Search

Shigella spp. are responsible for bacillary dysentery or shigellosis transmitted via the fecal–oral route, causing significant morbidity and mortality, especially among vulnerable populations. There are currently no licensed Shigella vaccines. Shigella spp. use a type III secretion system (T3SS) to invade host cells. We have shown that L-DBF, a recombinant fusion of the T3SS needle tip (IpaD) and translocator (IpaB) proteins with the LTA1 subunit of enterotoxigenic E. coli labile toxin, is broadly protective against Shigella spp. challenge in a mouse lethal pulmonary model. Here, we assessed the effect of LDBF, formulated with a unique TLR4 agonist called BECC470 in an oil-in-water emulsion (ME), on the murine immune response in a high-risk population (young and elderly) in response to Shigella challenge. Dual RNA Sequencing captured the transcriptome during Shigella infection in vaccinated and unvaccinated mice. Both age groups were protected by the L-DBF formulation, while younger vaccinated mice exhibited more adaptive immune response gene patterns. This preliminary study provides a step toward identifying the gene expression patterns and regulatory pathways responsible for a protective immune response against Shigella. Furthermore, this study provides a measure of the challenges that need to be addressed when immunizing an aging population. [ABSTRACT FROM AUTHOR]

Shigella flexneri , causative agent of bacillary dysentery (shigellosis), uses a type III secretion system (T3SS) as its primary virulence factor. The T3SS injectisome delivers effector proteins into host cells to promote entry and create an important intracellular niche. The injectisome's cytoplasmic sorting platform (SP) is a critical assembly that contributes to substrate selection and energizing secretion. The SP consists of oligomeric Spa33 "pods" that associate with the basal body via MxiK and connect to the Spa47 ATPase via MxiN. The pods contain heterotrimers of Spa33 with one full-length copy associated with two copies of a C-terminal domain (Spa33C). The structure of Spa33C is known, but the precise makeup and structure of the pods in situ remains elusive. We show here that recombinant wild-type Spa33 can be prepared as a heterotrimer that forms distinct stable complexes with MxiK and MxiN. In two-hybrid analyses, association of the Spa33 complex with these proteins occurs via the full-length Spa33 component. Furthermore, these complexes each have distinct biophysical properties. Based on these properties, new high-resolution cryo-electron tomography data and architectural similarities between the Spa33 and flagellar FliM-FliN complexes, we provide a preliminary model of the Spa33 heterotrimers within the SP pods. From these findings and evolving models of SP interfaces and dynamics in the Yersinia and Salmonella T3SS, we suggest a model for SP function in which two distinct complexes come together within the context of the SP to contribute to form the complete pod structures during the recruitment of T3SS secretion substrates. [ABSTRACT FROM AUTHOR]

Infections caused by the opportunistic pathogen Pseudomonas aeruginosa can be difficult to treat due to innate and acquired antibiotic resistance and this is exacerbated by the emergence of multi-drug resistant strains. Unfortunately, no licensed vaccine yet exists to prevent Pseudomonas infections. Here we describe a novel subunit vaccine that targets the P. aeruginosa type III secretion system (T3SS). This vaccine is based on the novel antigen PaF (Pa Fusion), a fusion of the T3SS needle tip protein, PcrV, and the first of two translocator proteins, PopB. Additionally, PaF is made self-adjuvanting by the N-terminal fusion of the A1 subunit of the mucosal adjuvant double-mutant heat-labile enterotoxin (dmLT). Here we show that this triple fusion, designated L-PaF, can activate dendritic cells in vitro and elicits strong IgG and IgA titers in mice when administered intranasally. This self-adjuvanting vaccine expedites the clearance of P. aeruginosa from the lungs of challenged mice while stimulating host expression of IL-17A, which may be important for generating a protective immune response in humans. L-PaF's protective capacity was recapitulated in a rat pneumonia model, further supporting the efficacy of this novel fusion vaccine. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The first reported structure for a T3SS SctK protein family member was solved for PscK from Pseudomonas aeruginosa and this allowed for modeling of the interface between this sorting platform protein and the cytoplasmic domain of PscD (a SctD protein family member). This allowed for identification of amino acid residues that may play a role in the interaction between these proteins. The interface appears to be dominated by electrostatic interactions and mutagenesis confirmed the importance of key residues in driving their interaction based on two-hybrid analysis. • The structures of Pseudomonas aeruginosa PscD (SctD) and PscK (SctK) were solved. • The interface between the T3SS basal body and sorting platform was modeled. • The crystal structure of PscK is the first for any SctK family member. • PscK represents a novel protein fold. • Site-directed mutagenesis supports a computational model of the PscD–PscK interface. Many Gram-negative bacterial pathogens use type III secretion systems (T3SS) to inject proteins into eukaryotic cells to subvert normal cellular functions. The T3SS apparatus (injectisome) shares a common architecture in all systems studied thus far, comprising three major components – the cytoplasmic sorting platform, envelope-spanning basal body and external needle with tip complex. The sorting platform consists of an ATPase (SctN) connected to "pods" (SctQ) having six-fold symmetry via radial spokes (SctL). These pods interface with the 24-fold symmetric SctD inner membrane ring (IR) via an adaptor protein (SctK). Here we report the first high-resolution structure of a SctK protein family member, PscK from Pseudomonas aeruginosa , as well as the structure of its interacting partner, the cytoplasmic domain of PscD (SctD). The cytoplasmic domain of PscD forms a forkhead-associated (FHA) fold, like that of its homologues from other T3SS. PscK, on the other hand, forms a helix-rich structure that does not resemble any known protein fold. Based on these structural findings, we present the first model for an interaction between proteins from the sorting platform and the IR. We also test the importance of the PscD residues predicted to mediate this electrostatic interaction using a two-hybrid analysis. The functional need for these residues in vivo was then confirmed by monitoring secretion of the effector ExoU. These structures will contribute to the development of atomic-resolution models of the entire sorting platform and to our understanding of the mechanistic interface between the sorting platform and the basal body of the injectisome. [ABSTRACT FROM AUTHOR]