Bacteria encode a wide range of survival and immunity systems, including CRISPR-Cas, restriction-modification systems, and toxin-antitoxin systems involved in defence against bacteriophages, as well as survival during challenging growth conditions or exposure to antibiotics. Toxin-antitoxin (TA) systems are small two- or three-gene cassettes consisting of a metabolic regulator (the \"toxin\") and its associated antidote (the \"antitoxin\"), which also often functions as a transcriptional regulator. TA systems are widespread in the genomes of pathogens but are also present in commensal bacterial species and on plasmids. For mobile elements such as plasmids, TA systems play a role in maintenance, and increasing evidence now points to roles of chromosomal toxin-antitoxin systems in anti-phage defence. Moreover, the widespread occurrence of toxin-antitoxin systems in the genomes of pathogens has been suggested to relate to survival during host infection as well as in persistence during antibiotic treatment. Upon repeated exposure to antibiotics, TA systems have been shown to acquire point mutations as well as more dramatic rearrangements such as in-frame deletions with potential relevance for bacterial survival and pathogenesis. In this review, we present an overview of the known functional and structural consequences of mutations and rearrangements arising in bacterial toxin-antitoxin systems and discuss their relevance for survival and persistence of pathogenic species.

The epidermal growth factor receptor (EGFR) is an important target for cancer therapies. Many head and neck cancer (HNC) cells have been reported to overexpress EGFR; therefore, anti-EGFR therapies have been attempted in patients with HNC. However, its clinical efficacy is limited owing to the development of drug resistance. In this study, we developed an EGFR-targeting immunotoxin consisting of a clinically proven anti-EGFR IgG (cetuximab; CTX) and a toxin fragment (LR-LO10) derived from Pseudomonas exotoxin A (PE) using a novel site-specific conjugation technology (peptide-directed photo-crosslinking reaction), as an alternative option. The immunotoxin (CTX-LR-LO10) showed specific binding to EGFR and properties of a typical IgG, such as stability, interactions with receptors of immune cells, and pharmacokinetics, and inhibited protein synthesis via modification of elongation factor-2. Treatment of EGFR-positive HNC cells with the immunotoxin resulted in apoptotic cell death and the inhibition of cell migration and invasion. The efficacy of CTX-LR-LO10 was evaluated in xenograft mouse models, and the immunotoxin exhibited much stronger tumor suppression than CTX or LR-LO10. Transcriptome analyses revealed that the immunotoxins elicited immune responses and altered the expression of genes related to its mechanisms of action. These results support the notion that CTX-LR-LO10 may serve as a new therapeutic agent targeting EGFR-positive cancers.

The genome of Mycobacterium tuberculosis encodes for a large repertoire of toxin-antitoxin systems. In the present study, MenT3 and MenT4 toxins belonging to MenAT subfamily of TA systems have been functionally characterized. We demonstrate that ectopic expression of these toxins inhibits bacterial growth and this is rescued upon co-expression of their cognate antitoxins. Here, we show that simultaneous deletion of menT3 and menT4 results in enhanced susceptibility of M. tuberculosis upon exposure to oxidative stress and attenuated growth in guinea pigs and mice. We observed reduced expression of transcripts encoding for proteins that are essential or required for intracellular growth in mid-log phase cultures of ΔmenT4ΔT3 compared to parental strain. Further, the transcript levels of proteins involved in efficient bacterial clearance were increased in lung tissues of ΔmenT4ΔT3 infected mice relative to parental strain infected mice. We show that immunization of mice and guinea pigs with ΔmenT4ΔT3 confers significant protection against M. tuberculosis infection. Remarkably, immunization of mice with ΔmenT4ΔT3 results in increased antigen-specific TH1 bias and activated memory T cell response. We conclude that MenT3 and MenT4 are important for M. tuberculosis pathogenicity and strains lacking menT3 and menT4 have the potential to be explored further as vaccine candidates.

