Preparation and Characterization of Translocator/Chaperone Complexes and Their Component Proteins from <i>Shigella flexneri</i>

Birket, Susan E.; Harrington, Amanda; Espina, Marianela; Smith, Nathan D.; Terry, Christina M.; Darboe, Numukunda; Markham, Aaron P.; Middaugh, C. Russell; Picking, Wendy L.; Picking, William D.

doi:10.1021/bi700099c

Cited by 49 publications

(110 citation statements)

References 31 publications

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“…In Shigella bacteria, IpgC is the chaperone for the T3SS translocon proteins IpaB and IpaD (32). Upon the secretion of IpaB and IpaD, IpgC is released and acts as a coactivator of the transcription factor MxiE for the transcription of virulence genes within the MxiE regulon (32,33,34). In Salmonella bacteria, SicA (a chaperone located in Salmonella pathogenicity island 1) interacts with InvF, a member of the AraC/XylS family of transcriptional activators.…”

Section: Discussionmentioning

confidence: 99%

EseE of Edwardsiella tarda Augments Secretion of Translocon Protein EseC and Expression of the escC - eseE Operon

et al. 2016

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Edwardsiella tarda is an important Gram-negative pathogen that employs a type III secretion system (T3SS) to deliver effectors into host cells to facilitate bacterial survival and replication. These effectors are translocated into host cells through a translocon complex composed of three secreted proteins, namely, EseB, EseC, and EseD. The secretion of EseB and EseD requires a chaperone protein called EscC, whereas the secretion of EseC requires the chaperone EscA. In this study, we identified a novel protein (EseE) that also regulates the secretion of EseC. An eseE deletion mutant secreted much less EseC into supernatants, accompanied by increased EseC levels within bacterial cells. We also demonstrated that EseE interacted directly with EseC in a pulldown assay. Interestingly, EseC, EseE, and EscA were able to form a ternary complex, as revealed by pulldown and gel filtration assays. Of particular importance, the deletion of eseE resulted in decreased levels of EseB and EseD proteins in both the bacterial pellet and supernatant fraction. Furthermore, real-time PCR assays showed that EseE positively regulated the transcription of the translocon operon escC-eseE, comprising escC, eseB, escA, eseC, eseD, and eseE. These effects of EseE on the translocon components/operon appeared to have a functional consequence, since the ⌬eseE strain was outcompeted by wild-type E. tarda in a mixed infection in blue gourami fish. Collectively, our results demonstrate that EseE not only functions as a chaperone for EseC but also acts as a positive regulator controlling the expression of the translocon operon escC-eseE, thus contributing to the pathogenesis of E. tarda in fish.T he type III secretion system (T3SS) is a contact-dependent translocation system. It forms a syringe-like structure spanning the inner and outer membranes of bacteria and induces pore formation on host cells (1). Through these pores, an array of bacterial proteins (effectors) are delivered into host cells, where they manipulate host cell signaling pathways to promote bacterial survival (2).Edwardsiella tarda is a Gram-negative intracellular pathogen that can cause hemorrhagic septicemia in fish (3), as well as gastroand extraintestinal infections in humans (4, 5). The T3SS is well known to be one of the most important virulence factors in E. tarda (6, 7). It facilitates the survival and replication of E. tarda in host cells (6-9). E. tarda T3SS contains 34 open reading frames (ORFs), which encode the apparatus, chaperones, effectors, and regulators (6, 10). The expression of E. tarda T3SS is controlled by many factors, such as the two-component system esrA-esrB (6) and esrC (11) inside the T3SS gene cluster, as well as phoP-phoQ (12) and phoR-phoB (13) located outside the T3SS gene cluster. Intriguingly, our group recently showed that a type III secretion system-secreted protein (EscE) also functions as a regulator controlling the injection of effectors and secretion of translocators (14).EseB, EseC, and EseD, secreted by the E. tarda T3SS, are homologous ...

