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Clostridium perfringens is a kind of anaerobic Gram-positive bacterium that widely exists in the intestinal tissue of humans and animals. And the main virulence factor in Clostridium perfringens is its exotoxins. Clostridium perfringens type C is the main strain of livestock disease, its exotoxins can induce necrotizing enteritis and enterotoxemia, which lead to the reduction in feed conversion, and a serious impact on breeding production performance. Our study found that treatment with exotoxins reduced cell viability and triggered intracellular reactive oxygen species (ROS) in human mononuclear leukemia cells (THP-1) cells. Through transcriptome sequencing analysis, we found that the levels of related proteins such as heme oxygenase 1 (HO-1) and ferroptosis signaling pathway increased significantly after treatment with exotoxins. To investigate whether ferroptosis occurred after exotoxin treatment in macrophages, we confirmed that the protein expression levels of antioxidant factors glutathione peroxidase 4/ferroptosis-suppressor-protein 1/the cystine/glutamate antiporter solute carrier family 7 member 11 (GPX4/FSP1/xCT), ferroptosis-related protein nuclear receptor coactivator 4/transferrin/transferrin receptor (NCOA4/TF/TFR)/ferritin and the level of lipid peroxidation were significantly changed. Based on the above results, our study suggested that Clostridium perfringens type C exotoxins can induce macrophage injury through oxidative stress and ferroptosis. [ABSTRACT FROM AUTHOR]

Type 3 Secretion System (T3SS) is a highly conserved virulence structure that plays an essential role in the pathogenesis of many Gram-negative pathogenic bacteria, including Pseudomonas aeruginosa. Exotoxin T (ExoT) is the only T3SS effector protein that is expressed in all T3SS-expressing P. aeruginosa strains. Here we show that T3SS recognition leads to a rapid phosphorylation cascade involving Abl / PKCδ / NLRC4, which results in NLRC4 inflammasome activation, culminating in inflammatory responses that limit P. aeruginosa infection in wounds. We further show that ExoT functions as the main anti-inflammatory agent for P. aeruginosa in that it blocks the phosphorylation cascade through Abl / PKCδ / NLRC4 by targeting CrkII, which we further demonstrate to be important for Abl transactivation and NLRC4 inflammasome activation in response to T3SS and P. aeruginosa infection.

The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.

Abstract Staphylococcus aureus is a frequent cause of infections worldwide. Methicillin-resistant S. aureus (MRSA) is one of the main causes of Gram-positive infections, and methicillin-susceptible strains (MSSA) primarily colonize and infect community hosts. Multiple virulence factors are involved, with toxins playing a significant role in several diseases. In this study, we assess the prevalence of toxin genes in 89 S. aureus clinical isolates (31 MRSA and 58 MSSA). We evaluated the discriminatory power of the association of internal transcribed spacer-PCR (ITS-PCR) and 3’- end coa gene (coa-PCR) when compared with other more commonly used and costly techniques. The isolates showed a high level of genetic diversity, and toxins were found in all the isolates. While most toxin classes displayed no statistically significant correlations and were equally distributed in isolates regardless of their resistance status, classic enterotoxins (sea-see) showed a positive correlation with MSSA isolates. The combination of coa-PCR with ITS-PCR showed a discriminatory index of 0.84, discriminating 22 genotypes that agree with previously determined data by PFGE and MLST. This association between the two PCR-based methods suggests that they can be useful for an initial molecular epidemiological investigation of S. aureus in hospitals, providing significant information while requiring fewer resources.

Recently, we demonstrated that Pseudomonas aeruginosa Exotoxin T (ExoT) employs two distinct mechanisms to induce potent apoptotic cytotoxicity in a variety of cancer cell lines. We further demonstrated that it can significantly reduce tumour growth in an animal model for melanoma. During these studies, we observed that melanoma cells that were transfected with ExoT failed to undergo mitosis, regardless of whether they eventually succumbed to ExoT-induced apoptosis or survived in ExoTs presence. In this report, we sought to investigate ExoTs antiproliferative activity in melanoma. We delivered ExoT into B16 melanoma cells by bacteria (to show necessity) and by transfection (to show sufficiency). Our data indicate that ExoT exerts a potent antiproliferative function in melanoma cells. We show that ExoT causes cell cycle arrest in G1 interphase in melanoma cells by dampening the G1/S checkpoint proteins. Our data demonstrate that both domains of ExoT; (the ADP-ribosyltransferase (ADPRT) domain and the GTPase activating protein (GAP) domain); contribute to ExoT-induced G1 cell cycle arrest in melanoma. Finally, we show that the ADPRT-induced G1 cell cycle arrest in melanoma cells likely involves the Crk adaptor protein. Our data reveal a novel virulence function for ExoT and further highlight the therapeutic potential of ExoT against cancer.

Linoleic acid (LA) is the most abundant polyunsaturated fatty acid found in the Western diet. Cytochrome P450-derived LA metabolites 9,10-epoxyoctadecenoic acid (9,10-EpOME), 12,13-epoxyoctadecenoic acid (12,13-EpOME), 9,10-dihydroxy-12Z-octadecenoic acid (9,10-DiHOME) and 12,13-dihydroxy-9Z-octadecenoic acid (12,13-DiHOME) have been studied for their association with various disease states and biological functions. Previous studies of the EpOMEs and DiHOMEs have focused on their roles in cytotoxic processes, primarily in the inhibition of the neutrophil respiratory burst. More recent research has suggested the DiHOMEs may be important lipid mediators in pain perception, altered immune response and brown adipose tissue activation by cold and exercise. The purpose of this review is to summarize the current understanding of the physiological and pathophysiological roles and modes of action of the EpOMEs and DiHOMEs in health and disease.

