Inhibitory effect and mechanism of violacein on planktonic growth, spore germination, biofilm formation and toxin production of Bacillus cereus and its application in grass carp preservation.

Bacillus cereus is a ubiquitous foodborne pathogen commonly found in various foods. Its ability to form spores, biofilms and diarrhoeal and/or emetic toxins further exacerbates the risk of food poisoning. Violacein is a tryptophan derivative with excellent antibacterial activity. However, the knowledge on the antibacterial action of violacein against B. cereus was lacking, and thus this study aimed to investigate the antibacterial activity and mechanism. The antibacterial results demonstrated that minimum inhibitory concentration and minimum bactericidal concentration of violacein were 3.125 mg/L and 12.50 mg/L, respectively. Violacein could effectively inhibit planktonic growth, spore germination and biofilm formation of B. cereus (P < 0.001). Meanwhile, violacein significantly downregulated the expression of toxin genes, including nheA (P < 0.05), nheB (P < 0.001), bceT (P < 0.01), cytK (P < 0.001), hblC (P < 0.001) and hblD (P < 0.001). Results of extracellular alkaline phosphatase, nucleotide and protein leakage assays and scanning and transmission electron microscopy observation tests showed violacein destroyed cell walls and membranes of B. cereus. In addition, 6.25 mg/kg of violacein could significantly inhibit B. cereus in grass carp fillets (P < 0.05). These results demonstrate that violacein has great potential as an effective natural antimicrobial preservative to control food contamination and poisoning events caused by B. cereus.

Alpha-hemolysin promotes internalization of Staphylococcus aureus into human lung epithelial cells via caveolin-1- and cholesterol-rich lipid rafts.

Staphylococcus aureus is a pathogen associated with severe respiratory infections. The ability of S. aureus to internalize into lung epithelial cells complicates the treatment of respiratory infections caused by this bacterium. In the intracellular environment, S. aureus can avoid elimination by the immune system and the action of circulating antibiotics. Consequently, interfering with S. aureus internalization may represent a promising adjunctive therapeutic strategy to enhance the efficacy of conventional treatments. Here, we investigated the host-pathogen molecular interactions involved in S. aureus internalization into human lung epithelial cells. Lipid raft-mediated endocytosis was identified as the main entry mechanism. Thus, bacterial internalization was significantly reduced after the disruption of lipid rafts with methyl-ß-cyclodextrin. Confocal microscopy confirmed the colocalization of S. aureus with lipid raft markers such as ganglioside GM1 and caveolin-1. Adhesion of S. aureus to α5ß1 integrin on lung epithelial cells via fibronectin-binding proteins (FnBPs) was a prerequisite for bacterial internalization. A mutant S. aureus strain deficient in the expression of alpha-hemolysin (Hla) was significantly impaired in its capacity to enter lung epithelial cells despite retaining its capacity to adhere. This suggests a direct involvement of Hla in the bacterial internalization process. Among the receptors for Hla located in lipid rafts, caveolin-1 was essential for S. aureus internalization, whereas ADAM10 was dispensable for this process. In conclusion, this study supports a significant role of lipid rafts in S. aureus internalization into human lung epithelial cells and highlights the interaction between bacterial Hla and host caveolin-1 as crucial for the internalization process.

Use of Bacterial Toxin-Antitoxin Systems as Biotechnological Tools in Plants.

Toxin-antitoxin (TA) systems in bacteria are key regulators of the cell cycle and can activate a death response under stress conditions. Like other bacterial elements, TA modules have been widely exploited for biotechnological purposes in diverse applications, such as molecular cloning and anti-cancer therapies. However, their use in plants has been limited, leaving room for the development of new approaches. In this study, we examined two TA systems previously tested in plants, MazEF and YefM-YoeB, and identified interesting differences between them, likely related to their modes of action. We engineered modifications to these specific modules to transform them into molecular switches that can be activated by a protease, inducing necrosis in the plant cells where they are expressed. Finally, we demonstrated the antiviral potential of the modified TA modules by using, as a proof-of-concept, the potyvirus plum pox virus as an activator of the death phenotype.

Refined egoist: The toxin-antitoxin immune system of T6SS.

