150 research outputs found
Antibody Response of Monkeys to Invasion Plasmid Antigen D after Infection with Shigella spp.
The antigen preparation most often used for determining the levels of antibodies to virulence-associated proteins of Shigella spp. consists of a mixture of proteins (including IpaB, IpaC, IpaD, and VirG*) extracted from virulent shigellae with water (water extract). To overcome the lack of specificity for individual antigens in the water-extract enzyme-linked immunosorbent assay (ELISA), the ipaD gene from S. flexneri has been cloned, expressed to a high level, and purified for use in a new ELISA for the determination of the levels of antibody against IpaD in monkeys and humans challenged with shigellae. The IpaD ELISA for serum immunoglobulins G and A correlated well with the water-extract ELISA in that monkeys infected with S. flexneri or S. sonnei responded with high serum antibody titers in both assays. The IpaD assay required less antigen per well, had much lower background levels, and did not require correction with antigens from an avirulent organism. In conjunction with the water-extract ELISA, it was possible to identify infected animals that did not respond to IpaD but did produce antibodies that reacted in the water-extract ELISA. This indicates that even though IpaB, IpaC, and IpaD are essential for the invasiveness phenotype, the infected host does not always produce antibodies against all components of the invasiveness apparatus
A synthetic alanyl-initiator tRNA with initiator tRNA properties as determined by fluorescence measurements: comparison to synthetic alanyl-elongator tRNA
Soluble invasion plasmid antigen C (IpaC) from Shigella flexneri elicits epithelial cell responses related to pathogen invasion
Shigella flexneri invades colonic epithelial cells by pathogen-induced phagocytosis. The three proposed effectors of S. flexneri internalization are invasion plasmid antigens B (IpaB), IpaC, and IpaD, which are encoded on the pathogen's 230-kb virulence plasmid and translocated to the extracellular milieu via the Mxi-Spa translocon. To date, there are no definitive functional data for any purified Ipa protein. Here, we describe the first characterization of highly purified recombinant IpaC, which elicits numerous epithelial cell responses related to events that take place during pathogen invasion. 125I-labeled IpaC binds cultured Henle 407 intestinal cells with an apparent dissociation constant in the low micromolar range. Moreover, incubation of epithelial cells with IpaC results in general changes in cellular phosphoprotein content, demonstrating this protein's ability to influence cellular protein kinase activities. These results contrast dramatically with those seen for recombinant IpaD, which does not bind to or induce detectable changes in the normal activities of cultured epithelial cells. In addition to influencing host cell activities, preincubation of epithelial cells with purified IpaC enhances uptake of S. flexneri by host cells. A similar result is seen when the cells are preincubated with a highly concentrated water extract of virulent S. flexneri 2a (strain 2457T). Interestingly, soluble IpaC also appears to promote uptake of the noninvasive S. flexneri 2a strain BS103. Purified IpaD failed to enhance the uptake of virulent S. flexneri and did not facilitate uptake of BS103. Taken together, the data suggest that IpaC is a potential effector of the host cell biological activities and may be responsible for entry of S. flexneri into target cells
The Shigella Type III Secretion System: An Overview from Top to Bottom
Shigella comprises four species of human-restricted pathogens causing bacillary dysentery. While Shigella possesses multiple genetic loci contributing to virulence, a type III secretion system (T3SS) is its primary virulence factor. The Shigella T3SS nanomachine consists of four major assemblies: the cytoplasmic sorting platform; the envelope-spanning core/basal body; an exposed needle; and a needle-associated tip complex with associated translocon that is inserted into host cell membranes. The initial subversion of host cell activities is carried out by the effector functions of the invasion plasmid antigen (Ipa) translocator proteins, with the cell ultimately being controlled by dedicated effector proteins that are injected into the host cytoplasm though the translocon. Much of the information now available on the T3SS injectisome has been accumulated through collective studies on the T3SS from three systems, those of Shigella flexneri, Salmonella typhimurium and Yersinia enterocolitica/Yersinia pestis. In this review, we will touch upon the important features of the T3SS injectisome that have come to light because of research in the Shigella and closely related systems. We will also briefly highlight some of the strategies being considered to target the Shigella T3SS for disease prevention
