Non-toxic Mutant Research Articles

Bioinformatic Analysis of the Structural Features of the H106P/N137A Mutant as a Potential Vaccine Candidate Against Clostridium perfringens.

The complex and expensive processes involved in the production of current clostridial toxoid vaccines require the design of new methods. The aim of this study was to introduce single and combined nontoxic mutants of Clostridium perfringens epsilon toxin (ETX) into the vaccine production. Antigenic properties, amino acid sequence, physicochemical characteristics, stability, and protein structure of individual and combined ETX mutants were investigated with bioinformatics software. Spatial structure prediction was done by using a homologous modeling method based on the structure of wild type ETX (1UYJ1A.pdb) and the validity of the drawn spatial model was evaluated by estimating the quality and accuracy of the spatial chemistry of the modeled mutant proteins by Ramachandran plot. The highest instability index was observed in H106P/N137A mutant. The results of the homology modeling did not show a clear structural change in any of the mutants compared to the wild ETX. The percentages of amino acids in the favored regions, allowed regions, and outlier regions were 92.81%, 5.03%, and 2.16%, respectively, which indicate the desirability of the proposed model for the three-dimensional structure of the H106P/N137A hybrid mutant. Recombinant H106P/N137A mutant of ETX can be considered a suitable candidate for the production of epsilon genetic toxoid, and this requires extensive laboratory evaluations.

Microcystin aids in cold temperature acclimation: Differences between a toxic Microcystis wildtype and non-toxic mutant

For Microcystis aeruginosa PCC 7806, temperature decreases from 26 °C to 19 °C double the microcystin quota per cell during growth in continuous culture. Here we tested whether this increase in microcystin provided M. aeruginosa PCC 7806 with a fitness advantage during colder-temperature growth by comparing cell concentration, cellular physiology, reactive oxygen species damage, and the transcriptomics-inferred metabolism to a non-toxigenic mutant strain M. aeruginosa PCC 7806 ΔmcyB. Photo-physiological data combined with transcriptomic data revealed metabolic changes in the mutant strain during growth at 19 °C, which included increased electron sinks and non-photochemical quenching. Increased gene expression was observed for a glutathione-dependent peroxiredoxin during cold treatment, suggesting compensatory mechanisms to defend against reactive oxygen species are employed in the absence of microcystin in the mutant. Our observations highlight the potential selective advantages of a longer-term defensive strategy in management of oxidative stress (i.e., making microcystin) vs the shorter-term proactive strategy of producing cellular components to actively dissipate or degrade oxidative stress agents.

CD95/Fas ligand mRNA is toxic to cells through more than one mechanism

CD95/Fas ligand (CD95L) induces apoptosis through protein binding to the CD95 receptor. However, CD95L mRNA also induces toxicity in the absence of CD95 through induction of DISE (Death Induced by Survival Gene Elimination), a form of cell death mediated by RNA interference (RNAi). We now report that CD95L mRNA processing generates a short (s)RNA nearly identical to shL3, a commercial CD95L-targeting shRNA that led to the discovery of DISE. Neither of the miRNA biogenesis proteins Drosha nor Dicer are required for this processing. Interestingly, CD95L toxicity depends on the core component of the RISC, Ago2, in some cell lines, but not in others. In the HCT116 colon cancer cell line, Ago 1–4 appear to function redundantly in RNAi. In fact, Ago 1/2/3 knockout cells retain sensitivity to CD95L mRNA toxicity. Toxicity was only blocked by mutation of all in-frame start codons in the CD95L ORF. Dying cells exhibited an enrichment of RISC bound (R)-sRNAs with toxic 6mer seed sequences, while expression of the non-toxic CD95L mutant enriched for loading of R-sRNAs with nontoxic 6mer seeds. However, CD95L is not the only source of these R-sRNAs. We find that CD95L mRNA may induce DISE directly and indirectly, and that alternate mechanisms may underlie CD95L mRNA processing and toxicity.

