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.     

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.     

The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri

Bacteria of Shigella spp. are the causative agents of shigellosis. The virulence traits of these pathogens include their ability to enter into epithelial cells and induce apoptosis in macrophages. Expression of these functions requires the Mxi-Spa type III secretion apparatus and the secreted IpaA-D proteins, all of which are encoded by a virulence plasmid. In wild-type strains, the activity of the secretion apparatus is tightly regulated and induced upon contact of bacteria with epithelial cells. To investigate the repertoire of proteins secreted by Shigella flexneri in conditions of active secretion, we determined the N-terminal sequence of 14 proteins that are secreted by a mutant in which secretion was deregulated. Sequencing of the virulence plasmid pWR100 of the S. flexneri strain M90T (serotype 5) has allowed us to identify the genes encoding these secreted proteins and suggests that approximately 25 proteins are secreted by the type III secretion apparatus. Analysis of the G+C content and the relative positions of genes and open reading frames carried by the plasmid, together with information concerning the localization and function of encoded proteins, suggests that pWR100 contains blocks of genes of various origins, some of which were initially carried by four different plasmids.     

The secreted IpaB and IpaC invasins and their cytoplasmic chaperone IpgC are required for intercellular dissemination of Shigella flexneri

Invasion of epithelial cells by Shigella flexneri involves entry and dissemination. The main effectors of entry, IpaB and IpaC, are also required for contact haemolytic activity and escape from the phagosome in infected macrophages. These proteins are stored in the cytoplasm in association with the chaperone IpgC, before their secretion by a type III secretion apparatus is activated by host cells. We used a His-tagged IpgC protein to purify IpgC-containing complexes and showed that only IpaB and IpaC are associated with IpgC. Plasmids expressing His6-IpgC either alone or together with IpaB or IpaC under the control of an IPTG-inducible lac promoter were introduced into ipgC , ipaB or ipaC mutants. Induction of expression of the recombinant plasmid-encoded proteins by IPTG allowed bacteria to enter epithelial cells, and the role of these proteins in dissemination was investigated by incubating infected cells in either the absence or the presence of IPTG. The size of plaques produced by recombinant strains on cell monolayers was regulated by IPTG, indicating that IpgC, IpaB and IpaC were each required for efficient dissemination. Electron microscopy analysis of infected cells indicated that these proteins were necessary for lysis of the membrane of the protrusions during cell-to-cell spread.     

The chaperone IpgC copurifies with the virulence regulator MxiE

The expression of a subset of Shigella flexneri virulence genes is dependent upon a cytoplasmic chaperone, IpgC, and an activator from the AraC/XylS family, MxiE. In this paper, we report that the chaperone forms a specific and stable heteromer with MxiE.     

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.     

Homologs of the Shigella IpaB and IpaC invasins are required for Salmonella typhimurium entry into cultured epithelial cells.

Entry into host cells is an essential feature in the pathogenicity of Salmonella spp. The inv locus of Salmonella typhimurium encodes several proteins which are components of a type III protein secretion system required for these organisms to gain access to host cells. We report here the identification of several proteins whose secretion into the culture supernatant of S. typhimurium is dependent on the function of the inv-encoded translocation apparatus. Nucleotide sequence analysis of the genes encoding two of these secreted proteins, SipB and SipC, indicated that they are homologous to the Shigella sp. invasins IpaB and IpaC, respectively. An additional gene was identified, sicA, which encodes a protein homologous to IpgC, a Shigella protein that serves as a molecular chaperone for the invasins IpaB and IpaC. Nonpolar mutations in sicA, sipB, and sipC rendered S. typhimurium unable to enter cultured epithelial cells, indicating that these genes are required for bacterial internalization.     

Identification of the bile salt binding site on IpaD from Shigella flexneri and the influence of ligand binding on IpaD structure

Type III secretion (TTS) is an essential virulence factor for Shigella flexneri, the causative agent of shigellosis. The Shigella TTS apparatus (TTSA) is an elegant nanomachine that is composed of a basal body, an external needle to deliver effectors into human cells, and a needle tip complex that controls secretion activation. IpaD is at the tip of the nascent TTSA needle where it controls the first step of TTS activation. The bile salt deoxycholate (DOC) binds to IpaD to induce recruitment of the translocator protein IpaB into the maturing tip complex. We recently used spectroscopic analyses to show that IpaD undergoes a structural rearrangement that accompanies binding to DOC. Here, we report a crystal structure of IpaD with DOC bound and test the importance of the residues that make up the DOC binding pocket on IpaD function. IpaD binds DOC at the interface between helices α3 and α7, with concomitant movement in the orientation of helix α7 relative to its position in unbound IpaD. When the IpaD residues involved in DOC binding are mutated, some are found to lead to altered invasion and secretion phenotypes. These findings suggest that adoption of a DOC bound structural state for IpaD primes the Shigella TTSA for contact with host cells. The data presented here and in the studies leading up to this work provide the foundation for developing a model of the first step in Shigella TTS activation.     

