Shiga Toxin Research Articles

Isolation and Identification of Shigella SonneiProducing Shiga Toxin from Children with Bloody Diarrhea and Evaluation of the Inhibition Effectiveness of Zinc oxide Nanoparticles

Shigella infection strains producing shiga toxin results in bloody diarrheais a dangerous symptom in children under five years of age that can even lead to death. Therefore, this study was conducted to isolate Shigella strains producing shiga toxin from 150 samples which were taken from bloody stool of children under the five years of age. They were suffering from the diarrhea during the period from March 2021 to March 2022 in Tikrit city, Iraq. The results showed the possibility to isolate six isolates of Shigella sonnei at ratio 4% of the total samples. The isolates producing shiga toxin were identified by using stx1 gene. The results showed two isolates of Shigella possessing stx1 gene, at ratio 33.33 of total S. sonnei strains. The two isolates producing shiga toxin have been submitted to NCBI, then accepted as Iraqi strains in NCBI under the registration numbers OK127759.1 and OK127760.1. The Iraqi strains registered in NCBI showed agreement with a global strain of 99-100% that were recorded by registration numbers in Malaysia (CP060117.1), China (CP000038.1), United Kingdom (CP066810.1), Hungary (CP019689.1), Somalia (CP023645.1), Spain (CP022672.1), Italy (CP035008.1), India (CP041322.1), Australia (CP045932.1), Nigeria (CP046286.1), Switzerland (CP049183.1), USA (CP053751.1) and South Korea (CP055292.1). As for the sensitivity test, the results showed that the strain OK127759.1 was resistant to antibiotics azithromycin, ceftazidime, cefotaxime, ceftriaxone, augmentin and tetracycline. And it was sensitive to gentamicin and intermediately sensitive to ampicillin, chloramphenicol and nalidixic acid. As for the strain OK127760.1, the results showed his strain was intermediately sensitive to gentamicin and resistant to the other antibiotics which were used in the test. ZnO-NPs with a concentration of 200 μg/ml showed the highest inhibitory effectiveness against S. sonnei strains OK127759.1 and OK127760.1 with inhibition diameter of 22 mm and 21 mm respectively.

Pathogenomes of Shiga Toxin Positive and Negative Escherichia coli O157:H7 Strains TT12A and TT12B: Comprehensive Phylogenomic Analysis Using Closed Genomes.

Shiga toxin-producing Escherichia coli are zoonotic pathogens that cause food-borne human disease. Among these, the O157:H7 serotype has evolved from an enteropathogenic O55:H7 ancestor through the displacement of the somatic gene cluster and recurrent toxigenic conversion by Shiga toxin-converting bacteriophages. However, atypical strains that lack the Shiga toxin, the characteristic virulence hallmark, are circulating in this lineage. For this study, we analyzed the pathogenome and virulence inventories of the stx+ strain, TT12A, isolated from a patient with hemorrhagic colitis, and its respective co-isolated stx- strain, TT12B. Sequencing the genomes to closure proved critical to the cataloguing of subtle strain differentiating sequence and structural polymorphisms at a high-level of phylogenetic accuracy and resolution. Phylogenomic profiling revealed SNP and MLST profiles similar to the near clonal outbreak isolates. Their prophage inventories, however, were notably different. The attenuated atypical non-shigatoxigenic status of TT12B is explained by the absence of both the ΦStx1a- and ΦStx2a-prophages carried by TT12A, and we also recorded further alterations in the non-Stx prophage complement. Phenotypic characterization indicated that culture growth was directly impacted by the strains' distinct lytic phage complement. Altogether, our phylogenomic and phenotypic analyses show that these intimately related isogenic strains are on divergent Stx(+/stx-) evolutionary paths.

A Comprehensive Review on Shiga Toxin Subtypes and Their Niche-Related Distribution Characteristics in Shiga-Toxin-Producing E. coli and Other Bacterial Hosts.