High-Level Production of Soluble Cross-Reacting Material 197 in Escherichia coli Cytoplasm Due to Fine Tuning of the Target Gene’s mRNA Structure

Cross-reacting material 197 (CRM197) is a non-toxic mutant of the diphtheria toxin and is widely used as a carrier protein in conjugate vaccines. This protein was first obtained from the supernatant of the mutant Corynebacterium diphtheriae strain. This pathogenic bacteria strain is characterized by a slow growth rate and a relatively low target protein yield, resulting in high production costs for CRM197. Many attempts have been made to establish high-yield protocols for the heterologous expression of recombinant CRM197 in different host organisms. In the present work, a novel CRM197-producing Escherichia coli strain was constructed. The target protein was expressed in the cytoplasm of SHuffle T7 E. coli cells without any additional tags and with a single potential mutation-an additional Met [-1]. The fine tuning of the mRNA structure (the disruption of the single hairpin in the start codon area) was sufficient to increase the CRM197 expression level several times, resulting in 150-270 mg/L (1.1-2.0 mg/g wet biomass) yields of pure CRM197 protein. Besides the high yield, the advantages of the obtained expression system include the absence of the necessity of CRM197 refolding or tag removal. Thus, an extensive analysis of the mRNA structure and the removal of the unwanted hairpins in the 5' area may significantly improve the target protein expression rate.

Identification of Novel Nontoxic Mutants of Diphtheria Toxin Unable to ADP-ribosylate EF2 Using Molecular Dynamics Simulations and Free Energy Calculations

Introduction: The Diphtheria vaccine is currently manufactured by chemical inactivation of Diphtheria toxin (DT) following large-scale cultivation of the pathogenic Corynebacterium diphtheriae. The development of non-toxic variants of DT in non-pathogenic recombinant hosts improves the process safety aspects considerably and is therefore desirable. The cytotoxicity of DT was exerted through ADPribosylation of elongation factor 2 (EF2) by the catalytic (C) domain of DT using NAD as the ADP donor. Aim: We, therefore, aim to design DT variants unable to perform ADP-ribosylation of EF2. Methods: We used molecular dynamics simulations (MDS) for docking of the C-domain of DT onto EF2 to identify interacting amino acid fluctuations using the root mean square fluctuation (RMSF). Amino acids of the C domain possessing low fluctuations and hence low flexibility were then specified and used for the mutant design. The C domain and the mutants were docked with NAD and ADENYLYL-3'-5'- PHOSPHO-URIDINE3'-MONOPHOSPHATE (APU) as a C domain inhibitor. G52E showing negligible NAD binding was also included in our study. Free binding energies of the complexes were calculated and used to select the desired mutants. Moreover, contact maps of the C domain and the selected mutants were compared to elucidate their structural differences. The SCRATCH tool was used to estimate their solubility upon recombinant expression in E. coli. ElliPro was further used to determine their B-cell epitopes. Results: Our results indicated that amino acids Y20, V80, V81, V83, and Y149 within the C domain showed low flexibility. The constructed mutants, including Y20E and Y80E were able to bind APU, whereas Y149E was not able to bind it. Both Y20E and Y149E were unable to bind NAD. Thus, these mutants did not have ADP-ribosylation activity and were nontoxic. These were structurally different from the C domain. However, their solubility in E. coli and their conformational B-cell epitopes were similar to those of the C domain. Conclusion: Therefore, Y20E and Y149E are applicable for the vaccine design with DT using a much safer process compared to the commercial process.

New Mutants of Epsilon Toxin from Clostridium perfringens with an Altered Receptor-Binding Site and Cell-Type Specificity.

Epsilon toxin (Etx) from Clostridium perfringens is the third most potent toxin after the botulinum and tetanus toxins. Etx is the main agent of enterotoxemia in ruminants and is produced by Clostridium perfringens toxinotypes B and D, causing great economic losses. Etx selectively binds to target cells, oligomerizes and inserts into the plasma membrane, and forms pores. A series of mutants have been previously generated to understand the cellular and molecular mechanisms of the toxin and to obtain valid molecular tools for effective vaccination protocols. Here, two new non-toxic Etx mutants were generated by selective deletions in the binding (Etx-ΔS188-F196) or insertion (Etx-ΔV108-F135) domains of the toxin. As expected, our results showed that Etx-ΔS188-F196 did not exhibit the usual Etx binding pattern but surprisingly recognized specifically an O-glycoprotein present in the proximal tubules of the kidneys in a wide range of animals, including ruminants. Although diminished, Etx-ΔV108-F135 maintained the capacity for binding and even oligomerization, indicating that the mutation particularly affected the pore-forming ability of the toxin.

ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification.

Several studies have searched for new antigens to produce pneumococcal vaccines that are more effective and could provide broader coverage, given the great number of serotypes causing pneumococcal diseases. One of the promising subunit vaccine candidates is untagged recombinant pneumococcal surface protein A (PspA4Pro), obtainable in high quantities using recombinant Escherichia coli as a microbial factory. However, lipopolysaccharides (LPS) present in E. coli cell extracts must be removed, in order to obtain the target protein at the required purity, which makes the downstream process more complex and expensive. Endotoxin-free E. coli strains, which synthesize a nontoxic mutant LPS, may offer a cost-effective alternative way to produce recombinant proteins for application as therapeutics. This paper presents an investigation of PspA4Pro production employing the endotoxin-free recombinant strain ClearColi® BL21(DE3) with different media (defined, auto-induction, and other complex media), temperatures (27, 32, and 37 °C), and inducers. In comparison to conventional E. coli cells in a defined medium, ClearColi presented similar PspA4Pro yields, with lower productivities. Complex medium formulations supplemented with salts favored PspA4Pro yields, titers, and ClearColi growth rates. Induction with isopropyl-β-d-thiogalactopyranoside (0.5 mM) and lactose (2.5 g/L) together in a defined medium at 32 °C, which appeared to be a promising cultivation strategy, was reproduced in 5 L bioreactor culture, leading to a yield of 146.0 mg PspA4Pro/g dry cell weight. After purification, the cell extract generated from ClearColi led to 98% purity PspA4Pro, which maintained secondary structure and biological function. ClearColi is a potential host for industrial recombinant protein production.Key points • ClearColi can produce as much PspA4Pro as conventional E. coli BL21(DE3) cells. • 10.5 g PspA4Pro produced in ClearColi bioreactor culture using a defined medium. • Functional PspA4Pro (98% of purity) was obtained in ClearColi bioreactor culture. Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-022-11758-9.

A Decade of Fighting Invasive Meningococcal Disease: A Narrative Review of Clinical and Real-World Experience with the MenACWY-CRM Conjugate Vaccine.

The quadrivalent A, C, W and Y meningococcal vaccine conjugated to nontoxic mutant of diphtheria toxin (MenACWY-CRM) has been licensed since 2010 for the prevention of invasive meningococcal disease (IMD), an uncommon but life-threatening condition. Here, we summarize the experience accrued with MenACWY-CRM during the first decade since its licensure, by providing an overview of clinical trials investigating the safety, immunogenicity and co-administration of MenACWY-CRM with other vaccines as well as presenting real-world evidence regarding the impact of MenACWY-CRM vaccination on carriage and IMD incidence. MenACWY-CRM has demonstrated an acceptable clinical safety profile across a wide range of age groups; no safety concerns have been reported in special populations, such as immunocompromised infants and toddlers, or pregnant women. MenACWY-CRM has also been proven to be immunogenic in various age groups and geographic settings, and a booster dose has been shown to elicit strong anamnestic responses in all studied populations, irrespective of the vaccine used for priming. With no clinically relevant vaccine interactions reported, MenACWY-CRM is being conveniently integrated into existing vaccination programs for various age and risk groups; this possibility of co-administration helps improving vaccine coverage and streamlining the healthcare process of fighting preventable infectious diseases. Vaccination of adolescents and adults has been proven to reduce nasopharyngeal carriage for serogroups C, W and Y, which is an important element in reducing transmission. Real-world evidence indicates that MenACWY-CRM can reduce IMD incidence even in high-exposure groups. When combined with vaccines against serogroup B meningococci, MenACWY-CRM can offer protection against five of the most common serogroups responsible for IMD, which is an important advantage in the continuously evolving landscape of meningococcal serogroup epidemiology.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40121-021-00519-2.

