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Wolbachia bacteria encompass noteworthy reproductive manipulators of their arthropod hosts. which influence host reproduction to favour their own transmission, also exploiting toxin-antitoxin systems. Recently, multiple other bacterial symbionts of arthropods have been shown to display comparable manipulative capabilities. Here, we wonder whether such phenomena are truly restricted to arthropod hosts. We focused on protists, primary models for evolutionary investigations on eukaryotes due to their diversity and antiquity, but still overall under-investigated. After a thorough re-examination of the literature on bacterial-protist interactions with this question in mind, we conclude that such bacterial 'addictive manipulators' of protists do exist, are probably widespread, and have been overlooked until now as a consequence of the fact that investigations are commonly host-centred, thus ineffective to detect such behaviour. Additionally, we posit that toxin-antitoxin systems are crucial in these phenomena of addictive manipulation of protists, as a result of recurrent evolutionary repurposing. This indicates intriguing functional analogy and molecular homology with plasmid-bacterial interplays. Finally, we remark that multiple addictive manipulators are affiliated with specific bacterial lineages with ancient associations with diverse eukaryotes. This suggests a possible role of addictive manipulation of protists in paving the way to the evolution of bacteria associated with multicellular organisms.

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Bacteria and their viral predators (phages) are constantly evolving to subvert one another. Many bacterial immune systems that inhibit phages are encoded on mobile genetic elements that can be horizontally transmitted to diverse bacteria. Despite the pervasive appearance of immune systems in bacteria, it is not often known if these immune systems function against phages that the host encounters in nature. Additionally, there are limited examples demonstrating how these phages counter-adapt to such immune systems. Here, we identify clinical isolates of the global pathogen Vibrio cholerae harboring a novel genetic element encoding the bacterial immune system DarTG and reveal the immune system's impact on the co-circulating lytic phage ICP1. We show that DarTG inhibits ICP1 genome replication, thus preventing ICP1 plaquing. We further characterize the conflict between DarTG-mediated defense and ICP1 by identifying an ICP1-encoded protein that counters DarTG and allows ICP1 progeny production. Finally, we identify this protein, AdfB, as a functional antitoxin that abrogates the toxin DarT likely through direct interactions. Following the detection of the DarTG system in clinical V. cholerae isolates, we observed a rise in ICP1 isolates with the functional antitoxin. These data highlight the use of surveillance of V. cholerae and its lytic phages to understand the co-evolutionary arms race between bacteria and their phages in nature.IMPORTANCEThe global bacterial pathogen Vibrio cholerae causes an estimated 1 to 4 million cases of cholera each year. Thus, studying the factors that influence its persistence as a pathogen is of great importance. One such influence is the lytic phage ICP1, as once infected by ICP1, V. cholerae is destroyed. To date, we have observed that the phage ICP1 shapes V. cholerae evolution through the flux of anti-phage bacterial immune systems. Here, we probe clinical V. cholerae isolates for novel anti-phage immune systems that can inhibit ICP1 and discover the toxin-antitoxin system DarTG as a potent inhibitor. Our results underscore the importance of V. cholerae and ICP1 surveillance to elaborate novel means by which V. cholerae can persist in both the human host and aquatic reservoir in the face of ICP1.

