In Staphylococcus aureus, the intracellular siderophore staphyloferrin B, which has been shown to chelate iron-bound to serum transferrin, is transported into cells by the SirABC system. In this ...work, we have analysed the role of the Sir transporter under stress conditions that resemble those imposed by the mammalian innate immune system. We show that exposure of S. aureus to oxidative and nitrosative stress generated by hydrogen peroxide and S-nitrosoglutathione, respectively, induced the expression of the sirA gene. The disruption of the sir operon led to a strain with lower viability and decreased resistance to oxidative stress. S. aureus sir null mutant was also analysed during infection of murine macrophages and shown to contribute to S. aureus survival inside macrophages. Altogether, our results indicate that the Sir transport system confers protection against reactive oxygen species, therefore, contributing to the virulence of S. aureus.
•High quality and functional human recombinant DLL1 proteins were generated.•Specific high affinity anti-DLL1 antibodies were identified by phage display.•Anti-DLL1 IgG-69 impairs DLL1-induced Notch ...signalling and growth in MCF-7 cells.•Anti-DLL1 IgG-69 reduces breast cancer stem cell subpopulation of MCF-7 cells.
Notch signalling is a well-established oncogenic pathway, and its ligand Delta-like 1 (DLL1) is overexpressed in estrogen receptor-positive (ER+) breast cancers and associated with poor patient prognosis. Hence, DLL1 has become an interesting therapeutic target for breast cancer. Here, the development of specific functional blocking anti-DLL1 antibodies with potential activity against ER+ breast cancer cells is reported. Human DLL1 proteins, containing the essential regions for binding to the Notch receptor and Notch signalling activation, were produced and used to select specific scFv antibody fragments by phage display. Fifteen unique scFvs were identified and reformatted into full IgGs. Characterization of these antibodies by ELISA, surface plasmon resonance and flow cytometry enabled selection of three specific anti-DLL1 IgGs, sharing identical VH regions, with nM affinities. Cellular assays on ER+ breast cancer MCF-7 cells showed that one of the IgGs (IgG-69) was able to partially impair DLL1-mediated activation of the Notch pathway, as determined by Notch reporter and RT-qPCR assays, and to attenuate cell growth. Treatment of MCF-7 cells with IgG-69 reduced mammosphere formation, suggesting that it decreases the breast cancer stem cell subpopulation. These results support the use of this strategy to develop and identify potential anti-DLL1 antibodies candidates against breast cancer.
RICs are a family of bacterial proteins involved in the repair of iron centers containing proteins damaged by the antimicrobial reactive species liberated by the innate immune system of infected ...hosts. Staphylococcus aureus is a human pathogen with increasing antibiotic resistance that also contains a RIC-like protein. In this work, we show that the survival of S. aureus within macrophages decreases upon inactivation of ric, and that the viability was restored to levels similar to the wild-type strain by reintroduction of ric via in trans complementation. Importantly, in macrophages that do not produce reactive oxygen species, the lower survival of the ric mutant was no longer observed. In lung epithelial cells, the intracellular viability of the S. aureus ric mutant was also shown to be lower than that of the wild-type. The wax moth larvae Galleria mellonella infected with S. aureus ric mutant presented an approximately 2.5-times higher survival when compared to the wild-type strain. Moreover, significantly lower bacterial loads were determined in the larvae hemolymph infected with strains not expressing ric, and complementation assays confirmed that this behavior was related to RIC. Furthermore, expression of the S. aureus ric gene within the larvae increased along the course of infection with a ~20-fold increase after 8 h of infection. Altogether, the data show that RIC is important for the virulence of S. aureus.
•Structural model of the E. coli RIC diiron centre is presented.•Conserved residues H84, H129, H160, H204, E133 and E208 are ligands of the diiron centre.•Residues E133 and E208 form two ...μ-carboxylate bridges essential for RIC assembly.
Repair of Iron Centres (RICs) are a widely-spread family of diiron proteins involved in the protection of iron–sulphur-containing enzymes from nitrosative and oxidative stress. Here, homology-based modelling was used to predict putative ligands of the RIC diiron centre in E. coli. Site-directed mutagenesis studies showed that several conserved residues modulate the spectroscopic properties of the diiron centre, and mutations in H129, E133 and E208 abrogated RIC ability to protect aconitase. Taken together, these data led to a structural model of a diiron centre inserted in a four-helix bundle fold and coordinated by H84, H129, H160, H204, E133 and E208. Moreover, two μ-carboxylate bridges involving E133 and E208 were found to be required for assembly of a stable diiron centre.
Trichomonas vaginalis, the causative parasite of one of the most prevalent sexually transmitted diseases is, so far, the only protozoan encoding two putative Repair of Iron Centres (RIC) proteins. ...Homologs of these proteins have been shown to protect bacteria from the chemical stress imposed by mammalian immunity. In this work, the biochemical and functional characterisation of the T. vaginalis RICs revealed that the two proteins have different properties. Expression of ric1 is induced by nitrosative stress but not by hydrogen peroxide, while ric2 transcription remained unaltered under similar conditions. T. vaginalis RIC1 contains a di-iron centre, but RIC2 apparently does not. Only RIC1 resembles bacterial RICs on spectroscopic profiling and repairing ability of oxidatively-damaged iron-sulfur clusters. Unexpectedly, RIC2 was found to bind DNA plasmid and T. vaginalis genomic DNA, a function proposed to be related with its leucine zipper domain. The two proteins also differ in their cellular localization: RIC1 is expressed in the cytoplasm only, and RIC2 occurs both in the nucleus and cytoplasm. Therefore, we concluded that the two RIC paralogs have different roles in T. vaginalis, with RIC2 showing an unprecedented DNA binding ability when compared with all other until now studied RICs.