Shiga toxin (Stx), the main virulence factor of Shiga-toxin-producing E. coli (STEC), was first discovered in Shigella dysenteriae strains. While several other bacterial species have since been reported to produce Stx, STEC poses the most significant risk to human health due to its widespread prevalence across various animal hosts that have close contact with human populations. Based on its biochemical and molecular characteristics, Shiga toxin can be grouped into two types, Stx1 and Stx2, among which a variety of variants and subtypes have been identified in various bacteria and host species. Interestingly, the different Stx subtypes appear to vary in their host distribution characteristics and in the severity of diseases that they are associated with. As such, this review provides a comprehensive overview on the bacterial species that have been recorded to possess stx genes to date, with a specific focus on the various Stx subtype variants discovered in STEC, their prevalence in certain host species, and their disease-related characteristics. This review provides a better understanding of the Stx subtypes and highlights the need for rapid and accurate approaches to toxin subtyping for the proper evaluation of the health risks associated with Shiga-toxin-related bacterial food contamination and human infections.

Structural basis to identify a target site in Shiga toxin for the inhibitor discovery against growth of Shiga toxin-producing E. coli

BackgroundCertain peptides that bind Shiga toxin 2 (Stx2) have been reported to treat Shiga toxin-producing Escherichia coli (STEC) infections. However, their mechanisms of action remain unknown. STEC infections lead to serious diseases, such as hemolytic uremic syndrome, in humans. Antibiotic therapy is usually not recommended because of the major challenges of antibiotic resistance and SOS repair. Currently, there is no human vaccine for STEC infection, leaving rehydration therapy as the recommended supportive therapy. Therefore, there is a need for targeted therapeutic intervention to inhibit STEC growth. The purpose of this study was to evaluate the interaction of five known peptides with Stx2 to identify a more suitable peptide based on structural changes. These peptides have been used to inhibit the growth of STEC.ResultsThe current study demonstrated that only tetravalent peptide (TVP) out of 5 common peptides interrupted the Y77-E259 interaction of Stx2, making it active by exposing active site, which ultimately leads to STEC cell death. We also demonstrated that amino acids R170 and F171 of Stx2 in the docked complex of Stx2 and TVP form a helix-loop-helix (HLH). This might lead to the differential expression of genes regulated by Stx2 and ultimately inhibit STEC growth. However, in the case of Stx2-ribosomal P-stalk, these residues did not form HLH. The 3D refined model of TVP showed a low MolProbity score and low energy zones in the ANOLEA profile compared to the original one. Moreover, the low radius of gyration of the refined TVP suggests that it is more compact than the original TVP. Therefore, TVP is a suitable drug candidate for the inhibition of STEC growth. However, the low antigenicity of TVP makes it unsuitable as a drug candidate. We also evaluated three antibiotics that have been used as active ingredients in FDA-approved peptides. Only Oritavancin diphosphate showed strong polar interactions with Y77-E259 and also had the highest binding affinity.ConclusionsPotential drug candidates that inhibit or interrupt the interaction between Y77-E259 and have high antigenicity, low toxicity, and no allergenicity should be explored against the growth of STEC.

A bacterial toxin co-opts caspase-3 to disable active gasdermin D and limit macrophage pyroptosis

During infections, host cells are exposed to pathogen-associated molecular patterns (PAMPs) and virulence factors that stimulate multiple signaling pathways that interact additively, synergistically, or antagonistically. The net effect of such higher-order interactions is a vital determinant of the outcome of host-pathogen interactions. Here, we demonstrate one such complex interplay between bacterial exotoxin- and PAMP-induced innate immune pathways. We show that two caspases activated during enterohemorrhagic Escherichia coli (EHEC) infection by lipopolysaccharide (LPS) and Shiga toxin (Stx) interact in a functionally antagonistic manner; cytosolic LPS-activated caspase-11 cleaves full-length gasdermin D (GSDMD), generating an active pore-forming N-terminal fragment (NT-GSDMD); subsequently, caspase-3 activated by EHEC Stx cleaves the caspase-11-generated NT-GSDMD to render it nonfunctional, thereby inhibiting pyroptosis and interleukin-1β maturation. Bacteria typically subvert inflammasomes by targeting upstream components such as NLR sensors or full-length GSDMD but not active NT-GSDMD. Thus, our findings uncover a distinct immune evasion strategy where a bacterial toxin disables active NT-GSDMD by co-opting caspase-3.