Clostridium perfringens is a kind of anaerobic Gram-positive bacterium that widely exists in the intestinal tissue of humans and animals. And the main virulence factor in Clostridium perfringens is its exotoxins. Clostridium perfringens type C is the main strain of livestock disease, its exotoxins can induce necrotizing enteritis and enterotoxemia, which lead to the reduction in feed conversion, and a serious impact on breeding production performance. Our study found that treatment with exotoxins reduced cell viability and triggered intracellular reactive oxygen species (ROS) in human mononuclear leukemia cells (THP-1) cells. Through transcriptome sequencing analysis, we found that the levels of related proteins such as heme oxygenase 1 (HO-1) and ferroptosis signaling pathway increased significantly after treatment with exotoxins. To investigate whether ferroptosis occurred after exotoxin treatment in macrophages, we confirmed that the protein expression levels of antioxidant factors glutathione peroxidase 4/ferroptosis-suppressor-protein 1/the cystine/glutamate antiporter solute carrier family 7 member 11 (GPX4/FSP1/xCT), ferroptosis-related protein nuclear receptor coactivator 4/transferrin/transferrin receptor (NCOA4/TF/TFR)/ferritin and the level of lipid peroxidation were significantly changed. Based on the above results, our study suggested that Clostridium perfringens type C exotoxins can induce macrophage injury through oxidative stress and ferroptosis.

Key Clinical Message: We present a case of a 20‐month‐old child with a history of atopic dermatitis that exhibited recurrent erythematous‐bullous lesions consistent with Staphylococcal Scalded Skin syndrome (SSSS). SSSS is an exfoliative toxin‐mediated skin disorder most commonly found in children. In this paper, we discuss the importance of recognizing the clinical symptomatology and progressive nature of SSSS, particularly in patients with a history of atopic dermatitis, to ensure prompt treatment and resolution of the syndrome. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa has assumed an increasingly prominent role as the aetiological agent in serious hard-to-treat infections in animals and humans. In this study, 271 P. aeruginosa strains collected from dogs and cats were investigated. The aim of the research was to screen these P. aeruginosa strains for antibiotic resistance and the presence of selected virulence factor genes. Antibiotic resistance was determined using the Kirby–Bauer method, while virulence genes were detected by polymerase chain reaction (PCR). The most frequently detected resistance was to fluoroquinolones, ranging in prevalence from 17.3% for ciprofloxacin up to 83% for enrofloxacin. The resistance to carbapenems was 14% and 4.8% for imipenem and meropenem, respectively. Almost all P. aeruginosa strains harboured the exoT (97.8%) and lasB (93.4%) genes, while the lowest prevalence was found for exoU (17.3%) and plcH (17.3%). P. aeruginosa strains isolated from dogs that harboured the toxA gene were more frequently resistant to ceftazidime (p = 0.012), while the presence of the exoU gene was found to be connected with resistance to marbofloxacin (p = 0.025) and amikacin (p = 0.056). In strains originating from cats, only the connection between the presence of the exoU gene and resistance to enrofloxacin (p = 0.054) was observed. The confirmation of associations between virulence-factor-encoding genes and antibiotic resistance indicates that problems of antibiotic resistance may not only cause complications at the level of antibiotic dosage but also lead to changes in the virulence of the bacteria; thus, further studies in this area are required. [ABSTRACT FROM AUTHOR]

Background and Aim:. Pseudomonas aeruginosa exotoxins are responsible for pathogenicity and antibiotic resistant. The aim of this study was to investigate the effects of the Ammi. visnaga (A. visnaga) extract on the P. aeruginosa exotoxin A and S genes expression. Materials and Methods: Antibacterial effects of the A. visnaga ethanolic extract were performed using the micro dilution technique in sequential dilutions. Then, the Real-Time- RT PCR method was used to analyze the level of expression of the exotoxin A and S genes of the bacterium. Real-Time PCR data were analyzed using Graph Pad Software Inc. San Diego (GRAPHPAD PRISM 6CA, USA). Results: Results showed that, the expression of Exotoxin A and S genes was significantly (P <0.001) reduced in comparing the expression level of the reference gene and control. It had a significant effect on the inhibition of bacterial growth at different concentrations but the lowest inhibitory concentration was observed at 3.125 µg/well. Conclusion: Accordingly, the extract of A. visnaga can decrease the expression of the Exotoxin A and S genes of P. aeruginosa and consequently it may decrease the pathogenicity of bacteria. These phenomena (exoA and exoS expression inhibition) may involve reducing the antibiotic resistance of P. aeruginosa. [ABSTRACT FROM AUTHOR]

Key Clinical Message We present a case of a 20‐month‐old child with a history of atopic dermatitis that exhibited recurrent erythematous‐bullous lesions consistent with Staphylococcal Scalded Skin syndrome (SSSS). SSSS is an exfoliative toxin‐mediated skin disorder most commonly found in children. In this paper, we discuss the importance of recognizing the clinical symptomatology and progressive nature of SSSS, particularly in patients with a history of atopic dermatitis, to ensure prompt treatment and resolution of the syndrome.

Sunflower trypsin inhibitor (SFTI) is a 14-amino-acid bicyclic peptide that contains a single internal disulfide bond. We initially constructed chimeras of SFTI with N-terminal secretion signals from the Escherichia coli OmpA and Pseudomonas aeruginosa ToxA, but only detected small amounts of protease inhibition resulting from these constructs. A substantially higher degree of protease inhibition was detected from a C-terminal SFTI fusion with E. coli YebF, which radiated more than a centimeter from an individual colony of E. coli using a culture-based inhibitor assay. Inhibitory activity was further improved in YebF-SFTI fusions by the addition of a trypsin cleavage signal immediately upstream of SFTI, and resulted in production of a 14-amino-acid, disulfide-bonded SFTI free in the culture supernatant. To assess the potential of the secreted SFTI to protect the ability of a cytotoxic protein to kill tumor cells, we utilized a tumor-selective form of the Pseudomonas ToxA (OTG-PE38K) alone and expressed as a polycistronic construct with YebF-SFTI in the tumor-targeted Salmonella VNP20009. When we assessed the ability of toxin-containing culture supernatants to kill MDA-MB-468 breast cancer cells, the untreated OTG-PE38K was able to eliminate all detectable tumor cells, while pretreatment with trypsin resulted in the complete loss of anticancer cytotoxicity. However, when OTG-PE38K was co-expressed with YebF-SFTI, cytotoxicity was completely retained in the presence of trypsin. These data demonstrate SFTI chimeras are secreted in a functional form and that co-expression of protease inhibitors with therapeutic proteins by tumor-targeted bacteria has the potential to enhance the activity of therapeutic proteins by suppressing their degradation within a proteolytic environment.