Immunogenicity Evaluation of N-Glycans Recognized by HIV Broadly Neutralizing Antibodies.

While the improved treatment of human immunodeficiency virus type 1 (HIV-1) infection is available, the development of an effective and safe prophylactic vaccine against HIV-1 is still an unrealized goal. Encouragingly, the discovery of broadly neutralizing antibodies (bNAbs) from HIV-1 positive patients that are capable of neutralizing a broad spectrum of HIV-1 isolates of various clades has accelerated the progress of vaccine development in the past few years. Some of these bNAbs recognize the N-glycans on the viral surface gp120 glycoprotein. We have been interested in using the glycan epitopes recognized by bNAbs for the development of vaccines to elicit bNAb-like antibodies with broadly neutralizing activities. Toward this goal, we have identified novel hybrid-type structures with subnanomolar avidity toward several bNAbs including PG16, PGT121, PGT128-3C, 2G12, VRC13, VRC-PG05, VRC26.25, VRC26.09, PGDM1400, 35O22, and 10-1074. Here, we report the immunogenicity evaluation of a novel hybrid glycan conjugated to carrier DTCRM197, a nontoxic mutant of the diphtheria toxin, for immunization in mice. Our results indicated that the IgG response was mainly against the chitobiose motif with nonspecific binding to a panel of N-glycans with reducing end GlcNAc-GlcNAc (chitobiose) printed on the glass slides. However, the IgM response was mainly toward the reducing end GlcNAc moiety. We further used the glycoconjugates of Man3GlcNAc2, Man5GlcNAc2, and Man9GlcNAc2 glycans for immunization, and a similar specificity pattern was observed. These findings suggest that the immunogenicity of chitobiose may interfere with the outcome of N-glycan-based vaccines, and modification may be necessary to increase the immunogenicity of the entire N-glycan epitope.

Glycoconjugation of Shigella flexneri type 2a O-polysaccharide with CRM197 as a potential vaccine candidate for shigellosis

Shigellosis, a diarrheal disorder caused by an entero-invasive bacterium Shigella, is a major concern among children often leading to mortality. As most of these strains have developed universal antibiotic resistance, the development of a vaccine is crucial in combating the infection. The O-specific polysaccharide (O-PSs) from S. flexneri type 2a is considered to be the major disease-causing antigen in shigellosis. Therefore, the O-PSs conjugated with carrier proteins, can serve as a potential high molecular weight vaccine candidate. Accordingly, in the present study, O-PS extracted from S. flexneri 2a is conjugated with Cross-Reactive Material (CRM197), a non-toxic mutant of diphtheria toxin. We derivatized CRM197 and O-PS separately with adipic acid dihydrazide (ADH) and reacted with their counterparts to probe the conjugation efficacy. Among the two strategies, the CRM197-ADH treated with O-PS has yielded a stable glycoconjugate of 311 kDa. The conjugation efficiency has been probed by estimating the free protein, free O-PS and O-PS:CRM197 ratio using slot-blot, size exclusion and high-performance anion exchange chromatography techniques. The conjugate exhibited enhanced shelf-life of three months. The cytotoxicity studies with Vero/MRC-5 cells have confirmed the non-toxicity of the conjugate, which makes the glycoconjugate a potential vaccine candidate for shigellosis.

Cytokine vaccine for asthma

Vaccines The cytokines interleukin-4 (IL-4) and IL-13 play important, nonredundant roles in the pathogenesis of asthma. Monoclonal antibodies against IL-4 receptor α (IL-4Rα), which inhibit both IL-4 and IL-13 signaling, can effectively ameliorate severe asthmatic exacerbations. However, monoclonal antibody therapy is expensive and requires regular injections. Conde et al. developed conjugate vaccines (“kinoids”) against IL-4 and IL-13 by coupling these cytokines with a nontoxic mutant of diptheria toxin. These kinoids were effective as both prophylactics and therapeutics in mouse models of allergic airway inflammation, durably reducing hallmarks of disease including serum immunoglobulin E, airway hyperresponsiveness, and mucus hypersecretion. Similar results were obtained with transgenic mice expressing human IL-4, IL-13, and IL-4Rα. Thus, dual IL-4/IL-13 vaccination may be a long-lasting and economical approach to the treatment of asthma and other allergic diseases. Nat. Commun. 12 , 2574 (2021).

