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The SARS-CoV-2 virus, the agent of COVID-19, caused unprecedented loss of lives and economic decline worldwide. Although the introduction of public health measures, vaccines, diagnostics, and therapeutics disrupted the spread of the SARS-CoV-2, the emergence of variants poses substantial threat. This study traced SARS-CoV-2 variants circulating in Uganda by July 2021 to inform the necessity for refinement of the intervention medical products. A comprehensivein silicoanalysis of the SARS-CoV-2 genomes detected in clinical samples collected from COVID-19 patients in Uganda revealed occurrence of structural protein variants with potential of escaping detection, resisting antibody therapy, or increased infectivity. The genome sequence dataset was retrieved from the GISAID database and the open reading frame encoding the spike, envelope, membrane, or nucleocapsid proteins was translated. The obtained protein sequences were aligned and inspected for existence of variants. The variant positions on each of the four alignment sets were mapped on predicted epitopes as well as the 3D structures. Additionally, sequences within each of the sets were clustered by family. A phylogenetic tree was constructed to assess relationship between the encountered spike protein sequences and Wuhan-Hu-1 wild-type, or theAlpha,Beta,DeltaandGammavariants of concern. Strikingly, the frequency of each of the spike protein point mutations F157L/Del, D614G and P681H/R was over 50%. The furin and the transmembrane serine protease 2 cleavage sites were unaffected by mutation. Whereas theDeltadominated the spike sequences (16.5%, 91/550),Gammawas not detected. The envelope protein was the most conserved with 96.3% (525/545) sequences being wild-type followed by membrane at 68.4% (397/580). Although the nucleocapsid protein sequences varied, the variant residue positions were less concentrated at the RNA binding domains. The dominant nucleocapsid sequence variant was S202N (34.5%, 205/595). These findings offer baseline information required for refining the existing COVID-19 vaccines, diagnostics, and therapeutics.

paaR2–paaA2–parE2 is a three-component toxin–antitoxin module found in prophage CP-993P of Escherichia coli O157:H7. Transcription regulation of this module occurs via the 123-amino-acid regulator PaaR2, which forms a large oligomeric structure. Despite appearing to be well folded, PaaR2 withstands crystallization, as does its N-terminal DNA-binding domain. Native mass spectrometry was used to screen for nanobodies that form a unique complex and stabilize the octameric structure of PaaR2. One such nanobody, Nb33, allowed crystallization of the protein. The resulting crystals belong to space group F432, with unit-cell parameter a = 317 Å, diffract to 4.0 Å resolution and are likely to contain four PaaR2 monomers and four nanobody monomers in the asymmetric unit. Crystals of two truncates containing the N-terminal helix–turn–helix domain also interact with Nb33, and the corresponding co-crystals diffracted to 1.6 and 1.75 Å resolution.

Salivarian trypanosomes comprise a group of extracellular anthroponotic and zoonotic parasites. The only sustainable method for global control of these infection is through vaccination of livestock animals. Despite multiple reports describing promising laboratory results, no single field-applicable solution has been successful so far. Conventionally, vaccine research focusses mostly on exposed immunogenic antigens, or the structural molecular knowledge of surface exposed invariant immunogens. Unfortunately, extracellular parasites (or parasites with extracellular life stages) have devised efficient defense systems against host antibody attacks, so they can deal with the mammalian humoral immune response. In the case of trypanosomes, it appears that these mechanisms have been perfected, leading to vaccine failure in natural hosts. Here, we provide two examples of potential vaccine candidates that, despite being immunogenic and accessible to the immune system, failed to induce a functionally protective memory response. First, trypanosomal enolase was tested as a vaccine candidate, as it was recently characterized as a highly conserved enzyme that is readily recognized during infection by the host antibody response. Secondly, we re-addressed a vaccine approach towards the Invariant Surface Glycoprotein ISG75, and showed that despite being highly immunogenic, trypanosomes can avoid anti-ISG75 mediated parasitemia control.

The implementation of prospective drug resistance (DR) studies in the R&D pipelines is a common practice for many infectious diseases, but not for Neglected Tropical Diseases. Here, we explored and demonstrated the importance of this approach, using as paradigms Leishmania donovani, the etiological agent of Visceral Leishmaniasis (VL), and TCMDC-143345, a promising compound of the GSK ‘Leishbox’ to treat VL. We experimentally selected resistance to TCMDC-143345 in vitro and characterized resistant parasites at genomic and phenotypic levels. We found that it took more time to develop resistance to TCMDC-143345 than to other drugs in clinical use and that there was no cross resistance to these drugs, suggesting a new and unique mechanism. By whole genome sequencing, we found two mutations in the gene encoding the L. donovani dynamin-1-like protein (LdoDLP1) that were fixed at highest drug pressure. Through phylogenetic analysis, we identified LdoDLP1 as a family member of the dynamin-related proteins, a group of proteins that impacts the shapes of biological membranes by mediating fusion and fission events, with a putative role in mitochondrial fission. We found that L. donovani lines genetically engineered to harbor the two identified LdoDLP1 mutations were resistant to TCMDC-143345 and displayed altered mitochondrial properties. By homology modeling, we showed how the two LdoDLP1 mutations may influence protein structure and function. Taken together, our data reveal a clear involvement of LdoDLP1 in the adaptation/resistance of L. donovani to TCMDC-143345.ImportanceHumans and their pathogens are continuously locked in a molecular arms race during which the eventual emergence of pathogen drug resistance (DR) seems inevitable. For neglected tropical diseases (NTDs), DR is generally studied retrospectively, once it has already been established in clinical settings. We previously recommended to keep one step ahead in the host-pathogen arms race and implement prospective DR studies in the R&D pipeline, a common practice for many infectious diseases, but not for NTDs. Here, using Leishmania donovani, the etiological agent of Visceral Leishmaniasis (VL), and TCMDC-143345, a promising compound of the GSK ‘Leishbox’ to treat VL, as paradigms, we experimentally selected resistance to the compound and proceeded to genomic and phenotypic characterization of DR parasites. The results gathered in the present study suggest a new DR mechanism involving the L. donovani dynamin-1 like protein (LdoDLP1) and demonstrate the practical relevance of prospective DR studies.

