DNA binding reveals hidden interdomain allostery of a MazE antitoxin from Mycobacterium tuberculosis.

Type II toxin-antitoxin (TA) systems are ubiquitously distributed genetic elements in prokaryotes and are crucial for cell maintenance and survival under environmental stresses. The antitoxin is a modular protein consisting of the disordered C-terminal region that physically contacts and neutralizes the cognate toxin and the well-folded N-terminal DNA binding domain responsible for autorepression of TA transcription. However, how the two functional domains communicate is largely unknown. Herein, we determined the crystal structure of the N-terminal domain of the type II antitoxin MazE-mt10 from Mycobacterium tuberculosis, revealing a homodimer of the ribbon-helix-helix (RHH) fold with distinct DNA binding specificity. NMR studies demonstrated that full-length MazE-mt10 forms the helical and coiled states in equilibrium within the C-terminal region, and that helical propensity is allosterically enhanced by the N-terminal binding to the cognate operator DNA. This coil-to-helix transition may promote toxin binding/neutralization of MazE-mt10 and further stabilize the TA-DNA transcription repressor. This is supported by many crystal structures of type II TA complexes in which antitoxins form an α-helical structure at the TA interface. The hidden helical state of free MazE-mt10 in solution, favored by DNA binding, adds a new dimension to the regulatory mechanism of type II TA systems. Furthermore, complementary approaches using X-ray crystallography and NMR allow us to study the allosteric interdomain interplay of many other full-length antitoxins of type II TA systems.

Salmonella Enteritidis antitoxin DinJ inhibits NLRP3-dependent canonical inflammasome activation in macrophages.

The inflammasome is a pivotal component of the innate immune system, acting as a multiprotein complex that plays an essential role in detecting and responding to microbial infections. Salmonella Enteritidis have evolved multiple mechanisms to regulate inflammasome activation and evade host immune system clearance. Through screening S. Enteritidis C50336ΔfliC transposon mutant library, we found that the insertion mutant of dinJ increased inflammasome activation. In this study, we demonstrated the genetic connection between the antitoxin DinJ and the toxin YafQ in S. Enteritidis, confirming their co-transcription. The deletion mutant ΔfliCΔdinJ increased cell death and IL-1ß secretion in J774A.1 cells. Western blotting analysis further showed elevated cleaved Caspase-1 product (p10 subunits) and IL-1ß secretion in cells infected with ΔfliCΔdinJ compared to cells infected with ΔfliC. DinJ was found to inhibit canonical inflammasome activation using primary bone marrow-derived macrophages (BMDMs) from Casp-/- C57BL/6 mice. Furthermore, DinJ specifically inhibited NLRP3 inflammasome activation, as demonstrated in BMDMs from Nlrp3-/- and Nlrc4-/- mice. Fluorescence resonance energy transfer (FRET) experiments confirmed the translocation of DinJ into host cells during infection. Finally, we revealed that DinJ could inhibit the secretion of IL-1ß and IL-18 in vivo, contributing to S. Enteritidis evading host immune clearance. In summary, our findings provide insights into the role of DinJ in modulating the inflammasome response during S. Enteritidis infection, highlighting its impact on inhibiting inflammasome activation and immune evasion.

Collaborative study for the establishment of Ph. Eur. Biological Reference Preparation for Human tetanus immunoglobulin batch 2.

This publication describes the outcome of a project to develop a replacement European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for Human tetanus immunoglobulin (TIg) as well as for the World Health Organization (WHO) International Standard (IS) for Tetanus Immunoglobulin, Human. Bulk TIg was kindly provided by a European manufacturer and was used to prepare the candidate standard. The candidate standard was freeze-dried and calibrated in an international collaborative study jointly co-ordinated by the Medicines & Healthcare products Regulatory Agency (MHRA) and the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe). The results of this study show that there was good agreement between laboratories for the potency estimates obtained for the candidate standard relative to the current WHO IS/Ph. Eur. BRP. The study also demonstrated that the candidate standard is suitable for use in Ph. Eur. assays for potency testing of TIg products and there was good agreement in the potency estimates obtained using the different assay methods included in the study. Accelerated degradation studies performed at the MHRA over a period of 4 years suggest that the freeze-dried candidate standard will be very stable. The candidate standard was established as Ph. Eur. BRP for Human tetanus immunoglobulin, batch 2 with an assigned potency of 45 IU/ampoule. The same preparation was also adopted by the WHO Expert Committee on Biological Standardization (ECBS) to serve as the WHO 2nd IS for Tetanus Immunoglobulin, Human (13/240).

