Modular capsid decoration boosts adenovirus vaccine-induced humoral immunity against SARS-CoV-2.

Adenovirus vector vaccines have been widely and successfully deployed in response to coronavirus disease 2019 (COVID-19). However, despite inducing potent T cell immunity, improvement of vaccine-specific antibody responses upon homologous boosting is modest compared with other technologies. Here, we describe a system enabling modular decoration of adenovirus capsid surfaces with antigens and demonstrate potent induction of humoral immunity against these displayed antigens. Ligand attachment via a covalent bond was achieved using a protein superglue, DogTag/DogCatcher (similar to SpyTag/SpyCatcher), in a rapid and spontaneous reaction requiring only co-incubation of ligand and vector components. DogTag was inserted into surface-exposed loops in the adenovirus hexon protein to allow attachment of DogCatcher-fused ligands on virus particles. Efficient coverage of the capsid surface was achieved using various ligands, with vector infectivity retained in each case. Capsid decoration shielded particles from vector neutralizing antibodies. In prime-boost regimens, adenovirus vectors decorated with the receptor-binding domain of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike induced >10-fold higher SARS-CoV-2 neutralization titers compared with an undecorated vector encoding spike. Importantly, decorated vectors achieved equivalent or superior T cell immunogenicity against encoded antigens compared with undecorated vectors. We propose capsid decoration using protein superglues as a novel strategy to improve efficacy and boostability of adenovirus-based vaccines and therapeutics.

Characterisation of Shigella Spa33 and Thermotoga FliM/N reveals a new model for C-ring assembly in T3SS.

Flagellar type III secretion systems (T3SS) contain an essential cytoplasmic-ring (C-ring) largely composed of two proteins FliM and FliN, whereas an analogous substructure for the closely related non-flagellar (NF) T3SS has not been observed in situ. We show that the spa33 gene encoding the putative NF-T3SS C-ring component in Shigella flexneri is alternatively translated to produce both full-length (Spa33-FL) and a short variant (Spa33-C), with both required for secretion. They associate in a 1:2 complex (Spa33-FL/C2) that further oligomerises into elongated arrays in vitro. The structure of Spa33-C2 and identification of an unexpected intramolecular pseudodimer in Spa33-FL reveal a molecular model for their higher order assembly within NF-T3SS. Spa33-FL and Spa33-C are identified as functional counterparts of a FliM-FliN fusion and free FliN respectively. Furthermore, we show that Thermotoga maritima FliM and FliN form a 1:3 complex structurally equivalent to Spa33-FL/C2 , allowing us to propose a unified model for C-ring assembly by NF-T3SS and flagellar-T3SS.

Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation.

The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins.

Nitrosothiols in bacterial pathogens and pathogenesis.

SIGNIFICANCE: The formation and degradation of S-nitrosothiols (SNOs) are important mechanisms of post-translational protein modification and appear to be ubiquitous in biology. These processes play well-characterized roles in eukaryotic cells, including a variety of pathologies and in relation to chronic conditions. We know little of the roles of these processes in pathogenic and other bacteria. RECENT ADVANCES: It is clear, mostly from growth and transcriptional studies, that bacteria sense and respond to exogenous SNOs. These responses are phenotypically and mechanistically distinct from the responses of bacteria to nitric oxide (NO) and NO-releasing agents, as well as peroxynitrite. Small SNOs, such as S-nitrosoglutathione (GSNO), are accumulated by bacteria with the result that intracellular S-nitrosoproteins (the 'S-nitrosoproteome') are detectable. Recently, conditions for endogenous SNO formation in enterobacteria have been described. CRITICAL ISSUES: The propensity of intracellular proteins to form SNOs is presumably constrained by the same rules of selectivity that have been discovered in eukaryotic systems, but is also influenced by uniquely bacterial NO detoxification systems, exemplified by the flavohemoglobin Hmp in enterobacteria and NO reductase of meningococci. Furthermore, the bacterial expression of such proteins impacts upon the formation of SNOs in mammalian hosts. FUTURE DIRECTIONS: The impairment during bacterial infections of specific SNO events in the mammalian host is of considerable interest in the context of proteins involved in innate immunity and intracellular signalling. In bacteria, numerous mechanisms of S-nitrosothiol degradation have been reported (e.g., GSNO reductase); others are thought to operate, based on consideration of their mammalian counterparts. The nitrosothiols of bacteria and particularly of pathogens warrant more intensive investigation.

The diversity of microbial responses to nitric oxide and agents of nitrosative stress close cousins but not identical twins.

Nitric oxide and related nitrogen species (reactive nitrogen species) now occupy a central position in contemporary medicine, physiology, biochemistry, and microbiology. In particular, NO plays important antimicrobial defenses in innate immunity but microbes have evolved intricate NO-sensing and defense mechanisms that are the subjects of a vast literature. Unfortunately, the burgeoning NO literature has not always been accompanied by an understanding of the intricacies and complexities of this radical and other reactive nitrogen species so that there exists confusion and vagueness about which one or more species exert the reported biological effects. The biological chemistry of NO and derived/related molecules is complex, due to multiple species that can be generated from NO in biological milieu and numerous possible reaction targets. Moreover, the fate and disposition of NO is always a function of its biological environment, which can vary significantly even within a single cell. In this review, we consider newer aspects of the literature but, most importantly, consider the underlying chemistry and draw attention to the distinctiveness of NO and its chemical cousins, nitrosonium (NO(+)), nitroxyl (NO(-), HNO), peroxynitrite (ONOO(-)), nitrite (NO(2)(-)), and nitrogen dioxide (NO(2)). All these species are reported to be generated in biological systems from initial formation of NO (from nitrite, NO synthases, or other sources) or its provision in biological experiments (typically from NO gas, S-nitrosothiols, or NO donor compounds). The major targets of NO and nitrosative damage (metal centers, thiols, and others) are reviewed and emphasis is given to newer "-omic" methods of unraveling the complex repercussions of NO and nitrogen oxide assaults. Microbial defense mechanisms, many of which are critical for pathogenicity, include the activities of hemoglobins that enzymically detoxify NO (to nitrate) and NO reductases and repair mechanisms (e.g., those that reverse S-nitrosothiol formation). Microbial resistance to these stresses is generally inducible and many diverse transcriptional regulators are involved-some that are secondary sensors (such as Fnr) and those that are "dedicated" (such as NorR, NsrR, NssR) in that their physiological function appears to be detecting primarily NO and then regulating expression of genes that encode enzymes with NO as a substrate. Although generally harmful, evidence is accumulating that NO may have beneficial effects, as in the case of the squid-Vibrio light-organ symbiosis, where NO serves as a signal, antioxidant, and specificity determinant. Progress in this area will require a thorough understanding not only of the biology but also of the underlying chemical principles.