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Section: Discussionmentioning

confidence: 99%

EseE of Edwardsiella tarda Augments Secretion of Translocon Protein EseC and Expression of the escC - eseE Operon

et al. 2016

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“…After purification of the IpaB/IpgC complex, IpgC was removed by the Dickenson et al addition of 0.5% OPOE, a nonionic detergent. 17 To further purify the isolated protein, IpaB in 0.5% OPOE (OPOE-IpaB) was then passed over a SEC column to give a stable, soluble protein preparation that existed as a multimeric complex. 20 While OPOE is a chemically heterogeneous detergent with a broad range of molecular sizes (see Supporting Information Fig.…”

Section: Ipab Purifies In Discrete Oligomeric Statesmentioning

confidence: 99%

“…17 We have recently solved the structure of a stable N-terminal core of IpaB (residues 74-224) at a 2.1 Å resolution. 18 A prominent feature of the IpaB N-terminal domain is an elongated coiled-coil.…”

Section: Introductionmentioning

confidence: 99%

“…17 Separation of IpaB from IpgC with 0.5% n-octyl-oligo-oxyethylene (OPOE) results in self assembly of IpaB into discrete oligomers while separation with 0.05% N,N-dimethyldodecylamine N-oxide (LDAO) maintains IpaB in a monomeric state. Here we define the biochemical and biophysical characteristics of the ''membrane active'' recombinant IpaB oligomer, comparing it to the ''membrane inactive'' monomer, to investigate the initial step of T3SA translocon pore formation in vitro.…”

Section: Introductionmentioning

confidence: 99%

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Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

et al. 2013

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The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein.We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n-octyl-oligooxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,Ndimethyldodecylamine N-oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size-dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose Abbreviations: AUC, analytical ultracentrifugation; CD, circular dichroism; CMC, critical micelle concentration; DGS-NTA, 1,2-dioleoylsn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid)succinyl]; DLS, dynamic light scattering; DMSO, dimethyl sulfoxide; DOPC, dioleoylphosphatidylcholine; DOPG, dioleoylphosphatidylglycerol; DSP, dithiobis[succinimidyl proprionate; FM, fluorescein maleimide; FRET, F-rster resonance energy transfer; Ipa, invasion plasmid antigen; Ipg, invasion plasmid gene; LDAO, N,N-dimethyldodecylamine N-oxide; Mxi, major exporter of Ipas; OPOE, n-Octyl-oligo-oxyethylene; SATA, N-succinimidyl-S-acetylthioacetate; SEC, size exclusion chromatography; SRB, sulforhodamine B; T m , thermal unfolding midpoint; TCEP, (tris (2-carboxyethyl)phosphine; TRITC DHPE, (N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine); T3SS, type-III secretion system; T3SA, type III secretion apparatus.Additional Supporting Information may be found in the online version of this article. that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore.

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“…Purification of large quantities of IpaB in E. coli requires coexpression with its cognate chaperone, IpgC, forming a heterodimer. 8,9 The IpaB/IpgC complex is then separated into an IpaB tetramer 10 and an IpgC dimer with mild detergent. 11 A major challenge in formulating a subunit vaccine involving the hydrophilic IpaD and hydrophobic IpaB is the maintenance of these vastly different protective antigens in stable, biologically active states for extended times to permit their storage and subsequent delivery to diverse clinical settings worldwide.…”

Section: Introductionmentioning

confidence: 99%

Studies of the conformational stability of invasion plasmid antigen B from Shigella

et al. 2013

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Shigella spp. are the causative agent of shigellosis, the second leading cause of diarrhea in children of ages 2-5. Despite many years of research, a protective vaccine has been elusive. We recently demonstrated that invasion plasmid antigens B and D (IpaB and IpaD) provide protection against S. flexneri and S. sonnei. These proteins, however, have very different properties which must be recognized and then managed during vaccine formulation. Herein, we employ spectroscopy to assess the stability of IpaB as well as IpgC (invasion protein gene), IpaB's cognate chaperone, and the IpaB/IpgC complex. The resulting data are mathematically summarized into a visual map illustrating the stability of the proteins and their complex as a function of pH and temperature. The IpaB/IpgC complex exhibits thermal stability at higher pH values but, though initially stable, quickly unfolds with increasing temperature when maintained at lower pH. In contrast, IpaB is a much more complex protein exhibiting increased stability at higher pH, but shows initial instability at lower pH values with pH 5 showing a distinct transition. IpgC precipitates at and below pH 5 and is stable above pH 7. Most strikingly, it is clear that complex formation results in stabilization of the two components. This work serves as a basis for the further development of IpaB as a vaccine candidate as well as extends our understanding of the structural stability of the Shigella type III secretion system.

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Preparation and Characterization of Translocator/Chaperone Complexes and Their Component Proteins from Shigella flexneri

Cited by 49 publications

References 31 publications

EseE of Edwardsiella tarda Augments Secretion of Translocon Protein EseC and Expression of the escC - eseE Operon

EseE of Edwardsiella tarda Augments Secretion of Translocon Protein EseC and Expression of the escC - eseE Operon

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

Studies of the conformational stability of invasion plasmid antigen B from Shigella

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