Trypanosoma evansi is a widely spread parasite that causes the debilitating disease &ldquo, surra&rdquo, in several types of ungulates. This severely challenges livestock rearing and heavily weighs on the socio-economic development in the affected areas, which include countries on five continents. Active case finding requires a sensitive and specific diagnostic test. In this paper, we describe the application of an unbiased immunization strategy to identify potential biomarkers for Nanobody (Nb)-based detection of T. evansi infections. Alpaca immunization with soluble lysates from different T. evansi strains followed by panning against T. evansi secretome resulted in the selection of a single Nb (Nb11). By combining Nb11-mediated immuno-capturing with mass spectrometry, the T. evansi target antigen was identified as the glycolytic enzyme enolase. Four additional anti-enolase binders were subsequently generated by immunizing another alpaca with the recombinant target enzyme. Together with Nb11, these binders were evaluated for their potential use in a heterologous sandwich detection format. Three Nb pairs were identified as candidates for the further development of an antigen-based assay for Nb-mediated diagnosis of T. evansi infection.

Trypanosoma evansi is a widely spread parasite that causes the debilitating disease “surra” in several types of ungulates. This severely challenges livestock rearing and heavily weighs on the socio-economic development in the affected areas, which include countries on five continents. Active case finding requires a sensitive and specific diagnostic test. In this paper, we describe the application of an unbiased immunization strategy to identify potential biomarkers for Nanobody (Nb)-based detection of T. evansi infections. Alpaca immunization with soluble lysates from different T. evansi strains followed by panning against T. evansi secretome resulted in the selection of a single Nb (Nb11). By combining Nb11-mediated immuno-capturing with mass spectrometry, the T. evansi target antigen was identified as the glycolytic enzyme enolase. Four additional anti-enolase binders were subsequently generated by immunizing another alpaca with the recombinant target enzyme. Together with Nb11, these binders were evaluated for their potential use in a heterologous sandwich detection format. Three Nb pairs were identified as candidates for the further development of an antigen-based assay for Nb-mediated diagnosis of T. evansi infection.

Animal African trypanosomosis (AAT), a disease affecting livestock, is caused by parasites of the Trypanosoma genus (mainly T. vivax and T. congolense). AAT is widespread in Sub-Saharan Africa, where it continues to impose a heavy socio-economic burden as it renders development of sustainable livestock rearing very strenuous. Active case-finding and the identification of infected animals prior to initiation of drug treatment requires the availability of sensitive and specific diagnostic tests. In this paper, we describe the development of two heterologous sandwich assay formats (ELISA and LFA) for T. congolense detection through the use of Nanobodies (Nbs). The immunisation of an alpaca with a secretome mix from two T. congolense strains resulted in the identification of a Nb pair (Nb44/Nb42) that specifically targets the glycolytic enzyme pyruvate kinase. We demonstrate that the Nb44/Nb42 ELISA and LFA can be employed to detect parasitaemia in plasma samples from experimentally infected mice and cattle and, additionally, that they can serve as ‘test-of-cure’ tools. Altogether, the findings in this paper present the development and evaluation of the first Nb-based antigen detection LFA to identify active T. congolense infections.

Purpose: Camelid single-domain antibody-fragments (sdAb) have beneficial pharmacokinetic properties, and those targeted to HER2 can be used for imaging of HER2-overexpressing cancer. Labeled with a therapeutic radionuclide, they may be used for HER2-targeted therapy. Here, we describe the generation of a 131I-labeled sdAb as a theranostic drug to treat HER2-overexpressing cancer.Experimental Design: Anti-HER2 sdAb 2Rs15d was labeled with 131I using [131I]SGMIB and evaluated in vitro. Biodistribution was evaluated in two HER2+ murine xenograft models by micro-SPECT/CT imaging and at necropsy, and under challenge with trastuzumab and pertuzumab. The therapeutic potential of [131I]SGMIB-2Rs15d was investigated in two HER2+ tumor mouse models. A single-dose toxicity study was performed in mice using unlabeled [127I]SGMIB-sdAb at 1.4 mg/kg. The structure of the 2Rs15d–HER2 complex was determined by X-ray crystallography.Results: [131I]SGMIB-2Rs15d bound specifically to HER2+ cells (Kd = 4.74 ± 0.39 nmol/L). High and specific tumor uptake was observed in both BT474/M1 and SKOV-3 tumor xenografted mice and surpassed kidney levels by 3 hours. Extremely low uptake values were observed in other normal tissues at all time points. The crystal structure revealed that 2Rs15d recognizes HER2 Domain 1, consistent with the lack of competition with trastuzumab and pertuzumab observed in vivo. [131I]SGMIB-2Rs15d alone, or in combination with trastuzumab, extended median survival significantly. No toxicity was observed after injecting [127I]SGMIB-2Rs15d.Conclusions: These findings demonstrate the theranostic potential of [131I]SGMIB-2Rs15d. An initial scan using low radioactive [*I]SGMIB-2Rs15d allows patient selection and dosimetry calculations for subsequent therapeutic [131I]SGMIB-2Rs15d and could thereby impact therapy outcome on HER2+ breast cancer patients. Clin Cancer Res; 23(21); 6616–28. ©2017 AACR.