Molecular chaperones facilitate soluble expression of recombinant non-toxic mutant CRM197 of diphtheria toxin in Escherichia coli

Diphtheria toxin is an ADP-ribosyltransferase toxic to human cells. Mutation of the active site in its catalytic domain eliminates the toxicity, but retains its immunogenicity. A non-toxic mutant of diphtheria toxin known as CRM197 protein has become an ideal carrier protein for conjugate vaccines. CRM197 can further improve its immunogenicity by cross-linking with other antigens, so it has good potential to find broad applications. Unfortunately, inclusion bodies are easily formed during the expression of recombinant CRM197 protein in Escherichia coli, which greatly reduces its yield. In order to address this problem, pG-KJE8 vector carrying molecular chaperones and plasmid pET28a-CRM197, were co-expressed in Escherichia coli. The results showed that the recombinant CRM197 protein was successfully expressed and appeared largely in inclusion bodies. The molecular chaperones DnaK, DnaJ, GrpE, GroES and GroEL5 expressed can facilitate correct and rapid folding of CRM197. Furthermore, it can also improve the recovery rate of soluble CRM197 protein. The soluble expression of CRM197 was maximized upon addition of 1.0 mmol/L IPTG, 0.5 mg L-arabinose, 5.0 ng/mL tetracycline and induction at 20oC for 16 h. The soluble CRM197 protein shows good immunoreactivity, demonstrating the molecular chaperones expressed from pG-KJE8 facilitated the soluble expression of CRM197 protein in E. coli.

Catalytic Cross Talk between Key Peptide Fragments That Couple Alzheimer's Disease with Amyotrophic Lateral Sclerosis.

Protein aggregation is a common feature in prominent neurodegenerative diseases, usually thought to be due to the assembly of a single peptide or protein. Recent studies have challenged this notion and suggested several proteins may be involved in promoting and amplifying disease. For example, the TDP-43 protein associated with Amyotrophic Lateral Sclerosis has been found in the brain along with Aβ assemblies associated with Alzheimer's disease, and those patients that show the presence of TDP-43 are 10 times more likely to demonstrate cognitive impairment compared to TDP-43-negative Alzheimer's patients. Here we examine the interactions between the amyloidogenic core of TDP-43, TDP-43307-319, and a neurotoxic physiologically observed fragment of Aβ, Aβ25-35. Utilizing ion mobility mass spectrometry in concert with atomic force microscopy and molecular dynamics simulations, we investigate which oligomers are involved in seeding aggregation across these two different protein systems and gain insight into which structures initiate and result from these interactions. Studies were conducted by mixing Aβ25-35 with the toxic wild type TDP-43307-319 peptide and with the nontoxic synthetic TDP-43307-319 mutant, G314V. Our findings identify a strong catalytic effect of TDP-43307-319 WT monomer in the acceleration of Aβ25-35 aggregation to its toxic cylindrin and β barrel forms. This observation is unprecedented in both its speed and specificity. Interestingly, the nontoxic G314V mutant of TDP-43307-319 and dimers or higher order oligomers of WT TDP-43307-319 do not promote aggregation of Aβ25-35 but rather dissociate preformed toxic higher order oligomers of Aβ25-35. Reasons for these very different behaviors are reported.

In vivo nanoscale analysis of the dynamic synergistic interaction of Bacillus thuringiensis Cry11Aa and Cyt1Aa toxins in Aedes aegypti.