Production of monoclonal antibodies against botulinum neurotoxin in Nicotiana benthamiana.

Botulism is a fatal neurologic disease caused by the botulinum toxin (BoNT) produced by Clostridium botulinum. It is a rare but highly toxic disease with symptoms, such as cramps, nausea, vomiting, diarrhea, dysphagia, respiratory failure, muscle weakness, and even death. Currently, two types of antitoxin are used: equine-derived heptavalent antitoxin and human-derived immunoglobulin (BabyBIG®). However, heptavalent treatment may result in hypersensitivity, whereas BabyBIG®, has a low yield. The present study focused on the development of three anti-BoNT monoclonal antibodies (mAbs), 1B18, C25, and M2, in Nicotiana benthamiana. The plant-expressed mAbs were purified and examined for size, purity and integrity by SDS-PAGE, western blotting and size-exclusion chromatography. Analysis showed that plant-produced anti-BoNT mAbs can fully assemble in plants, can be purified in a single purification step, and mostly remain as monomeric proteins. The efficiency of anti-BoNT mAbs binding to BoNT/A and B was then tested. Plant-produced 1B18 retained its ability to recognize both mBoNT/A1 and ciBoNT/B1. At the same time, the binding specificities of two other mAbs were determined: C25 for mBoNT/A1 and M2 for ciBoNT/B1. In conclusion, our results confirm the use of plants as an alternative platform for the production of anti-BoNT mAbs. This plant-based technology will serve as a versatile system for the development botulism immunotherapeutics.

[Clinical characteristics and prognosis of 8 cases of severe infant botulism].

Objective: To summarize the clinical characteristics and prognosis of severe infant botulism and evaluate the therapeutic effect of botulinum antitoxin in the pediatric intensive care unit (PICU). Methods: The clinical data of 8 cases diagnosed with infantile botulism were retrospectively analyzed in the PICU of Beijing Children's Hospital from October 2019 to August 2023. Data of basic demographic information, clinical manifestations, laboratory tests, treatment and prognosis of each child were collected and analyzed using descriptive statistical methods. Results: Eight laboratory-confirmed cases of infant botulism were included in this study, all of which were male infants with an age of 6.0 (3.3,6.8) months. Three of the children were from Inner Mongolia Autonomous Region, 2 of them were from Hebei, and the other 3 were from Beijing, Shandong and Xinjiang Uyghur Autonomous Region, respectively. All the patients were previously healthy. In 4 of these cases, the possible cause was the ingestion of either honey and its products or sealed pickled food by the mother or child before the onset of the disease. The first symptom was poor milk intake (4 cases), followed by shallow shortness of breath (7 cases), limb weakness (7 cases) and so on. The typical signs were bilateral dilated pupils (8 cases) and decreased limb muscle strength (8 cases). The main subtype was type B (7 cases), and only 1 case was classified as type A. Six of the children were treated with antitoxin therapy for a duration of 24 (19, 49) d. Seven of them had invasive mechanical ventilation. All the patients survived upon discharge with a follow-up period of 29 d to 3 years and 8 months. Six patients had fully recovered, and 2 recently discharged patients were gradually recovering. Conclusions: For infants with suspected contact or ingestion of botulinum and presented with bilateral pupillary paralysis, muscle weakness and clear consciousness, the stool should be collected for diagnostic testing using a mouse bioassay as soon as possible. Type B was the most common type. The antitoxin treatment was effectiveness and the prognosis was well.

Gene Editing of Candida glycerinogenes by Designed Toxin-Antitoxin Cassette.

