The long-term antibody response after SARS-CoV-2 prime-boost vaccination in healthy individuals. The positive influence of extended between-dose intervals and heterologous schedule.

Introduction: Anti-COVID vaccination in Argentina was carried out using different protocols and variations in periods between administrations, as well as combinations of different vaccine platforms. Considering the relevance of the antibody response in viral infections, we analyzed anti-S antibodies in healthy people at different points of time following the Sputnik immunization procedure. Methods: We attended the vaccination centers in the city of Rosario, which had shorter versus longer intervals between both doses. A total of (1021) adults with no COVID-compatible symptoms (throughout the study period) were grouped according to the gap between both vaccine doses: 21 (Group A, n=528), 30 (Group B, n=147), and 70 days (Group C, n=82), as well as an additional group of individuals with heterologous vaccination (Sputnik/Moderna, separated by a 107-day interval, group D, n=264). Results and conclusions: While there were no between-group differences in baseline levels of specific antibodies, data collected several weeks after administering the second dose showed that group D had the highest amounts of specific antibodies, followed by values recorded in Groups C, B, and A. The same pattern of group differences was seen when measuring anti-S antibodies at 21 or 180 days after the first and second doses, respectively. Delayed between-dose intervals coexisted with higher antibody titers. This happened even more when using a prime-boost heterologous schedule.

From the notebook to recombinant protein production in Escherichia coli: Design of expression vectors and gene cloning.

Research in recombinant protein expression in microorganism hosts spans half a century. The field has evolved from mostly trial-and-error approaches to more rational strategies, including careful design of the expression vectors and the coding sequence for the protein of interest. It is important to reflect on many aspects about vector construction, such as codon usage, integration site, coding sequence mutagenesis and many others. In this chapter, we overview methods and considerations to generate a suitable construct and anticipate possible experimental roadblocks.

Starting a new recombinant protein production project in Escherichia coli.

One of the goals in recombinant protein production in Escherichia coli is to maximize productivity. High volumetric and specific yields can be reached after careful selection of expression strains and optimization of cultivation parameters. In this chapter, we review the many tools available to make the most out of this versatile microbial cell factory. Useful guidelines and options for troubleshooting production are presented.

A new catalytic mechanism of bacterial ferredoxin-NADP+ reductases due to a particular NADP+ binding mode.

Ferredoxin-NADP+ reductases (FNRs) are ubiquitous flavoenzymes involved in redox metabolisms. FNRs catalyze the reversible electron transfer between NADP(H) and ferredoxin or flavodoxin. They are classified as plant- and mitochondrial-type FNR. Plant-type FNRs are divided into plastidic and bacterial classes. The plastidic FNRs show turnover numbers between 20 and 100 times higher than bacterial enzymes and these differences have been related to their physiological functions. We demonstrated that purified Escherichia coli FPR (EcFPR) contains tightly bound NADP+ , which does not occur in plastidic type FNRs. The three-dimensional structure of EcFPR evidenced that NADP+ interacts with three arginines (R144, R174, and R184) which could generate a very high affinity and structured site. These arginines are conserved in other bacterial FNRs but not in the plastidic enzymes. We have cross-substituted EcFPR arginines with residues present in analogous positions in the Pisum sativum FNR (PsFNR) and replaced these amino acids by arginines in PsFNR. We analyzed all proteins by structural, kinetic, and stability studies. We found that EcFPR mutants do not contain bound NADP+ and showed increased Km for this nucleotide. The EcFPR activity was inhibited by NADP+ but this behavior disappeared as arginines were removed. A NADP+ analog of the nicotinamide portion produced an activating effect on EcFPR and promoted the NADP+ release. Our results give evidence for a new model of NADP+ binding and catalysis in bacterial FNRs.We propose that this tight NADP+ binding constitutes an essential catalytic and regulatory mechanism of bacterial FNRs involved in redox homeostasis.

Structural features of the plant N-recognin ClpS1 and sequence determinants in its targets that govern substrate selection.

In the N-degron pathway of protein degradation of Escherichia coli, the N-recognin ClpS identifies substrates bearing N-terminal phenylalanine, tyrosine, tryptophan, or leucine and delivers them to the caseinolytic protease (Clp). Chloroplasts contain the Clp system, but whether chloroplastic ClpS1 adheres to the same constraints is unknown. Moreover, the structural underpinnings of substrate recognition are not completely defined. We show that ClpS1 recognizes canonical residues of the E. coli N-degron pathway. The residue in second position influences recognition (especially in N-terminal ends starting with leucine). N-terminal acetylation abrogates recognition. ClpF, a ClpS1-interacting partner, does not alter its specificity. Substrate binding provokes local remodeling of residues in the substrate-binding cavity of ClpS1. Our work strongly supports the existence of a chloroplastic N-degron pathway.