Soluplus® nanomicelles enhance IgG neutralizing properties against Shiga toxin type 2

Shiga toxin (Stx) is the main virulence factor of Stx-producing Escherichia coli, a pathogen responsible for developing Hemolytic Uremic Syndrome. Nowadays, no specific treatment exists for this disease, and nanomicelles may be an excellent platform for the delivery of specific compounds, including immunoglobulins (IgG), for therapeutic or diagnostic strategies. In this work, Soluplus® nanomicelles (NM) were associated with IgG against Stx type 2 (NM-IgG-Stx2). Physicochemical evaluation of NM-IgG-Stx2 was performed by Dynamic Light Scattering, Uv–Vis, Fluorescence, and Circular Dichroism spectroscopy. Morphology was analyzed by transmission electron microscopy. An in silico analysis of NM-IgG-Stx2 association was performed, and neutralization properties against purified Stx2 and Stx-derived supernatants were analyzed on Vero and HGEC cells. Our results suggest that IgG-Stx2 assemble with NM, and the interaction improves the Stx2 neutralization capacity of the antibody. These results suggest that NM may enhance IgG-Stx2 paratope availability and may be an excellent platform for the recognition and neutralization of Stx2.

Determination of Virulence Factors and Antibiotic Resistance Profile of E. coli O157:H7 in some Marine and Freshwater Fish from Samsun, Türkiye

In this study, Escherichia coli O157:H7 contamination was investigated in Engraulis encrasicolus, Merlangius euxmus, Trachurus mediterraneus, Mullus barbutus, Tinca tinca, Cyprinus carpio and Oncorchynhus mykiss (total of 300 samples). The presence of certain virulence genes such as shiga toxin 1 (stx1), shiga toxin 2 (stx2), intimin (eaeA), and hemolysin (hlyA) was determined. The E-test method was employed to determine the antibiotic resistance profile and minimum inhibitory concentration (MIC) values of the isolates. Out of a total 141 freshwater fish samples, E. coli O157:H7 was detected in 5 of them (3.5%). In 159 marine fish, 2 of them (1.25%) were E. coli O157 positive. In terms of virulence characteristics, the E. coli O157:H7 isolate obtained from the Oncorhynchus mykiss sample had the stx1, eaeA, and hlyA genes. Both eaeA and hlyA genes were determined as positive in the E. coli O157 isolate identified from the Merlangius euxmus sample. Resistance to tetracycline, cefotaxime, cefepime, ciprofloxacin, gentamicin, ertapenem and cefoxitin was demonstrated in both samples. In this study, the presence of E. coli O157 in fish, detection of virulence genes and antibiotic resistance profile indicate the potential risk of infection from contaminated seafood.

Pathogenomes and virulence profiles of representative big six non-O157 serogroup Shiga toxin-producing Escherichia coli.

Shiga toxin (Stx)-producing Escherichia coli (STEC) of non-O157:H7 serotypes are responsible for global and widespread human food-borne disease. Among these serogroups, O26, O45, O103, O111, O121, and O145 account for the majority of clinical infections and are colloquially referred to as the "Big Six." The "Big Six" strain panel we sequenced and analyzed in this study are reference type cultures comprised of six strains representing each of the non-O157 STEC serogroups curated and distributed by the American Type Culture Collection (ATCC) as a resource to the research community under panel number ATCC MP-9. The application of long- and short-read hybrid sequencing yielded closed chromosomes and a total of 14 plasmids of diverse functions. Through high-resolution comparative phylogenomics, we cataloged the shared and strain-specific virulence and resistance gene content and established the close relationship of serogroup O26 and O103 strains featuring flagellar H-type 11. Virulence phenotyping revealed statistically significant differences in the Stx-production capabilities that we found to be correlated to the strain's individual stx-status. Among the carried Stx1a, Stx2a, and Stx2d phages, the Stx2a phage is by far the most responsive upon RecA-mediated phage mobilization, and in consequence, stx2a + isolates produced the highest-level of toxin in this panel. The availability of high-quality closed genomes for this "Big Six" reference set, including carried plasmids, along with the recorded genomic virulence profiles and Stx-production phenotypes will provide a valuable foundation to further explore the plasticity in evolutionary trajectories in these emerging non-O157 STEC lineages, which are major culprits of human food-borne disease.