The insecticidal Cry11Aa and Cyt1Aa proteins are produced by Bacillus thuringiensis as crystal inclusions. They work synergistically inducing high toxicity against mosquito larvae. It was proposed that these crystal inclusions are rapidly solubilized and activated in the gut lumen, followed by pore formation in midgut cells killing the larvae. In addition, Cyt1Aa functions as a Cry11Aa binding receptor, inducing Cry11Aa oligomerization and membrane insertion. Here, we used fluorescent labeled crystals, protoxins or activated toxins for in vivo localization at nano-scale resolution. We show that after larvae were fed solubilized proteins, these proteins were not accumulated inside the gut and larvae were not killed. In contrast, if larvae were fed soluble non-toxic mutant proteins, these proteins were found inside the gut bound to gut-microvilli. Only feeding with crystal inclusions resulted in high larval mortality, suggesting that they have a role for an optimal intoxication process. At the macroscopic level, Cry11Aa completely degraded the gastric caeca structure and, in the presence of Cyt1Aa, this effect was observed at lower toxin-concentrations and at shorter periods. The labeled Cry11Aa crystal protein, after midgut processing, binds to the gastric caeca and posterior midgut regions, and also to anterior and medium regions where it is internalized in ordered "net like" structures, leading finally to cell break down. During synergism both Cry11Aa and Cyt1Aa toxins showed a dynamic layered array at the surface of apical microvilli, where Cry11Aa is localized in the lower layer closer to the cell cytoplasm, and Cyt1Aa is layered over Cry11Aa. This array depends on the pore formation activity of Cry11Aa, since the non-toxic mutant Cry11Aa-E97A, which is unable to oligomerize, inverted this array. Internalization of Cry11Aa was also observed during synergism. These data indicate that the mechanism of action of Cry11Aa is more complex than previously anticipated, and may involve additional steps besides pore-formation activity.

Direct Observation of β-Barrel Intermediates in the Self-Assembly of Toxic SOD128-38 and Absence in Nontoxic Glycine Mutants.

Soluble low-molecular-weight oligomers formed during the early stage of amyloid aggregation are considered the major toxic species in amyloidosis. The structure-function relationship between oligomeric assemblies and the cytotoxicity in amyloid diseases are still elusive due to the heterogeneous and transient nature of these aggregation intermediates. To uncover the structural characteristics of toxic oligomeric intermediates, we compared the self-assembly dynamics and structures of SOD128-38, a cytotoxic fragment of the superoxide dismutase 1 (SOD1) associated with the amyotrophic lateral sclerosis, with its two nontoxic mutants G33V and G33W using molecular dynamics simulations. Single-point glycine substitutions in SOD128-38 have been reported to abolish the amyloid toxicity. Our simulation results showed that the toxic SOD128-38 and its nontoxic mutants followed different aggregation pathways featuring distinct aggregation intermediates. Specifically, wild-type SOD128-38 initially self-assembled into random-coil-rich oligomers, among which fibrillar aggregates composed of well-defined curved single-layer β-sheets were nucleated via coil-to-sheet conversions and the formation of β-barrels as intermediates. In contrast, the nontoxic G33V/G33W mutants readily assembled into small β-sheet-rich oligomers and then coagulated with each other into cross-β fibrils formed by two-layer β-sheets without forming β-barrels as the intermediates. The direct observation of β-barrel oligomers during the assembly of toxic SOD128-38 fragments but not the nontoxic glycine-substitution mutants strongly supports β-barrels as the toxic oligomers in amyloidosis, probably via interactions with the cell membrane and forming amyloid pores. With well-defined structures, the β-barrel might serve as a novel therapeutic target against amyloid-related diseases.

The use of pore-forming toxins to image lipids and lipid domains.

Very few proteins are reported to bind specific lipids. Because of the high selectivity and strong binding to specific lipids, lipid-targeting pore forming toxins (PFTs) have been employed to study the distribution of lipids in cell- and model-membranes. Non-toxic and monomeric PFT-derivatives are especially useful to study living cells. In this chapter we highlight sphingomyelin (SM)-binding PFT, lysenin (Lys), its derivatives, and newly identified SM/cholesterol binding protein, nakanori. We describe the preparation of non-toxic mutant of Lys (NT-Lys) and its application in optical and super resolution microscopy. We also discuss the observation of nanometer scale lipid domains labeled with nakanori and maltose-binding protein (MBP)-Lys in electron microscopy.