This is a pivotal, multicenter, open-label study of moxetumomab pasudotox, a recombinant CD22-targeting immunotoxin, in hairy cell leukemia (HCL), a rare B cell malignancy with high CD22 expression. The study enrolled patients with relapsed/refractory HCL who had ≥2 prior systemic therapies, including ≥1 purine nucleoside analog. Patients received moxetumomab pasudotox 40 µg/kg intravenously on days 1, 3, and 5 every 28 days for ≤6 cycles. Blinded independent central review determined disease response and minimal residual disease (MRD) status. Among 80 patients (79% males; median age, 60.0 years), durable complete response (CR) rate was 30%, CR rate was 41%, and objective response rate (CR and partial response) was 75%; 64 patients (80%) achieved hematologic remission. Among complete responders, 27 (85%) achieved MRD negativity by immunohistochemistry. The most frequent adverse events (AEs) were peripheral edema (39%), nausea (35%), fatigue (34%), and headache (33%). Treatment-related serious AEs of hemolytic uremic syndrome (7.5%) and capillary leak syndrome (5%) were reversible and generally manageable with supportive care and treatment discontinuation (6 patients; 7.5%). Moxetumomab pasudotox treatment achieved a high rate of independently assessed durable response and MRD eradication in heavily pretreated patients with HCL, with acceptable tolerability.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause critical cellular damage and subvert the immune response to promote its survival. Among the numerous virulence factors of P. aeruginosa, the type III secretion system (T3SS) is involved in host cell pathogenicity. Using a needle-like structure, T3SS detects eukaryotic cells and injects toxins directly into their cytosol, thus highlighting its ability to interfere with the host immune response. In this mini-review, we discuss how the T3SS and bacterial effectors secreted by this pathway not only activate the immune response but can also manipulate it to promote the establishment of P. aeruginosa infections. [ABSTRACT FROM AUTHOR]

Hemolysins are lytic exotoxins expressed in most strains of S. aureus , but hemolytic activity varies between strains. We have previously reported several novel anti-virulence compounds that disrupt the S. aureus transcriptome, including hemolysin gene expression. This report delves further into our two lead compounds, loratadine and a structurally related brominated carbazole, and their effects on hemolysin production in methicillin-resistant S. aureus (MRSA). To gain understanding into how these compounds affect hemolysis, we analyzed these exotoxins at the DNA, RNA, and protein level after in vitro treatment. While lysis of red blood cells varied between strains, DNA sequence variation did not account for it. We hypothesized that our compounds would modulate gene expression of multiple hemolysins in two hospital-acquired strains of MRSA, both with staphylococcal cassette chromosome mec (SCC mec ) type II. RNA-seq analysis of differential gene expression in untreated and compound-treated cultures revealed hundreds of differentially expressed genes, with a significant enrichment in genes involved in hemolysis. The brominated carbazole and loratadine both displayed the ability to reduce hemolysis in strain 43300 but displayed differential activity in strain USA100. These results corroborate gene expression studies as well as western blots of alpha hemolysin. Together, this work suggests that small molecules may alter exotoxin production in MRSA but that the directionality and/or magnitude of the difference are likely strain dependent.IMPORTANCEMethicillin-resistant S. aureus (MRSA) is a deadly human pathogen. In addition to evading antibiotics, these bacteria produce a wide range of toxins that negatively affect the host. Our work aims to identify and characterize novel compounds that can decrease the pathogenic effects of MRSA. Two lead compounds investigated in this study triggered changes in the production of multiple toxins. These changes were specific to the strain of MRSA investigated. Specifically, this work sheds light on novel compounds that decrease MRSA's ability to lyse host red blood cells. Importantly, it also highlights the importance of examining strain-specific differences in response to therapeutic interventions that could ultimately affect clinical outcomes.

Background: The use of adjunctive antibiotics directed against exotoxin production in Staphylococcus aureus bacteremia (SAB) is widespread, and it is recommended in many guidelines, but this is based on limited evidence. Existing guidelines are based on the theoretical premise of toxin suppression, as many strains of S. aureus produce toxins such as leukocidins (eg, Panton-Valentine leukocidin, toxic shock syndrome toxin 1, exfoliative toxins, and various enterotoxins). Many clinicians therefore believe that limiting exotoxin production release by S. aureus could reduce its virulence and improve clinical outcomes. Clindamycin, a protein synthesis inhibitor antibiotic, is commonly used for this purpose. We report the domain-specific protocol, embedded in a large adaptive, platform trial, seeking to definitively answer this question., Methods and Analysis: The Staphylococcus aureus Network Adaptive Platform (SNAP) trial is a pragmatic, randomized, multicenter adaptive platform trial that aims to compare different SAB therapies, simultaneously, for 90-day mortality rates. The adjunctive treatment domain aims to test the effectiveness of adjunctive antibiotics, initially comparing clindamycin to no adjunctive antibiotic, but future adaptations may include other agents. Individuals will be randomized to receive either 5 days of adjunctive clindamycin (or lincomycin) or no adjunctive antibiotic therapy alongside standard-of-care antibiotics. Most participants with SAB (within 72 hours of index blood culture and with no contraindications) will be eligible to participate in this domain. Prespecified analyses are defined in the statistical appendix to the core protocol, and domain-specific secondary analyses will be adjusted for resistance to clindamycin, disease phenotype (complicated or uncomplicated SAB) and Panton-Valentine leukocidin-positive isolate., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Pseudomonas aeruginosa is an opportunistic pathogen that can cause critical cellular damage and subvert the immune response to promote its survival. Among the numerous virulence factors of P. aeruginosa, the type III secretion system (T3SS) is involved in host cell pathogenicity. Using a needle-like structure, T3SS detects eukaryotic cells and injects toxins directly into their cytosol, thus highlighting its ability to interfere with the host immune response. In this mini-review, we discuss how the T3SS and bacterial effectors secreted by this pathway not only activate the immune response but can also manipulate it to promote the establishment of P. aeruginosa infections.