Endotoxicosis Induced by Coxiella burnetii Lipopolysaccharide Stimulates a Ribosomal Protein S6 Kinase: Some Properties of the Partially Purified Enzyme
Guinea pig endotoxicosis induced by lipopolysaccharide from Coxiella burnetii Nine Mile phase I stimulates phosphorylation of liver ribosomal protein S6, with a 50% increase at 12 h postinoculation. The responsible protein kinase (S6PK) has been partially purified from liver; its activity is independent of cyclic AMP and of Ca2+ plus phosphatidyl serine or diacylglycerol. The preparation has an apparent optimum concentration of 20 mM Mg2+, while Ca2+ and Mn2+ are each inhibitory at 2 mM. The apparent Km for ATP is 30 microM with intact ribosomes. Because of the central role of phosphorylation in metabolic regulation and a purported role of phosphorylated S6 in protein synthesis, the lipopolysaccharide-induced stimulation of S6PK suggests a significant regulatory role of such enzymes in the pathobiochemistry of Q fever infection and endotoxicosis
Influence of Oligomerization State on the Structural Properties of Invasion Plasmid Antigen B (IpaB) from Shigella flexneri in the Presence and Absence of Phospholipid Membranes
Shigella flexneri causes bacillary dysentery, an important cause of mortality among children in the developing world. Shigella secretes effector proteins via its type III secretion system (T3SS) to promote bacterial uptake into human colonic epithelial cells. The T3SS basal body spans the bacterial cell envelope anchoring a surface-exposed needle. A pentamer of invasion plasmid antigen D (IpaD) lies at the nascent needle tip and IpaB is recruited into the needle tip complex upon exposure to bile salts. From here, IpaB forms a translocon pore in the host cell membrane. Although the mechanism by which IpaB inserts into the membrane is unknown, it was recently shown that recombinant IpaB can exist as either a monomer or tetramer. Both of these forms of IpaB associate with membranes, however, only the tetramer forms pores in liposomes. To reveal differences between these membrane-binding events, Cys mutations were introduced throughout IpaB, allowing site-specific fluorescence labeling. Fluorescence quenching was used to determine the influence of oligomerization and/or membrane association on the accessibility of different IpaB regions to small solutes. The data show that the hydrophobic region of tetrameric IpaB is more accessible to solvent relative to the monomer. The hydrophobic region appears to promote membrane interaction for both forms of IpaB, however, more of the hydrophobic region is protected from solvent for the tetramer after membrane association. Limited proteolysis demonstrated that changes in IpaB’s oligomeric state may determine the manner by which it associates with phospholipid membranes and the subsequent outcome of this association
Interaction of Shigella flexneri IpaC with Model Membranes Correlates with Effects on Cultured Cells
Invasion of enterocytes by Shigella flexneri requires the properly timed release of IpaB and IpaC at the host-pathogen interface; however, only IpaC has been found to possess quantifiable activities in vitro. We demonstrate here that when added to cultured cells, purified IpaC elicits cytoskeletal changes similar to those that occur during Shigella invasion. This IpaC effect may correlate with its ability to interact with model membranes at physiological pH and to promote entry by an ipaC mutant of S. flexneri
Vaccination With Mouse Dendritic Cells Loaded With an IpaD-IpaB Fusion Provides Protection Against Shigellosis