Candida glycerinogenes is an industrial yeast with excellent multistress resistance. However, due to the diploid genome and the lack of meiosis and screening markers, its molecular genetic operation is limited. Here, a gene editing system using the toxin-antitoxin pair relBE from the type II toxin-antitoxin system in Escherichia coli as a screening marker was constructed. The RelBE complex can specifically and effectively regulate cell growth and arrest through a conditionally controlled toxin RelE switch, thereby achieving the selection of positive recombinants. The constructed editing system achieved precise gene deletion, replacement, insertion, and gene episomal expression in C. glycerinogenes. Compared with the traditional amino acid deficiency complementation editing system, this editing system produced higher biomass and the gene deletion efficiency was increased by 3.5 times. Using this system, the production of 2-phenylethanol by C. glycerinogenes was increased by 11.5-13.5% through metabolic engineering and tolerance engineering strategies. These results suggest that the stable gene editing system based on toxin-antitoxin pairs can be used for gene editing of C. glycerinogenes to modify metabolic pathways and promote industrial applications. Therefore, the constructed gene editing system is expected to provide a promising strategy for polyploid industrial microorganisms lacking gene manipulation methods.

Serum immunoglobulin or albumin binding single-domain antibodies that enable tailored half-life extension of biologics in multiple animal species.

Single-domain antibody fragments (sdAbs) can be isolated from heavy-chain-only antibodies that occur in camelids or the heavy chain of conventional antibodies, that also occur in camelids. Therapeutic application of sdAbs is often complicated by their low serum half-life. Fusion to sdAb that bind to long-lived serum proteins albumin or IgG can prolong serum half-life of fusion partners. Such studies mostly focused on human application. For half-life prolongation in multiple animal species novel species cross-reacting sdAb are needed. We here describe the isolation from immunized llamas of sdAbs G6 and G13 that bound IgG of 9-10 species analysed, including horse, dog, cat, and swine, as well as sdAb A12 that bound horse, dog, swine and cat albumin. A12 bound albumin with 13 to 271 nM affinity dependent on the species. G13 affinity was difficult to determine by biolayer interferometry due to low and heterogeneous signals. G13 and G6 compete for the same binding domain on Fab fragments. Furthermore, they both lack the hallmark residues typical of camelid sdAbs derived from heavy-chain antibodies and had sequence characteristics typical of human sdAbs with high solubility and stability. This suggests they are derived from conventional llama antibodies. They most likely bind IgG through pairing with VL domains at the VH-VL interface rather than a paratope involving complementarity determining regions. None of the isolated sdAb interfered with FcRn binding to albumin or IgG, and thus do not prevent endosomal albumin/IgG-sdAb complex recycling. Fusions of albumin-binding sdAb A12 to several tetanus neurotoxin (TeNT) binding sdAbs prolonged the terminal serum half-life in piglets to about 4 days, comparable to authentic swine albumin. However, G13 conferred a much lower half-life of 0.84 days. Similarly, in horse, G13 prolonged half-life to only 1.2 days whereas A12 fused to two TeNT binding domains (T6T16A12) had a half-life of 21 days. The high half-life of T6T16A12, which earlier proved to be a highly potent TeNT antitoxin, further supports its therapeutic value. Furthermore, we have identified several additional sdAbs that enable tailored half-life extension of biologicals in multiple animal species.

Long-Term Pulmonary Damage in Surviving Antitoxin-Treated Mice following a Lethal Ricin Intoxication.

Ricin, a highly potent plant-derived toxin, is considered a potential bioterrorism weapon due to its pronounced toxicity, high availability, and ease of preparation. Acute damage following pulmonary ricinosis is characterized by local cytokine storm, massive neutrophil infiltration, and edema formation, resulting in respiratory insufficiency and death. A designated equine polyclonal antibody-based (antitoxin) treatment was developed in our laboratory and proved efficacious in alleviating lung injury and increasing survival rates. Although short-term pathogenesis was thoroughly characterized in antitoxin-treated mice, the long-term damage in surviving mice was never determined. In this study, long-term consequences of ricin intoxication were evaluated 30 days post-exposure in mice that survived antitoxin treatment. Significant pulmonary sequelae were demonstrated in surviving antitoxin-treated mice, as reflected by prominent histopathological changes, moderate fibrosis, increased lung hyperpermeability, and decreased lung compliance. The presented data highlight, for the first time to our knowledge, the possibility of long-term damage development in mice that survived lethal-dose pulmonary exposure to ricin due to antitoxin treatment.