Biochemical characterization of ClpB3, a chloroplastic disaggregase from Arabidopsis thaliana.

KEY MESSAGE: The first biochemical characterization of a chloroplastic disaggregase is reported (Arabidopsis thaliana ClpB3). ClpB3 oligomerizes into active hexamers that resolubilize aggregated substrates using ATP and without the aid of partners. Disaggregases from the Hsp100/Clp family are a type of molecular chaperones involved in disassembling protein aggregates. Plant cells are uniquely endowed with ClpB proteins in the cytosol, mitochondria and chloroplasts. Chloroplastic ClpB proteins have been implicated in key processes like the unfolded protein response; however, they have not been studied in detail. In this study, we explored the biochemical properties of a chloroplastic ClpB disaggregase, in particular, ClpB3 from A. thaliana. ClpB3 was produced recombinantly in Escherichia coli and affinity-purified to near homogeneity. ClpB3 forms a hexameric complex in the presence of MgATP and displays intrinsic ATPase activity. We demonstrate that ClpB3 has ATPase activity in a wide range of pH and temperature values and is particularly resistant to heat. ClpB3 specifically targets unstructured polypeptides and mediates the reactivation of heat-denatured model substrates without the aid of the Hsp70 system. Overall, this work represents the first in-depth biochemical description of a ClpB protein from plants and strongly supports its role as the putative disaggregase chaperone in chloroplasts.

A novel Xanthomonas citri subsp. citri NADPH quinone reductase involved in salt stress response and virulence.

BACKGROUND: Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker is maintained as an epiphyte on citrus leaves until entering the plant tissue. During epiphytic survival, bacteria may encounter low water availability that challenges the infection process. Proteomics analyses of Xcc under saline stress, mimicking the conditions found during epiphytic survival, showed increased abundance of a putative NAD(P)H dehydrogenase encoded by XAC2229. METHODS: Expression levels of XAC2229 and a Xcc mutant in XAC2229 were analyzed in salt and oxidative stress and during plant-pathogen interaction. An Escherichia coli expressing XAC2229 was obtained, and the role of this protein in oxidative stress resistance and in reactive oxygen species production was studied. Finally, Xac2229 protein was purified, spectrophotometric and cofactor analyses were done and enzymatic activities determined. RESULTS: XAC2229 was expressed under salt stress and during plant-pathogen interaction. ΔXAC2229 mutant showed less number of cankers and impaired epiphytic survival than the wild type strain. ΔXAC2229 survived less in the presence of H2O2 and produced more reactive oxygen species and thiobarbituric acid-reactive substances than the wild type strain. Similar results were observed for E. coli expressing XAC2229. Xac2229 is a FAD containing flavoprotein, displays diaphorase activity with an optimum at pH 6.0 and has quinone reductase activity using NADPH as an electron donor. CONCLUSIONS: A FAD containing flavoprotein from Xcc is a new NADPH quinone reductase required for bacterial virulence, particularly in Xcc epiphytic survival on citrus leaves. GENERAL SIGNIFICANCE: A novel protein involved in the worldwide disease citrus canker was characterized.

New tools for recombinant protein production in Escherichia coli: A 5-year update.

The production of proteins in sufficient amounts is key for their study or use as biotherapeutic agents. Escherichia coli is the host of choice for recombinant protein production given its fast growth, easy manipulation, and cost-effectiveness. As such, its protein production capabilities are continuously being improved. Also, the associated tools (such as plasmids and cultivation conditions) are subject of ongoing research to optimize product yield. In this work, we review the latest advances in recombinant protein production in E. coli.

A novel method for removing contaminant Hsp70 molecular chaperones from recombinant proteins.

The production of recombinant proteins in bacteria has increased significantly in recent years, becoming a common tool for both research and the industrial production of proteins. One of the requirements of this methodology is to obtain the desired protein without contaminants. However, this goal cannot always be readily achieved. Multiple strategies have been developed to improve the quality of the desired protein product. Nevertheless, contamination with molecular chaperones is one of the recalcitrant problems that still affects the quality of the obtained proteins. The ability of chaperones to bind to unfolded proteins or to regions where the polypeptide chain is exposed make the removal of the contamination during purification challenging to achieve. This work aimed to develop a strategy to remove contaminating DnaK, one of the homologous Hsp70 molecular chaperones found in Escherichia coli, from purified recombinant proteins. For this purpose, we developed a methodology that captures the DnaK from the contaminating proteins by co-incubation with a GST-cleanser protein that has free functional binding sites for the chaperone. The cleanser protein can then be easily removed together with the captured DnaK. Here, we demonstrated the utility of our system by decontaminating a Histidine-tagged recombinant protein in a batch process. The addition of the GST-cleanser protein in the presence of ATP-Mg eliminates the DnaK contamination substantially. Thus, our decontaminant strategy results versatile and straightforward and can be applied to proteins obtained with different expression and purifications systems as well as to small samples or large volume preparations.