The Type VI secretory system (T6SS) is a key regulatory network in the bacterial system, which plays an important role in host-pathogen interactions and maintains cell homeostasis by regulating the release of effector proteins in specific competition. T6SS causes cell lysis or competitive inhibition by delivering effector molecules, such as toxic proteins and nucleic acids, directly from donor bacterial cells to eukaryotic or prokaryotic targets. Additionally, it orchestrates synthesis of immune effectors that counteract toxins thus preventing self-intoxication or antagonistic actions by competing microbes. Even so, the mechanism of toxin-antitoxin regulation in bacteria remains unclear. In response, this review discusses the bacterial T6SS's structure and function and the mechanism behind toxin-antitoxin secretion and the T6SS's expression in order to guide the further exploration of the pathogenic mechanism of the T6SS and the development of novel preparations for reducing and replacing toxins and antitoxins.

Relevance of charged and polar amino acids for functionality of membrane toxin TisB.

Bacterial dormancy is marked by reduced cellular activity and the suspension of growth. It represents a valuable strategy to survive stressful conditions, as exemplified by the long-term tolerance towards antibiotics that is attributable to a fraction of dormant cells, so-called persisters. Here, we investigate the membrane toxin TisB (29 amino acids) from the chromosomal toxin-antitoxin system tisB/istR-1 in Escherichia coli. TisB depolarizes the inner membrane in response to DNA damage, which eventually promotes a stress-tolerant state of dormancy within a small fraction of the population. Using a plasmid-based system for moderate tisB expression and single amino acid substitutions, we dissect the importance of charged and polar amino acids. We observe that the central amino acids lysine 12 and glutamine 19 are of major importance for TisB functionality, which is further validated for lysine 12 in the native context upon treatment with the DNA-damaging antibiotic ciprofloxacin. Finally, we apply a library-based approach to test additional TisB variants in higher throughput, revealing that at least one positive charge at the C-terminus (either lysine 26 or 29) is mandatory for TisB-mediated dormancy. Our study provides insights into the molecular basis for TisB functionality and extends our understanding of bacterial membrane toxins.

De novo design of mini-protein binders broadly neutralizing Clostridioides difficile toxin B variants.

Clostridioides difficile toxin B (TcdB) is the key virulence factor accounting for C. difficile infection-associated symptoms. Effectively neutralizing different TcdB variants with a universal solution poses a significant challenge. Here we present the de novo design and characterization of pan-specific mini-protein binders against major TcdB subtypes. Our design successfully binds to the first receptor binding interface (RBI-1) of the varied TcdB subtypes, exhibiting affinities ranging from 20 pM to 10 nM. The cryo-electron microscopy (cryo-EM) structures of the mini protein binder in complex with TcdB1 and TcdB4 are consistent with the computational design models. The engineered and evolved variants of the mini-protein binder and chondroitin sulfate proteoglycan 4 (CSPG4), another natural receptor that binds to the second RBI (RBI-2) of TcdB, better neutralize major TcdB variants both in cells and in vivo, as demonstrated by the colon-loop assay using female mice. Our findings provide valuable starting points for the development of therapeutics targeting C. difficile infections (CDI).

Inhibition of neutrophil swarming by type I interferon promotes intracellular bacterial evasion.

Listeria monocytogenes (LM) possesses the ability to breach multiple barriers and elicit intricate immune responses. However, there remains a lack of explicit understanding regarding how LM evades innate immune surveillance within the body. Here, we utilized liver intravital imaging to elucidate the dynamic process of LM during infection in the liver. We discovered that LM can rapidly escape from Kupffer cells (KCs) through listeriolysin O (LLO) and proliferate within hepatocytes. Upon LM exposure to the hepatic sinusoids, neutrophils rapidly aggregate at the site of infection. Subsequently, LM can induce type I interferon (IFN-I) production primarily in the spleen, which acts systemically on neutrophils to hamper their swarming by deactivating the ERK pathway, thus evading neutrophil-mediated eradication. Furthermore, our findings suggest that virus-induced IFN-I suppresses neutrophil swarming, and COVID-19 patients exhibit impaired neutrophil aggregation function. In conclusion, our findings provide compelling evidence demonstrating that intracellular bacteria represented by LM can hijack host defense mechanisms against viral infections to evade immune surveillance. Additionally, impaired neutrophil swarming caused by IFN-I is one of the significant factors contributing to the increased susceptibility to bacterial infections following viral infections.