Background Animal trypanosomosis caused by Trypanosoma evansi is known as “surra” and is a widespread neglected tropical disease affecting wild and domestic animals mainly in South America, the Middle East, North Africa and Asia. An essential necessity for T. evansi infection control is the availability of reliable and sensitive diagnostic tools. While DNA-based PCR detection techniques meet these criteria, most of them require well-trained and experienced users as well as a laboratory environment allowing correct protocol execution. As an alternative, we developed a recombinase polymerase amplification (RPA) test for Type A T. evansi. The technology uses an isothermal nucleic acid amplification approach that is simple, fast, cost-effective and is suitable for use in minimally equipped laboratories and even field settings. Methodology/Principle findings An RPA assay targeting the T. evansi RoTat1.2 VSG gene was designed for the DNA-based detection of T. evansi. Comparing post-amplification visualization by agarose gel electrophoresis and a lateral flow (LF) format reveals that the latter displays a higher sensitivity. The RPA-LF assay is specific for RoTat1.2-expressing strains of T. evansi as it does not detect the genomic DNA of other trypanosomatids. Finally, experimental mouse infection trials demonstrate that the T. evansi specific RPA-LF can be employed as a test-of-cure tool. Conclusions/Significance Compared to other DNA-based parasite detection methods (such as PCR and LAMP), the T. evansi RPA-LF (TevRPA-LF) described in this paper is an interesting alternative because of its simple read-out (user-friendly), short execution time (15 minutes), experimental sensitivity of 100 fg purified genomic T. evansi DNA, and ability to be carried out at a moderate, constant temperature (39°C). Therefore, the TevRPA-LF is an interesting tool for the detection of active T. evansi infections., Author summary Neglected tropical diseases (NTDs) affecting humans and/or domestic animals severely impair the socio-economic development of endemic areas. One of these diseases, animal trypanosomosis, affects livestock and is caused by the parasites of the Trypanosoma genus. The most widespread causative agent of animal trypanosomosis is T. evansi, which is found in large parts of the world (Africa, Asia, South America, Middle East, and the Mediterranean). Proper control and treatment of the disease requires the availability of reliable and sensitive diagnostic tools. DNA-based detection techniques are powerful and versatile in the sense that they can be tailored to achieve a high specificity and usually allow the reliable detection of low amounts of parasite genetic material. However, many DNA-based methodologies (such as PCR) require trained staff and well-equipped laboratories, which is why the research community has actively investigated in developing amplification strategies that are simple, fast, cost-effective and are suitable for use in minimally equipped laboratories and field settings. In this paper, we describe the development of a diagnostic test under a dipstick format for the specific detection of T. evansi, based on a DNA amplification principle (Recombinase Polymerase Amplification aka RPA) that meets the above-mentioned criteria.

Kinetoplastids are the causative agents of leishmaniasis, human African trypanosomiasis, and American trypanosomiasis. They are responsible for high mortality and morbidity in (sub)tropical regions. Adequate treatment options are limited and have several drawbacks, such as toxicity, need for parenteral administration, and occurrence of treatment failure and drug resistance. Therefore, there is an urgency for the development of new drugs. Phenotypic screening already allowed the identification of promising new chemical entities with anti-kinetoplastid activity potential, but knowledge on their mode-of-action (MoA) is lacking due to the generally applied whole-cell based approach. However, identification of the drug target is essential to steer further drug discovery and development. Multiple complementary techniques have indeed been used for MoA elucidation. In this review, the different ‘omics’ approaches employed to define the MoA or mode-of-resistance of current reference drugs and some new anti-kinetoplastid compounds are discussed.

H-NS proteins act as osmotic sensors translating changes in osmolarity into altered DNA binding properties, thus, regulating enterobacterial genome organization and genes transcription. The molecular mechanism underlying the switching process and its conservation among H-NS family members remains elusive.Here, we focus on the H-NS family protein MvaT from P. aeruginosa and demonstrate experimentally that its protomer exists in two different conformations, corresponding to two different functional states. In the half-opened state (dominant at low salt) the protein forms filaments along DNA, in the fully opened state (dominant at high salt) the protein bridges DNA. This switching is a direct effect of ionic strengths on electrostatic interactions between the appositively charged DNA binding and N-terminal domains of MvaT. The asymmetric charge distribution and intramolecular interactions are conserved among the H-NS family of proteins. Therefore, our study establishes a general paradigm for the molecular mechanistic basis of the osmosensitivity of H-NS proteins.

PaaR2 is a putative transcription regulator encoded by a three-component parDE-like toxin-antitoxin module from Escherichia coli O157:H7. Although this module's toxin, antitoxin, and toxin-antitoxin complex have been more thoroughly investigated, little remains known about its transcription regulator PaaR2. Using a wide range of biophysical techniques (circular dichroism spectroscopy, size-exclusion chromatography-multiangle laser light scattering, dynamic light scattering, small-angle x-ray scattering, and native mass spectrometry), we demonstrate that PaaR2 mainly consists of α-helices and displays a concentration-dependent octameric build-up in solution and that this octamer contains a global shape that is significantly nonspherical. Thermal unfolding of PaaR2 is reversible and displays several transitions, suggesting a complex unfolding mechanism. The unfolding data obtained from spectroscopic and calorimetric methods were combined into a unifying thermodynamic model, which suggests a five-state unfolding trajectory. Furthermore, the model allows the calculation of a stability phase diagram, which shows that, under physiological conditions, PaaR2 mainly exists as a dimer that can swiftly oligomerize into an octamer depending on local protein concentrations. These findings, based on a thorough biophysical and thermodynamic analysis of PaaR2, may provide important insights into biological function such as DNA binding and transcriptional regulation., SCOPUS: ar.j, info:eu-repo/semantics/published

Nanobodies (Nbs) are single-domain antibodies with highly desirable properties employed as versatile tools in structural biology, biochemistry and biomedical sciences. Nbs also have a significant potential for the development of highly specific and user-friendly diagnostic assays. These immuno-assays often employ gold nanoparticles (AuNPs) as a detection label. Despite great advances in Nb technology and the immuno-sensor field, the functionalization of AuNPs with Nbs mostly remains unchartered territory. In this paper, we present a study of the functionalization of AuNPs with Nbs through physical adsorption. We show that Nb pI, solution pH, additives and protein engineering strategies have profound effects on the generation of stable Nb-AuNPs. Our results further demonstrate that physical adsorption can be used to produce functional Nb-AuNPs that could be employed in a wide array of applications.