A bacterial 2[4Fe4S] ferredoxin as redox partner of the plastidic-type ferredoxin-NADP+ reductase from Leptospira interrogans.

BACKGROUND: Ferredoxins are small iron-sulfur proteins that participate as electron donors in various metabolic pathways. They are recognized substrates of ferredoxin-NADP+ reductases (FNR) in redox metabolisms in mitochondria, plastids, and bacteria. We previously found a plastidic-type FNR in Leptospira interrogans (LepFNR), a parasitic bacterium of animals and humans. Nevertheless, we did not identify plant-type ferredoxins or flavodoxins, the common partners of this kind of FNR. METHODS: Sequence alignment, phylogenetical analyses and structural modeling were performed for the identification of a 2[4Fe4S] ferredoxin (LepFd2) as a putative redox partner of LepFNR in L. interrogans. The gene encoding LepFd2 was cloned and the protein overexpressed and purified. The functional properties of LepFd2 and LepFNR-LepFd2 complex were analyzed by kinetic and mutagenesis studies. RESULTS: We succeeded in expressing and purifying LepFd2 with its FeS cluster properly bound. We found that LepFd2 exchanges electrons with LepFNR. Moreover, a unique structural subdomain of LepFNR (loop P75-Y91), was shown to be involved in the recognition and binding of LepFd2. This structural subdomain is not found in other FNR homologs. CONCLUSIONS: We report for the first time a redox pair in L. interrogans in which a plastidic FNR exchanges electron with a bacterial 2[4Fe4S] ferredoxin. We characterized this reaction and proposed a model for the productive LepFNR-LepFd2 complex. GENERAL SIGNIFICANCE: Our findings suggest that the interaction of LepFNR with the iron-sulfur protein would be different from the one previously described for the homolog enzymes. This knowledge would be useful for the design of specific LepFNR inhibitors.

A Gatekeeper Residue of ClpS1 from Arabidopsis thaliana Chloroplasts Determines its Affinity Towards Substrates of the Bacterial N-End Rule.

Proteins that are to be eliminated must be proficiently recognized by proteolytic systems so that inadvertent elimination of useful proteins is avoided. One mechanism to ensure proper recognition is the presence of N-terminal degradation signals (N-degrons) that are targeted by adaptor proteins (N-recognins). The members of the caseinolytic protease S (ClpS) family of N-recognins identify targets bearing an N-terminal phenylalanine, tyrosine, tryptophan or leucine residue, and then present them to a protease system. This process is known as the 'bacterial N-end rule'. The presence of a ClpS protein in Arabidopsis thaliana chloroplasts (AtClpS1) prompted the hypothesis that the bacterial N-end rule exists in this organelle. However, the specificity of AtClpS1 is unknown. Here we show that AtClpS1 has the ability to recognize bacterial N-degrons, albeit with low affinity. Recognition was assessed by the effect of purified AtClpS1 on the degradation of fluorescent variants bearing bacterial N-degrons. In many bacterial ClpS proteins, a methionine residue acts as a 'gatekeeper' residue, fine-tuning the specificity of the N-recognin. In plants, the amino acid at that position is an arginine. Replacement of this arginine for methionine in recombinant AtClpS1 allows for high-affinity binding to classical N-degrons of the bacterial N-end rule, suggesting that the arginine residue in the substrate-binding site may also act as a gatekeeper for plant substrates.

TAT-mediated transduction of bacterial redox proteins generates a cytoprotective effect on neuronal cells.

Cell penetrating peptides, also known as protein transduction domains, have the capacity to ubiquitously cross cellular membranes carrying many different cargos with negligible cytotoxicity. As a result, they have emerged as a powerful tool for macromolecular delivery-based therapies. In this study, catalytically active bacterial Ferredoxin-NADP+ reductase (LepFNR) and Heme oxygenase (LepHO) fused to the HIV TAT-derived protein transduction peptide (TAT) were efficiently transduced to neuroblastoma SHSY-5Y cells. Proteins entered the cells through an endocytic pathway showing a time/concentration dependent mechanism that was clearly modulated by the nature of the cargo protein. Since ferredoxin-NADP+ reductases and heme oxygenases have been implicated in mechanisms of oxidative stress defense, neuroblastoma cells simultaneously transduced with TAT-LepFNR and TAT-LepHO were challenged by H2O2 incubations to judge the cytoprotective power of these bacterial enzymes. Accumulation of reactive oxygen species was significantly reduced in these transduced neuronal cells. Moreover, measurements of metabolic viability, membrane integrity, and cell survival indicated that these cells showed a better tolerance to oxidative stress. Our results open the possibility for the application of transducible active redox proteins to overcome the damage elicited by oxidative stress in cells and tissues.