Enhanced Staphylococcus aureus protection by uncoupling of the α-toxin-ADAM10 interaction during murine neonatal vaccination.

Staphylococcus aureus remains a leading global cause of bacterial infection-associated mortality and has eluded prior vaccine development efforts. S. aureus α-toxin (Hla) is an essential virulence factor in disease, impairing the T cell response to infection. The anti-Hla antibody response is a correlate of human protective immunity. Here we observe that this response is limited early in human life and design a vaccine strategy to elicit immune protection against Hla in a neonatal mice. By targeted disruption of the interaction of Hla with its receptor ADAM10, we identify a vaccine antigen (HlaH35L/R66C/E70C, HlaHRE) that elicits an ~100-fold increase in the neutralizing anti-Hla response. Immunization with HlaHRE enhances the T follicular helper (TFH) cell response to S. aureus infection, correlating with the magnitude of the neutralizing anti-toxin response and disease protection. Furthermore, maternal HlaHRE immunization confers protection to offspring. Together, these findings illuminate a path for S. aureus vaccine development at the maternal-infant interface.

Structure of a Rhs effector clade domain provides mechanistic insights into type VI secretion system toxin delivery.

The type VI secretion system (T6SS) is a molecular machine utilised by many Gram-negative bacteria to deliver antibacterial toxins into adjacent cells. Here we present the structure of Tse15, a T6SS Rhs effector from the nosocomial pathogen Acinetobacter baumannii. Tse15 forms a triple layered ß-cocoon Rhs domain with an N-terminal α-helical clade domain and an unfolded C-terminal toxin domain inside the Rhs cage. Tse15 is cleaved into three domains, through independent auto-cleavage events involving aspartyl protease activity for toxin self-cleavage and a nucleophilic glutamic acid for N-terminal clade cleavage. Proteomic analyses identified that significantly more peptides from the N-terminal clade and toxin domains were secreted than from the Rhs cage, suggesting toxin delivery often occurs without the cage. We propose the clade domain acts as an internal chaperone to mediate toxin tethering to the T6SS machinery. Conservation of the clade domain in other Gram-negative bacteria suggests this may be a common mechanism for delivery.

Review of advances in molecular structure and biological function of alpha toxin of Clostridium perfringens.

Alpha toxin has become the subject of research in recent years. The objective of this article was to review and summarize recent research on the molecular structure and biological function of the alpha toxin of Clostridium perfringens. This includes the work of our research team, as well as that of other researchers. Clostridium perfringens is an anaerobic, spore-forming, Gram-positive bacillus. It can cause various intestinal diseases, such as gas gangrene, food poisoning, non-foodborne diarrhea, and enteritis. Clostridium perfringens can be classified into 5 toxinotypes A, B, C, D, and E, based on the production of major toxins. Each type of C. perfringens produces alpha toxin, which is one of the most important lethal and dermonecrotic toxins and is considered a primary virulence factor. Alpha toxin is a multifunctional metalloenzyme with phospholipase C and sphingomyelinase activities that simultaneously hydrolyze phosphatidylcholine and sphingomyelin. It can therefore destroy the integrity of cell membranes and eventually cause cell lysis. The clinical effects of alpha toxins are characterized by cytotoxicity, hemolytic activity, lethality, skin necrosis, platelet aggregation, and increased vascular permeability. Future research will concentrate on the pathogenesis of a lpha toxin exposure, clarifying the interaction between alpha toxin and the cell membrane and investigating the mechanism of activating platelet function. This research will have substantial theoretical and practical value in controlling disease progression, identifying targeted therapeutic sites, and reducing the toxic effects of vaccines.