RES-Xre toxin-antitoxin locus knaAT maintains the stability of the virulence plasmid in Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae isolates have been increasingly reported worldwide, especially hypervirulent drug-resistant variants owing to the acquisition of a mobilizable virulence plasmid by a carbapenem-resistant strain. This pLVPK-like mobilizable plasmid encodes various virulence factors; however, information about its genetic stability is lacking. This study aimed to investigate the type II toxin-antitoxin (TA) modules that facilitate the virulence plasmid to remain stable in K. pneumoniae. More than 3,000 TA loci in 2,000 K. pneumoniae plasmids were examined for their relationship with plasmid cargo genes. TA loci from the RES-Xre family were highly correlated with virulence plasmids of hypervirulent K. pneumoniae. Overexpression of the RES toxin KnaT, encoded by the virulence plasmid-carrying RES-Xre locus knaAT, halts the cell growth of K. pneumoniae and E. coli, whereas co-expression of the cognate Xre antitoxin KnaA neutralizes the toxicity of KnaT. knaA and knaT were co-transcribed, representing the characteristics of a type II TA module. The knaAT deletion mutation gradually lost its virulence plasmid in K. pneumoniae, whereas the stability of the plasmid in E. coli was enhanced by adding knaAT, which revealed that the knaAT operon maintained the genetic stability of the large virulence plasmid in K. pneumoniae. String tests and mouse lethality assays subsequently confirmed that a loss of the virulence plasmid resulted in reduced pathogenicity of K. pneumoniae. These findings provide important insights into the role of the RES-Xre TA pair in stabilizing virulence plasmids and disseminating virulence genes in K. pneumoniae.

Diverse genetic contexts of HicA toxin domains propose a role in anti-phage defense.

Toxin-antitoxin (TA) modules are prevalent in prokaryotic genomes, often in substantial numbers. For instance, the Mycobacterium tuberculosis genome alone harbors close to 100 TA modules, half of which belong to a singular type. Traditionally ascribed multiple biological roles, recent insights challenge these notions and instead indicate a predominant function in phage defense. TAs are often located within Defense Islands, genomic regions that encode various defense systems. The analysis of genes within Defense Islands has unveiled a wide array of systems, including TAs that serve in anti-phage defense. Prokaryotic cells are equipped with anti-phage Viperins that, analogous to their mammalian counterparts, inhibit viral RNA transcription. Additionally, bacterial Structural Maintenance of Chromosome (SMC) proteins combat plasmid intrusion by recognizing foreign DNA signatures. This study undertakes a comprehensive bioinformatics analysis of genetic elements encoding the HicA double-stranded RNA-binding domain, complemented by protein structure modeling. The HicA toxin domains are found in at least 14 distinct contexts and thus exhibit a remarkable genetic diversity. Traditional bicistronic TA operons represent eight of these contexts, while four are characterized by monocistronic operons encoding fused HicA domains. Two contexts involve hicA adjacent to genes that encode bacterial Viperins. Notably, genes encoding RelE toxins are also adjacent to Viperin genes in some instances. This configuration hints at a synergistic enhancement of Viperin-mediated anti-phage action by HicA and RelE toxins. The discovery of a HicA domain merged with an SMC domain is compelling, prompting further investigation into its potential roles.IMPORTANCEProkaryotic organisms harbor a multitude of toxin-antitoxin (TA) systems, which have long puzzled scientists as "genes in search for a function." Recent scientific advancements have shed light on the primary role of TAs as anti-phage defense mechanisms. To gain an overview of TAs it is important to analyze their genetic contexts that can give hints on function and guide future experimental inquiries. This article describes a thorough bioinformatics examination of genes encoding the HicA toxin domain, revealing its presence in no fewer than 14 unique genetic arrangements. Some configurations notably align with anti-phage activities, underscoring potential roles in microbial immunity. These insights robustly reinforce the hypothesis that HicA toxins are integral components of the prokaryotic anti-phage defense repertoire. The elucidation of these genetic contexts not only advances our understanding of TAs but also contributes to a paradigm shift in how we perceive their functionality within the microbial world.

Single-cell analysis reveals that cryptic prophage protease LfgB protects Escherichia coli during oxidative stress by cleaving antitoxin MqsA.