Development of a Rapid and Sensitive CANARY Biosensor Assay for the Detection of Shiga Toxin 2 from Escherichia coli.

Shiga-toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). The current Food Safety Inspection Service (FSIS) testing methods for STEC use the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) protocol, which includes enrichment, cell plating, and genomic sequencing and takes time to complete, thus delaying diagnosis and treatment. We wanted to develop a rapid, sensitive, and potentially portable assay that can identify STEC by detecting Shiga toxin (Stx) using the CANARY (Cellular Analysis and Notification of Antigen Risks and Yields) B-cell based biosensor technology. Five potential biosensor cell lines were evaluated for their ability to detect Stx2. The results using the best biosensor cell line (T5) indicated that this biosensor was stable after reconstitution with assay buffer covered in foil at 4 °C for up to 10 days with an estimated limit of detection (LOD) of ≈0.1-0.2 ng/mL for days up to day 5 and ≈0.4 ng/mL on day 10. The assay detected a broad range of Stx2 subtypes, including Stx2a, Stx2b, Stx2c, Stx2d, and Stx2g but did not cross-react with closely related Stx1, abrin, or ricin. Additionally, this assay was able to detect Stx2 in culture supernatants of STEC grown in media with mitomycin C at 8 and 24 h post-inoculation. These results indicate that the STEC CANARY biosensor developed in this study is sensitive, reproducible, specific, rapid (≈3 min), and may be applicable for surveillance of the environment and food to protect public health.

Synthesis of biologically active Shiga toxins in cell-free systems

Shiga toxins (Stx) produced by pathogenic bacteria can cause mild to severe diseases in humans. Thus, the analysis of such toxins is of utmost importance. As an AB5 toxin, Stx consist of a catalytic A-subunit acting as a ribosome-inactivating protein (RIP) and a B-pentamer binding domain. In this study we synthesized the subunits and holotoxins from Stx and Stx2a using different cell-free systems, namely an E. coli- and CHO-based cell-free protein synthesis (CFPS) system. The functional activity of the protein toxins was analyzed in two ways. First, activity of the A-subunits was assessed using an in vitro protein inhibition assay. StxA produced in an E. coli cell-free system showed significant RIP activity at concentrations of 0.02 nM, whereas toxins synthesized in a CHO cell-free system revealed significant activity at concentrations of 0.2 nM. Cell-free synthesized StxA2a was compared to StxA2a expressed in E. coli cells. Cell-based StxA2a had to be added at concentrations of 20 to 200 nM to yield a significant RIP activity. Furthermore, holotoxin analysis on cultured HeLa cells using an O-propargyl-puromycin assay showed significant protein translation reduction at concentrations of 10 nM and 5 nM for cell-free synthesized toxins derived from E. coli and CHO systems, respectively. Overall, these results show that Stx can be synthesized using different cell-free systems while remaining functionally active. In addition, we were able to use CFPS to assess the activity of different Stx variants which can further be used for RIPs in general.

Structure-Function Analysis of the A1 Subunit of Shiga Toxin 2 with Peptides That Target the P-Stalk Binding Site and Inhibit Activity.