La toxine alpha est devenue l'objet de recherches ces dernières années. L'objectif de cet article était de passer en revue et de résumer les recherches récentes sur la structure moléculaire et la fonction biologique de la toxine alpha de Clostridium perfringens. Cela inclut les travaux de notre équipe de recherche, ainsi que ceux d'autres chercheurs. Clostridium perfringens est un bacille anaérobie, sporulé et à Gram positif. Il peut provoquer diverses maladies intestinales, telles que la gangrène gazeuse, une intoxication alimentaire, de la diarrhée non alimentaire et une entérite. Clostridium perfringens peut être classé en 5 toxinotypes A, B, C, D et E, en fonction de la production des principales toxines. Chaque type de C. perfringens produit de la toxine alpha, qui est l'une des toxines létales et dermonécrotiques les plus importantes et est considérée comme un facteur de virulence primaire. La toxine alpha est une métalloenzyme multifonctionnelle possédant des activités de phospholipase C et de sphingomyélinase qui hydrolysent simultanément la phosphatidylcholine et la sphingomyéline. Elle peut donc détruire l'intégrité des membranes cellulaires et éventuellement provoquer une lyse cellulaire. Les effets cliniques des toxines alpha sont caractérisés par une cytotoxicité, une activité hémolytique, une létalité, une nécrose cutanée, une agrégation plaquettaire et une augmentation de la perméabilité vasculaire. Les recherches futures se concentreront sur la pathogénèse de l'exposition à la toxine alpha, en clarifiant l'interaction entre la toxine alpha et la membrane cellulaire et en étudiant le mécanisme d'activation de la fonction plaquettaire. Ces recherches auront une valeur théorique et pratique substantielle pour contrôler la progression de la maladie, identifier les sites thérapeutiques ciblés et réduire les effets toxiques des vaccins.(Traduit par Docteur Serge Messier).

A tetravalent peptide efficiently inhibits the intestinal toxicity of heat-labile enterotoxin by targeting the receptor-binding region of the B-subunit pentamer.

Infection by enterotoxigenic Escherichia coli (ETEC) causes severe watery diarrhea and dehydration in humans. Heat-labile enterotoxin (LT) is a major virulence factor produced by ETEC. LT is one of AB5-type toxins, such as Shiga toxin (Stx) and cholera toxin (Ctx), and the B-subunit pentamer is responsible for high affinity binding to the LT-receptor, ganglioside GM1, through multivalent interaction. In this report, we found that Glu51 of the B-subunit plays an essential role in receptor binding compared with other amino acids, such as Glu11, Arg13, and Lys91, all of which were previously shown to be involved in the binding. By targeting Glu51, we identified four tetravalent peptides that specifically bind to the B-subunit pentamer with high affinity by screening tetravalent random-peptide libraries, which were tailored to bind to the B-subunit through multivalent interaction. One of these peptides, GGR-tet, efficiently inhibited the cell-elongation phenotype and the elevation of cellular cAMP levels, both induced by LT. Furthermore, GGR-tet markedly inhibited LT-induced fluid accumulation in the mouse ileum. Thus, GGR-tet represents a novel therapeutic agent against ETEC infection.

Deciphering the role of VapBC13 and VapBC26 toxin antitoxin systems in the pathophysiology of Mycobacterium tuberculosis.

The expansion of VapBC TA systems in M. tuberculosis has been linked with its fitness and survival upon exposure to stress conditions. Here, we have functionally characterized VapBC13 and VapBC26 TA modules of M. tuberculosis. We report that overexpression of VapC13 and VapC26 toxins in M. tuberculosis results in growth inhibition and transcriptional reprogramming. We have also identified various regulatory proteins as hub nodes in the top response network of VapC13 and VapC26 overexpression strains. Further, analysis of RNA protection ratios revealed potential tRNA targets for VapC13 and VapC26. Using in vitro ribonuclease assays, we demonstrate that VapC13 and VapC26 degrade serT and leuW tRNA, respectively. However, no significant changes in rRNA cleavage profiles were observed upon overexpression of VapC13 and VapC26 in M. tuberculosis. In order to delineate the role of these TA systems in M. tuberculosis physiology, various mutant strains were constructed. We show that in comparison to the parental strain, ΔvapBC13 and ΔvapBC26 strains were mildly susceptible to oxidative stress. Surprisingly, the growth patterns of parental and mutant strains were comparable in aerosol-infected guinea pigs. These observations imply that significant functional redundancy exists for some TA systems from M. tuberculosis.