Flavohemoglobins and flavodiiron proteins are two families of enzymes involved in nitrosative detoxification. However, the physiological oxygen-related conditions under which they work and their ...relative role are still a matter of debate. To address this question we analyzed the function of the putative flavohemoprotein of
Staphylococcus aureus, an organism that lacks a flavodiiron-like gene. In this report we show that the recombinant protein contains all features typical of canonical flavohemoglobins and that the transcription of flavohemoglobin gene was upregulated by nitrosative stress in an oxygen-dependent manner. However, and in contrast to other bacterial flavohemoglobins, the
S.
aureus protein has no apparent role in aerobic nitrosative protection, being only beneficial when cells of
S.
aureus are submitted to nitrosative stress in a microaerophilic environment. The in vivo data corroborates the proposal that Hmp acts physiologically as a denitrosylase.
Serological assays are valuable tools to study SARS‐CoV‐2 spread and, importantly, to identify individuals that were already infected and would be potentially immune to a virus reinfection. ...SARS‐CoV‐2 Spike protein and its receptor binding domain (RBD) are the antigens with higher potential to develop SARS‐CoV‐2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the development of COVID‐19 therapeutics. Thus, it is urgent that significant amounts of this protein became available at the highest quality. In this study, we produced Spike and RBD in two human derived cell hosts: HEK293‐E6 and Expi293F™. We evaluated the impact of different and scalable bioprocessing approaches on Spike and RBD production yields and, more importantly, on these antigens' quality attributes. Using negative and positive sera collected from human donors, we show an excellent performance of the produced antigens, assessed in serologic enzyme‐linked immunosorbent assay (ELISA) tests, as denoted by the high specificity and sensitivity of the test. We show robust Spike productions with final yields of approx. 2 mg/L of culture that were maintained independently of the production scale or cell culture strategy. To the best of our knowledge, the final yield of 90 mg/L of culture obtained for RBD production, was the highest reported to date. An in‐depth characterization of SARS‐CoV‐2 Spike and RBD proteins was performed, namely the antigen's oligomeric state, glycosylation profiles, and thermal stability during storage. The correlation of these quality attributes with ELISA performance show equivalent reactivity to SARS‐CoV‐2 positive serum, for all Spike and RBD produced, and for all storage conditions tested. Overall, we provide straightforward protocols to produce high‐quality SARS‐CoV‐2 Spike and RBD antigens, that can be easily adapted to both academic and industrial settings; and integrate, for the first time, studies on the impact of bioprocess with an in‐depth characterization of these proteins, correlating antigen's glycosylation and biophysical attributes to performance of COVID‐19 serologic tests.
Spike and RBD were produced in human derived cells hosts. Shake flasks, stirred tank and wave bioreactors were compared. The impact of these different scalable bioprocessing approaches on Spike and RBD production yields and quality attributes was evaluated. An in‐depth characterization of SARS‐CoV‐2 Spike and RBD proteins was performed, namely the antigen s oligomeric state, glycosylation profiles and thermal stability during storage. The correlation of these quality attributes with ELISA performance is discussed.
Intracellular free iron, is under aerobic conditions and via the Fenton reaction a catalyst for the formation of harmful reactive oxygen species. In this article, we analyzed the relation between ...intracellular iron storage and oxidative stress response in the sulfate reducing bacterium Desulfovibrio vulgaris Hildenborough, an anaerobe that is often found in oxygenated niches. To this end, we investigated the role of the iron storage protein bacterioferritin using transcriptomic and physiological approaches. We observed that transcription of bacterioferritin is strongly induced upon exposure of cells to an oxygenated atmosphere. When grown in the presence of high concentrations of oxygen the D. vulgaris bacterioferritin mutant exhibited, in comparison with the wild type strain, lower viability and a higher content of intracellular reactive oxygen species. Furthermore, the bacterioferritin gene is under the control of the oxidative stress response regulator D. vulgaris PerR. Altogether the data revealed a previously unrecognized ability for the iron storage bacterioferritin to contribute to the oxygen tolerance exhibited by D. vulgaris.
► The bacterioferritin gene is induced by oxygen. ► Deletion of bacterioferritin makes Desulfovibrio vulgaris less resistant to oxygen. ► The bacterioferritin mutant has a higher content of reactive oxygen species. ► The transcription of bacterioferritin gene is controlled by PerR.
Biotically, bacteria encounter nitrogen-reactive species in environments where denitrification occurs or when nitric oxide (NO) is generated by the mammal NO synthase, particularly during the ...infectious processes. In bacteria, flavohemoglobins have been shown to be one of the major systems responsible for the scavenging of these chemical species, either in aerobic or in anaerobic environments. Staphylococcus aureus, a pathogenic bacterium with high impact on human heath, also contains a gene encoding a homologue of a flavohemoprotein. The study of the recombinant protein and of the knockout mutant strain allowed us to conclude that, in spite of sharing similar physicochemical properties with the other known flavohemoglobins, the S. aureus flavohemoglobin requires microaerobic conditions to protect this bacterium from the nocive effects of the nitrosative stress.
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