Shiga toxin 2a (Stx2a) is the virulence factor of Escherichia coli (STEC), which is associated with hemolytic uremic syndrome, the leading cause of pediatric kidney failure. The A1 subunit of Stx2a (Stx2A1) binds to the conserved C-terminal domain (CTD) of the ribosomal P-stalk proteins to remove an adenine from the sarcin-ricin loop (SRL) in the 28S rRNA, inhibiting protein synthesis. There are no antidotes against Stx2a or any other ribosome-inactivating protein (RIP). The structural and functional details of the binding of Stx2A1 to the P-stalk CTD are not known. Here, we carry out a deletion analysis of the conserved P-stalk CTD and show that the last eight amino acids (P8) of the P-stalk proteins are the minimal sequence required for optimal affinity and maximal inhibitory activity against Stx2A1. We determined the first X-ray crystal structure of Stx2A1 alone and in complex with P8 and identified the exact binding site. The C-terminal aspartic acid of the P-stalk CTD serves as an anchor, forming key contacts with the conserved arginine residues at the P-stalk binding pocket of Stx2A1. Although the ricin A subunit (RTA) binds to the P-stalk CTD, the last aspartic acid is more critical for the interaction with Stx2A1, indicating that RIPs differ in their requirements for the P-stalk. These results demonstrate that the catalytic activity of Stx2A1 is inhibited by blocking its interactions with the P-stalk, providing evidence that P-stalk binding is an essential first step in the recruitment of Stx2A1 to the SRL for depurination.

Re-analysis of an outbreak of Shiga toxin-producing Escherichia coli O157:H7 associated with raw drinking milk using Nanopore sequencing

The aim of this study was to compare Illumina and Oxford Nanopore Technology (ONT) sequencing data to quantify genetic variation to assess within-outbreak strain relatedness and characterise microevolutionary events in the accessory genomes of a cluster of 23 genetically and epidemiologically linked isolates related to an outbreak of Shiga toxin-producing Escherichia coli O157:H7 caused by the consumption of raw drinking milk. There were seven discrepant variants called between the two technologies, five were false-negative or false-positive variants in the Illumina data and two were false-negative calls in ONT data. After masking horizontally acquired sequences such as prophages, analysis of both short and long-read sequences revealed the 20 isolates linked to the outbreak in 2017 had a maximum SNP distance of one SNP between each other, and a maximum of five SNPs when including three additional strains identified in 2019. Analysis of the ONT data revealed a 47 kbp deletion event in a terminal compound prophage within one sample relative to the remaining samples, and a 0.65 Mbp large chromosomal rearrangement (inversion), within one sample relative to the remaining samples. Furthermore, we detected two bacteriophages encoding the highly pathogenic Shiga toxin (Stx) subtype, Stx2a. One was typical of Stx2a-phage in this sub-lineage (Ic), the other was atypical and inserted into a site usually occupied by Stx2c-encoding phage. Finally, we observed an increase in the size of the pO157 IncFIB plasmid (1.6 kbp) in isolates from 2019 compared to those from 2017, due to the duplication of insertion elements within the plasmids from the more recently isolated strains. The ability to characterize the accessory genome in this way is the first step to understanding the significance of these microevolutionary events and their impact on the genome plasticity and virulence between strains of this zoonotic, foodborne pathogen.

Shiga Toxin-Producing Escherichia coli Testing in New York 2011-2022 Reveals Increase in Non-O157 Identifications.

Shiga toxin-producing Escherichia coli (STEC) are an important cause of bacterial enteric infection. STEC strains cause serious human gastrointestinal disease, which may result in life-threatening complications such as hemolytic uremic syndrome. They have the potential to impact public health due to diagnostic challenges of identifying non-O157 strains in the clinical laboratory. The Wadsworth Center (WC), the public health laboratory of the New York State Department of Health, has isolated and identified non-O157 STEC for decades. A shift from initially available enzyme immunoassay testing to culture-independent diagnostic tests (CIDTs) has increased the uptake of testing at clinical microbiology laboratories. This testing change has resulted in an increased number of specimen submissions to WC. During a 12-year period between 2011 and 2022, WC received 5037 broths and/or stool specimens for STEC confirmation from clinical microbiology laboratories. Of these, 3992 were positive for Shiga toxin genes (stx1 and/or stx2) by real-time PCR. Furthermore, culture methods were utilized to isolate, identify, and characterize 2925 STEC from these primary specimens. Notably, WC observed a >200% increase in the number of STEC specimens received in 2021-2022 compared with 2011-2012 and an 18% increase in the number of non-O157 STEC identified using the same methodologies. During the past decade, the WC testing algorithm has been updated to manage the increase in specimens received, while also navigating the novel COVID-19 pandemic, which took priority over other testing for a period of time. This report summarizes updated methods for confirmation, surveillance, and outbreak detection of STEC and describes findings that may be related to our algorithm updates and the increased use of CIDTs, which is starting to elucidate the true incidence of non-O157 STEC.