Systematic discovery of antibacterial and antifungal bacterial toxins.

Microorganisms use toxins to kill competing microorganisms or eukaryotic cells. Polymorphic toxins are proteins that encode carboxy-terminal toxin domains. Here we developed a computational approach to identify previously undiscovered, conserved toxin domains of polymorphic toxins within 105,438 microbial genomes. We validated nine short toxins, showing that they cause cell death upon heterologous expression in either Escherichia coli or Saccharomyces cerevisiae. Five cognate immunity genes that neutralize the toxins were also discovered. The toxins are encoded by 2.2% of sequenced bacteria. A subset of the toxins exhibited potent antifungal activity against various pathogenic fungi but not against two invertebrate model organisms or macrophages. Experimental validation suggested that these toxins probably target the cell membrane or DNA or inhibit cell division. Further characterization and structural analysis of two toxin-immunity protein complexes confirmed DNase activity. These findings expand our knowledge of microbial toxins involved in inter-microbial competition that may have the potential for clinical and biotechnological applications.

The epsilon toxin from Clostridium perfringens stimulates calcium-activated chloride channels, generating extracellular vesicles in Xenopus oocytes.

The epsilon toxin (Etx) from Clostridium perfringens has been identified as a potential trigger of multiple sclerosis, functioning as a pore-forming toxin that selectively targets cells expressing the plasma membrane (PM) myelin and lymphocyte protein (MAL). Previously, we observed that Etx induces the release of intracellular ATP in sensitive cell lines. Here, we aimed to re-examine the mechanism of action of the toxin and investigate the connection between pore formation and ATP release. We examined the impact of Etx on Xenopus laevis oocytes expressing human MAL. Extracellular ATP was assessed using the luciferin-luciferase reaction. Activation of calcium-activated chloride channels (CaCCs) and a decrease in the PM surface were recorded using the two-electrode voltage-clamp technique. To evaluate intracellular Ca2+ levels and scramblase activity, fluorescent dyes were employed. Extracellular vesicles were imaged using light and electron microscopy, while toxin oligomers were identified through western blots. Etx triggered intracellular Ca2+ mobilization in the Xenopus oocytes expressing hMAL, leading to the activation of CaCCs, ATP release, and a reduction in PM capacitance. The toxin induced the activation of scramblase and, thus, translocated phospholipids from the inner to the outer leaflet of the PM, exposing phosphatidylserine outside in Xenopus oocytes and in an Etx-sensitive cell line. Moreover, Etx caused the formation of extracellular vesicles, not derived from apoptotic bodies, through PM fission. These vesicles carried toxin heptamers and doughnut-like structures in the nanometer size range. In conclusion, ATP release was not directly attributed to the formation of pores in the PM, but to scramblase activity and the formation of extracellular vesicles.

Analysis of factors affecting microbial degradation of cyanobacterial toxins based on theoretical calculations.

Cyanobacterial toxins are the most common algal toxins, which are highly toxic and can persist in the aquatic environment without easy degradation, posing risks to the ecosystem and human health that cannot be ignored. Although microbiological methods for the removal of cyanobacterial toxins from aqueous environments are highly efficient, their degradation efficiency is susceptible to many abiotic environmental factors. In this paper, Microcystin-LR (MC-LR) and its microbial degrading enzymes were selected to study the effects of common environmental factors (temperature (T), NO3-, NH4+, Cu2+, Zn2+) and their levels during microbial degradation of cyanobacterial toxins in aqueous environments by using molecular docking, molecular dynamics simulation, analytical factor design, and the combined toxicokinetics of TOPKAT (toxicity prediction). It was found that the addition of T, NO3- and Cu2+ to the aqueous environment promoted the microbial degradation of MC-LR, while the addition of NH4+ and Zn2+ inhibited the degradation; The level effect study showed that the microbial degradation of MC-LR was promoted by increasing levels of added T and NO3- in the aqueous environment, whereas it was inhibited and then promoted by increasing levels of NH4+, Cu2+ and Zn2+. In addition, the predicted toxicity of common Microcystins (MCs) showed that MC-LR, Microcystin-RR (MC-RR) and Microcystin-YR (MC-YR) were not carcinogenic, developmentally toxic, mutagenic or ocular irritants in humans. MC-LR and MC-RR are mild skin irritants and MC-YR is not a skin irritant. MC-YR has a higher chronic and acute toxicity in humans than MC-LR and MC-RR. Acute/chronic toxicity intensity for aquatic animals: MC-YR > MC-LR > MC-RR and for aquatic plants: MC-LR > MC-YR > MC-RR. This suggests that MC-YR also has a high environmental health risk. This paper provides theoretical support for optimizing the environmental conditions for microbial degradation of cyanobacterial toxins by studying the effects of common environmental factors and their level effects in the aquatic environment.