Combination of two separate binding domains defines stoichiometry between type III secretion system chaperone IpgC and translocator protein IpaB

Type III secretion systems (TTSSs) utilized by enteropathogenic bacteria require the presence of small, acidic virulence-associated chaperones for effective host cell infection. We adopted a combination of biochemical and cellular techniques to define the chaperone binding domains (CBDs) in the translocators IpaB and IpaC associated with the chaperone IpgC from Shigella flexneri. We identified a novel CBD in IpaB and furthermore precisely mapped the boundaries of the CBDs in both translocator proteins. In IpaC a single binding domain associates with IpgC. In IpaB, we show that the binding of the newly characterized CBD is essential in maintaining the ternary arrangement of chaperone-translocator complex. This hitherto unknown function is reflected in the co-crystal structure as well, with an IpgC dimer bound to an IpaB fragment comprising both CBDs. Moreover, in the absence of this novel CBD the IpaB/IpgC complex aggregates. This dual-recognition of a domain in the protein by the chaperone in facilitating the correct chaperone-substrate organization describes a new function for the TTSS associated chaperone-substrate complexes.     

The needle component of the type III secreton of Shigella regulates the activity of the secretion apparatus

Gram-negative bacteria commonly interact with eukaryotic host cells by using type III secretion systems (TTSSs or secretons). TTSSs serve to transfer bacterial proteins into host cells. Two translocators, IpaB and IpaC, are first inserted with the aid of IpaD by Shigella into the host cell membrane. Then at least two supplementary effectors of cell invasion, IpaA and IpgD, are transferred into the host cytoplasm. How TTSSs are induced to secrete is unknown, but their activation appears to require direct contact of the external distal tip of the apparatus with the host cell. The extracellular domain of the TTSS is a hollow needle protruding 60 nm beyond the bacterial surface. The monomeric unit of the Shigella flexneri needle, MxiH, forms a superhelical assembly. To probe the role of the needle in the activation of the TTSS for secretion, we examined the structure-function relationship of MxiH by mutagenesis. Most point mutations led to normal needle assembly, but some led to polymerization or possible length control defects. In other mutants, secretion was constitutively turned "on." In a further set, it was "constitutively on" but experimentally "uninducible." Finally, upon induction of secretion, some mutants released only the translocators and not the effectors. Most types of mutants were defective in interactions with host cells. Together, these data indicate that the needle directly controls the activity of the TTSS and suggest that it may be used to "sense" host cells.     

Preparation and characterization of translocator/chaperone complexes and their component proteins from Shigella flexneri

Shigella flexneri causes a severe form of bacillary dysentery also known as shigellosis. Onset of shigellosis requires bacterial invasion of colonic epithelial cells which is initiated by the delivery of translocator and effector proteins to the host cell membrane and cytoplasm, respectively, by the Shigella type III secretion system (TTSS). The Shigella translocator proteins, IpaB and IpaC, form a pore complex in the host cell membrane to facilitate effector delivery; however, prior to their secretion IpaB and IpaC are partitioned in the bacterial cytoplasm by association with the cytoplasmic chaperone IpgC. To determine their structural and biophysical properties, recombinant IpaB/IpgC and IpaC/IpgC complexes were prepared for their first detailed in vitro analysis. Both IpaB/IpgC and IpaC/IpgC complexes are highly stable and soluble heterodimers whose formation prevents IpaB-IpaC interaction as well as Ipa-dependent disruption of phospholipid membranes. Circular dichroism spectroscopy shows that IpgC binding has a detectable influence on IpaC secondary/tertiary structure and stability. In contrast, IpaB structure is not as dramatically affected by chaperone binding. To more precisely ascertain the influence of chaperone binding on IpaC structure and stability, single tryptophan mutants were generated for detailed fluorescence spectroscopy analysis. These mutants provide a low-resolution picture of how IpaC exists in the Shigella cytoplasm with chaperone binding possibly involving distinct regions within the N- and C-terminal halves of IpaC. This preliminary assessment of the IpaC-IpgC interaction is supported by initial deletion mutagenesis studies. The data provide the first structural analysis of IpgC association with IpaB and IpaC.     

Binding affects the tertiary and quaternary structures of the Shigella translocator protein IpaB and its chaperone IpgC

Shigella flexneri uses its type III secretion system (T3SS) to promote invasion of human intestinal epithelial cells as the first step in causing shigellosis, a life-threatening form of dysentery. The Shigella type III secretion apparatus (T3SA) consists of a basal body that spans the bacterial envelope and an exposed needle that injects effector proteins into target cells. The nascent Shigella T3SA needle is topped with a pentamer of the needle tip protein invasion plasmid antigen D (IpaD). Bile salts trigger recruitment of the first hydrophobic translocator protein, IpaB, to the tip complex where it senses contact with a host membrane. In the bacterial cytoplasm, IpaB exists in a complex with its chaperone IpgC. Several structures of IpgC have been determined, and we recently reported the 2.1 ? crystal structure of the N-terminal domain (IpaB(74.224)) of IpaB. Like IpgC, the IpaB N-terminal domain exists as a homodimer in solution. We now report that when the two are mixed, these homodimers dissociate and form heterodimers having a nanomolar dissociation constant. This is consistent with the equivalent complexes copurified after they had been co-expressed in Escherichia coli. Fluorescence data presented here also indicate that the N-terminal domain of IpaB possesses two regions that appear to contribute additively to chaperone binding. It is also likely that the N-terminus of IpaB adopts an alternative conformation as a result of chaperone binding. The importance of these findings within the functional context of these proteins is discussed.