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Objective: Persister cells are a specific subset of bacteria capable of surviving exposure to lethal doses of antibiotics, leading to antibiotic therapy failures and infection relapses. This research explores the utilization of drug repositioning to target the Lon protease in Salmonella Typhimurium. Method: In this study, FDA-approved drugs sourced from the Drug Bank database were screened to identify existing pharmaceuticals with the potential to combat the Lon protease. The formation of persister cells in the presence of antibiotics, as well as the combination of antibiotics with potential Lon protease inhibitors, was examined. Furthermore, the expression of type II toxin-antitoxin system genes was analyzed to enhance our comprehension of the inhibitors' effects. Result: Molecular docking analysis revealed that Diosmin and Nafcillin exhibited strong binding affinity to the Lon protease. Molecular dynamics simulation trajectories analysis demonstrated that the interaction of these ligands with the enzyme did not induce instability; rather, the enzyme's structure remained stable. Combinations of ceftazidime and ciprofloxacin with either Nafcillin or Diosmin led to significant reductions in bacterial cell counts. Furthermore, the effectiveness of these combinations, when compared to antibiotics alone, highlighted the substantial impact of Nafcillin and Diosmin in reducing type II TA system gene expression. Conclusion: These findings suggest promising prospects for developing novel therapeutic approaches targeting persister cells to mitigate treatment failures in Salmonella infections.

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The toxin-antitoxin (TA) system regulates many physiological processes in free-living bacteria. One such TA system in Escherichia coli comprises an RNA toxin SdsR and an antitoxin RyeA. An overabundance of SdsR is toxic to the cells. RyeA normalizes SdsR abundance and helps the cells to adapt to altered conditions. The current study showed that a novel small RNA (sRNA) regulator GcvB directly interacts with RyeA to maintain its abundance in the cells under normal or low pH conditions. The deletion of the gcvB allele in the E. coli chromosome resulted in a ∼3-fold decrease in intrabacterial RyeA accumulation. An ectopic expression of GcvB in ΔgcvB strain reinstated RyeA abundance to its normal level. Induction of GcvB in the cells upon exposure to low pH resulted in a simultaneous increase in intracellular RyeA. While GcvB increases RyeA abundance in the cells, SdsR accumulation is divergently regulated by GcvB. The absence of the gcvB gene in E. coli leads to upregulation of SdsR and vice versa. The GcvB-mediated decrease of SdsR accumulation stems from the increased RyeA-driven normalization of SdsR. This study delineates a novel mechanism for the regulation of the expression of an RNA toxin SdsR by another sRNA regulator GcvB through a feed-forward control.

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The CcdAB system expressed in the E.coli cells is a prototypical example of the bacterial toxin-antitoxin (TA) systems that ensure the survival of the bacterial population under adverse environmental conditions. The solution and crystal structures of CcdA, CcdB and of CcdB in complex with the toxin-binding C-terminal domain of CcdA have been reported. Our interest lies in the dynamics of CcdB-CcdA complex formation. Solution NMR studies have shown that CcdB_G100T, in presence of saturating concentrations of CcdA-c, a truncated C-terminal fragment of CcdA exists in equilibrium between two major populations. Sequence specific backbone resonance assignments of both equilibrium forms of the ~ 27 kDa complex, have been obtained from a suite of triple resonance NMR experiments acquired on 2H, 13C, 15N enriched samples of CcdB_G100T. Analysis of 1H, 13Cα, 13Cß secondary chemical shifts, shows that both equilibrium forms of CcdB_G100T have five beta-strands and one alpha-helix as the major secondary structural elements in the tertiary structure. The results of these studies are presented below.

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Botulism, caused by Clostridium botulinum, continues to pose a significant threat to public health. This review explores the historical context and contemporary relevance of botulism, emphasizing its potential lethality and evolving medical applications. In recent years, the use of botulinum toxin in medical procedures, particularly in cosmetic and therapeutic applications, has increased the risk of iatrogenic botulism. The rise in iatrogenic cases underscores the importance of vigilance among health-care providers, especially those in emergency departments, where prompt diagnosis and intervention are critical. This review underscores the necessity for health-care professionals to consider botulism in the differential diagnosis of patients presenting with relevant symptoms, given the potential severity of the condition.