Structural and mutational analyses of the Leptospira interrogans virulence-related heme oxygenase provide insights into its catalytic mechanism.

Heme oxygenase from Leptospira interrogans is an important virulence factor. During catalysis, redox equivalents are provided to this enzyme by the plastidic-type ferredoxin-NADP+ reductase also found in L. interrogans. This process may have evolved to aid this bacterial pathogen to obtain heme-iron from their host and enable successful colonization. Herein we report the crystal structure of the heme oxygenase-heme complex at 1.73 Å resolution. The structure reveals several distinctive features related to its function. A hydrogen bonded network of structural water molecules that extends from the catalytic site to the protein surface was cleared observed. A depression on the surface appears to be the H+ network entrance from the aqueous environment to the catalytic site for O2 activation, a key step in the heme oxygenase reaction. We have performed a mutational analysis of the F157, located at the above-mentioned depression. The mutant enzymes were unable to carry out the complete degradation of heme to biliverdin since the reaction was arrested at the verdoheme stage. We also observed that the stability of the oxyferrous complex, the efficiency of heme hydroxylation and the subsequent conversion to verdoheme was adversely affected. These findings underscore a long-range communication between the outer fringes of the hydrogen-bonded network of structural waters and the heme active site during catalysis. Finally, by analyzing the crystal structures of ferredoxin-NADP+ reductase and heme oxygenase, we propose a model for the productive association of these proteins.

Khellin and Visnagin, Furanochromones from Ammi visnaga (L.) Lam., as Potential Bioherbicides.

Plants constitute a source of novel phytotoxic compounds to be explored in searching for effective and environmentally safe herbicides. From a previous screening of plant extracts for their phytotoxicity, a dichloromethane extract of Ammi visnaga (L.) Lam. was selected for further study. Phytotoxicity-guided fractionation of this extract yielded two furanochromones, khellin and visnagin, for which herbicidal activity had not been described before. Khellin and visnagin were phytotoxic to model species lettuce (Lactuca sativa) and duckweed (Lemna paucicostata), with IC50 values ranging from 110 to 175 µM. These compounds also inhibited the growth and germination of a diverse group of weeds at 0.5 and 1 mM. These weeds included five grasses [ryegrass (Lolium multiflorum), barnyardgrass (Echinocloa crus-galli), crabgrass (Digitaria sanguinalis), foxtail (Setaria italica), and millet (Panicum sp.)] and two broadleaf species [morningglory (Ipomea sp.) and velvetleaf (Abutilon theophrasti)]. During greenhouse studies visnagin was the most active and showed significant contact postemergence herbicidal activity on velvetleaf and crabgrass at 2 kg active ingredient (ai) ha-1. Moreover, its effect at 4 kg ai ha-1 was comparable to the bioherbicide pelargonic acid at the same rate. The mode of action of khellin and visnagin was not a light-dependent process. Both compounds caused membrane destabilization, photosynthetic efficiency reduction, inhibition of cell division, and cell death. These results support the potential of visnagin and, possibly, khellin as bioherbicides or lead molecules for the development of new herbicides.

Heme-iron utilization by Leptospira interrogans requires a heme oxygenase and a plastidic-type ferredoxin-NADP(+) reductase.

BACKGROUND: Heme oxygenase catalyzes the conversion of heme to iron, carbon monoxide and biliverdin employing oxygen and reducing equivalents. This enzyme is essential for heme-iron utilization and contributes to virulence in Leptospira interrogans. METHODS: A phylogenetic analysis was performed using heme oxygenases sequences from different organisms including saprophytic and pathogenic Leptospira species. L. interrogans heme oxygenase (LepHO) was cloned, overexpressed and purified. The structural and enzymatic properties of LepHO were analyzed by UV-vis spectrophotometry and (1)H NMR. Heme-degrading activity, ferrous iron release and biliverdin production were studied with different redox partners. RESULTS: A plastidic type, high efficiently ferredoxin-NADP(+) reductase (LepFNR) provides the electrons for heme turnover by heme oxygenase in L. interrogans. This catalytic reaction does not require a ferredoxin. Moreover, LepFNR drives the heme degradation to completeness producing free iron and α-biliverdin as the final products. The phylogenetic divergence between heme oxygenases from saprophytic and pathogenic species supports the functional role of this enzyme in L. interrogans pathogenesis. CONCLUSIONS: Heme-iron scavenging by LepHO in L. interrogans requires only LepFNR as redox partner. Thus, we report a new substrate of ferredoxin-NADP(+) reductases different to ferredoxin and flavodoxin, the only recognized protein substrates of this flavoenzyme to date. The results presented here uncover a fundamental step of heme degradation in L. interrogans. GENERAL SIGNIFICANCE: Our findings contribute to understand the heme-iron utilization pathway in Leptospira. Since iron is required for pathogen survival and infectivity, heme degradation pathway may be relevant for therapeutic applications.

