Search Results (176)

Hemolytic Uremic Syndrome (HUS) is a severe clinical manifestation primarily triggered by Shiga toxin-producing Escherichia coli (STEC). While Shiga toxins (Stx) are central to the development of systemic endothelial damage, current recombinant antibody developments have overwhelmingly focused on neutralizing the Stx2 subtype. However, numerous STEC isolates produce Stx1 either independently or alongside Stx2, revealing a critical need to diversify the antibody repertoire for comprehensive antitoxin therapies. To address this, we characterized two novel, fully human recombinant Fabs targeting Stx1 (FabB6:Stx1 and FabC8:Stx1) selected from a synthetic library via phage display. We evaluated their binding specificity and neutralizing activity in Vero and human proximal tubular epithelial (HK-2) cells, as well as in primary human glomerular endothelial cells (HGEC exposed to HUS-derived STEC supernatants. Both Fabs exhibited high specificity and nanomolar affinity for Stx1. Notably, they displayed cell-type-dependent neutralization profiles, with FabC8:Stx1 demonstrating superior and more consistent neutralization in HK-2 cells. Crucially, when evaluated alongside previously characterized anti-Stx2 antibodies (FabC11:Stx1/Stx2 and FabF8:Stx2), the Stx1-specific Fabs conferred complementary protection against clinical STEC isolates. These findings support the inclusion of Stx1-targeting recombinant antibodies into broader multi-toxin neutralization strategies, thereby expanding the therapeutic potential against STEC-associated diseases. Full article

Antibiotic resistance is a growing global health threat. However, its evolution cannot be fully understood without considering antibiotic tolerance and persistence. Persister cells are phenotypic variants that survive lethal antibiotic exposure without heritable resistance, primarily through growth arrest, metabolic slowdown, and stress-adaptive states. Although persistence has been viewed as a transient survival phenomenon, increasing evidence suggests that it may also have a genetic basis by preserving populations during antibiotic-induced bottlenecks and enabling regrowth, mutation, and selection under certain conditions. This review examines the molecular mechanisms underlying persister formation, including toxin–antitoxin systems, stringent-response signaling, ATP depletion, translational arrest, and stress-response networks. We discuss how persistence contributes to antibiotic tolerance in biofilms, host environments, and recurrent infections, and how repeated antibiotic exposure may promote stepwise evolution from phenotypic survival to stable resistance in specific contexts. Evidence from experimental evolution, clinical observations, and system-level analyses supports a potential but context-dependent link between persistence and resistance. We also highlight therapeutic strategies targeting persister cells, including antipersister compounds, metabolic activation, combination therapies, bacteriophages, and alternative approaches. Finally, we outline future research directions, emphasizing single-cell technologies, systems biology, longitudinal clinical studies, and evolution-informed treatment design. A comprehensive understanding of persistence and its evolutionary implications is essential for improving treatment efficacy and limiting the emergence of long-term antibiotic resistance. Full article

Salmonella persistence contributes to infection relapse, chronic carriage, and reduced antibiotic efficacy. Traditionally viewed as dormant subpopulations that passively survive antibiotic exposure, persister cells are now increasingly recognized as dynamic, heterogeneous, and context-dependent physiological states shaped by bacterial regulatory programs and host microenvironmental pressures. This review examines Salmonella persistence from a multiscale perspective. We first clarify key antibiotic survival phenotypes, including resistance, heteroresistance, tolerance, persistence, and viable but non-culturable states. We then discuss how host-derived stressors, such as phagosomal acidification, nutritional restriction, metal perturbation, and reactive oxygen and nitrogen species, promote growth-restricted, persistence-associated bacterial states. At the bacterial level, we summarize stress-response networks involving the stringent response, SOS response, toxin–antitoxin systems, and auxiliary regulators that coordinate metabolic remodeling, growth restriction, and antibiotic survival. At the host level, we highlight how organ reservoirs, immune cell subsets, metabolic cues, and Salmonella-mediated immune niche remodeling shape persistence-associated phenotypes in vivo. Finally, we discuss clinical and translational implications, including endogenous relapse, resistance evolution, and emerging anti-persistence strategies. Together, this review provides a framework for understanding Salmonella persistence as a multiscale, niche-dependent process relevant to recurrent and chronic infection. Full article