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Mycobacterium indicus pranii (MIP), a benign saprophyte with potent immunomodulatory attributes, holds a pivotal position in mycobacterial evolution, potentially serving as the precursor to the pathogenic Mycobacterium avium complex (MAC). Despite its established immunotherapeutic efficacy against leprosy and notable outcomes in gram-negative sepsis and COVID-19 cases, the genomic and biochemical features of MIP remain largely elusive. This study explores the uncharted territory of toxin-antitoxin (TA) systems within MIP, hypothesizing their role in mycobacterial pathogenicity regulation. Genome-wide screening, employing diverse databases, unveils putative TA modules in MIP, setting the stage for a comparative analysis with known modules in Mycobacterium tuberculosis, Mycobacterium smegmatis, Escherichia coli, and Vibrio cholerae. The study further delves into the TA network of MAC and Mycobacterium intracellulare, unraveling interactive properties and family characteristics of identified TA modules in MIP. This comprehensive exploration seeks to illuminate the contribution of TA modules in regulating virulence, habitat diversification, and the evolutionary pathogenicity of mycobacteria. The insights garnered from this investigation not only enhance our understanding of MIP's potential as a vaccine candidate but also hold promise in optimizing tuberculosis drug regimens for expedited recovery.

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Background: The toxin-antitoxin (TA) system plays a vital role in the virulence and pathogenicity of Mycobacterium tuberculosis (M. tuberculosis). However, the regulatory mechanisms and the impact of gene mutations on M. tuberculosis transmission remain poorly understood. Objective: To investigate the influence of gene mutations in the toxin-antitoxin system on M. tuberculosis transmission dynamics. Method: We performed whole-genome sequencing on the analyzed strains of M. tuberculosis. The genes associated with the toxin-antitoxin system were obtained from the National Center for Biotechnology Information (NCBI) Gene database. Mutations correlating with enhanced transmission within the genes were identified by using random forest, gradient boosting decision tree, and generalized linear mixed models. Results: A total of 13,518 M. tuberculosis isolates were analyzed, with 42.29% (n = 5,717) found to be part of genomic clusters. Lineage 4 accounted for the majority of isolates (n = 6488, 48%), followed by lineage 2 (n = 5133, 37.97%). 23 single nucleotide polymorphisms (SNPs) showed a positive correlation with clustering, including vapB1 G34A, vapB24 A76C, vapB2 T171C, mazF2 C85T, mazE2 G104A, vapB31 T112C, relB T226A, vapB11 C54T, mazE5 T344C, vapB14 A29G, parE1 (C103T, C88T), and parD1 C134T. Six SNPs, including vapB6 A29C, vapB31 T112C, parD1 C134T, vapB37 G205C, Rv2653c A80C, and vapB22 C167T, were associated with transmission clades across different countries. Notably, our findings highlighted the positive association of vapB6 A29C, vapB31 T112C, parD1 C134T, vapB37 G205C, vapB19 C188T, and Rv2653c A80C with transmission clades across diverse regions. Furthermore, our analysis identified 32 SNPs that exhibited significant associations with clade size. Conclusion: Our study presents potential associations between mutations in genes related to the toxin-antitoxin system and the transmission dynamics of M. tuberculosis. However, it is important to acknowledge the presence of confounding factors and limitations in our study. Further research is required to establish causation and assess the functional significance of these mutations. These findings provide a foundation for future investigations and the formulation of strategies aimed at controlling TB transmission.

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The use of the serum or plasma of patients or animals who have recovered from an infectious disease, or had been immunized with a relevant antigen, to treat or prevent the same infection in others began in the late 1880s when French and German scientists uncovered, one step at a time, several of the elements of the immune system's response to infection. A key finding was that the damage caused by some bacteria depends upon their secreted toxins which can be neutralized by biologic agents. Antitoxins to diphtheria and tetanus began to be manufactured in large animals in France, Germany, and the US in the 1890s and were soon being used worldwide. The impact of diphtheria antitoxin on childhood mortality was profound. Shortly after the development of antitoxins, convalescent serum began to be used for its anti-bactericidal properties thus addressing serious infections caused by non-toxin-producing organisms. The effectiveness of antitoxins and antisera was demonstrated by examining mortality rates in hospitals before and after the introduction of antitoxins, by comparisons of treated and untreated patients, by comparing early and late treatment and dosage, by examining vital data mortality trends, and by several randomized and alternate assignment trials. Antitoxins continue to have a role in the rare cases of diphtheria and other conditions largely eradicated by immunization, but serum therapy nearly disappeared from the medical armamentarium with the development of antibiotics in the 1940s. Inasmuch as new human pathogens are now emerging with unprecedented regularity as seen in the recent COVID-19 pandemic, and because specific therapies are unlikely to be available for them, plasma-based antibody therapies are likely to again carve out a niche in infectious disease control.