This work is licensed under a Creative Commons Attribution 4.0 International License.Diarrheal diseases are a major cause of morbidity and mortality worldwide. They are most prevalent in settings with inadequate sanitation, poor hygiene and contaminated water. An important diarrheal pathogen in such settings is Shigella. No commercially available vaccine exists against shigellosis and immunity to the pathogen is serotype-restricted. We have previously shown that a polypeptide fusion of the Type Three Secretion Apparatus (T3SA) proteins IpaB and IpaD (named DBF) was efficacious as a vaccine against Shigella. Vaccination using different administration routes indicated that protection conferred by DBF did not fully correlate with antibodies. To define the immune responses involved in protection, we studied cellular responses to intranasal immunization with the DBF and the adjuvant dmLT. We found dendritic cell (DC) activation at the nasal associated lymphoid tissue (NALT). Activation markers CD86 and MHCII significantly increase in cells from immunized mice. Antigen exposure in vitro further confirmed the upregulation of CD80 and CD40 in primary dendritic cells. Animals immunized with antigen-primed dendritic cells were protected against Shigella infection, at levels comparable to the efficacy of immunization with the protein vaccine formulation. Therefore, we show that antigen-primed DCs are enough to provide immunity, and propose a mechanism of protection against Shigella spp. based on DC-mediated antigen presentation to T cells.NIH R01 AI 007337
Identification of the MxiH Needle Protein Residues Responsible for Anchoring Invasion Plasmid Antigen D to the Type III Secretion Needle Tip
The pathogenesis of Shigella flexneri requires a functional type III secretion apparatus to serve as a conduit for injecting host-altering effector proteins into the membrane and cytoplasm of the targeted cell. The type III secretion apparatus is composed of a basal body and an exposed needle that is an extended polymer of MxiH with a 2.0-nm inner channel. Invasion plasmid antigen D (IpaD) resides at the tip of the needle to control type III secretion. The atomic structures of MxiH and IpaD have been solved. MxiH (8.3 kDa) is a helix-turn-helix, whereas IpaD (36.6 kDa) has a dumbbell shape with two globular domains flanking a central coiled-coil that stabilizes the protein. These structures alone, however, have not been sufficient to produce a workable in silico model by which IpaD docks at the needle tip. Thus, the work presented here provides an initial step in understanding this important protein-protein interaction. We have identified key MxiH residues located in its PSNP loop and the contiguous surface that uniquely contribute to the formation of the IpaD-needle interface as determined by NMR chemical shift mapping. Mutation of Asn-43, Leu-47, and Tyr-50 residues severely affects the stable maintenance of IpaD at the Shigella surface and thus compromises the invasive phenotype of S. flexneri. Other residues could be mutated to give rise to intermediate phenotypes, suggesting they have a role in tip complex stabilization while not being essential for tip complex formation. Initial in vitro fluorescence polarization studies confirmed that specific amino acid changes adversely affect the MxiH-IpaD interaction. Meanwhile, none of the mutations appeared to have a negative effect on the MxiH-MxiH interactions required for efficient needle assembly
Preformulation Characterization and the Effect of Ionic Excipients on the Stability of a Novel DB Fusion Protein
Shigella ssp cause bacillary dysentery (shigellosis) which has high global morbidity in young children and the elderly. The virulence of Shigella relies upon a type III secretion system (T3SS) which injects host altering effector proteins into targeted intestinal cells. The Shigella T3SS contains two components, invasion plasmid antigen D (IpaD) and invasion plasmid antigen B (IpaB), that were previously identified as broadly protective antigens. When IpaD and IpaB were co-expressed to give the DB fusion (DBF) protein, vaccine efficacy was further improved. Biophysical characterization under various pH conditions showed that DBF is most stable at pH 7 and 8 and loses its conformational integrity at 48 and 50 °C respectively. Forced degradation studies revealed significant effects on the secondary structure, tertiary structure and conformational stability of DBF. In the presence of phosphate buffers as well as other anionic excipients, DBF demonstrated a concentration dependent conformational stabilization. Molecular docking revealed potential polyanion binding sites in DBF that may interact with phytic acid. These sites can be exploited to stabilize the DBF protein. This work highlights potential destabilizing and stabilizing factors, which not only improves our understanding of the DBF protein but helps in future development of a stable Shigella vaccine
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