Type II toxin–antitoxin (TA) systems are significantly expanded in the Mycobacterium tuberculosis complex; however, the functional role of the VapBC40 system in Mycobacterium bovis (M. bovis) pathogenesis remains poorly characterized. This study aimed to investigate the role of VapBC40 in mycobacterial virulence and evaluate its potential as a target for rational vaccine attenuation. We performed evolutionary analysis and yeast two-hybrid assays to characterize VapBC40 system specificity, conducted in vitro macrophage infection models and in vivo murine studies to assess virulence contribution, and evaluated the immunoprotective efficacy of a VapC40 knockout strain. Evolutionary analysis revealed progressive sequence conservation and stringent homologous pairing specificity within the VapBC40 system. The VapC40 toxin correlates with enhanced intracellular bacterial survival, increased host cell death, and more severe pulmonary pathology with systemic dissemination. Based on these findings, we evaluated the vaccine potential of a vapC40 knockout strain. Immunization with this attenuated strain elicited a Th1 cellular immune response, characterized by enhanced IFN-γ production and increased frequency of CD4⁺IFN-γ⁺ T cells. Upon challenge with virulent M. bovis, the knockout strain conferred superior protection compared to the conventional BCG vaccine, significantly reducing lung pathology and restricting extrapulmonary bacterial dissemination. Although the molecular mechanisms underlying VapC40-mediated effects remain to be fully elucidated, our findings suggest an important role of the VapBC40 system in mycobacterial-host interactions and support its potential as a target for next-generation tuberculosis vaccine development. Full article

Bacterial persistence has been extensively studied as a possible explanation for strain survival under stress; however, in Enterococcus spp., this ability is still an understudied phenomenon. In this study, 40 Enterococcus spp. isolates of human clinical (n = 10), veterinary commensal (n = 10), veterinary clinical (n = 10) and veterinary environmental (hospital surfaces) (n = 10) origins, were exposed to a high concentration of ciprofloxacin. Time–kill curves were established, after which antimicrobial susceptibility profiles were reassessed. Subsequently, the only presumptive persister was selected for Whole-Genome Sequencing, together with one isolate showing no evidence of persister formation. Comparative genomic analyses were conducted to identify genetic variations between exposed and non-exposed isolates and to explore potential genetic determinants associated with persistence. Observed genetic features present in the persister isolate included toxin–antitoxin systems, a cold-shock protein and the tyrosine-type recombinase/integrase XerC, which may represent putative candidates for further investigation. Interestingly, the majority of toxin–antitoxin system-associated genes were found in plasmids. This study represents an important step towards a better understanding of persistence development in Enterococcus spp.; however, validation using other methodologies such as RNA-sequencing is an important next step. Full article

A recent publication proposed that the main biological function of chromosomal toxin–antitoxin systems (TASs) in free-living bacteria is to optimize fitness by mediating K Sensing and Control via a Nutrient-Responsive Cybernetic System. Viable cell density data were consistent with analog (continuous) regulation of population dynamics and cellular physiology throughout the life cycle; however, exactly how bacteria utilize TASs to regulate this was not explained in that publication. Two different concepts of injury have been proposed in the field of microbiology: (1) injury due to external physical and chemical stresses, which lead to sublethal (reversible) or lethal (irreversible) injury depending on the degree of injury, and (2) injury due to internal, self-inflicted stresses mediated by TA toxins. While self-inflicted injury due to TA toxins has been recognized as playing a role in growth arrest and dormancy, which can be reversed by repair, there is little support for TA toxins causing irreversible programmed cell death under normal physiological conditions. The purpose of the present paper was to explain how merging the above two concepts of injury might reveal how TASs optimize the fitness of free-living bacteria under normal physiological conditions by continuously regulating the ratio of injury: repair throughout the life cycle. Full article