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Herein, we investigated the toxicity and membrane-permeabilizing capabilities of Lpt and Lpt-like peptides, belonging to type I toxin-antitoxin systems carried by plasmid DNA of Lacticaseibacillus strains. These 29 amino acid peptides are predicted to form α-helical structures with a conserved central hydrophobic sequence and differently charged hydrophilic termini. Like Lpt, the expression of Lpt-like in E. coli induced growth arrest, nucleoid condensation, and cell membrane damage, suggesting membrane interaction as the mode of action. The membrane permeabilization activity of both peptides was evaluated by using liposome leakage assays, dynamic light scattering, and CD spectroscopy. Lpt and Lpt-like showed liposome leakage activity, which did not lead to liposome disruption but depended on peptide concentration. Lpt was generally more effective than Lpt-like, probably due to different physical chemical properties. Leakage was significantly reduced in larger liposomes and increased with negatively charged PCPS liposomes, indicating that electrostatic interactions and membrane curvature influence peptide activity. Contrary to most membrane-active peptides, Lpt an Lpt-like progressively lost their α-helical structure upon interaction with liposomes. Our data are inconsistent with the formation of membrane-spanning peptide pores but support a mechanism relying on the transient failure of the membrane permeability barrier possibly through the formation of "lipid pores".

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Current biotechnology relies on a few well-studied model organisms, such as Escherichia coli and Saccharomyces cerevisiae, for which abundant information and efficient toolkits are available for genetic manipulation, but which lack industrially favorable characteristics. Non-model industrial microorganisms usually do not have effective and/or efficient genome-engineering toolkits, which hampers the development of microbial cell factories to meet the fast-growing bioeconomy. In this study, using the non-model ethanologenic bacterium Zymomonas mobilis as an example, we developed a workflow to mine and temper the elements of restriction-modification (R-M), CRISPR/Cas, toxin-antitoxin (T-A) systems, and native plasmids, which are hidden within industrial microorganisms themselves, as efficient genome-editing toolkits, and established a genome-wide iterative and continuous editing (GW-ICE) system for continuous genome editing with high efficiency. This research not only provides tools and pipelines for engineering the non-model polyploid industrial microorganism Z. mobilis efficiently, but also sets a paradigm to overcome biotechnological limitations in other genetically recalcitrant non-model industrial microorganisms.

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Botulinum toxin (BoNT) is a potent neurotoxin with broad applications in medicine and cosmetics. Cases of botulism toxicity from aesthetic injections have been spiking in recent months across the United States. Here, we present a patient who presented with bulbar paralysis and increasing respiratory distress following BoNT injections performed at her home by an unlicensed aesthetician. The patient presented with ptosis, dysphagia, dysarthria, shortness of breath, and proximal muscle weakness. The patient was given botulism antitoxin and showed marked improvement in the following days. The pathophysiology of iatrogenic botulism is unknown but may be due to improper injections with large doses or direct injection into the bloodstream. Counterfeit BoNT injections have been reported and may include unsafe components or dangerous levels of BoNT that surpass the therapeutic dose.