Escherichia coli MazF (EcMazF) is the archetype of a large family of ribonucleases involved in bacterial stress response. The crystal structure of EcMazF in complex with a 7-nucleotide substrate mimic explains the relaxed substrate specificity of the E. coli enzyme relative to its Bacillus subtilis counterpart and provides a framework for rationalizing specificity in this enzyme family. In contrast to a conserved mode of substrate recognition and a conserved active site, regulation of enzymatic activity by the antitoxin EcMazE diverges from its B. subtilis homolog. Central in this regulation is an EcMazE-induced double conformational change as follows: a rearrangement of a crucial active site loop and a relative rotation of the two monomers in the EcMazF dimer. Both are induced by the C-terminal residues Asp-78-Trp-82 of EcMazE, which are also responsible for strong negative cooperativity in EcMazE-EcMazF binding. This situation shows unexpected parallels to the regulation of the F-plasmid CcdB activity by CcdA and further supports a common ancestor despite the different activities of the MazF and CcdB toxins. In addition, we pinpoint the origin of the lack of activity of the E24A point mutant of EcMazF in its inability to support the substrate binding-competent conformation of EcMazF., SCOPUS: ar.j, info:eu-repo/semantics/published

Many bacterial pathogens modulate their metabolic activity, virulence and pathogenicity through so-called "toxin-antitoxin" (TA) modules. The genome of the human pathogen Escherichia coli O157 contains two three-component TA modules related to the known parDE module. Here, we show that the toxin EcParE2 maps in a branch of the RelE/ParE toxin superfamily that is distinct from the branches that contain verified gyrase and ribosome inhibitors. The structure of EcParE2 closely resembles that of Caulobacter crescentus ParE but shows a distinct pattern of conserved surface residues, in agreement with its apparent inability to interact with GyrA. The antitoxin EcPaaA2 is characterized by two α-helices (H1 and H2) that serve as molecular recognition elements to wrap itself around EcParE2. Both EcPaaA2 H1 and H2 are required to sustain a high-affinity interaction with EcParE2 and for the inhibition of EcParE2-mediated killing in vivo. Furthermore, evidence demonstrates that EcPaaA2 H2, but not H1, determines specificity for EcParE2. The initially formed EcPaaA2-EcParE2 heterodimer then assembles into a hetero-hexadecamer, which is stable in solution and is formed in a highly cooperative manner. Together these findings provide novel data on quaternary structure, TA interactions and activity of a hitherto poorly characterized family of TA modules.

Trypanosoma are blood-borne parasites and are the causative agents of neglected tropical diseases (NTDs) affecting both humans and animals. These parasites mainly rely on glycolysis for their energy production within the mammalian host, which is why trypanosomal glycolytic enzymes have been pursued as interesting targets for the development of trypanocidal drugs. The structure–function relationships of pyruvate kinases (PYKs) from trypanosomatids (Trypanosoma and Leishmania) have been well-studied within this context. In this paper, we describe the structural and enzymatic characterization of PYK from T. congolense (TcoPYK), the main causative agent of Animal African Trypanosomosis (AAT), by employing a combination of enzymatic assays, thermal unfolding studies and X-ray crystallography.

Toxin-antitoxin (TA) modules are stress response elements that are ubiquitous in the genomes of bacteria and archaea. Production and subsequent purification of individual TA proteins is anything but straightforward as over-expression of the toxin gene is lethal to bacterial and eukaryotic cells and over-production of the antitoxin leads to its proteolytic degradation because of its inherently unstructured nature. Here we describe an effective production and purification strategy centered on an on-column denaturant-induced dissociation of the toxin-antitoxin complex. The success of the method is demonstrated by its application on four different TA families, encoding proteins with distinct activities and folds. A series of biophysical and in vitro activity tests show that the purified proteins are of high quality and suitable for structural studies.

Sepsis-leading to septic shock-is the leading cause of death in intensive care units. The systemic inflammatory response to infection, which is initiated by activated myeloid cells, plays a key role in the lethal outcome. Macrophage migration inhibitory factor (MIF) is an upstream immunoregulatory mediator, released by myeloid cells, that underlies a common genetic susceptibility to different infections and septic shock. Accordingly, strategies that are aimed at inhibiting the action of MIF have therapeutic potential. Here, we report the isolation and characterization of tailorable, small, affinity-matured nanobodies (Nbs; single-domain antigen-binding fragments derived from camelid heavy-chain Abs) directed against MIF. Of importance, these bioengineered Nbs bind both human and mouse MIFs with nanomolar affinity. NbE5 and NbE10 inhibit key MIF functions that can exacerbate septic shock, such as the tautomerase activity of MIF (by blocking catalytic pocket residues that are critical for MIF's conformation and receptor binding), the TNF-inducing potential, and the ability of MIF to antagonize glucocorticoid action. A lead NbE10, tailored to be a multivalent, half-life extended construct (NbE10-NbAlb8-NbE10), attenuated lethality in murine endotoxemia when administered via single injection, either prophylactically or therapeutically. Hence, Nbs, with their structural and pharmacologic advantages over currently available inhibitors, may be an effective, novel approach to interfere with the action of MIF in septic shock and other conditions of inflammatory end-organ damage.-Sparkes, A., De Baetselier, P., Brys, L., Cabrito, I., Sterckx, Y. G.-J., Schoonooghe, S., Muyldermans, S., Raes, G., Bucala, R., Vanlandschoot, P., Van Ginderachter, J. A., Stijlemans, B. Novel half-life extended anti-MIF nanobodies protect against endotoxic shock.