Background: Persistence represents a critical evolutionary reservoir for the development of antimicrobial resistance in Acinetobacter baumannii (Ab). Understanding the basal mechanisms that enable this survival strategy is crucial for elucidating how high-risk clones evolve resistance during therapy. Methods: High-dose colistin time-kill assays were performed in ten ST2 clinical colistin-susceptible (COL-S) Carbapenem-Resistant Ab (CRAB) developing in vivo stable and full-colistin resistance to detect persisters. Genomics and basal transcriptomics of chromosomal/plasmid toxin–antitoxin systems (T/As) were performed, as duplicates for each sample, in two ST2 COL-S CRAB to investigate the genomics and basal T/A transcriptomic backgrounds. Results: Phenotypically, all strains showed a persistent subpopulation (~1% survival at 8 h) under 5× COL MIC exposure. Genomics identified 10 type-II and one type-IV T/A systems. Basal transcriptomics revealed active expression patterns mainly of GNAT superfamily T/A systems, with consistently low toxin mRNA levels associated with toxin- or antitoxin-directed asRNAs in chromosomal modules. This architecture defined new dual-combined regulatory models in which asRNAs acted as primary T/A mRNA balance modulators, putatively impacting on the T/A mRNA ratio. Conversely, the plasmid-encoded BrnT/A module showed a highly balanced expression. Conclusions: Our findings revealed, for the first time, the role of the type-II GNAT T/A superfamily as putative molecular switchers via a fine-tuning transcript balance regulation, impacting the transition from a metabolically active cell state to a dormant one in developing colistin persistence and in vivo resistance CRAB. Full article

Brucella anthropi is an emerging opportunistic pathogen characterized by intrinsic resistance to most $\beta$-lactams. However, the acquisition of carbapenem resistance in this species has rarely been documented in environmental, animal, or clinical settings. In this study, a multidrug-resistant strain, SBA01, was isolated from wastewater-irrigated soil. SBA01 exhibited phenotypic resistance to carbapenems and colistin, the latter being independent of mcr genes. Genomic analysis localized bla_IMP-1 on a stable 21 kb plasmid maintained by a Type II toxin–antitoxin system. While non-self-transmissible, this plasmid was mobilized to Escherichia coli and Klebsiella pneumoniae via an unclassified 50 kb helper plasmid. Additionally, a 217 kb prophage-bearing megaplasmid was identified, enhancing genomic plasticity. Genomic screening identified 32 putative virulence determinants, including markers associated with host interaction. Risk profiling indicated an elevated hazard index for SBA01, driven by the convergence of multidrug resistance, cryptic mobilization capacity, and opportunistic survival traits. These findings position B. anthropi as a resilient environmental reservoir for clinically relevant carbapenemases. Expanding surveillance frameworks to include such adaptive hosts is necessary to better evaluate potential occupational exposures at the wastewater–soil interface. Full article

Background: Salmonella is a major zoonotic foodborne pathogen. Conventional poultry vaccines may present limitations in terms of efficacy, safety, and practicality. Objectives: This study focuses on enhancing the immunogenicity and improving the safety of a novel oral vaccination employing inducible toxin–antitoxin (TA) systems, which lead to self-destruction of virulent Salmonella Enteritidis. Methods: A Hok/Sok (HS) TA system was designed to induce cell death upon absence of arabinose. Point mutations were introduced to the Hok toxin promoter to moderate toxin production. A combination of HS and CeaB/CeiB (CC) TA systems was designed to induce cell death both in low di-cation levels or anaerobic conditions. Survival of Salmonella-carrying TA systems was tested in culture and in the Raw264.7 macrophage cell line. One-day old chicks were inoculated with Salmonella carrying the TA system to evaluate bacterial persistence and induction of a protective immune response. Results: Attenuation of the Hok toxin promoter prolonged bacterial survival in vitro. Salmonella carrying the combined TA systems was eliminated completely both in vitro and in inoculated chickens, eliciting high levels of antibodies and conferring protection against challenge with wild-type Salmonella. Conclusions: These findings highlight the potential of the adaptable TA-based vaccination platform to generate safe and efficacious Salmonella vaccines for poultry, contributing to reduced transmission in the food chain. Full article