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Bacterial ubiquitous Toxin-Antitoxin (TA) systems are considered to be important survival mechanisms during stress conditions. In regular environmental conditions, the antitoxin blocks the toxin, whereas during imbalanced conditions, the antitoxin concentration decreases, exposing the bacteria cell to a range of toxic events. The most evident consequence of this disequilibrium is cell growth arrest, which is the reason why TAs are generally described as active in the function of bacterial growth kinetics. Virulence-associated proteins B and C (VapBC) are a family of type II TA system, in which VapC is predicted to display the toxic ribonuclease activity while VapB counteracts this activity. Previously, using in silico data, we designated four VapBC TA modules in Leptospira interrogans serovar Copenhageni, the main etiological agent of human leptospirosis in Brazil. The present study aimed to obtain the proteins and functionally characterize the VapBC-1 module. The expression of the toxin gene vapC in E. coli did not decrease the cell growth rate in broth culture, as was expected to happen within active TA modules. However, interestingly, when the expression of the toxin was compared to that of the complexed toxin and antitoxin, cell viability was strongly affected, with a decrease of three orders of magnitude in colony forming unity (CFU). The assumption of the affinity between the toxin and the antitoxin was confirmed in vivo through the observation of their co-purification from cultivation of E. coli co-expressing vapB-vapC genes. RNAse activity assays showed that VapC-1 cleaves MS2 RNA and ribosomal RNA from L. interrogans. Our results indicate that the VapBC-1 module is a potentially functional TA system acting on targets that involve specific functions. It is very important to emphasize that the common attribution of the functionality of TA modules cannot be defined based merely on their ability to inhibit bacterial growth in a liquid medium.

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Mycobacterium phage Adephagia is a cluster K phage that infects Mycobacterium smegmatis and some strains of Mycobacterium pathogens. Adephagia has a siphoviral virion morphology and is temperate. Its genome is 59,646 bp long and codes for one tRNA gene and 94 predicted protein-coding genes; most genes not associated with virion structure and assembly are functionally ill-defined. Here, we determined the Adephagia gene expression patterns in lytic and lysogenic growth and used structural predictions to assign additional gene functions. We characterized 66 nonstructural genes for their toxic phenotypes when expressed in M. smegmatis, and we show that 25 of these (38%) are either toxic or strongly inhibit growth, resulting in either reduced viability or small colony sizes. Some of these genes are predicted to be involved in DNA metabolism or regulation, but others are of unknown function. We also characterize the HicAB-like toxin-antitoxin (TA) system encoded by Adephagia (gp91 and gp90, respectively) and show that the gp90 antitoxin is lysogenically expressed, abrogates gp91 toxicity, and is required for normal lytic and lysogenic growth.

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Some microbial toxins also target the producer species itself, necessitating a means of self-protection. The M2 double-stranded RNA (dsRNA) killer virus in Saccharomyces cerevisiae contains a single open reading frame (ORF) encoding both the secreted pore-forming toxin K2 as well as a cognate immunity factor. Here, we show that expression of a 49-amino acid N-terminal peptide from the K2 precursor is both necessary and sufficient for immunity. This immunity peptide simultaneously functions as a signal peptide for toxin secretion and protects the cell against the cytotoxic K2 α subunit. The K2 toxin and immunity factor can be functionally separated into two ORFs, yielding a modular toxin-immunity system. This case further shows how a (signal) peptide can carry the potential for providing cellular protection against an antimicrobial toxin.