Pseudomonas aeruginosa has assumed an increasingly prominent role as the aetiological agent in serious hard-to-treat infections in animals and humans. In this study, 271 P. aeruginosa strains collected from dogs and cats were investigated. The aim of the research was to screen these P. aeruginosa strains for antibiotic resistance and the presence of selected virulence factor genes. Antibiotic resistance was determined using the Kirby–Bauer method, while virulence genes were detected by polymerase chain reaction (PCR). The most frequently detected resistance was to fluoroquinolones, ranging in prevalence from 17.3% for ciprofloxacin up to 83% for enrofloxacin. The resistance to carbapenems was 14% and 4.8% for imipenem and meropenem, respectively. Almost all P. aeruginosa strains harboured the exoT (97.8%) and lasB (93.4%) genes, while the lowest prevalence was found for exoU (17.3%) and plcH (17.3%). P. aeruginosa strains isolated from dogs that harboured the toxA gene were more frequently resistant to ceftazidime (p = 0.012), while the presence of the exoU gene was found to be connected with resistance to marbofloxacin (p = 0.025) and amikacin (p = 0.056). In strains originating from cats, only the connection between the presence of the exoU gene and resistance to enrofloxacin (p = 0.054) was observed. The confirmation of associations between virulence-factor-encoding genes and antibiotic resistance indicates that problems of antibiotic resistance may not only cause complications at the level of antibiotic dosage but also lead to changes in the virulence of the bacteria; thus, further studies in this area are required.

Diamondback moth, Pluella xylostella is an important insect pest of cabbage and cauliflower that causes significant yield losses. Several chemicals insecticides are being used by the farmers to manage this pest. Due to indiscriminate usage of insecticides, the minimum residue levels of these chemicals were identified above threshold levels. Therefore, an eco-fiendly approach and naturally available insecticidal compounds were explored in this study. A soil inhibitant actinomycetes bacterial exotoxins were evaluated for their larvicidal activity against P. xylostella. An ethyl acetate extract of 95 actinomycete isolates containing exotoxins was tested against third instar larvae of P. xylostella by leaf disc bioassay method under laboratory conditions. Thirty-two isolates of actinomycete demonstrated entomotoxic nature by achieving larval mortality of > 50%. Isolate AUDT-240 (92%) showed the highest and significant larvicidal activity. The 16S rDNA sequence data of AUDT-240 isolate was matched (99%) with Streptomyces sp. HNGL19 strain. The ethyl acetate extract of AUDT-240 isolate was subjected to Gas Chromatography-Mass Spectroscopy that revealed, three insecticidal (= larvicidal) exotoxins viz., methyl formate, 3,9-Epoxy-p-mentha-1,8(10) diene and trans-p-mentha-2,8-dienol. In addition to them, two new exotoxins—Cyclohexene, 4-ethenyl-1,4-dimethyl and 2-Cycloxen-1-one, 2-methyl-5-(1-methylethenyl) which were presumed to have insecticidal action. Insecticidal exotoxins from AUDT-240 isolate can be further exploited as a potential microbial based insecticide for eco-friendly management of diamondback moth. [ABSTRACT FROM AUTHOR]

The article presents the discussion on bacterial toxins bypassing the rate-limiting steps in systemic inflammation triggering harmful levels of T-lymphocyte activation.

Streptococcus pyogenes is one of the most important species among beta-haemolytic streptococci, causing human infections of different localization. It is isolated from clinical specimens relatively frequently. In this study, the frequency and co-occurrence of toxin genes (speA, speB, speC, speH, speJ, speK) among 147 S. pyogenes strains were evaluated, using real-time PCR. In addition, the relationship between the occurrence of these genes and the origin of S. pyogenes strains from selected clinical material was assessed. The speB gene was present with the highest incidence (98.6%), while the speK gene was the least frequent (8.2%) among the tested strains. Based on the presence of the detected genes, the distribution of 17 genotypes was determined. The most common (21.8%), was speA (−) speB (+) speC (−) speH (−) speJ (−) speK (−) genotype. Furthermore, significant variation in the presence of some genes and genotypes of toxins in S. pyogenes strains isolated from different types of clinical material was found. There is a considerable variety and disproportion between the frequency of individual genes and genotypes of toxins in S. pyogenes strains. The relationship between the origin of S. pyogenes isolates and the presence of toxins genes indicates their pathogenic potential in the development of infections of selected localization. [ABSTRACT FROM AUTHOR]

Pathogens attack host cells by deploying toxins that perturb core host processes. Recent findings from the nematode C. elegans and other metazoans indicate that surveillance or "effector-triggered" pathways monitor functioning of these core processes and mount protective responses when they are perturbed. Despite a growing number of examples of surveillance immunity, the signaling components remain poorly defined. Here, we show that CEBP-2, the C. elegans ortholog of mammalian CCAAT-enhancer-binding protein gamma, is a key player in surveillance immunity. We show that CEBP-2 acts together with the bZIP transcription factor ZIP-2 in the protective response to translational block by P. aeruginosa Exotoxin A as well as perturbations of other processes. CEBP-2 serves to limit pathogen burden, promote survival upon P. aeruginosa infection, and also promote survival upon Exotoxin A exposure. These findings may have broad implications for the mechanisms by which animals sense pathogenic attack and mount protective responses.