[Background] Variable domains of camelid heavy-chain antibodies, commonly named nanobodies, have highbiotechnological potential. In view of their broad range of applications in research, diagnostics and therapy,engineering their stability is of particular interest. One important aspect is the improvement of thermostability,because it can have immediate effects on conformational stability, protease resistance and aggregation pro-pensity of the protein, [Methods] We analyzed the sequences and thermostabilities of 78 purified nanobody binders. From this data,potentially stabilizing amino acid variations were identified and studied experimentally.Results:Some mutations improved the stability of nanobodies by up to 6.1 °C, with an average of 2.3 °C acrosseight modified nanobodies. The stabilizing mechanism involves an improvement of both conformational stabilityand aggregation behavior, explaining the variable degree of stabilization in individual molecules. In some in-stances, variations predicted to be stabilizing actually led to thermal destabilization of the proteins. The reasonsfor this contradiction between prediction and experiment were investigated., [Conclusions] The results reveal a mutational strategy to improve the biophysical behavior of nanobody bindersand indicate a species-specificity of nanobody architecture, [General significance] This study illustrates the potential and limitations of engineering nanobody thermostabilityby merging sequence information with stability data, an aspect that is becoming increasingly important with therecent development of high-throughput biophysical methods, We thank NanoTemper Technologies in Munich for its generous support with free DSF measurements. Funding by the European Union(grantnumber: Health-F4-2010-241481) as part of the Affinomics consortium is gratefully acknowledged.

Bacteria can become transiently tolerant to several classes of antibiotics. This phenomenon known as persistence is regulated by small genetic elements called toxin-antitoxin modules with intricate yet often poorly understood self-regulatory features. Here, we describe the structures of molecular complexes and interactions that drive the transcription regulation of the ccdAB toxin-antitoxin module. Low specificity and affinity of the antitoxin CcdA2 for individual binding sites on the operator are enhanced by the toxin CcdB2, which bridges the CcdA2 dimers. This results in a unique extended repressing complex that spirals around the operator and presents equally spaced DNA binding sites. The multivalency of binding sites induces a digital on-off switch for transcription, regulated by the toxin:antitoxin ratio. The ratio at which this switch occurs is modulated by non-specific interactions with the excess chromosomal DNA. Altogether, we present the molecular mechanisms underlying the ratio-dependent transcriptional regulation of the ccdAB operon. ispartof: NUCLEIC ACIDS RESEARCH vol:45 issue:6 pages:2937-2950 ispartof: location:England status: published

Nanobodies (Nbs) are the smallest antigen-binding, single domain fragments derived from heavy-chain-only antibodies from Camelidae. Among the several advantages over conventional monoclonal antibodies, their small size (12−15 kDa) allows them to extravasate rapidly, to show improved tissue penetration, and to clear rapidly from blood, which are important characteristics for cancer imaging and targeted radiotherapy. Herein, we identified Nbs against CD33, a marker for acute myeloid leukemia (AML). A total of 12 Nbs were generated against recombinant CD33 protein, out of which six bound natively CD33 protein, expressed on the surface of acute myeloid leukemia THP-1 cells. The equilibrium dissociation constants (KD) of these six Nbs and CD33 range from 4 to 270 nM, and their melting temperature (Tm) varies between 52.67 and 67.80 °C. None of these Nbs showed leukemogenicity activity in vitro. The selected six candidates were radiolabeled with 99mTc, and their biodistribution was evaluated in THP-1-tumor-bearing mice. The imaging results demonstrated the fast tumor-targeting capacity of the Nbs in vivo. Among the anti-CD33 Nbs, Nb_7 showed the highest tumor uptake (2.53 ± 0.69 % injected activity per gram (IA/g), with low background signal, except in the kidneys and bladder. Overall, Nb_7 exhibits the best characteristics to be used as an anti-CD33 targeting vehicle for future diagnostic or therapeutic applications.

ThepaaR2–paaA2–parE2operon is a three-component toxin–antitoxin module encoded in the genome of the human pathogenEscherichia coliO157. The toxin (ParE2) and antitoxin (PaaA2) interact to form a nontoxic toxin–antitoxin complex. In this paper, the crystallization and preliminary characterization of two variants of the ParE2–PaaA2 toxin–antitoxin complex are described. Selenomethionine-derivative crystals of the full-length ParE2–PaaA2 toxin–antitoxin complex diffracted to 2.8 Å resolution and belonged to space groupP41212 (orP43212), with unit-cell parametersa=b= 90.5,c= 412.3 Å. It was previously reported that the full-length ParE2–PaaA2 toxin–antitoxin complex forms a higher-order oligomer. In contrast, ParE2 and PaaA213–63, a truncated form of PaaA2 in which the first 12 N-terminal residues of the antitoxin have been deleted, form a heterodimer as shown by analytical gel filtration, dynamic light scattering and small-angle X-ray scattering. Crystals of the PaaA213–63–ParE2 complex diffracted to 2.7 Å resolution and belonged to space groupP6122 (orP6522), with unit-cell parametersa=b= 91.6,c= 185.6 Å.

Uropathogenic Escherichia coli cause urinary tract infections by adhering to mannosylated receptors on the human urothelium via the carbohydrate-binding domain of the FimH adhesin (FimHL). Numerous α-d-mannopyranosides, including α-d-heptyl mannose (HM), inhibit this process by interacting with FimHL. To establish the molecular basis of the high-affinity HM binding, we solved the solution structure of the apo form and the crystal structure of the FimHL-HM complex. NMR relaxation analysis revealed that protein dynamics were not affected by the sugar binding, yet HM addition promoted protein dimerization, which was further confirmed by small-angle X-ray scattering. Finally, to address the role of Y48, part of the "tyrosine gate" believed to govern the affinity and specificity of mannoside binding, we characterized the FimHL Y48A mutant, whose conformational, dynamical, and HM binding properties were found to be very similar to those of the wild-type protein.