A plasmid-mediated type III secretion system associated with invasiveness and diarrheagenicity of Providencia rustigianii.

We have recently described a clinical isolate of Providencia rustigianii strain JH-1 carrying the genes for cytolethal distending toxin (CDT) in a conjugative plasmid. A cdtB mutant of strain JH-1, which lost CDT activity, was still found to retain invasiveness and diarrheagenicity. The strain was subjected to phenotypic and genetic analyses including whole genome sequencing (WGS) to explore the genetic determinants of the observed invasiveness and diarrheagenic properties. Analysis and annotation of WGS data revealed the presence of two distinct type III secretion systems (T3SS) in strain JH-1, one of which was located on the chromosome designated as cT3SS (3,992,833 bp) and the other on a mega-plasmid designated as pT3SS (168,819 bp). Comparative genomic analysis revealed that cT3SS is generally conserved in Providencia spp. but pT3SS was limited to a subset of Providencia spp., carrying cdt genes. Strain JH-1 was found to invade HeLa cells and induce fluid accumulation with characteristic pathological lesions in rabbit ileal loops. Remarkably, these phenomena were associated with the pT3SS but not cT3SS. The plasmid could be transferred by conjugation from strain JH-1 to other strains of P. rustigianii, Providencia rettgeri, and Escherichia coli with concomitant transfer of these virulence properties. This is the first report of a functional and mobile T3SS in P. rustigianii and its association with invasiveness and diarrheagenicity of this bacterium. These data suggest that P. rustigianii and other CDT-producing Providencia strains might carry T3SS and exert their diarrheagenic effect by exploiting the T3SS nano-machinery.IMPORTANCEThe precise mechanism of virulence of Providencia rustigianii is unclear, although some strains produce cytolethal distending toxin as a putative virulence factor. We have detected the presence of a type III secretion system (T3SS) for the first time on a plasmid in a P. rustigianii strain. Plasmid-mediated T3SS seems to be directly involved in virulence of P. rustigianii and may serve as a means of horizontal transfer of T3SS genes. Our results may have implication in understanding the mechanism of emergence of new pathogenic strains of P. rustigianii.

A membrane localized RTX-like protein mediates physiochemical properties of the Pantoea stewartii subsp. stewartii cell envelope that impact surface adhesion, cell surface hydrophobicity and plant colonization.

Pantoea stewartii subsp. stewartii (Pnss), is the bacterial causal agent of Stewart's wilt of sweet corn. Disease symptoms include systemic wilting and foliar, water-soaked lesions. A Repeat-in-toxin (RTX)-like protein, RTX2, causes cell leakage and collapse in the leaf apoplast of susceptible corn varieties and is a primary mediator of water-soaked lesion formation in the P. stewartii-sweet corn pathosystem. RTX toxins comprise a large family of proteins, which are widely distributed among Gram-negative bacteria. These proteins are generally categorized as cellulolysins, but the Biofilm-Associated Proteins (Bap) subfamily of RTX toxins are implicated in surface adhesion and other biofilm behaviors. The Pnss RTX2 is most phylogenetically related to other Bap proteins suggesting that Pnss RTX2 plays a dual role in adhesion to host surfaces in addition to mediating the host cell lysis that leads to water-soaked lesion formation. Here we demonstrated that RTX2 localizes to the bacterial cell envelope and influences physiochemical properties of the bacterial cell envelope that impact bacterial cell length, cell envelope integrity and overall cellular hydrophobicity. Interestingly, the role of RTX2 as an adhesin was only evident in absence of exopolysaccharide (EPS) production suggesting that RTX2 plays a role as an adhesin early in biofilm development before EPS production is fully induced. However, deletion of rtx2 severely impacted Pnss' colonization of the xylem suggesting that the dual role of RTX2 as a cytolysin and adhesin is a mechanism that links the apoplastic water-soaked lesion phase of infection to the wilting phase of the infection in the xylem.