Shaymaa H Abdel-Rhman,1,2 Dina E Rizk,1 Eman S Abdelmegeed1 1Department of Microbiology and Immunology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt; 2Department of Pharmaceutics and Pharmaceutical Biotechnology, Faculty of Pharmacy, Taibah University, AlMadinah Al Munawwarah, Saudi ArabiaCorrespondence: Dina E RizkDepartment of Microbiology and Immunology, Faculty of Pharmacy, Mansoura University, Mansoura 35516, EgyptTel +201002605440Fax +20502247496Email dena@mans.edu.egIntroduction: Pseudomonas aeruginosa is considered a dangerous pathogen, as it causes many human diseases, besides that it is resistant to almost all types of antibacterial agents. So, new strategies to overcome P. aeruginosa infection have evolved to attenuate its virulence factors and inhibit its quorum-sensing (QS) activity.Purpose: This study investigated the effect of tyrosol and EDTA as anti-quorum-sensing and antivirulence agents against P. aeruginosa PAO1.Methods: Anti-quorum activity of sub-minimum inhibitory concentrations (sub-MICs) of tyrosol and EDTA was tested using Chromobacterium violaceum (CV 12,472) biosensor bioassay. Miller assay was used to assess the inhibition of QS signal molecules by β-galactosidase activity determination. Also, their effects on the production of protease, lipase, lecithinase, and motility were tested. The inhibitory effects of these molecules on QS regulatory genes and exotoxins genes expression were evaluated by real-time PCR.Results: Tyrosol and EDTA at sub-MICs inhibited the production of violacein pigment. Both compounds inhibited QS molecules production and their associated virulence factors (protease, lipase, lecithinase, and motility) (P≤ 0.05). Besides, the expression levels of QS regulatory genes (lasI, lasR, rhƖI, rhIR, pqsA, and pqsR) and exotoxins genes (exoS and exoY) were significantly reduced (P≤ 0.05).Conclusion: Both tyrosol and EDTA can be used to fight P. aeruginosa infection as anti-quorum-sensing and antivirulence agents at their sub-MICs.Keywords: PAO1, C. violaceum, signal molecules, real-time PCR, exotoxins, QS regulatory genes

Three family members attended their general practice and emergency department over a 3-month period with recurrent skin and soft tissue infections (SSTIs) such as paronychia, submandibular carbuncle and groin and gluteal abscess requiring surgical drainage. Only when two family members were concurrently admitted with abscesses requiring drainage under general anaesthetic was the definitive diagnosis reached. The wound swabs identified methicillin-resistant Staphylococcus aureus (MRSA) and subsequent identification of the exotoxin Panton-Valentine leukocidin (PVL). Following MRSA decolonisation therapy with mupirocin and octenidine, only one family member has had one recurrence of an SSTI with MRSA isolated from the wound. When patients present with a history of recurrent SSTIs or a family all have had similar presentations, the clinician should consider MRSA with PVL exotoxin infection. Then patients must be referred for confirmation to ensure management is effective for the SSTI and prescribe MRSA decolonisation therapy concurrently to reduce recurrence., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Athletes are vulnerable to Staphylococcus aureus infections due to skin-to-skin contact and skin abrasions during training and competitions involving sharied sport equipment or toiletries, which promote the spread of the bacteria between athletes and within sport teams. This results not only in higher prevalence of S.aureus carriage among athletes compared to the general population, but also in outbreaks of infections, particularly skin infections, within sports teams. To limit the spread of S. aureus among athletes, a decolonization protocol can be applied when clustered cases of S. aureus infections occur, especially if Panton-Valentine leukocidin-producing strains are implicated. Finally, to avoid exposing athletes to S.aureus transmission/colonization, it is recommended to establish strict and clearly formulated individual and collective hygiene rules and to regularly disinfect shared sports equipment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

The aim of this study is to present the first nationwide microbiological and epidemiological study of invasive group A Streptococcus (iGAS) disease in Spain. One thousand eight hundred ninety-three iGAS isolates were analyzed over 2007–2019. emm typing was performed by sequencing the gene's variable 5' end, exotoxin genes were identified by PCR, and antimicrobial susceptibility explored via the E test and disk diffusion. Five hundred twenty-three isolates were associated with sepsis, 292 with cellulitis, 232 with scarlet fever, 153 with pneumonia, 141 with streptococcal toxic shock syndrome, and 94 with necrotizing fasciitis. The most prevalent emm types were emm1 (449/1893 isolates), emm89 (210/1893), emm3 (208/1893), emm4 (150/1893), emm12 (112/1893) emm6 (107/1893), emm87 (89/1893), emm28 (88/1893), emm75 (78/1893), emm77 (78/1893), emm11 (58/1893), and emm22 (35/1893). emm1, emm3, emm4, and emm6 were the predominant types affecting children (mostly respiratory infections), while emm11, emm77, and emm89 prevailed in the elderly (mostly skin infections). Each emm type was associated with one or more exotoxin gene (spe, sme, and ssa) profiles. speA was detected in 660 isolates, speB in 1829, speC in 1014, speF in 1826, speG in 1651, speJ in 716, speH in 331, smeZ in 720, and ssa in 512. Isolates with speA were associated with the most severe infections. Penicillin susceptibility was universal. Two hundred twenty-four isolates were resistant to tetracycline, 169 to erythromycin, and 81 to clindamycin. Tetracycline, erythromycin, and clindamycin resistance rates declined over the study period. The above information could serve as the basis for continued surveillance efforts designed to control disease cause by this bacterium. [ABSTRACT FROM AUTHOR]

A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.

Group A Streptococcus (GAS) is a leading human pathogen producing a diverse array of infections from simple pharyngitis ("strep throat") to invasive conditions, including necrotizing fasciitis and toxic shock syndrome. The surface-anchored GAS M1 protein is a classical virulence factor that promotes phagocyte resistance and exaggerated inflammation by binding host fibrinogen (Fg) to form supramolecular networks. In this study, we used a virulent WT M1T1 GAS strain and its isogenic M1-deficient mutant to examine the role of M1-Fg binding in a proximal step in GAS infection-interaction with the pharyngeal epithelium. Expression of the M1 protein reduced GAS adherence to human pharyngeal keratinocytes by 2-fold, and this difference was increased to 4-fold in the presence of Fg. In stationary phase, surface M1 protein cleavage by the GAS cysteine protease SpeB eliminated Fg binding and relieved its inhibitory effect on GAS pharyngeal cell adherence. In a mouse model of GAS colonization of nasal-associated lymphoid tissue, M1 protein expression was associated with an average 6-fold decreased GAS recovery in isogenic strain competition assays. Thus, GAS M1 protein-Fg binding reduces GAS pharyngeal cell adherence and colonization in a fashion that is counterbalanced by SpeB. Inactivation of SpeB during the shift to invasive GAS disease allows M1-Fg binding, increasing pathogen phagocyte resistance and proinflammatory activities.