Identification of GB3 as a Novel Biomarker of Tumor-Derived Vasculature in Neuroblastoma Using a Stiffness-Based Model.

Neuroblastoma (NB) is a childhood cancer in sympathetic nervous system cells. NB exhibits cellular heterogeneity, with adrenergic and mesenchymal states displaying distinct tumorigenic potentials. NB is highly vascularized, and blood vessels can form through various mechanisms, including endothelial transdifferentiation, leading to the development of tumor-derived endothelial cells (TECs) associated with chemoresistance. We lack specific biomarkers for TECs. Therefore, identifying new TEC biomarkers is vital for effective NB therapies. A stiffness-based platform simulating human arterial and venous stiffness was developed to study NB TECs in vitro. Adrenergic cells cultured on arterial-like stiffness transdifferentiated into TECs, while mesenchymal state cells did not. The TECs derived from adrenergic cells served as a model to explore new biomarkers, with a particular focus on GB3, a glycosphingolipid receptor implicated in angiogenesis, metastasis, and drug resistance. Notably, the TECs unequivocally expressed GB3, validating its novelty as a marker. To explore targeted therapeutic interventions, nanoparticles functionalized with the non-toxic subunit B of the Shiga toxin were generated, because they demonstrated a robust affinity for GB3-positive cells. Our results demonstrate the value of the stiffness-based platform as a predictive tool for assessing NB aggressiveness, the discovery of new biomarkers, and the evaluation of the effectiveness of targeted therapeutic strategies.

85 Evaluation of a Saccharomyces cerevisiae fermentation product on performance, plasma metabolites, rumen parameters, and fecal pathogen shedding in feedlot steers

Abstract The objective of the experiment was to evaluate a commercial Saccharomyces cerevisiae fermentation product (SCFP; NaturSafe, Dimond V, Cedar Rapids, IA) on performance, plasma metabolites, rumen parameters, and fecal pathogen shedding in feedlot steers. Crossbred steers (n = 61; 271 ± 48.1 kg) were sourced from 3 different research units at Oklahoma State University and assigned to 1 of 3 experimental treatments in a randomized complete block design. Steers were group housed in pens equipped with an automated individual feed intake system. Treatments consisted of a negative control (CON), CON with tylosin phosphate (TYL; 9.9 mg · kg-1 of diet DM), or CON with SCFP (1.63 g · kg-1 of diet DM). Body weight and feed efficiency did not differ among treatments (P ≥ 0.12). However, steers fed SCFP tended to have greater ADG from d 35 to 56 (P = 0.10). In addition, steers fed SCFP exhibited increased DMI from d 56 to 112 (P = 0.03), a tendency for increased DMI from d 112 to final (P = 0.07), and increased DMI overall (P = 0.05). There was no treatment × time interaction for pH, pH change, or lactate concentrations in rumen fluid (P ≥ 0.11), but all were impacted by time (P < 0.01). A treatment × time interaction (P < 0.01) was detected for fecal pH and fecal pH change, with the pH being greater in TYL fed steers on d 56 and in CON fed steers on the final day. Steers that consumed CON had a reduced fecal pH (P < 0.01) and steers consuming SCFP tended to have a reduced fecal pH (P = 0.08) from d 35 to 56 compared with TYL fed steers, while both CON and SCFP fed steers experienced an increase in fecal pH from d 56 to 112 (P < 0.01). A time effect was detected (P ≤ 0.04) for plasma glucose, lactate, urea nitrogen, and non-esterified fatty acid concentrations among treatment groups. A treatment × time interaction was observed (P < 0.02) for stx2 genes of Shiga toxin producing Escherichia coli (STEC) in individual fecal samples where SCFP and TYL fed steers tended to exhibit an increased concentration compared with CON fed steers on d 14, while on d 190 CON and SCFP fed steers exhibited an increased concentration compared with TYL fed steers. Overall, STEC, E. coli O157:H7, and Salmonella concentrations were all impacted by time (P < 0.001). This experiment suggests that SCFP could be utilized as a potential alternative to TYL in feedlot cattle without adversely affecting cattle performance. However, SCFP did not impact pathogen shedding. In addition, CON fed steers performed similarly to both SCFP and TYL fed steers.