Genotype and host microbiome alter competitive interactions between Microcystis aeruginosa and Chlorella sorokiniana

Cyanobacterial harmful algal blooms (cyanoHABs) continue to increase in frequency and magnitude, threatening global freshwater ecosystems and services. In north-temperate lakes cyanobacteria appear in early summer, succeeding green algae as the dominant phytoplankton group, a pattern thought to be mediated by changes in temperature and bioavailable nutrients. To understand additional drivers of this successional pattern our study used reciprocal invasion experiments to examine the competitive interaction between Microcystis aeruginosa, a dominant contributor to cyanoHABs, and the green alga Chlorella sorokiniana. We considered two factors that may impact these interactions: (1) strain variation, with a specific emphasis on the presence or absence of the gene for the hepatotoxin microcystin, and (2) host-associated bacteria. We used toxic M. aeruginosa PCC 7806 (microcystin producing strain), a non-toxic mutant of PCC 7806, non-toxic M. aeruginosa PCC 9701 (non-microcystin producing strain), and C. sorokiniana. Each organism was available free of all bacteria (i.e., axenic) and with a re-introduced defined bacterial community to generate their xenic counterparts. Competitive interactions were assessed with reciprocal invasion experiments between paired xenic and paired axenic populations of C. sorokiniana and one of the two Microcystis strains, each assessed separately. Flow cytometry and random forest models were used to rapidly discriminate and quantify phytoplankton population densities with 99% accuracy. We found that M. aeruginosa PCC 7806, but not strain PCC 9701, could proliferate from low abundance in a steady-state population of C. sorokiniana. Further, the presence of bacteria allowed M. aeruginosa PCC 7806 to grow to a higher population density into an established C. sorokiniana population than when grown axenic. Conversely, when M. aeruginosa was dominant, C. sorokiniana was only able to proliferate from low density into the PCC 9701 strain, and only when axenic. The mutant of PCC 7806 lacking the ability to produce microcystin behaved similarly to the toxic wild-type, implying microcystin is not responsible for the difference in competitive abilities observed between the two wild-type strains. Quantification of microcystins (MCs) when PCC 7806 M. aeruginosa was introduced into the C. sorokiniana culture showed two-fold more MCs per cell when host-associated bacteria were absent compared to present in both species cultures. Our results show that the ability of M. aeruginosa to compete with C. sorokiniana is determined by genomic differences beyond genes involved in microcystin toxin generation and indicate an important role of host-associated bacteria in mediating phytoplankton interspecies interactions. These results expand our understanding of the key drivers of phytoplankton succession and the establishment and persistence of freshwater harmful cyanobacterial blooms.

Etx-Y71A as a non-toxic mutant of Clostridium perfringens epsilon toxin induces protective immunity in mice and sheep

Epsilon toxin (Etx) is an extremely potent toxin produced by Clostridium perfringens toxinotypes B and D, which cause fatal enterotoxemia in many livestock species, mainly sheep and goats. Our previous study demonstrated that the aromatic amino acid (AA) residue at position 71 in domain III of Etx is needed for its cytotoxic activity toward MDCK cells. Here, we first determined that Etx mutants with non-aromatic AA substitutions at Tyr71 lost lethality in mice, indicating that the aromatic AA residue at position 71 is a toxicity determinant of Etx in vivo. After intravenous injection with a high dose of the trypsin-activated Etx-Y71A mutant, mice did not show any histopathological lesions, and confocal microscopy observations further showed that Etx-Y71A lost the ability to cross the blood–brain barrier of the mice. These results suggested that the Etx-Y71A mutant is sufficiently safe in vivo to be a vaccine candidate. Furthermore, the immune efficacy of Etx-Y71A was evaluated in model and host animals. Mice inoculated with this mutant produced high levels of neutralizing antibodies and were completely protected from a 100 LD50 of trypsin-activated Etx challenge. Sheep immunized with Etx-Y71A produced high levels of neutralizing antibodies that provided protection in mice against an activated Etx challenge, and lambs could receive passive immunity through immunization of pregnant ewes. Additionally, homology modeling and circular dichroism analysis showed that Etx-Y71A has structural similarity to Etx, which provides a structural basis for Etx-Y71A retaining the immunogenicity of Etx. Taken together, these results suggest that Etx-Y71A is a potential vaccine candidate against Etx-inducing enterotoxemia.