Abstract Background Exotoxins are important virulence factors in Staphylococcus aureus. Clindamycin, a protein synthesis inhibitor antibiotic, is thought to limit exotoxin production and improve outcomes in severe S. aureus infections. However, randomised prospective data to support this are lacking. Methods An open-label, multicentre, randomised controlled trial (RCT) will compare outcome differences in severe S. aureus infection between standard treatment (flucloxacillin/cefazolin in methicillin-susceptible S. aureus; and vancomycin/daptomycin in methicillin-resistant S. aureus) and standard treatment plus an additional clindamycin given for 7 days. We will include a minimum of 60 participants (both adult and children) in the pilot study. Participants will be enrolled within 72 h of an index culture. Severe infections will include septic shock, necrotising pneumonia, or multifocal and non-contiguous skin and soft tissue/osteoarticular infections. Individuals who are immunosuppressed, moribund, with current severe diarrhoea or Clostridiodes difficile infection, pregnant, and those with anaphylaxis to β-lactams or lincosamides will be excluded. The primary outcomes measure is the number of days alive and free (1 or 0) of systemic inflammatory response syndrome (SIRS) within the first 14 days post randomisation. The secondary outcomes measure will include all-cause mortality at 14, 42, and 90 days, time to resolution of SIRS, proportion with microbiological treatment failure, and rate of change of C-reactive protein over time. Impacts of inducible clindamycin resistance, strain types, methicillin susceptibility, and presence of various exotoxins will also be analysed. Discussion This study will assess the effect of adjunctive clindamycin on patient-centred outcomes in severe, toxin-mediated S. aureus infections. The pilot study will provide feasibility for a much larger RCT. Trial registration Australian New Zealand Clinical Trials Registry, ACTRN12617001416381p. Registered on 6 October 2017.

Objectives: Outbreaks of scarlet fever in Mexico in 1999, Hong Kong and mainland China in 2011, and England in 2014-2016 have received global attention, and the number of notified cases in Korean children, including in Jeju Province, has also increased since 2010. To identify relevant hypotheses regarding this emerging outbreak, an age-period-cohort (APC) analysis of scarlet fever incidence was conducted among children in Jeju Province, Korea. Methods: This study analyzed data from the nationwide insurance claims database administered by the Korean National Health Insurance Service. The inclusion criteria were children aged ≤14 years residing in Jeju Province, Korea who received any form of healthcare for scarlet fever from 2002 to 2016. The age and year variables were categorized into 5 groups, respectively. After calculating the crude incidence rate (CIR) for age and calendar year groups, the intrinsic estimator (IE) method was applied to conduct the APC analysis. Results: In total, 2345 cases were identified from 2002 to 2016. Scarlet fever was most common in the 0-2 age group, and boys presented more cases than girls. Since the CIR decreased with age between 2002 and 2016, the age and period effect decreased in all observed years. The IE coefficients suggesting a cohort effect shifted from negative to positive in 2009. Conclusions: The results suggest that the recent outbreak of scarlet fever among children in Jeju Province might be explained through the cohort effect. As children born after 2009 showed a higher risk of scarlet fever, further descriptive epidemiological studies are needed.

Objective(s): Honey’s ability to kill microorganisms and even eradication of chronic infections with drug-resistant pathogens has been documented by numerous studies. The present study is focused on the action of honey in its sub-inhibitory levels to impact on the pathogens coordinated behaviors rather than killing them. Materials and Methods: The impact of local honey on the quorum sensing related virulence of multidrug resistant Pseudomonas aeruginosa burn isolates was investigated by detection its effect on the virulence, biofilm formation and expression of quorum sensing related and exotoxin A genes.Results: Experiments to characterise and quantify the impact of honey on the P. aeruginosa quorum sensing networks showed that the expression of exotoxin A ( ETA), las and rhl glucons reduced by low concentrations of honey including the associated virulence factors. Conclusion: Our results indicated that honey fights infections either by its bactericidal components which vigorously kill cells or by weakening bacterial coordination and virulence through interruption of quorum sensing system.

Staphylococcal scalded skin syndrom is a bullous dermatosis induced by exfoliating staphylococcal exotoxins. Children are most often affected. We report the case of a 6-month-old infant who had angina in the few days before leading up to bullous erythroderma and whose skin biopsy showed characteristic appearance of staphylococcal scalded skin syndrom. The development was rapidly unfavourable and the infant died in a refractory septic shock chart, despite the introduction of norepinephrine and anti-SAMR antibiotic therapy. The term staphylococcal scalded skin syndrome (SSSS) was separated from the toxic or allergic epidermal necrolysis by Lyell into the opposite anatomical aspect of these two entities: in scalded skin syndrome, Skin detachment is done by cleavage of the superficial part of the epidermis at the granular layer, while in toxic Lyell syndrome, the cleavage sits deeper at the level of the mucous body. [ABSTRACT FROM AUTHOR]

Streptococcus pyogenes is one of the most important species among beta-haemolytic streptococci, causing human infections of different localization. It is isolated from clinical specimens relatively frequently. In this study, the frequency and co-occurrence of toxin genes (speA, speB, speC, speH, speJ, speK) among 147 S. pyogenes strains were evaluated, using real-time PCR. In addition, the relationship between the occurrence of these genes and the origin of S. pyogenes strains from selected clinical material was assessed. The speB gene was present with the highest incidence (98.6%), while the speK gene was the least frequent (8.2%) among the tested strains. Based on the presence of the detected genes, the distribution of 17 genotypes was determined. The most common (21.8%), was speA (−) speB (+) speC (−) speH (−) speJ (−) speK (−) genotype. Furthermore, significant variation in the presence of some genes and genotypes of toxins in S. pyogenes strains isolated from different types of clinical material was found. There is a considerable variety and disproportion between the frequency of individual genes and genotypes of toxins in S. pyogenes strains. The relationship between the origin of S. pyogenes isolates and the presence of toxins genes indicates their pathogenic potential in the development of infections of selected localization.