Interaction of Carbon Nanotubes, Capped Carbon Nanotubes, CNT2–5, C60, C70, HO-C60, [C60]2, and [C60]3 Fullerenes with Virulence Factors of Gram-Negative and Gram-Positive Bacteria: Potential Applications for 3D-Printed Scaffolds

The antimicrobial application of carbon nanomaterials, such as carbon nanotubes (CNTs), capped CNTs, CNT2–5, C60, C70, HO-C60, [C60]2, and [C60]3 fullerenes, is increasing, owing to their low cytotoxicity properties compared to other nanomaterials such as metallic nanoparticles. Enhanced mechanical properties and antibacterial activity can be caused by the incorporation of CNTs in 3-dimensional (3D) printed nanocomposites (NCs). The interruption of the bacterial membrane resulting from the cylindrical shape and high aspect ratio properties has been found to be the most prominent antibacterial mechanism of CNTs. However, the unraveling interaction of CNTs, capped CNTs, CNT2–5, C60, C70, HO-C60, [C60]2, and [C60]3 fullerenes with virulence factors of the main bacterial pathogenesis has not yet been understood. Therefore, in the present study, interactions of these carbon-based nanomaterials with the eight virulence factors, including protein kinase A and (ESX)-secreted protein B of Mycobacterium tuberculosis, pseudomonas elastase and exotoxin A of Pseudomonas aeruginosa, alpha-hemolysin and penicillin-binding protein 2a of Staphylococcus aureus, and shiga toxin 2a and heat-labile enterotoxin of Escherichia coli, were evaluated with the molecular docking method of AutoDock Vina. This study disclosed that the binding affinity was highest for CNT2–5 and [C60]3 toward alpha-hemolysin, with binding energies of −32.7 and −26.6 kcal/mol, respectively. The stability of the CNT2–5–alpha-hemolysin complex at different times was obtained according to the normal mode analysis of ElNémo and iMOD servers.

Effects of Nano-Electrospray Ionization Emitter Position on Unintentional In-Source Activation of Peptide and Protein Ions.

Native ion mobility-mass spectrometry (IM-MS) typically introduces protein ions into the gas phase through nano-electrospray ionization (nESI). Many nESI setups have mobile stages for tuning the ion signal and extent of co-solute and salt adduction. However, tuning the position of the emitter capillary in nESI can have unintended downstream consequences for collision-induced unfolding or collision-induced dissociation (CIU/D) experiments. Here, we show that relatively small variations in the nESI emitter position can shift the midpoint (commonly called the "CID50" or "CIU50") potential of CID breakdown curves and CIU transitions by as much as 8 V on commercial instruments. A spatial "map" of the shift in CID50 for the loss of heme from holomyoglobin onto the emitter position on a Waters Synapt G2-Si mass spectrometer shows that emitter positions closer to the instrument inlet can result in significantly greater in-source activation, whereas different effects are found on an Agilent 6545XT instrument for the ions studied. A similar effect is observed for CID of the singly protonated leucine enkephalin peptide and Shiga toxin 1 subunit B homopentamer on the Waters Synapt G2-Si instrument. In-source activation effects on a Waters Synapt G2-Si are also investigated by examining the RMSD between CIU fingerprints acquired at different emitter positions and the shifts in CIU50 for structural transitions of bovine serum albumin and NIST monoclonal antibody.

