William D. Picking

The bacterial type III secretion system (T3SS) uses a membrane-embedded injectisome assembly to export effector proteins into host cells. While atomic-level structural details have been revealed for much of the T3SS apparatus, the model of the cytoplasmic sorting platform remains largely low-resolution. A central structural element of the sorting platform is the so-called "pod" protein, SctQ, which anchors the sorting platform to the inner membrane via interaction with the adaptor protein SctK, and connects to the central ATPase via the spoke protein SctL. SctQ proteins also interact with alternatively translated homodimers of their C-terminal SPOA2 domains. Low resolution electron density maps have provided an outline of the sorting platform architecture, and fluorescence microscopy studies have suggested a 1:4:2 SctK:SctQ:SctL stoichiometry. While there are experimental and AlphaFold structures of the individual components or complexes of sorting platform pod, there is currently no model for the pod structure that adequately fits the electron density or accounts for the proposed stoichiometry. Here we use AlphaFold to generate a model of the Shigella pod complex in which two copies of the SctQ protein, Spa33, bind the adaptor protein MxiK, with each copy of Spa33 bound to an alternately translated SPOA2-SPOA2 domain. We show through mutation of energetically critical interface residues, predicted by computational mutant scanning, that both Spa33 binding sites on MxiK are required for T3SS activity in Shigella flexneri , as well as binding of Spa33 to the SPOA2-SPOA2 homodimer. We find that this model fits well to the upper two-thirds of the pod electron density, albeit in a manner that places the protein components slightly closer to the inner membrane than traditionally presented. Further, cryogenic electron tomography of mutant injectisomes reveals the lack of a complete sorting platform and may suggest an alternative model featuring four copies of Spa33 that still fits the upper pod electron density but better explains unfilled density in the lower third of the pod.

Shigella flexneri uses its type III secretion system (T3SS) to invade human enterocytes. The T3SS injectisome is controlled by proteins at the tip of an exposed needle that sense host cell contact. Substrate selection and powering of secretion is controlled by a cytoplasmic assembly called the sorting platform (SP). The Shigella SP possesses six pod structures linked to a central ATPase via radial spokes. The SP associates with the injectisome inner membrane ring (IR) via the adaptor protein MxiK. The major IR component is MxiG, whose globular periplasmic domain (MxiG^P) packs with MxiJ in a 24-fold symmetry. MxiG also has a transmembrane helix attached to a small cytoplasmic domain (MxiG^C) via a flexible linker peptide. Change from the IR’s 24-fold symmetry to six-fold symmetry for the SP in Shigella occurs via MxiG^C pairs that associate with MxiK. The intervening pairs shift to the center of the IR/SP assembly, which is distinct from what is seen for Salmonella. This implicates the linker in dynamic motions at the IR-SP interface, but the functional importance of the linker is unknown. Using a library of mutants, we found that the linker can accept diverse mutations without eliminating injectisome function. However, some mutants were found to give rise to subpopulations able to form needles and secrete effectors in the absence of a stably assembled SP. Mutants lacking the entire linker could not secrete any effector proteins (e.g. the IpaD tip protein) and had no T3SS-related virulence functions, however, there were subpopulations that could still secrete MxiH and assemble visible needles. In contrast, a very short linker could export IpaD to the needle tip, but could not rapidly respond to external secretion signals and were thus unable to quickly enter epithelial cells. These findings implicate the MxiG linker in signaling processes that are sensed at the needle tip. Our findings suggest that the native MxiG linker peptide has evolved to maximize T3SS function at steps beyond needle formation, while needle formation can occur even when the SP is highly destabilized.

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.

Shigellosis is a severe gastrointestinal disease that annually affects approximately 270 million individuals globally. It has particularly high morbidity and mortality in low-income regions; however, it is not confined to these regions and occurs in high-income nations when conditions allow. The ill effects of shigellosis are at their highest in children ages 2 to 5, with survivors often exhibiting impaired growth due to infection-induced malnutrition. The escalating threat of antibiotic resistance further amplifies shigellosis as a serious public health concern. This review explores Shigella pathology, with a primary focus on the status of Shigella vaccine candidates. These candidates include killed whole-cells, live attenuated organisms, LPS-based, and subunit vaccines. The strengths and weaknesses of each vaccination strategy are considered. The discussion includes potential Shigella immunogens, such as LPS, conserved T3SS proteins, outer membrane proteins, diverse animal models used in Shigella vaccine research, and innovative vaccine development approaches. Additionally, this review addresses ongoing challenges that necessitate action toward advancing effective Shigella prevention and control measures.

DNA polymerases replicate cellular genomes and/or participate in the maintenance of genome integrity. DNA polymerases sharing high sequence homology with E. coli DNA polymerase I (pol I) have been grouped in Family A. Pol I participates in Okazaki fragment maturation and in bacterial genome repair. Since its discovery in 1956, pol I has been extensively studied, primarily to gain deeper insights into the mechanism of DNA replication. As research on DNA polymerases advances, many novel functions of this group of polymerases are being uncovered. For example, human DNA polymerase θ (a Family A DNA pol) has been shown to synthesize DNA using RNA as a template, a function typically attributed to retroviral reverse transcriptase. Increased interest in drug discovery against pol θ has emerged due to its roles in cancer. Likewise, Pol I family enzymes also appear attractive as drug-development targets against microbial infections. Development of antimalarial compounds targeting apicoplast apPOL, an ortholog of Pol I, further extends the targeting of this family of enzymes. Here, we summarize reported drug-development efforts against Family A polymerases and future perspective regarding these enzymes as antibiotic targets. Recently developed techniques, such as artificial intelligence, can be used to facilitate the development of new drugs.

DNA polymerases replicate cell genomes and/or participate in the maintenance of genome integrity. DNA polymerases sharing high sequence homology with E. coli DNA polymerase I (pol I), have been grouped in Family A. Pol I participates in Okazaki fragment maturation and in bacterial genome repair. Since its discovery in 1956, pol I has been extensively studied, primarily to get deeper insights into the mechanism of DNA replication. As research on DNA polymerases advances, many novel functions of this group of polymerases are uncovered. For example, human DNA polymerase θ (a Family A DNA pol) has been shown to synthesize DNA using RNA as a template, a function typically attributed to retroviral reverse transcriptase. A heightened interest in drug discovery against pol θ has emerged due to its roles in cancer. Likewise, Pol I family enzymes also appear attractive drug-development targets against microbial infections. Development of antimalarial compounds targeting apicoplast apPOL, an ortholog of Pol I, further extends the druggability of this family of enzymes. Here, we summarize reported drug-development efforts against Family A polymerases, and future perspective regarding these enzymes as antibiotic targets. Current techniques such artificial intelligence can be used to facilitate the goal of new drugs.

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 (Spa33^C). The structure of Spa33^C 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.

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 (Spa33 ^C ). The structure of Spa33 ^C 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.

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.