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Toxin-antitoxin (TA) modules are widely found in the genomes of pathogenic bacteria. They regulate vital cellular functions like transcription, translation, and DNA replication, and are therefore essential to the survival of bacteria under stress. With a focus on the type II parDE modules, this study thoroughly examines TAome in Pseudomonas aeruginosa, a bacterium well-known for its adaptability and antibiotic resistance. We explored the TAome in three P. aeruginosa strains: ATCC 27,853, PAO1, and PA14, and found 15 type II TAs in ATCC 27,853, 12 in PAO1, and 13 in PA14, with significant variation in the associated mobile genetic elements. Five different parDE homologs were found by further TAome analysis in ATCC 27,853, and their relationships were confirmed by sequence alignments and precise genomic positions. After comparing these ParDE modules' sequences to those of other pathogenic bacteria, it was discovered that they were conserved throughout many taxa, especially Proteobacteria. Nucleic acids were predicted as potential ligands for ParD antitoxins, whereas ParE toxins interacted with a wide range of small molecules, indicating a diverse functional repertoire. The interaction interfaces between ParDE TAs were clarified by protein-protein interaction networks and docking studies, which also highlighted important residues involved in binding. This thorough examination improves our understanding of the diversity, evolutionary dynamics, and functional significance of TA systems in P. aeruginosa, providing insights into their roles in bacterial physiology and pathogenicity.

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RNase R (encoded by the rnr gene) is a highly processive 3' → 5' exoribonuclease essential for the growth of the psychrotrophic bacterium Pseudomonas syringae Lz4W at low temperature. The cell death of a rnr deletion mutant at low temperature has been previously attributed to processing defects in 16S rRNA, defective ribosomal assembly, and inefficient protein synthesis. We recently showed that RNase R is required to protect P. syringae Lz4W from DNA damage and oxidative stress, independent of its exoribonuclease activity. Here, we show that the processing defect in 16S rRNA does not cause cell death of the rnr mutant of P. syringae at low temperature. Our results demonstrate that the rnr mutant of P. syringae Lz4W, complemented with a RNase R deficient in exoribonuclease function (RNase RD284A), is defective in 16S rRNA processing but can grow at 4 °C. This suggested that the processing defect in ribosomal RNAs is not a cause of the cold sensitivity of the rnr mutant. We further show that the rnr mutant accumulates copies of the indigenous plasmid pLz4W that bears a type II toxin-antitoxin (TA) system (P. syringae antitoxin-P. syringae toxin). This phenotype was rescued by overexpressing antitoxin psA in the rnr mutant, suggesting that activation of the type II TA system leads to cold sensitivity of the rnr mutant of P. syringae Lz4W. Here, we report a previously unknown functional relationship between the cold sensitivity of the rnr mutant and a type II TA system in P. syringae Lz4W.

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Botulism is a neuroparalytic syndrome resulting from the systemic effects of an exoneurotoxin produced by gram-positive, rod-shaped, spore-forming, obligate anaerobic bacterium Clostridium botulinum. Here, we present the case of a 40-year-old male, presenting with a sudden onset of abdominal pain associated with vomiting. He was admitted for conservative management once the CT of the abdomen and pelvis revealed partial small bowel obstruction with no signs of bowel perforation or ischemia. However, the next day, the patient had a cardiac arrest thought to be secondary to respiratory arrest. The return of spontaneous circulation was achieved after two cycles of cardiopulmonary resuscitation. The patient developed quadriplegia, areflexia, and bilateral ophthalmoplegia. He was empirically treated with pyridostigmine, intravenous immunoglobulin (IVIG), and botulinum antitoxin. Stool polymerase chain reaction (PCR) testing resulted positive for C. botulinum toxin type F. The patient ultimately recovered with botulinum antitoxin and a month of physical and speech therapy. Our case highlights that clinicians should consider botulism as a differential and emphasize the importance of early diagnosis for effective management and prognosis.

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The application of nanotechnology has significantly advanced the development of novel platforms that enhance disease treatment and diagnosis. A key innovation in this field is the creation of antitoxin nanoparticles (ATNs), designed to address toxin exposure. These precision-engineered nanosystems have unique physicochemical properties and selective binding capabilities, allowing them to effectively capture and neutralize toxins from various biological, chemical, and environmental sources. In this review, we thoroughly examine their therapeutic and diagnostic potential for managing toxin-related challenges. We also explore recent advancements and offer critical insights into the design and clinical implementation of ATNs.

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