The NMR structure of the Orf63 lytic developmental protein from lambda bacteriophage

The orf63 gene resides in a region of the lambda bacteriophage genome between the exo and xis genes and is among the earliest genes transcribed during infection. In lambda phage and Shiga toxin (Stx) producing phages found in enterohemorrhagic Escherichia coli (EHEC) associated with food poisoning, Orf63 expression reduces the host survival and hastens the period between infection and lysis thereby giving it pro-lytic qualities. The NMR structure of dimeric Orf63 reveals a fold consisting of two helices and one strand that all make extensive intermolecular contacts. Structure-based data mining failed to identify any Orf63 homolog beyond the family of temperate bacteriophages. A machine learning approach was used to design an amphipathic helical ligand that bound a hydrophobic cleft on Orf63 with micromolar affinity. This approach may open a new path towards designing therapeutics that antagonize the contributions of Stx phages in EHEC outbreaks.

Shiga toxin type 2 B subunit protects mice against toxin challenge when leashed and bundled by a stable pentameric coiled-coil molecule

Vaccines against Shiga toxin (Stx)-producing Escherichia coli (STEC) have not yet been developed. Two immunologically distinct serotypes of Stx (Stx1 and Stx2) are the main virulence factors of STEC. Thus, blocking their B subunits (StxB) from binding to the cell surface receptor globotriaosylceramide (Gb3) efficiently prevents the action of these toxins. We expressed Stx1B and Stx2B in E. coli inclusion bodies and reassembled them into pentamers by a stepwise dialysis. Stx1B pentamer fully protected mice against Stx1 challenge, but Stx2B pentamer failed to protect mice against Stx2 challenge. To explain those observations, we proposed that the pentamer of Stx2B readily dissociates into its constituent monomers, especially under in vivo conditions, thus being unable to induce pentamer-specific immunity. To increase pentamer stability, we fused the B subunit to a pentameric coiled-coil domain of the cartilage oligomeric matrix protein (COMP). This “five-to-five” fusion hybrid molecule (Stx2B–COMP) was shown to be protective against Stx2 challenge, demonstrating that the Stx2B subunit when leashed and bundled by a rigid pentameric coiled-coil domain mount a pentamer-specific immune response and efficiently neutralize the toxin both in vitro and in vivo. Our data strongly suggest that the Stx2B subunit moiety fluctuates between a pentameric and monomeric state within the fusion protein, which may increase the likelihood of the immune system recognizing the pentameric conformation for toxin neutralization.

Detection and Molecular Characterization of Shiga-toxin-producing Escherichia coli from Fecal Samples of Cattle Farms Al-Buḥayrah Governorate Egypt

Escherichia coli that produce the Shiga toxin (STEC) has become one of the most significant foodborne pathogens linked to serious illnesses. Our study was aimed to detect the existence, antimicrobial profiles, and virulence repertoire of different STEC serotypes from fecal samples of dairy cattle.
 We collected 19 fecal samples (12 diarrheic and 7 non-diarrheic) from dairy cattle. E. coli isolation and identification were performed. Shiga toxins genes were detected and the antimicrobial resistance was investigated.
 E. coli was detected at the rate of 13/19 (68.4%) from the fecal samples. The frequency of Stx2a represented 12/13 (92.3%), Stx2 represented 3/13 (23.14%), and Stx1d, Stx2f, Stx2g, and eae represented 2/13 (15.4%). While the virulence genes Stx1, Stx1c, Stx2c, Stx2d, Stx2e, and ehxA represented 0/13 (0%). The most effective antimicrobials were tulathithromycin 30 (53.84%) followed by ceftiofur 30 and marbofloxacin (46.16%), amikacin (15.38%), and the least resistance was recorded for nalidixic acid and oxytetracycline (7.70%). The resistant antibiotics with percentages reached to 100% were Amoxicillin/ Clavulanic acid, Ceftriaxone, Ciprofloxacin, Doxycycline, Erythromycin, Gentamicin, Ninezolid, Nitrofurantoin, Penicillin G, Streptomycin, and Trimethoprim/Sulfamethoxazole, followed by chloramphenicol, nalidixic acid, oxytetracycline, and Tylosine (92.30%), Amikacin and Ampicillin (84.62%), Ceftiofur (23.07%), Marbofloxacin (15.38%) and Tulathithromycin 30 (7.70%).
 The findings demonstrated that E. coli that are resistant to numerous groups of antimicrobials spread through feces and may pose a risk to human and animal health.