Background Animal African trypanosomosis (AAT) is a neglected tropical disease which imposes a heavy burden on the livestock industry in Sub-Saharan Africa. Its causative agents are Trypanosoma parasites, with T. congolense and T. vivax being responsible for the majority of the cases. Recently, we identified a Nanobody (Nb474) that was employed to develop a homologous sandwich ELISA targeting T. congolense fructose-1,6-bisphosphate aldolase (TcoALD). Despite the high sequence identity between trypanosomatid aldolases, the Nb474-based immunoassay is highly specific for T. congolense detection. The results presented in this paper yield insights into the molecular principles underlying the assay’s high specificity. Methodology/Principal findings The structure of the Nb474-TcoALD complex was determined via X-ray crystallography. Together with analytical gel filtration, the structure reveals that a single TcoALD tetramer contains four binding sites for Nb474. Through a comparison with the crystal structures of two other trypanosomatid aldolases, TcoALD residues Ala77 and Leu106 were identified as hot spots for specificity. Via ELISA and surface plasmon resonance (SPR), we demonstrate that mutation of these residues does not abolish TcoALD recognition by Nb474, but does lead to a lack of detection in the Nb474-based homologous sandwich immunoassay. Conclusions/Significance The results show that the high specificity of the Nb474-based immunoassay is not determined by the initial recognition event between Nb474 and TcoALD, but rather by its homologous sandwich design. This (i) provides insights into the optimal set-up of the assay, (ii) may be of great significance for field applications as it could explain the potential detection escape of certain T. congolense strains, and (iii) may be of general interest to those developing similar assays., Author summary Sub-Saharan Africa is plagued by many diseases, which impede its socio-economical development. One of these diseases, Animal African Trypanosomosis, affects livestock and is caused by the parasites of the Trypanosoma genus (T. vivax and T. congolense). Animal African Trypanosomosis leads to considerable economic losses and renders sustainable livestock industry in Sub-Saharan Africa very difficult. In order to proceed with the selective treatment of infected animals, they need to be properly diagnosed. We recently described the use of an assay to specifically detect T. congolense infections in both experimentally and naturally infected animals. The diagnostic assay employs a Nanobody (Nb), which is the smallest antigen-binding unit derived from camelid heavy-chain only antibodies. Our previous results showed that the Nb-based diagnostic test specifically recognizes T. congolense fructose-1,6-bisphosphate aldolase, a glycolytic enzyme that is well conserved amongst other Trypanosoma species. In this paper, we studied the molecular mechanisms underlying the high specificity of the Nb-based diagnostic assay. The principles derived from this work may be important for the design and improvement of similar diagnostic tests.

Background Infectious diseases pose a severe worldwide threat to human and livestock health. While early diagnosis could enable prompt preventive interventions, the majority of diseases are found in rural settings where basic laboratory facilities are scarce. Under such field conditions, point-of-care immunoassays provide an appropriate solution for rapid and reliable diagnosis. The limiting steps in the development of the assay are the identification of a suitable target antigen and the selection of appropriate high affinity capture and detection antibodies. To meet these challenges, we describe the development of a Nanobody (Nb)-based antigen detection assay generated from a Nb library directed against the soluble proteome of an infectious agent. In this study, Trypanosoma congolense was chosen as a model system. Methodology/Principal Findings An alpaca was vaccinated with whole-parasite soluble proteome to generate a Nb library from which the most potent T. congolense specific Nb sandwich immunoassay (Nb474H-Nb474B) was selected. First, the Nb474-homologous sandwich ELISA (Nb474-ELISA) was shown to detect experimental infections with high Positive Predictive Value (98%), Sensitivity (87%) and Specificity (94%). Second, it was demonstrated under experimental conditions that the assay serves as test-of-cure after Berenil treatment. Finally, this assay allowed target antigen identification. The latter was independently purified through immuno-capturing from (i) T. congolense soluble proteome, (ii) T. congolense secretome preparation and (iii) sera of T. congolense infected mice. Subsequent mass spectrometry analysis identified the target as T. congolense glycosomal aldolase. Conclusions/Significance The results show that glycosomal aldolase is a candidate biomarker for active T. congolense infections. In addition, and by proof-of-principle, the data demonstrate that the Nb strategy devised here offers a unique approach to both diagnostic development and target discovery that could be widely applied to other infectious diseases., Author Summary The lack of diagnostic tests that are sensitive, affordable and user-friendly is the impediment to early detection and containment of infectious diseases. For these reasons, African trypanosomosis continues to pose serious threat to the communities that are unable to access laboratory services. Assays that are able to address above-mentioned challenges would greatly reduce the disease burden. Although few relatively sensitive agglutination assays for some forms of African trypanosomosis have been adapted to field conditions, the tests are not reliable indicators of active infections given the fact that they only detect antibodies. The barrier in the development of alternative tests capable of revealing ongoing infections through antigen detection is the lack of potent monoclonal antibodies that can compete with infection-induced host antibodies for the circulating parasite antigens. Using T. congolense as a model system, we demonstrate that Nanobodies (Nbs) targeting the parasite glycosomal aldolase can detect active infections. The strategy described addresses the technical shortcoming of conventional monoclonal antibody-based assay development by adopting an unbiased proteome screening approach combined with a phage panning strategy that is adapted to avoid interference of the infection-induced host antibody response. Hence, this study shows prospect for future development of Nb-based tests for other infectious diseases.