Type I toxin-antitoxin systems (T1TAs) are bipartite bacterial loci encoding a growth-inhibitory toxin and an antitoxin small RNA (sRNA). In many of these systems, the transcribed toxin mRNA is translationally inactive, but becomes translation-competent upon ribonucleolytic processing. The antitoxin sRNA targets the processed mRNA to inhibit its translation. This two-level control mechanism prevents cotranscriptional translation of the toxin and allows its synthesis only when the antitoxin is absent. Contrary to this, we found that the timP mRNA of the timPR T1TA locus does not undergo enzymatic processing. Instead, the full-length timP transcript is both translationally active and can be targeted by the antitoxin TimR. Thus, tight control in this system relies on a noncanonical mechanism. Based on the results from in vitro binding assays, RNA structure probing, and cell-free translation experiments, we suggest that timP mRNA adopts mutually exclusive structural conformations. The active form uniquely possesses an RNA pseudoknot structure which is essential for translation initiation. TimR preferentially binds to the active conformation, which leads to pseudoknot destabilization and inhibited translation. Based on this, we propose a model in which \"structural processing\" of timP mRNA enables tight inhibition by TimR in nonpermissive conditions, and TimP synthesis only upon TimR depletion.

Trueperella pyogenes is an important opportunistic pathogenic bacterium widely distributed in the environment. Pyolysin (PLO) is a primary virulence factor of T. pyogenes and capable of lysing many different cells. PLO is a member of the cholesterol-dependent cytolysin (CDC) family of which the primary structure only presents a low level of homology with other members from 31% to 45%. By deeply studying PLO, we can understand the overall pathogenic mechanism of CDC family proteins. This study established a mouse muscle tissue model infected with recombinant PLO (rPLO) and its single-point mutations, rPLO N139K and rPLO F240A, and explored its mechanism of causing inflammatory damage. The inflammatory injury abilities of rPLO N139K and rPLO F240A are significantly reduced compared to rPLO. This study elaborated on the inflammatory mechanism of PLO by examining its unit point mutations in detail. Our data also provide a theoretical basis and practical significance for future research on toxins and bacteria.

The pregnane X receptor (PXR) is a nuclear hormone receptor that plays a pivotal role in regulating gene expression in response to various ligands, particularly xenobiotics. In this context, the aim of this study was to shed light on the ligand affinity and functions of four NR1J1 paralogs identified in the marine mussel Mytilus galloprovincialis, employing a dual-luciferase reporter assay. To achieve this, the activation patterns of these paralogs in response to various toxins, including freshwater cyanotoxins (Anatoxin-a, Cylindrospermopsin, and Microcystin-LR, -RR, and -YR) and marine algal toxins (Nodularin, Saxitoxin, and Tetrodotoxin), alongside natural compounds (Saint John\'s Wort, Ursolic Acid, and 8-Methoxypsoralene) and microalgal extracts (Tetraselmis, Isochrysis, LEGE 95046, and LEGE 91351 extracts), were studied. The investigation revealed nuanced differences in paralog response patterns, highlighting the remarkable sensitivity of MgaNR1J1γ and MgaNR1J1δ paralogs to several toxins. In conclusion, this study sheds light on the intricate mechanisms of xenobiotic metabolism and detoxification, particularly focusing on the role of marine mussel NR1J1 in responding to a diverse array of compounds. Furthermore, comparative analysis with human PXR revealed potential species-specific adaptations in detoxification mechanisms, suggesting evolutionary implications. These findings deepen our understanding of PXR-mediated metabolism mechanisms, offering insights into environmental monitoring and evolutionary biology research.

Single-domain antibodies (sdAbs), such as VHHs, are increasingly being developed for gastrointestinal (GI) applications against pathogens to strengthen gut health. However, what constitutes a suitable developability profile for applying these proteins in a gastrointestinal setting remains poorly explored. Here, we describe an in vitro methodology for the identification of sdAb derivatives, more specifically divalent VHH constructs, that display extraordinary developability properties for oral delivery and functionality in the GI environment. We showcase this by developing a heterodivalent VHH construct that cross-inhibits the toxic activity of the glycosyltransferase domains (GTDs) from three different toxinotypes of cytotoxin B (TcdB) from lineages of Clostridium difficile. We show that the VHH construct possesses high stability and binding activity under gastric conditions, in the presence of bile salts, and at high temperatures. We suggest that the incorporation of early developability assessment could significantly aid in the efficient discovery of VHHs and related constructs fit for oral delivery and GI applications.

