A Plant-Made Vaccine Candidate To Protect Ruminants Against Shiga Toxin-Producing Escherichia Coli

Shiga toxin-producing Escherichia coli (STEC) are enteropathogens colonizing the digestive tracts of humans and animals worldwide. STEC are shed in the manure of cattle and frequently contaminate beef products, vegetables, and drinking water. Immunizing cattle herds against STEC is a promising strategy to reduce STEC colonization in cattle and therefore decrease contamination in the food supply. The goal of this project is to produce a plant-made vaccine to protect ruminants against STEC. Several recombinant proteins were designed as vaccine candidates and expressed transiently in Nicotiana benthamiana and transplastomically Nicotiana tabacum. One vaccine candidate, an E. coli secreted protein A (EspA) fusion, accumulated in transplastomic lines up to 220.7 mg/kg of fresh leaf weight. Leaf tissue containing the antigen was lyophilized and will be orally administered to a small ruminant model to test vaccine efficacy

Co-expression with the Type 3 Secretion Chaperone CesT from Enterohemorrhagic E. coli Increases Accumulation of Recombinant Tir in Plant Chloroplasts

Type 3 secretion systems (T3SSs) are utilized by pathogenic Escherichia coli to infect their hosts and many proteins from these systems are affected by chaperones specific to T3SS-containing bacteria. Toward developing a recombinant vaccine against enterohaemorrhagic E. coli (EHEC), we expressed recombinant T3SS and related proteins from predominant EHEC serotypes in Nicotiana chloroplasts. Nicotiana benthamiana were transiently transformed to express chloroplast-targeted Tir, NleA, and EspD from the EHEC serotype O157:H7; a fusion of EspA proteins from serotypes O157:H7 and O26:H11; and a fusion of epitopes of Tir (Tir-ep) from serotypes O157:H7, O26:H11, O45:H2, and O111:H8. C-terminal GFP reporter fusion constructs were also developed and transiently expressed to confirm subcellular localization and quantify relative expression levels in situ. Recombinant proteins were co-expressed with chaperones specific to each T3SS protein with the goal of increasing their accumulation in the chloroplast. We found that co-expression with the chloroplast-targeted chaperone CesT significantly increases accumulation of recombinant Tir when the latter is either transiently expressed in the nucleus and targeted to the chloroplast of N. benthamiana or stably expressed in transplastomic Nicotiana tabacum. CesT also helped maintain higher levels of Tir:GFP fusion protein over time both in vivo and ex vivo, indicating that the favorable effect of CesT on accumulation of Tir is not specific to a single time point or to fresh material. By contrast, T3SS chaperones CesT, CesAB, CesD, and CesD2 did not increase accumulation of NleA:GFP, EspA:GFP, or EspD:GFP, which suggests dissimilar functioning of these chaperone–substrate combinations. CesT did not increase accumulation of Tir-ep:GFP, which may be due to the absence of the CesT binding domain from this fusion protein. The fusion to GFP improved accumulation of Tir-ep relative to the unfused protein, but not for the other recombinant proteins. These results emphasize the importance of native chaperones and stabilizing fusions as potential tools for the production of higher levels of recombinant proteins in plants; and may have implications for understanding interactions between T3SS chaperones and their substrates. In particular, our findings highlight the potential of T3SS chaperones to increase accumulation of recombinant T3SS proteins in heterologous systems

Glioma Through the Looking GLASS: Molecular Evolution of Diffuse Gliomas and the Glioma Longitudinal AnalySiS Consortium

Adult diffuse gliomas are a diverse group of brain neoplasms that inflict a high emotional toll on patients and their families. The Cancer Genome Atlas (TCGA) and similar projects have provided a comprehensive understanding of the somatic alterations and molecular subtypes of glioma at diagnosis. However, gliomas undergo significant cellular and molecular evolution during disease progression. We review the current knowledge on the genomic and epigenetic abnormalities in primary tumors and after disease recurrence, highlight the gaps in the literature, and elaborate on the need for a new multi-institutional effort to bridge these knowledge gaps and how the Glioma Longitudinal AnalySiS Consortium (GLASS) aims to systemically catalog the longitudinal changes in gliomas. The GLASS initiative will provide essential insights into the evolution of glioma toward a lethal phenotype, with the potential to reveal targetable vulnerabilities, and ultimately, improved outcomes for a patient population in need