Antibiotic persistence is a transient phenotype in which a subset of genetically susceptible bacteria survives lethal antibiotic exposure without acquiring resistance. However, survival alone does not define a persister cell—only cells that successfully recover, resume growth, and produce viable progeny complete the persister cycle. Recent studies in Escherichia coli show that persister awakening is a multistage process shaped by dormancy depth, metabolic state, and antibiotic-induced damage. Upstream induction mechanisms, including stringent-response signaling and toxin–antitoxin–mediated growth arrest, primarily determine dormancy depth but do not directly control awakening kinetics. During the lag phase, persister cells undergo coordinated recovery involving detoxification of residual antibiotics, ATP restoration, dissolution of protein aggregates, and ribosome reactivation. After exposure to fluoroquinolones, awakening additionally requires SOS-driven DNA repair via homologous recombination or transcription-coupled repair. In contrast, β-lactam–exposed persister cells rely mainly on efflux-mediated detoxification and asymmetric damage partitioning. Failure to restore proteostasis or resolve damage results in abortive recovery or cell death. Only after damage processing and metabolic reactivation can persister cells resume division and generate viable progeny. This review integrates current molecular insights into persister cell recovery in E. coli, highlighting the lag phase as the critical barrier between survival and true persistence. Full article

Bacteria encode a broad range of survival and defence systems, including CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas systems, restriction-modification systems, and toxin–antitoxin (TA) systems, which are involved in bacterial regulation and immunity. The traditional view holds that CRISPR-Cas systems and TA systems are two independent defense lines in prokaryotes. However, groundbreaking studies in recent years have revealed multi-level functional coupling between them. This review systematically elaborates on this mechanism, focusing on three types of TA systems that mediate the core correlation of CRISPR-Cas systems: CreTA maintains the evolutionary stability of CRISPR-Cas systems through an addiction mechanism; CreR enables self-regulation of CRISPR-Cas expression; and CrePA provides herd immunity by triggering abortive infection after the CRISPR-Cas system has been destroyed by Anti-CRISPRS protein. Additionally, we discuss the evolutionary homology between the type III toxin AbiF and the type VI CRISPR effector Cas13, offering a new perspective for understanding the origin of CRISPR-Cas systems. These findings not only reveal the functional coupling of prokaryotic defense systems but also provide a powerful theoretical framework and practical solutions for addressing stability challenges in CRISPR technology applications. Full article

Toxin–antitoxin (TA) modules represent sophisticated regulatory networks that have evolved from simple plasmid maintenance factors into multifunctional genetic modules orchestrating bacterial stress responses, pathogenesis, and ecological adaptation. This review highlights a compelling correlation between the abundance of toxin–antitoxin (TA) modules and bacterial pathogenicity, as exemplified by Mycobacterium tuberculosis (M.tb), which encodes 118 TA loci—significantly more than the fewer than 10 found in closely related saprophytic species. The clinical significance of TA modules extends beyond traditional stress response roles to encompass antimicrobial persistence, where systems like VapBC and MazEF facilitate dormant subpopulations that survive antibiotic therapy while maintaining chronic infections. Recent discoveries have revealed TA modules as sophisticated bacterial defense mechanisms against bacteriophage infection, with DarTG and ToxIN systems representing novel antiviral immunity components that complement CRISPR-Cas and restriction–modification systems. The immunomodulatory capacity of TA modules demonstrates their role in host–pathogen interactions, where systems such as VapC12 in M.tb promote macrophage polarization toward permissive M2 phenotypes while inducing anti-inflammatory cytokine production. Large-scale genomic analyses reveal that TA modules function as drivers of horizontal gene transfer networks, with their signatures enabling accurate prediction of plasmid community membership and serving as determinants of microbial community structure. The biotechnological applications of TA modules have expanded to include genetic circuit stabilization, biocontainment device construction, and multi-species microbial community engineering, while therapeutic strategies focus on developing multi-target inhibitors against conserved TA protein families as promising approaches for combating drug-resistant bacterial infections. The evolutionary conservation of TA modules across diverse bacterial lineages underscores their fundamental importance as central organizing principles in bacterial adaptation strategies, where their multifunctional nature reflects complex selective pressures operating across environmental niches and host-associated ecosystems. This review provides an integrated perspective on TA modules as dynamic regulatory elements that support bacterial persistence, immune evasion, and ecological versatility, establishing them as genetic elements with truly “many faces and functions” in prokaryotic biology. Full article