The rise in cyanobacterial blooms due to eutrophication and climate change has increased cyanotoxin presence in water. Most current water treatment plants do not effectively remove these toxins, posing a potential risk to public health. This study introduces a water treatment approach using nanostructured beads containing magnetic nanoparticles (MNPs) for easy removal from liquid suspension, coated with different adsorbent materials to eliminate cyanotoxins. Thirteen particle types were produced using activated carbon, CMK-3 mesoporous carbon, graphene, chitosan, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidised cellulose nanofibers (TOCNF), esterified pectin, and calcined lignin as an adsorbent component. The particles\' effectiveness for detoxification of microcystin-LR (MC-LR), cylindrospermopsin (CYN), and anatoxin-A (ATX-A) was assessed in an aqueous solution. Two particle compositions presented the best adsorption characteristics for the most common cyanotoxins. In the conditions tested, mesoporous carbon nanostructured particles, P1-CMK3, provide good removal of MC-LR and Merck-activated carbon nanostructured particles, P9-MAC, can remove ATX-A and CYN with high and fair efficacy, respectively. Additionally, in vitro toxicity of water treated with each particle type was evaluated in cultured cell lines, revealing no alteration of viability in human renal, neuronal, hepatic, and intestinal cells. Although further research is needed to fully characterise this new water treatment approach, it appears to be a safe, practical, and effective method for eliminating cyanotoxins from water.

Clostridioides difficile, a Gram-positive anaerobic bacterium, is the leading cause of hospital-acquired antibiotic-associated diarrhea worldwide. The severity of C. difficile infection (CDI) varies, ranging from mild diarrhea to life-threatening conditions such as pseudomembranous colitis and toxic megacolon. Central to the pathogenesis of the infection are toxins produced by C. difficile, with toxin A (TcdA) and toxin B (TcdB) as the main virulence factors. Additionally, some strains produce a third toxin known as C. difficile transferase (CDT). Toxins damage the colonic epithelium, initiating a cascade of cellular events that lead to inflammation, fluid secretion, and further tissue damage within the colon. Mechanistically, the toxins bind to cell surface receptors, internalize, and then inactivate GTPase proteins, disrupting the organization of the cytoskeleton and affecting various Rho-dependent cellular processes. This results in a loss of epithelial barrier functions and the induction of cell death. The third toxin, CDT, however, functions as a binary actin-ADP-ribosylating toxin, causing actin depolymerization and inducing the formation of microtubule-based protrusions. In this review, we summarize our current understanding of the interaction between C. difficile toxins and host cells, elucidating the functional consequences of their actions. Furthermore, we will outline how this knowledge forms the basis for developing innovative, toxin-based strategies for treating and preventing CDI.

The pathophysiology of Lyme disease, especially in its persistent form, remains to be determined. As many of the neurologic symptoms are similar to those seen in other toxin-associated disorders, a hypothesis was generated that B. burgdorferi, the causative agent of Lyme disease, may produce a neurotoxin to account for some of the symptoms. Using primers against known conserved bacterial toxin groups, and PCR technology, a candidate neurotoxin was discovered. The purified protein was temporarily named BbTox, and was subsequently found to be identical to BB0755, a protein deduced from the genome sequence of B. burgdorferi that has been annotated as a Z ribonuclease. BbTox has cytotoxic activity against cells of neural origin in tissue culture. Its toxic activity appears to be directed against cytoskeletal elements, similar to that seen with toxins of Clostridioides difficile and Clostridioides botulinum, but differing from that of cholera and E. coli toxins, and other toxins. It remains to be determined whether BbTox has direct cytotoxic effects on neural or glial cells in vivo, or its activity is primarily that of a ribonuclease analogous to other bacterial ribonucleases that are involved in antibiotic tolerance remains to be determined.