YjbH contributes to Staphylococcus aureus skin pathology and immune response through Agr-mediated α-toxin regulation.

Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs) with Methicillin-Resistant S. aureus (MRSA) strains being a major contributor in both community and hospital settings. S. aureus relies on metabolic diversity and a large repertoire of virulence factors to cause disease. This includes α-hemolysin (Hla), an integral player in tissue damage found in various models, including SSTIs. Previously, we identified a role for the Spx adapter protein, YjbH, in the regulation of several virulence factors and as an inhibitor of pathogenesis in a sepsis model. In this study, we found that YjbH is critical for tissue damage during SSTI, and its absence leads to decreased proinflammatory chemokines and cytokines in the skin. We identified no contribution of YjbI, encoded on the same transcript as YjbH. Using a combination of reporters and quantitative hemolysis assays, we demonstrated that YjbH impacts Hla expression and activity both in vitro and in vivo. Additionally, expression of Hla from a non-native promoter reversed the tissue damage phenotype of the ΔyjbIH mutant. Lastly, we identified reduced Agr activity as the likely cause for reduced Hla production in the ΔyjbH mutant. This work continues to define the importance of YjbH in the pathogenesis of S. aureus infection as well as identify a new pathway important for Hla production.

The role of male hormones in bacterial infections: enhancing Staphylococcus aureus virulence through testosterone-induced Agr activation.

Staphylococcus aureus is a notorious pathogen predominantly involved in skin and soft tissue infections, exhibiting a distinct innate sex bias. This study explores the influence of testosterone on the virulence of S. aureus and elucidates its underlying mechanisms. Utilizing a skin abscess model in intact and castrated male mice, we assessed the effects of testosterone on S. aureus pathogenicity. Compared to controls, castrated mice showed significantly reduced abscess sizes and decreased bacterial loads, highlighting the role of testosterone in modulating the severity of S. aureus infections. In vitro experiments revealed that testosterone enhances the hemolytic activity, cytotoxicity, and oxidative stress resistance of S. aureus. Real-time quantitative PCR analysis showed a significant upregulation of the genes encoding α-hemolysin (hla) and phenol-soluble modulin (psmα). Importantly, testosterone treatment significantly enhanced the expression of the accessory gene regulator (Agr) quorum-sensing system components (agrC, agrA, agrB, agrD), while the SaeRS system (saeR, saeS, and sbi) exhibited only slight changes. Gene knockout experiments revealed that deletion of agrC, rather than saeRS and agrBD, abolishes the testosterone-induced enhancement of hemolysis and gene expression, underscoring the key role of AgrC. Molecular docking simulations indicated a direct interaction between testosterone and AgrC protein, with a strong binding affinity at the active site residue SER201. This study provides new insights into the mechanistic basis of how testosterone enhances the pathogenicity of S. aureus, potentially contributing to increased male susceptibility to S. aureus infections and offering a targeted approach for therapeutic interventions.

Using nucleolytic toxins as restriction enzymes enables new RNA applications.

Over the past five decades, DNA restriction enzymes have revolutionized biotechnology. While these enzymes are widely used in DNA research and DNA engineering, the emerging field of RNA and mRNA therapeutics requires sequence-specific RNA endoribonucleases. Here, we describe EcoToxN1, a member of the type III toxin-antitoxin family of sequence-specific RNA endoribonucleases, and its use in RNA and mRNA analysis. This enzyme recognizes a specific pentamer in a single-stranded RNA and cleaves the RNA within this sequence. The enzyme is neither dependent on annealing of guide RNA or DNA oligos to the template nor does it require magnesium. Furthermore, it performs over a wide range of temperatures. With its unique functions and characteristics, EcoToxN1 can be classified as an RNA restriction enzyme. EcoToxN1 enables new workflows in RNA analysis and biomanufacturing, meeting the demand for faster, cheaper, and more robust analysis methods.