Comparing Structure and Conformation of Diphtheria Toxin With Its Non-toxic Mutant (E349K) at 300 K Using Molecular Dynamics Simulations

Background: The molecular dynamics simulations have provided detailed information on the fluctuations and conformational changes of proteins. Mutation of GLu349 of diphtheria toxin (DT) to Lys inhibits the molecular cytotoxicity in mammalian cells. In this work, we thus aimed to evaluate the effects of the mutation on the structure and the conformation of DT using molecular dynamic simulations. Methods: PYMOL system was used for introducing the mutant amino acid to DT. Three dimensional structures were visualized using YASARA. The protonation fixing process was done using H++ server. SPD viewer was then applied to retrieve missing hydrogen atoms. Molecular dynamic simulations were also carried out using GROMACS software. Finally, RMSF, Rg, and RMSD graphs were drawn by Sigma Plot using GROMACS outputs. Results: The results of our analysis indicated that amino acid residue fluctuations in the catalytic domain (C) of DT were greater than those of its mutant (E349K). Moreover, residue fluctuations in the region, including Ile344- val347, Tyr514-Ser525, and Ile533-Lys534 of DT were greater than those of the mutant (E349K). However, residue fluctuations in the region including Cys186-Cys201 and Glu349-Val351 of DT were lower than those of E349K. In addition, the disulfide bridge (Cys186–Cys201) was formed in DT, whereas it was not observed in the mutant. The secondary structure analysis showed that the beta-sheet content of E349K decreased compared with DT. Additionally, the conformation of DT was different from that of E349K in the hinge loop regions (Ala379–Thr386 and Tyr514-Ser525). The radius of gyration (Rg) and the root mean square deviation (RMSD) of E349K were greater than those of DT. The conformational stability and compactness of DT were higher than those of E349K. Conclusions: The method used in this study can demonstrate the structural details of toxins as important aspects to predict activities of the receptor binding and the catalytic domains. Therefore, it can be used to evaluate conformational changes of toxins as well as other vaccine candidates.

Hepatitis E vaccine candidate harboring a non-particulate immunogen of E2 fused with CRM197 fragment A

The Hepatitis E vaccine (Hecolin, licensed in China) harbors a potent particulate immunogen, p239, designed from a 26-aa N-terminal extension of its poorly immunogenic parental protein, E2. Although an effective vaccine, we sought to design a fusion protein in a non-particulate form that could improve the delivery and immunogenicity of E2 epitopes. The non-toxic mutant of diphtheria toxin, CRM197 (Cross-Reacting Material 197) has been successfully used as a carrier protein for conjugated vaccines to enhance the immunogenicity of polysaccharides. Here, we designed a fusion non-particulate protein of E2 and the catalytic domain (fragment A) of CRM197 and evaluated its antigenicity, immunogenicity and disease prevention efficacy in primates. This fusion protein, named CRM197(A)-E2, was bacterially expressed and purified by chromatography. CRM197(A)-E2 presented as a homodimer in solution, similar to its parental E2 protein, and exhibited excellent antigenicity against representative neutralizing monoclonal antibodies, like E2 and p239. However, CRM197(A)-E2 manifested higher immunogenicity in mice compared with that achieved by the particulate p239, as indicated by the 10-times lower ED50 value and 2-log higher HEV-specific antibody level that could persist for at least 28 weeks. In addition, both the 1 μg and 10 μg doses of CRM197(A)-E2 adjuvanted with aluminum could protect vaccinated monkeys against HEV challenge, matching that achieved with only the higher (10 μg) dose of the p239 vaccine. These results suggest that the CRM197 fragment A alone serves as an intra-molecular adjuvant to remarkably enhance the immunogenicity of the target of interest in a non-particulate form. These findings may pave the way for rational vaccine design, especially in cases where particulates are not accessible.