Background Linezolid is recommended for treatment of pneumonia and other invasive infections caused by methicillin-resistant Staphylococcus aureus (MRSA). The premise underlying this recommendation is that linezolid inhibits in vivo production of potent staphylococcal exotoxins, including Panton-Valentine leukocidin (PVL) and α-hemolysin (Hla), although supporting evidence is lacking.Methods A rabbit model of necrotizing pneumonia using MRSA clone USA300 was used to compare therapeutic effects of linezolid (50 mg/kg 3 times/day) and vancomycin (30 mg/kg 2 times/day) administered 1.5, 4, and 9 hours after infection on host survival outcomes and in vivo bacterial toxin production.Results Mortality rates were 100% for untreated rabbits and 83%-100% for vancomycin-treated rabbits. In contrast, mortality rates were 25%, 50%, and 100% for rabbits treated with linezolid 1.5, 4, and 9 hours after infection, respectively. Compared with untreated and vancomycin-treated rabbits, improved survival of rabbits treated 1.5 hours after infection with linezolid was associated with a significant decrease in bacterial counts, suppressed bacterial production of PVL and Hla, and reduced production of the neutrophil-chemoattractant interleukin 8 in the lungs.Conclusions Across the study interval, only early treatment with linezolid resulted in significant suppression of exotoxin synthesis and improved survival outcomes in a rabbit model of MRSA necrotizing pneumonia.

ObjectivesTo determine whether cytotoxic and invasive Pseudomonas aeruginosa strains differentially influence clinical presentation, outcomes, or therapeutic response in bacterial keratitis.MethodsPseudomonas aeruginosa isolates from the National Eye Institute-funded Steroids for Corneal Ulcers Trial were subtyped as cytotoxic or invasive strains. The main outcome measure compared between the 2 subtypes was change in visual acuity at 3 months using Huber robust regression, adjusting for topical corticosteroid treatment.ResultsOf 101 confirmed P aeruginosa isolates from the Steroids for Corneal Ulcers Trial, 74 had a classically cytotoxic or invasive genotype. While corneal ulcers caused by genotypically invasive P aeruginosa strains were associated at presentation with significantly better visual acuity than corneal ulcers caused by genotypically cytotoxic P aeruginosa strains when adjusting for the effect of ulcer location (P= .008), invasive ulcers had improved significantly less than cytotoxic ulcers at 3 months (0.35; 95% CI, 0.04-0.66 logMAR; P= .03 [3.5-line difference]). Compared with topical moxifloxacin alone, adjunctive treatment with topical corticosteroids was associated with significantly more improvement in visual acuity in the invasive subgroup (P= .04) but was associated with less improvement in visual acuity in the cytotoxic subgroup (P= .07).ConclusionsRational profiling of differentially expressed virulence determinants (eg, cytotoxicity and invasiveness for P aeruginosa) could be used as a tool for decision making in the management of infections to optimize outcomes.

Background: Based on gas chromatography - mass spectrometry (GC-MS) results of a previous study, six metabolites including alpha-terpineol, geranyl acetate, linalool, myrcenol, terpinolene, and thymol showed significantly higher amounts relative to other metabolites., Methods: A continuation of the previous study, the interaction of these metabolites with the main virulence factors of P. aeruginosa (pseudomonas elastase and exotoxin A), Staphylococcus aureus (alpha-hemolysin and protein 2a), Mycobacterium tuberculosis (ESX-secreted protein B and the serine/threonine protein kinase), and Escherichia coli (heat-labile enterotoxin and Shiga toxin) were evaluated by molecular docking study and molecular simulation., Results: In the case of Shiga toxin, higher and lower binding affinities were related to alpha-terpinolene and zincite with values of -5.8 and -2.6 kcal/mol, respectively. For alpha-hemolysin, terpinolene and alpha-terpinolene demonstrated higher binding affinities with similar energies of -5.9 kcal/mol. Thymol and geranyl acetate showed lower binding energy of -5.7 kcal/mol toward protein 2a. Furthermore, thymol had a higher binding affinity toward heat-labile enterotoxin and ESX-secreted protein B with values of -5.9 and -6.1 kcal/mol, respectively., Conclusions: It is concluded that the availability of secondary metabolites of A. haussknechtii surrounding zinc oxide (ZnO) NPs can hinder P. aeruginosa by inactivating Pseudomonas elastase and exotoxin.

Delayed wound healing caused by bacterial infection remains a major challenge in clinical treatment. Exotoxins incorporated in bacterial extracellular vesicles play a key role as the disease-causing virulence factors. Safe and specific antivirulence agents are expected to be developed as an effective anti-bacterial infection strategy, instead of single antibiotic therapy. Plant-derived extracellular vesicle-like nanoparticles have emerged as promising therapeutic agents for skin diseases, but the elucidations of specific mechanisms of action and clinical transformation still need to be advanced. Here, dandelion-derived extracellular vesicle-like nanoparticles (TH-EVNs) are isolated and exert antivirulence activity through specifically binding to Staphylococcus aureus (S. aureus) exotoxins, thereby protecting the host cell from attack. The neutralization of TH-EVNs against exotoxins has considerable binding force and stability, showing complete detoxification effect in vivo. Then gelatin methacryloyl hydrogel is developed as TH-EVNs-loaded dressing for S. aureus exotoxin-invasive wounds. Hydrogel dressings demonstrate good physical and mechanical properties, thus achieving wound retention and controlled release of TH-EVNs, in addition to promoting cell proliferation and migration. In vivo results show accelerated re-epithelialization, promotion of collagen maturity and reduction of inflammation after treatment. Collectively, the developed TH-EVNs-laden hydrogel dressings provide a potential therapeutic approach for S. aureus exotoxin- associated trauma., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study aimed to determine resistance to antimicrobials of Staphylococcus aureus strains isolated from clinical specimens in Lithuanian hospitals and to identify the genes conferring resistance and virulence. The study was carried out from June 2019 to September 2021. S. aureus strains were isolated from skin, soft tissues, blood, lower respiratory tract, urine and other specimens. Antibiotic susceptibility testing was performed using the disc diffusion method according to EUCAST guidelines. All isolates were analyzed for detection of the ermA , ermC , mecA , mecC , tetK , tetM , and lukF-PV genes by multiplex real-time PCR. The 16S rRNA coding sequence was applied as an internal PCR control. Altogether, 745 S. aureus strains were analyzed. Antimicrobial susceptibility testing revealed that all isolates were susceptible to rifampin and vancomycin. Of the 745 strains, 94.8% were susceptible to tetracycline, 94.5% to clindamycin, and 88.3% to erythromycin. The lowest susceptibility rate was found for penicillin (25.8%). Six percent of the tested strains were methicillin-resistant S. aureus (MRSA). The majority of methicillin-resistant strains were isolated from skin and soft tissues (73.3%), with a smaller portion isolated from blood (17.8%) and respiratory tract (8.9%). The ermC gene was detected in 41.1% of erythromycin-resistant S. aureus strains, whereas ermA was detected in 32.2% of erythromycin-resistant S. aureus strains. 69.2% of tetracycline-resistant S. aureus strains had tetK gene, and 28.2% had tetM gene. 7.3% of S. aureus isolates harbored lukF-PV gene. The frequency of the pvl gene detection was significantly higher in MRSA isolates than in methicillin-susceptible S. aureus isolates ( p < 0.0001)., (© 2024 Agnė Kirkliauskienė et al., published by Sciendo.)