Toxin-antitoxin (TA) modules are small operons associated with stress response of bacteria. F-plasmid CcdB(F) was the first TA toxin for which its target, gyrase, was identified. Plasmidic and chromosomal CcdBs belong to distinct families. Conserved residues crucial for gyrase poisoning activity of plasmidic CcdBs are not conserved among these families. Here we show that the chromosomal CcdB(Vfi) from Vibrio fischeri is an active gyrase poison that interacts with its target via an alternative energetic mechanism. Changes in the GyrA14-binding surface of the Vibrio and F-plasmid CcdB family members illustrate neutral drift where alternative interactions can be used to achieve the same functionality. Differences in affinity between V. fischeri and F-plasmid CcdB for gyrase and their corresponding CcdA antitoxin possibly reflect distinct roles for TA modules located on plasmids and chromosomes.

DNA gyrase is an essential bacterial enzyme required for the maintenance of chromosomal DNA topology. This enzyme is the target of several protein toxins encoded in toxin-antitoxin (TA) loci as well as of man-made antibiotics such as quinolones. The genome of Vibrio cholerae, the cause of cholera, contains three putative TA loci that exhibit modest similarity to the RK2 plasmid-borne parDE TA locus, which is thought to target gyrase although its mechanism of action is uncharacterized. Here we investigated the V. cholerae parDE2 locus. We found that this locus encodes a functional proteic TA pair that is active in Escherichia coli as well as V. cholerae. ParD2 co-purified with ParE2 and interacted with it directly. Unlike many other antitoxins, ParD2 could prevent but not reverse ParE2 toxicity. ParE2, like the unrelated F-encoded toxin CcdB and quinolones, targeted the GyrA subunit and stalled the DNA-gyrase cleavage complex. However, in contrast to other gyrase poisons, ParE2 toxicity required ATP, and it interfered with gyrase-dependent DNA supercoiling but not DNA relaxation. ParE2 did not bind GyrA fragments bound by CcdB and quinolones, and a set of strains resistant to a variety of known gyrase inhibitors all exhibited sensitivity to ParE2. Together, our findings suggest that ParE2 and presumably its many plasmid- and chromosome-encoded homologues inhibit gyrase in a different manner than previously described agents.

Type II toxin-antitoxin (TA) systems are considered as protein pairs in which a specific toxin is associated with a specific antitoxin. We have identified a novel antitoxin family (paaA) that is associated with parE toxins. The paaA-parE gene pairs form an operon with a third component (paaR) encoding a transcriptional regulator. Two paralogous paaR-paaA-parE systems are found in E. coli O157:H7. Deletions of the paaA-parE pairs in O157:H7 allowed us to show that these systems are expressed in their natural host and that PaaA antitoxins specifically counteract toxicity of their associated ParE toxin. For the paaR2-paaA2-parE2 system, PaaR2 and Paa2-ParE2 complex are able to regulate the operon expression and both are necessary to ensure complete repression. The paaR2-paaA2-parE2 system mediates ClpXP-dependent post-segregational killing. The PaaR2 regulator appears to be essential for this function, most likely by maintaining an appropriate antitoxin : toxin ratio in steady-state conditions. Ectopic overexpression of ParE2 is bactericidal and is not resuscitated by PaaA2 expression. ParE2 colocalizes with the nucleoid, while it is diffusely distributed in the cytoplasm when PaaA2 is coexpressed. This indicates that ParE2 interacts with DNA-gyrase cycling on DNA and that coexpression of PaaA2 antitoxin sequesters ParE2 away from its target by protein-protein complex formation.

Background The malaria vaccine candidate RTS,S/AS01 (GSK Vaccines) induces high IgG concentration against the circumsporozoite protein (CSP) of Plasmodium falciparum. In human vaccine recipients circulating anti-CSP antibody concentrations are associated with protection against infection but appear not to be the correlate of protection. However, in a humanized mouse model of malaria infection prophylactic administration of a human monoclonal antibody (MAL1C), derived from a RTS,S/AS01-immunized volunteer, directed against the CSP repeat region, conveyed full protection in a dose-dependent manner suggesting that antibodies alone are able to prevent P. falciparum infection when present in sufficiently high concentrations. A competition ELISA was developed to measure the presence of MAL1C-like antibodies in polyclonal sera from RTS,S/AS01 vaccine recipients and study their possible contribution to protection against infection. Results MAL1C-like antibodies present in polyclonal vaccine-induced sera were evaluated for their ability to compete with biotinylated monoclonal antibody MAL1C for binding sites on the capture antigen consisting of the recombinant protein encompassing 32 NANP repeats of CSP (R32LR). Serum samples were taken at different time points from participants in two RTS,S/AS01 vaccine studies (NCT01366534 and NCT01857869). Vaccine-induced protection status of the study participants was determined based on the outcome of experimental challenge with infected mosquito bites after vaccination. Optimal conditions were established to reliably detect MAL1C-like antibodies in polyclonal sera. Polyclonal anti-CSP antibodies and MAL1C-like antibody content were measured in 276 serum samples from RTS,S/AS01 vaccine recipients using the standard ELISA and MAL-1C competition ELISA, respectively. A strong correlation was observed between the results from these assays. However, no correlation was found between the results of either assay and protection against infection. Conclusions The competition ELISA to measure MAL1C-like antibodies in polyclonal sera from RTS,S/AS01 vaccine recipients was robust and reliable but did not reveal the elusive correlate of protection. Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1596-8) contains supplementary material, which is available to authorized users.