Toxin-antitoxin (TA) systems, ubiquitous in bacterial and archaeal genomes, play pivotal roles in responding to environmental stresses, forming biofilms, defending against phages, and influencing pathogen virulence. The marine environment harbors Earth’s most diverse and abundant microbial communities, where microorganisms have evolved unique genetic adaptations and specialized metabolic processes to thrive amid distinct environmental challenges. Research on the presence and function of TA systems in marine bacteria lags significantly behind that in model bacteria and pathogens. Here, we explored the diversity of the TA system in marine bacteria, including species from the Global Ocean Microbiome Catalogue (GOMC) and the Mariana Trench Environment and Ecology Research (MEER) databases. Our findings revealed that types I to VII (featuring protein toxins) of eight types of TA systems are prevalent in these microorganisms, with unidentified TA combinations diverging from previously characterized systems. Interestingly, some toxins or antitoxins lack canonical counterparts, indicating evolutionary divergence. Additionally, previously uncharacterized potential TA systems have been identified in extremophilic bacteria from the deep-sea Mariana Trench. These results highlight the adaptive importance of marine TA systems, which are likely operating through unconventional mechanisms. Full article

Antibiotic resistance, especially among Gram-negative bacterial strains, places a massive burden on global healthcare systems as resistance development has outpaced antibiotic discovery. Protein–protein interactions, successful in other therapeutic contexts, are emerging as promising, yet underexplored, targets for the development of novel classes of antibacterials. Pathogen-specific protein–protein interactions are attractive targets because they are often structurally and functionally distinct from host proteins and are less likely to elicit rapid resistance. This review summarizes recent developments in targeting protein–protein interactions in Gram-negative bacteria, focusing on the modulation of five critical cellular processes: membrane regulation, replication, transcription, translation, and toxin-antitoxin systems. We highlight the design and discovery of both small-molecule and peptide-based inhibitors. While many identified modulators exhibit potent in vitro activity against their respective targets, achieving effective penetration of the complex Gram-negative cell envelope remains a major challenge. Nevertheless, the diverse and essential nature of these bacteria-specific protein–protein interactions represents an attractive strategy for developing next-generation antimicrobials to combat drug-resistant pathogens. Full article

Live-attenuated vaccines face a critical challenge in balancing immunogenicity with safety. To address this, we engineered programmable finite-replicated organisms (FROs) by depositing a limited number of indispensable components (such as noncanonical amino acids, ncAAs) within the cell, consuming the coenabling precise control of bacterial replication capability while preserving antigenic breadth. Two strategies were adopted to achieve the following purposes: (1) encoding ncAA in essential genes; (2) encoding ncAA in antitoxin of toxin–antitoxin (TA) systems. As noncanonical amino acids, 3,5-dichlorotyrosine (Cl2Y) was encoded by the amber codon (TAG) and inserted into the essential genes (e.g., serS, murG, and dnaA) or antitoxin genes. After optimizing expression and the number of amber codons in the storage genes, the FRO cells can grow up to six generations, achieving amplification approaching 100 times after depletion of the ncAA in the growth medium. The escape frequencies are 10⁻⁵ to 10⁻⁷, which need to be optimized by combining multiple storage genes in the same genome in the future. This work holds the potential to amplify the amounts of antigens for vaccines, potentially accelerating the development of next-generation vaccines against antibiotic-resistant threats. Full article