. More generally, this work represents the first directed search for TTS effectors not based on genetic information or sequence similarity. It reveals the abundance of proteins secreted in the host cell by chlamydiae.     

Biology of type II secretion

The type II secretion pathway or the main terminal branch of the general secretion pathway, as it has also been referred to, is widely distributed among Proteobacteria, in which it is responsible for the extracellular secretion of toxins and hydrolytic enzymes, many of which contribute to pathogenesis in both plants and animals. Secretion through this pathway differs from most other membrane transport systems, in that its substrates consist of folded proteins. The type II secretion apparatus is composed of at least 12 different gene products that are thought to form a multiprotein complex, which spans the periplasmic compartment and is specifically required for translocation of the secreted proteins across the outer membrane. This pathway shares many features with the type IV pilus biogenesis system, including the ability to assemble a pilus-like structure. This review discusses recent findings on the organization of the secretion apparatus and the role of its various components in secretion. Different models for pilus-mediated secretion through the gated pore in the outer membrane are also presented, as are the possible properties that determine whether a protein is recognized and secreted by the type II pathway.