Although toxin/antitoxin (TA) systems are ubiquitous, beyond phage inhibition and mobile element stabilization, their role in host metabolism is obscure. One of the best-characterized TA systems is MqsR/MqsA of Escherichia coli, which has been linked previously to protecting gastrointestinal species during the stress it encounters from the bile salt deoxycholate as it colonizes humans. However, some recent whole-population studies have challenged the role of toxins such as MqsR in bacterial physiology since the mqsRA locus is induced over a hundred-fold during stress, but a phenotype was not found upon its deletion. Here, we investigate further the role of MqsR/MqsA by utilizing single cells and demonstrate that upon oxidative stress, the TA system MqsR/MqsA has a heterogeneous effect on the transcriptome of single cells. Furthermore, we discovered that MqsR activation leads to induction of the poorly characterized yfjXY ypjJ yfjZF operon of cryptic prophage CP4-57. Moreover, deletion of yfjY makes the cells sensitive to H2O2, acid, and heat stress, and this phenotype was complemented. Hence, we recommend yfjY be renamed to lfgB (less fatality gene B). Critically, MqsA represses lfgB by binding the operon promoter, and LfgB is a protease that degrades MqsA to derepress rpoS and facilitate the stress response. Therefore, the MqsR/MqsA TA system facilitates the stress response through cryptic phage protease LfgB.IMPORTANCEThe roles of toxin/antitoxin systems in cell physiology are few and include phage inhibition and stabilization of genetic elements; yet, to date, there are no single-transcriptome studies for toxin/antitoxin systems and few insights for prokaryotes from this novel technique. Therefore, our results with this technique are important since we discover and characterize a cryptic prophage protease that is regulated by the MqsR/MqsA toxin/antitoxin system in order to regulate the host response to oxidative stress.

Arbitrium communication controls phage lysogeny through non-lethal modulation of a host toxin-antitoxin defence system.

Temperate Bacillus phages often utilize arbitrium communication to control lysis/lysogeny decisions, but the mechanisms by which this control is exerted remains largely unknown. Here we find that the arbitrium system of Bacillus subtilis phage ϕ3T modulates the host-encoded MazEF toxin-antitoxin system to this aim. Upon infection, the MazF ribonuclease is activated by three phage genes. At low arbitrium signal concentrations, MazF is inactivated by two phage-encoded MazE homologues: the arbitrium-controlled AimX and the later-expressed YosL proteins. At high signal, MazF remains active, promoting lysogeny without harming the bacterial host. MazF cleavage sites are enriched on transcripts of phage lytic genes but absent from the phage repressor in ϕ3T and other Spß-like phages. Combined with low activation levels of MazF during infections, this pattern explains the phage-specific effect. Our results show how a bacterial toxin-antitoxin system has been co-opted by a phage to control lysis/lysogeny decisions without compromising host viability.

Collaborative study for the establishment of Ph. Eur. Biological Reference Preparation for Human tetanus immunoglobulin batch 2.

This publication describes the outcome of a project to develop a replacement European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for Human tetanus immunoglobulin (TIg) as well as for the World Health Organization (WHO) International Standard (IS) for Tetanus Immunoglobulin, Human. Bulk TIg was kindly provided by a European manufacturer and was used to prepare the candidate standard. The candidate standard was freeze-dried and calibrated in an international collaborative study jointly co-ordinated by the Medicines & Healthcare products Regulatory Agency (MHRA) and the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe). The results of this study show that there was good agreement between laboratories for the potency estimates obtained for the candidate standard relative to the current WHO IS/Ph. Eur. BRP. The study also demonstrated that the candidate standard is suitable for use in Ph. Eur. assays for potency testing of TIg products and there was good agreement in the potency estimates obtained using the different assay methods included in the study. Accelerated degradation studies performed at the MHRA over a period of 4 years suggest that the freeze-dried candidate standard will be very stable. The candidate standard was established as Ph. Eur. BRP for Human tetanus immunoglobulin, batch 2 with an assigned potency of 45 IU/ampoule. The same preparation was also adopted by the WHO Expert Committee on Biological Standardization (ECBS) to serve as the WHO 2nd IS for Tetanus Immunoglobulin, Human (13/240).

The Chlamydia-related Waddlia chondrophila encodes functional type II toxin-antitoxin systems.