A plant-produced bacteriophage tailspike protein for the control of Salmonella

The receptor binding domain of the tailspike protein Gp9 from the P22 bacteriophage was recently shown to reduce colonization in the chicken gut. In this study, we transiently expressed the receptor binding domain of the Gp9 tailspike protein in Nicotiana benthamiana, and targeted it to the endoplasmic reticulum (ER) or to the chloroplast. Gp9 was also fused to either an elastin-like polypeptide (ELP) or hydrophobin I (HFBI) tag, which were previously described to improve accumulation levels of recombinant proteins. The highest levels of recombinant protein accumulation occurred when unfused Gp9 was targeted to the ER. Lower levels of chloroplast-targeted Gp9 were also detected. ELP-fused Gp9 was purified and demonstrated to bind to Salmonella enterica serovar Typhimurium in vitro. Upon oral administration of lyophilized leaves expressing Gp9-ELP to newly hatched chicks, we found that this tailspike protein has the potential to be used as a prophylactic to control Salmonella contamination in chickens

PickYOLO: Fast deep learning particle detector for annotation of cryo electron tomograms

Particle localization (picking) in digital tomograms is a laborious and time-intensive step in cryogenic electron tomography (cryoET) analysis often requiring considerable user involvement, thus becoming a bottleneck for automated cryoET subtomogram averaging (STA) pipelines. In this paper, we introduce a deep learning framework called PickYOLO to tackle this problem. PickYOLO is a super-fast, universal particle detector based on the deep-learning real-time object recognition system YOLO (You Only Look Once), and tested on single particles, filamentous structures, and membrane-embedded particles. After training with the centre coordinates of a few hundred representative particles, the network automatically detects additional particles with high yield and reliability at a rate of 0.24–3.75 s per tomogram. PickYOLO can automatically detect number of particles comparable to those manually selected by experienced microscopists. This makes PickYOLO a valuable tool to substantially reduce the time and manual effort needed to analyse cryoET data for STA, greatly aiding in high-resolution cryoET structure determination

A Plant-produced candidate subunit vaccine reduces shedding of Enterohemorrhagic <em>Escherichia col</em>i in ruminants

Enterohemorrhagic Escherichia coli (EHEC) are commonly present in the gastrointestinal tract of cattle and cause serious infectious disease in humans. Immunizing cattle against EHEC is a promising strategy to decrease the risk of food contamination; however, veterinary vaccines against EHEC such as Econiche have not been widely adopted by the agricultural industry, and have been discontinued, prompting the need for more cost-effective EHEC vaccines. The objective of this project is to develop a platform to produce plant-made antigens for oral vaccination of ruminants against EHEC. Five recombinant proteins were designed as vaccine candidates and expressed transiently in Nicotiana benthamiana and transplastomically in Nicotiana tabacum. Three of these EHEC proteins, NleA, Stx2b, and a fusion of EspA accumulated when transiently expressed. Transient protein accumulation was the highest when EHEC proteins were fused to an elastin-like polypeptide (ELP) tag. In the transplastomic lines, EspA accumulated up to 479mgkg(-1) in lyophilized leaf material. Sheep that were administered leaf tissue containing recombinant EspA shed less E. coli O157:H7 when challenged, as compared to control animals. These results suggest that plant-made, transgenic EspA has the potential to reduce EHEC shedding in ruminants

Substrate-engaged type III secretion system structures reveal gating mechanism for unfolded protein translocation

Many bacterial pathogens rely on virulent type III secretion systems (T3SSs) or injectisomes to translocate effector proteins in order to establish infection. The central component of the injectisome is the needle complex which assembles a continuous conduit crossing the bacterial envelope and the host cell membrane to mediate effector protein translocation. However, the molecular principles underlying type III secretion remain elusive. Here, we report a structure of an active Salmonella enterica serovar Typhimurium needle complex engaged with the effector protein SptP in two functional states, revealing the complete 800Å-long secretion conduit and unraveling the critical role of the export apparatus (EA) subcomplex in type III secretion. Unfolded substrates enter the EA through a hydrophilic constriction formed by SpaQ proteins, which enables side chain-independent substrate transport. Above, a methionine gasket formed by SpaP proteins functions as a gate that dilates to accommodate substrates while preventing leaky pore formation. Following gate penetration, a moveable SpaR loop first folds up to then support substrate transport. Together, these findings establish the molecular basis for substrate translocation through T3SSs and improve our understanding of bacterial pathogenicity and motility