Recombinant protein expression in Escherichia coli: advances and challenges.

Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field.

Crystal structure of the FAD-containing ferredoxin-NADP+ reductase from the plant pathogen Xanthomonas axonopodis pv. citri.

We have solved the structure of ferredoxin-NADP(H) reductase, FPR, from the plant pathogen Xanthomonas axonopodis pv. citri, responsible for citrus canker, at a resolution of 1.5 Å. This structure reveals differences in the mobility of specific loops when compared to other FPRs, probably unrelated to the hydride transfer process, which contributes to explaining the structural and functional divergence between the subclass I FPRs. Interactions of the C-terminus of the enzyme with the phosphoadenosine of the cofactor FAD limit its mobility, thus affecting the entrance of nicotinamide into the active site. This structure opens the possibility of rationally designing drugs against the X. axonopodis pv. citri phytopathogen.

Redox proteins as targets for drugs development against pathogens.

Antimicrobial drug resistance in pathogens is an increasing human health problem. The rapid loss of effectiveness in antibiotics treatments and the accumulation of multi-resistant microbial strains are increasing worldwide threats. Moreover, several infectious diseases have been neglected for years and new antimicrobial treatments are lacking. In other cases, complexity of infectious organisms has exceeded the efforts to find new drugs to control them. Thus, strategies for the proper development of specific drugs are critically needed. Redox metabolism has already been proved to be a useful target for drug development. During the last years a significant number of electron carriers, enzymes, proteins and protein complexes have been studied and some of them were found to be essential for survival of several microbial pathogens. This review will focus on three major redox metabolic pathways which may provide promising strategies to fight against pathogens: the non-mevalonate pathway for isoprenoids biosynthesis, the iron metabolism and the iron-sulfur proteins.The common attractive link of all these processes is the plant-type ferredoxin-NADP+ reductase, an enzyme that participates in numerous electron transfer reactions and has no homologous enzyme in humans. Research in these redox pathways will open new perspectives for the rational design of drugs against infectious diseases.

Chloroplastic Hsp100 chaperones ClpC2 and ClpD interact in vitro with a transit peptide only when it is located at the N-terminus of a protein.

BACKGROUND: Clp/Hsp100 chaperones are involved in protein quality control. They act as independent units or in conjunction with a proteolytic core to degrade irreversibly damaged proteins. Clp chaperones from plant chloroplasts have been also implicated in the process of precursor import, along with Hsp70 chaperones. They are thought to pull the precursors in as the transit peptides enter the organelle. How Clp chaperones identify their substrates and engage in their processing is not known. This information may lie in the position, sequence or structure of the Clp recognition motifs. RESULTS: We tested the influence of the position of the transit peptide on the interaction with two chloroplastic Clp chaperones, ClpC2 and ClpD from Arabidopsis thaliana (AtClpC2 and AtClpD). The transit peptide of ferredoxin-NADP+ reductase was fused to either the N- or C-terminal end of glutathione S-transferase. Another fusion with the transit peptide interleaved between two folded proteins was used to probe if AtClpC2 and AtClpD could recognize tags located in the interior of a polypeptide. We also used a mutated transit peptide that is not targeted by Hsp70 chaperones (TP1234), yet it is imported at a normal rate. The fusions were immobilized on resins and the purified recombinant chaperones were added. After a washing protocol, the amount of bound chaperone was assessed. Both AtClpC2 and AtClpD interacted with the transit peptides when they were located at the N-terminal position of a protein, but not when they were allocated to the C-terminal end or at the interior of a polypeptide. CONCLUSIONS: AtClpC2 and AtClpD have a positional preference for interacting with a transit peptide. In particular, the localization of the signal sequence at the N-terminal end of a protein seems mandatory for interaction to take place. Our results have implications for the understanding of protein quality control and precursor import in chloroplasts.