Bacterial toxin-antitoxin (TA) systems are widespread in chromosomes and plasmids of free-living microorganisms, but only a few have been identified in obligate intracellular species. We found seven putative type II TA modules in Waddlia chondrophila, a Chlamydia-related species that is able to infect a very broad series of eukaryotic hosts, ranging from protists to mammalian cells. The RNA levels of Waddlia TA systems are significantly upregulated by iron starvation and novobiocin, but they are not affected by antibiotics such as ß-lactams and glycopeptides, which suggests different mechanisms underlying stress responses. Five of the identified TA modules, including HigBA1 and MazEF1, encoded on the Waddlia cryptic plasmid, proved to be functional when expressed in a heterologous host. TA systems have been associated with the maintenance of mobile genetic elements, bacterial defense against bacteriophages, and persistence upon exposure to adverse conditions. As their RNA levels are upregulated upon exposure to adverse conditions, Waddlia TA modules may be involved in survival to stress. Moreover, as Waddlia can infect a wide range of hosts including free-living amoebae, TA modules could also represent an innate immunity system to fight against bacteriophages and other microorganisms with which Waddlia has to share its replicative niche.IMPORTANCEThe response to adverse conditions, such as exposure to antibiotics, nutrient starvation and competition with other microorganisms, is essential for the survival of a bacterial population. TA systems are modules composed of two elements, a toxic protein and an antitoxin (protein or RNA) that counteracts the toxin. Although many aspects of TA biological functions still await to be elucidated, TAs have often been implicated in bacterial response to stress, including the response to nutrient starvation, antibiotic treatment and bacteriophage infection. TAs are ubiquitous in free-living bacteria but rare in obligate intracellular species such as chlamydiae. We identified functional TA systems in Waddlia chondrophila, a chlamydial species with a strikingly broad host range compared to other chlamydiae. Our work contributes to understand how obligate intracellular bacteria react to adverse conditions that might arise from competition with other viruses/bacteria for the same replicative niche and would threaten their ability to replicate.

Toxin:antitoxin ratio sensing autoregulation of the Vibrio cholerae parDE2 module.

The parDE family of toxin-antitoxin (TA) operons is ubiquitous in bacterial genomes and, in Vibrio cholerae, is an essential component to maintain the presence of chromosome II. Here, we show that transcription of the V. cholerae parDE2 (VcparDE) operon is regulated in a toxin:antitoxin ratio-dependent manner using a molecular mechanism distinct from other type II TA systems. The repressor of the operon is identified as an assembly with a 6:2 stoichiometry with three interacting ParD2 dimers bridged by two ParE2 monomers. This assembly docks to a three-site operator containing 5'- GGTA-3' motifs. Saturation of this TA complex with ParE2 toxin results in disruption of the interface between ParD2 dimers and the formation of a TA complex of 2:2 stoichiometry. The latter is operator binding-incompetent as it is incompatible with the required spacing of the ParD2 dimers on the operator.

Production and purification of Clostridium perfringens type C beta-toxin and IgG and IgY antitoxins.

OBJECTIVES: This study aimed to produce and purify Clostridium perfringens type C beta-toxin, sheep anti-beta toxin immunoglobulin G (IgG) and chicken immunoglobulin Y (IgY). METHODS: Two methods were used for beta-toxin purification: single-step metal affinity chromatography (MAC) using zinc as a chelator and ion exchange chromatography (IEX). The purified and inactivated beta-toxoids were then administered to sheep and chickens in order to produce IgG and IgY. RESULTS: All assays using the IEX failed. In contrast, MAC purified more than 21 mg of toxin per run in a single-step protocol. The purified and inactivated beta-toxoids were then administered to sheep and chickens, and IgG and IgY were purified with a high yield, medium antibody titer of 50 IU/mL, and high avidity (73.2 %). CONCLUSIONS: C. perfringens type C beta-toxin and sheep or chicken anti-beta toxin IgG and IgY antibodies were successfully produced and purified using a simple protocol. This protocol can be used for the production of components used in the diagnosis and research of necrotic enteritis caused by C. perfringens type C, as well as for the evaluation of existing vaccines and the development of new preventive methods against this disease.

Toxin/antitoxin systems induce persistence and work in concert with restriction/modification systems to inhibit phage.