Staphylococcus aureus is a leading cause of bloodstream infections worldwide. In the United States, many of these infections are caused by a strain known as USA300. Although progress has been made, our understanding of the S. aureus molecules that promote survival in human blood and ultimately facilitate metastases is incomplete. To that end, we analyzed the USA300 transcriptome during culture in human blood, human serum, and trypticase soy broth (TSB), a standard laboratory culture media. Notably, genes encoding several cytolytic toxins were up-regulated in human blood over time, and hlgA, hlgB, and hlgC (encoding gamma-hemolysin subunits HlgA, HlgB, and HlgC) were among the most highly up-regulated genes at all time points. Compared to culture supernatants from a wild-type USA300 strain (LAC), those derived from an isogenic hlgABC-deletion strain (LACΔhlgABC) had significantly reduced capacity to form pores in human neutrophils and ultimately cause neutrophil lysis. Moreover, LACΔhlgABC had modestly reduced ability to cause mortality in a mouse bacteremia model. On the other hand, wild-type and LACΔhlgABC strains caused virtually identical abscesses in a mouse skin infection model, and bacterial survival and neutrophil lysis after phagocytosis in vitro was similar between these strains. Comparison of the cytolytic capacity of culture supernatants from wild-type and isogenic deletion strains lacking hlgABC, lukS/F-PV (encoding PVL), and/or lukDE revealed functional redundancy among two-component leukotoxins in vitro. These findings, along with a requirement of specific growth conditions for leukotoxin expression, may explain the apparent limited contribution of any single two-component leukotoxin to USA300 immune evasion and virulence.

ABSTRACT The clostridial neurotoxins (CNTs) comprise tetanus toxin (TT) and botulinum neurotoxin (BoNT [BT]) serotypes (A to G and X) and several recently identified CNT-like proteins, including BT/En and the mosquito BoNT-like toxin Pmp1. CNTs are produced as single proteins cleaved to a light chain (LC) and a heavy chain (HC) connected by an interchain disulfide bond. LC is a zinc metalloprotease (cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]), while HC contains an N-terminal translocation domain (HCN) and a C-terminal receptor binding domain (HCC). HCN-mediated LC translocation is the least understood function of CNT action. Here, β-lactamase (βlac) was used as a reporter in discovery-based live-cell assays to characterize TT-mediated LC translocation. Directed mutagenesis identified a role for a charged loop (767DKE769) connecting α15 and α16 (cis-loop) within HCN in LC translocation; aliphatic substitution inhibited LC translocation but not other toxin functions such as cell binding, intracellular trafficking, or HCN-mediated pore formation. K768 was conserved among the CNTs. In molecular simulations of the HCN with a membrane, the cis-loop did not bind with the cell membrane. Taken together, the results of these studies implicate the cis-loop in LC translocation, independently of pore formation. IMPORTANCE How protein toxins translocate their catalytic domain across a cell membrane is the least understood step in toxin action. This study utilized a reporter, β-lactamase, that was genetically fused to full-length, nontoxic tetanus toxin (βlac-TT) in discovery-based live-cell assays to study LC translocation. Directed mutagenesis identified a role for K768 in LC translocation. K768 was located between α15 and α16 (termed the cis-loop). Cellular assays showed that K768 did not interfere with other toxin functions, including cell binding, intracellular trafficking, and pore formation. The equivalent K768 is conserved among the clostridial neurotoxin family of proteins as a conserved structural motif. The cis-loop appears to contribute to LC translocation.

BackgroundMany gram-positive bacteria produce pore-forming exotoxins that contain a highly conserved, 12-residue domain (ECTGLAWEWWRT) that binds cholesterol. This domain is usually flanked N-terminally by arginine and C-terminally by valine. We used this 14-residue sequence as a template to create a small library of peptides that bind cholesterol and other lipids.Methodology/resultsSeveral of these peptides manifested anti-inflammatory properties in a predictive in vitro monocyte chemotactic assay, and some also diminished the pro-inflammatory effects of low-density lipoprotein in apoE-deficient mice. The most potent analog, Oxpholipin-11D (OxP-11D), contained D-amino acids exclusively and was identical to the 14-residue design template except that diphenylalanine replaced cysteine-3. In surface plasmon resonance binding studies, OxP-11D bound oxidized (phospho)lipids and sterols in much the same manner as D-4F, a widely studied cardioprotective apoA-I-mimetic peptide with anti-inflammatory properties. In contrast to D-4F, which adopts a stable alpha-helical structure in solution, the OxP-11D structure was flexible and contained multiple turn-like features.ConclusionGiven the substantial evidence that oxidized phospholipids are pro-inflammatory in vivo, OxP-11D and other Oxpholipins may have therapeutic potential.