The complex of yeast cytochrome c peroxidase and cytochrome c is a paradigm of the biological electron transfer (ET). Building on seven decades of research, two different models have been proposed to explain its functional redox activity. One postulates that the intermolecular ET occurs only in the dominant, high-affinity protein–protein orientation, while the other posits formation of an additional, low-affinity complex, which is much more active than the dominant one. Unlike the high-affinity interaction—extensively studied by X-ray crystallography and NMR spectroscopy—until now the binding of cytochrome c to the low-affinity site has not been observed directly, but inferred mainly from kinetics experiments. Here we report the structure of this elusive, weak protein complex and show that it consists of a dominant, inactive bound species and an ensemble of minor, ET-competent protein–protein orientations, which summarily account for the experimentally determined value of the ET rate constant., The redox activity of cytochrome c in complex with its peroxidase has been rationalized by two possible models; a readily observable high-affinity complex and a more elusive but potentially more active low-affinity complex. Here, the authors provide an NMR-based structural mapping of this low-affinity complex.

oxin-antitoxin (TA) modules are pairs of genes essential for bacterial regulation upon environmental stresses. The mazEF module encodes the MazF toxin and its cognate MazE antitoxin. The highly dynamic MazE possesses an N-terminal DNA binding domain through which it can negatively regulate its own promoter. Despite being one of the first TA systems studied, transcriptional regulation of Escherichia coli mazEF remains poorly understood. This paper presents the solution structure of C-terminal truncated E. coli MazE and a MazE-DNA model with a DNA palindrome sequence about 10 bp upstream of the mazEF promoter. The work has led to a transcription regulator-DNA model, which has remained elusive thus far in the E. coli toxin-antitoxin family. Multiple complementary techniques including NMR, SAXS and ITC show that the long intrinsically disordered C-termini in MazE, required for MazF neutralization, does not affect the interactions between the antitoxin and its operator. Rather, the MazE C-terminus plays an important role in the MazF binding, which was found to increase the MazE affinity for the palindromic single site operator.

Yeast cytochrome c peroxidase (CcP) is a heme enzyme that reduces hydroperoxides using the electrons provided by its physiological partner cytochrome c (Cc). Contributing to the resistance against the oxidative stress associated with the aerobic metabolism, the Cc-CcP complex has been widely studied and became a paradigm for biological electron transfer. The heme-free, enzymatically inactive apo CcP is the natural precursor of the mature, cofactor-bound holo protein. Despite its physiological relevance, apo CcP is not well characterized, and at present, little is known about its structure or the interaction with Cc. Using a range of biophysical techniques, here we show that, while holo CcP binds Cc with micromolar affinity, the interaction between apo CcP and Cc is completely abolished. Characterized by small-angle X-ray scattering, solution nuclear magnetic resonance spectroscopy, and equilibrium unfolding experiments, apo and holo CcP exhibit very similar structural, hydrodynamic, and thermodynamic properties. However, detailed analysis reveals that apo CcP is more expanded in solution, displays a number of characteristics associated with a molten globule state, and, unlike the holo protein, does not form an unfolding intermediate during thermal and chemical denaturation. Overall, our data suggest that the Cc binding site present in the holo protein is disrupted in the apo form, explaining the inability of the latter to interact with Cc. We argue that the observed difference in Cc binding is physiologically relevant and suggest why abolishing the apo CcP-Cc interaction is beneficial to the organism.

International audience; Antitoxins from prokaryotic type II toxin-antitoxin modules are characterized by a high degree of intrinsic disorder. The description of such highly flexible proteins is challenging because they cannot be represented by a single structure. Here, we present a combination of SAXS and NMR data to describe the conformational ensemble of the PaaA2 antitoxin from the human pathogen E. coli O157. The method encompasses the use of SAXS data to filter ensembles out of a pool of conformers generated by a custom NMR structure calculation protocol and the subsequent refinement by a block jackknife procedure. The final ensemble obtained through the method is validated by an established residual dipolar coupling analysis. We show that the conformational ensemble of PaaA2 is highly compact and that the protein exists in solution as two preformed helices, connected by a flexible linker, that probably act as molecular recognition elements for toxin inhibition.

Escherichia coli O157 paaR2-paaA2-parE2 constitutes a unique three-component toxin-antitoxin (TA) module encoding a toxin (ParE2) related to the classic parDE family but with an unrelated antitoxin called PaaA2. The complex between PaaA2 and ParE2 was purified and characterized by analytical gel filtration, dynamic light scattering and small-angle X-ray scattering. It consists of a particle with a radius of gyration of 3.95 nm and is likely to form a heterododecamer. Crystals of the ParE2-PaaA2 complex diffract to 3.8 Å resolution and belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 142.9, c = 87.5 Å. The asymmetric unit is consistent with a particle of around 125 kDa, which is compatible with the solution data. Therefore, the ParE2-PaaA2 complex is the largest toxin-antitoxin complex identified to date and its quaternary arrangement is likely to be of biological significance.

The phd/doc family is one the smallest families of toxin-antitoxin modules and was first discovered as a plasmid addiction module on E. coli bacteriophage P1. The toxin Doc interacts with the ribosome, competes with hygromycin and inhibits translation. Structurally, Doc resembles Fic domains, which are known to transfer an AMP moiety to the hydroxyphenyl group of a tyrosine in the target protein. Although much of the AMP/ATP binding site of Fic is conserved in Doc, no specific enzymatic activity has yet been linked to Doc. Nevertheless, mutations in the loop corresponding to the active site loop of Fic render Doc inactive. Regulation of the P1 phd/doc operon involves conditional co-operativity and is understood in terms of a detailed molecular mechanism. The N-terminal domain of Phd forms the DNA binding unit and represents a common DNA binding fold that is also shared with a number of antitoxins from different TA families, among which YefM is the best studied. Enhancement of the DNA binding affinity of Phd by Doc stems both from allosteric coupling between the Doc- and DNA-binding sites on Phd and from an avidity affect due to Doc-mediated bridging of two Phd dimers bound to the operator site. Activation of the system at high Doc to Phd ratios stems from a low-to-high affinity switch in the interaction between Phd and Doc.

The antitoxin Phd from the phd/doc module of bacteriophage P1 was crystallized in two distinct crystal forms. Crystals of His-tagged Phd contain a C-terminally truncated version of the protein and diffract to 2.20 A resolution. Crystals of untagged Phd purified from the Phd-Doc complex diffract to 2.25 A resolution. These crystals are partially merohedrally twinned and contain the full-length version of the protein.