IMPORTANCE: To date, there are no reports of phage infection-inducing persistence. Therefore, our results are important since we show for the first time that a phage-defense system, the MqsRAC toxin/antitoxin system, allows the host to survive infection by forming persister cells, rather than inducing cell suicide. Moreover, we demonstrate that the MqsRAC system works in concert with restriction/modification systems. These results imply that if phage therapy is to be successful, anti-persister compounds need to be administered along with phages.

The crystal structure of insecticidal protein Txp40 from Xenorhabdus nematophila reveals a two-domain unique binary toxin with homology to the toxin-antitoxin (TA) system.

Txp40 is a ubiquitous, conserved, and novel toxin from Xenorhabdus and Photorhabdus bacteria, toxic to a wide range of insect pests. However, the three-dimensional structure and toxicity mechanism for Txp40 or any of its sequence homologs are not yet known. Here, we are reporting the crystal structure of the insecticidal protein Txp40 from Xenorhabdus nematophila at 2.08 Å resolution. The Txp40 was structurally distinct from currently known insecticidal proteins. Txp40 consists of two structurally different domains, an N-terminal domain (NTD) and a C-terminal domain (CTD), primarily joined by a 33-residue long linker peptide. Txp40 displayed proteolytic propensity. Txp40 gets proteolyzed, removing the linker peptide, which is essential for proper crystal packing. NTD adopts a novel fold composed of nine amphipathic helices and has no shared sequence or structural homology to any known proteins. CTD has structural homology with RNases of type II toxin-antitoxin (TA) complex belonging to the RelE/ParE toxin domain superfamily. NTD and CTD were individually toxic to Galleria mellonella larvae. However, maximal toxicity was observed when both domains were present. Our results suggested that the Txp40 acts as a two-domain binary toxin, which is unique and different from any known binary toxins and insecticidal proteins. Txp40 is also unique because it belongs to the prokaryotic RelE/ParE toxin family with a toxic effect on eukaryotic organisms, in contrast to other members of the same family. Broad insect specificity and unique binary toxin complex formation make Txp40 a viable candidate to overcome the development of resistance in insect pests.

Evaluation of different strategies to produce Vibrio cholerae ParE2 toxin.

Toxin-antitoxin (TA) systems are small operons that are omnipresent in bacteria and archaea with suggested roles in stabilization of mobile genetic elements, bacteriophage protection, stress response and possibly persister formation. A major bottleneck in the study of TA toxins is the production of sufficient amounts of well-folded, functional protein. Here we examine alternative approaches for obtaining the VcParE2 toxin from Vibrio cholerae. VcParE2 can be successfully produced via bacterial expression in presence of its cognate antitoxin VcParD2, followed by on-column unfolding and refolding. Alternatively, the toxin can be expressed in Spodoptera frugiperda (Sf9) insect cells. The latter requires disruption of the VcparE2 gene via introduction of an insect cell intron. Both methods provide protein with similar structural and functional characteristics.

Standardization of the Japanese National Standard, Equine Botulinum Antitoxin Type A, and Factors Affecting Standardization.

Equine botulinum antitoxin is one of the most popular countermeasures for human botulism. The unitage of the antitoxin product is defined according to national minimum requirement or pharmacopoeia in each country by referring to national standard antitoxins for four types (A, B, E, and F). With the expected depletion of the national standard antitoxins, replacement national standard antitoxins are produced and standardized through collaboration of the National Control Laboratory and other participants, including manufacturer(s). Therefore, Japanese National Standard Botulinum Antitoxin Type A, Equine, was replaced according to the results of a collaborative study involving the National Institute of Infectious Diseases and KM Biologics Co., Ltd. The unitage of the replacement material was determined through mouse neutralization tests, which involved toxin-antitoxin mixture injection at pH 7.0. Potency value of 440 units/vial was obtained. However, the Japanese Minimum Requirement for Biological Products was revised, and the neutralization reactions were repeated at pH 6.0, for which considerably different potency value (656 units/vial) and survival profile of mice were obtained. In September 2021, the replacement material, Japanese National Standard Botulinum Antitoxin Type A, Equine, lot 2, was established with potency value of 656 Units/vial. The impact of pH-dependent change in potency on antitoxin quality control is discussed.