Peroxynitrite stress is exacerbated by flavohaemoglobin-derived oxidative stress in Salmonella Typhimurium and is relieved by nitric oxide.

Oxidative and nitrosative stresses including nitric oxide (NO), superoxide (O2⁻) and peroxynitrite play key roles in determining the outcome of bacterial infections. In order to survive within the host and allow proliferation within immune cells such as macrophages, Salmonella isolates have a number of inducible proteins that are able to detoxify these highly reactive species, notably the anoxically functioning NO reductase NorVW, and the aerobically functioning flavohaemoglobin, Hmp, which catalyses the reaction between oxygen and NO to produce relatively inert nitrate. However, in the absence of NO but in the presence of reducing substrates and oxygen, O2⁻ is generated from Hmp-mediated electron transfer to bound oxygen and may form a variety of further oxidative species. Hence, Hmp expression is under tight negative regulation by the transcription factor NsrR, abolition of which causes an increase in the production of Hmp. In a previous study, this increase in Hmp levels conferred resistance to the nitrosating agent S-nitrosoglutathione but, perhaps surprisingly, the organism became more sensitive to killing by macrophages. Here, we report that an nsrR mutant that constitutively overexpresses Hmp is also hypersensitive to peroxynitrite in vitro. This sensitivity is alleviated by deletion of the hmp gene or pre-incubation of growing bacteria with NO-releasing agents. We hypothesize that Hmp-expressing cells, in the absence of NO, generate reactive oxygen species, the toxicity of which is exacerbated by peroxynitrite in vitro and in macrophages. RT-PCR confirmed that peroxynitrite causes oxidative stress and upregulation of katG and ahpC, whilst hmp and norV expression are affected very little. The katG gene upregulated by peroxynitrite encodes a catalase peroxidase enzyme with well-established roles in detoxifying peroxides. Here, we report that KatG is also able to enhance the breakdown of peroxynitrite, suggesting that the protective role of this enzyme may be wider than previously thought. These data suggest that spatial and temporal fluctuations in the levels of NO and reactive oxygen species will have important consequences for bacterial survival in the macrophage.

Peroxynitrite toxicity in Escherichia coli K12 elicits expression of oxidative stress responses and protein nitration and nitrosylation.

Peroxynitrite is formed in macrophages by the diffusion-limited reaction of superoxide and nitric oxide. This highly reactive species is thought to contribute to bacterial killing by interaction with diverse targets and nitration of protein tyrosines. This work presents for the first time a comprehensive analysis of transcriptional responses to peroxynitrite under tightly controlled chemostat growth conditions. Up-regulation of the cysteine biosynthesis pathway and an increase in S-nitrosothiol levels suggest S-nitrosylation to be a consequence of peroxynitrite exposure. Genes involved in the assembly/repair of iron-sulfur clusters also show enhanced transcription. Unexpectedly, arginine biosynthesis gene transcription levels were also elevated after treatment with peroxynitrite. Analysis of the negative regulator for these genes, ArgR, showed that post-translational nitration of tyrosine residues within this protein is responsible for its degradation in vitro. Further up-regulation was seen in oxidative stress response genes, including katG and ahpCF. However, genes known to be up-regulated by nitric oxide and nitrosating agents (e.g. hmp and norVW) were unaffected. Probabilistic modeling of the transcriptomic data identified five altered transcription factors in response to peroxynitrite exposure, including OxyR and ArgR. Hydrogen peroxide can be present as a contaminant in commercially available peroxynitrite preparations. Transcriptomic analysis of cells treated with hydrogen peroxide alone also revealed up-regulation of oxidative stress response genes but not of many other genes that are up-regulated by peroxynitrite. Thus, the cellular responses to peroxynitrite and hydrogen peroxide are distinct.

KatG from Salmonella typhimurium is a peroxynitritase.

Pathogenic bacteria elicit protective responses to oxidative and nitrosative stresses. Although such responses are generally distinct, it was recently reported in Mycobacterium tuberculosis that catalase-peroxidase (KatG), a classical defence against peroxides, also exhibits peroxynitritase activity. Here, the katG gene from Salmonella Typhimurium was cloned and protein purified and characterised. An increase in the rate of decomposition of peroxynitrite was observed for KatG from the enterobacterium with a second-order rate constant of 4.2x10(4)M(-1)s(-1) at pH 7.4, 25 degrees C. This enzyme was able to reduce dihydrorhodamine oxidation by peroxynitrite to approximately 83%. Given the peroxynitritase activity demonstrated here it is likely that KatG may play a wider role in the detoxification of oxidative stresses than previously thought.