Nanostructured Magnetic Particles for Removing Cyanotoxins: Assessing Effectiveness and Toxicity In Vitro.

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

Mulberry Leaf-Derived Morin Activates ß-Catenin by Binding to Frizzled7 to Promote Intestinal Stem Cell Expansion upon Heat-Stable Enterotoxin b Injury.

Intestinal stem cells (ISCs) sustain epithelial renewal by dynamically altering behaviors of proliferation and differentiation in response to various nutrition and stress inputs. However, how ISCs integrate bioactive substance morin cues to protect against heat-stable enterotoxin b (STb) produced by Escherichia coli remains an uncertain question with implications for treating bacterial diarrhea. Our recent work showed that oral mulberry leaf-derived morin improved the growth performance in STb-challenged mice. Furthermore, morin supplementation reinstated the impaired small-intestinal epithelial structure and barrier function by stimulating ISC proliferation and differentiation as well as supporting intestinal organoid expansion ex vivo. Importantly, the Wnt/ß-catenin pathway, an ISC fate commitment signal, was reactivated by morin to restore the jejunal crypt-villus architecture in response to STb stimulation. Mechanically, the extracellular morin-initiated ß-catenin axis is dependent or partially dependent on the Wnt membrane receptor Frizzled7 (FZD7). Our data reveal an unexpected role of leaf-derived morin, which represents molecular signaling targeting the FZD7 platform instrumental for controlling ISC regeneration upon STb injury.

Isolation and identification of Clostridium perfringens and its toxins from mutton in Lahore City, Punjab, Pakistan.

The present study was aimed to detect C. perfringens and identify its toxins in mutton samples collected from Lahore City in the Punjab Province of Pakistan. A total of 40 samples of minced and non­minced mutton were collected from local butcher and retail shops representing four areas of the city. The samples were subjected to ELISA for the detection of C. perfringens alpha, beta and epsilon toxins. The samples were simultaneously processed for bacterial isolation. The isolates were confirmed both by biochemical testing and a multiplex PCR targeting alpha, beta and epsilon toxin genes of C. perfringens. While 10% (4/40) of the samples were positive for C. perfringens alpha toxins, 17.5% (7/40) of the samples were positive for the alpha toxin gene. The present study indicated that the samples collected from the local butcher shops were contaminated with C. perfringens and its toxins. Interestingly, no such contamination was detected in any of the samples collected from retail meat shops. In conclusion, improper hygienic conditions at butcher shops could lead to the contamination of mutton with C. perfringens and its toxins.

An outbreak of food poisoning due to Escherichia coli serotype O7:H4 carrying astA for enteroaggregative E. coli heat-stable enterotoxin1 (EAST1).

In June 2020, a large-scale food poisoning outbreak involving about 3000 elementary and junior high school students occurred in Yashio, Saitama, Japan. A school lunch was the only food stuff ingested by all of the patients. Escherichia coli serotype O7:H4 carrying the astA gene for enteroaggregative E. coli (EAggEC) heat-stable enterotoxin 1 (EAST1) was detected in faecal specimens from the patients, and sample inspection revealed its presence in a seaweed salad and red seaweed (Gigartina tenella) as one of the raw materials. Analysis of the antibiotic sensitivity of the isolates revealed resistance to ampicillin and cefotaxime. All isolates were confirmed to be of the same origin by pulsed-field gel electrophoresis after digestion with the restriction enzyme XbaI, and single nucleotide polymorphism analysis using whole genome sequencing. To our knowledge, this is the first report of a large-scale food poisoning caused by E. coli O7:H4, which lacks well-characterized virulence genes other than astA.

Systematic Evaluation of Parameters Important for Production of Native Toxin A and Toxin B from Clostridioides difficile.

In the attempt to improve the purification yield of native toxin A (TcdA) and toxin B (TcdB) from Clostridioides difficile (C. difficile), we systematically evaluated culture parameters for their influence on toxin production. In this study, we showed that culturing C. difficile in a tryptone-yeast extract medium buffered in PBS (pH 7.5) that contained 5 mM ZnCl2 and 10 mM glucose supported the highest TcdB production, measured by the sandwich ELISA. These culture conditions were scalable into 5 L and 15 L dialysis tube cultures, and we were able to reach a TcdB concentration of 29.5 µg/mL of culture. Furthermore, we established a purification protocol for TcdA and TcdB using FPLC column chromatography, reaching purities of >99% for both toxins with a yield around 25% relative to the starting material. Finally, by screening the melting temperatures of TcdA and TcdB in various buffer conditions using differential scanning fluorimetry, we found optimal conditions for improving the protein stability during storage. The results of this study present a complete protocol for obtaining high amounts of highly purified native TcdA and TcdB from C. difficile.

In vitro Evaluation of Programmed Cell Death in the Immune System of Pacific Oyster Crassostrea gigas by the Effect of Marine Toxins.

Programmed cell death (PCD) is an essential process for the immune system's development and homeostasis, enabling the remotion of infected or unnecessary cells. There are several PCD's types, depending on the molecular mechanisms, such as non-inflammatory or pro-inflammatory. Hemocytes are the main component of cellular immunity in bivalve mollusks. Numerous infectious microorganisms produce toxins that impair hemocytes functions, but there is little knowledge on the role of PCD in these cells. This study aims to evaluate in vitro whether marine toxins induce a particular type of PCD in hemocytes of the bivalve mollusk Crassostrea gigas during 4 h at 25°C. Hemocytes were incubated with two types of marine toxins: non-proteinaceous toxins from microalgae (saxitoxin, STX; gonyautoxins 2 and 3, GTX2/3; okadaic acid/dynophysistoxin-1, OA/DTX-1; brevetoxins 2 and 3, PbTx-2,-3; brevetoxin 2, PbTx-2), and proteinaceous extracts from bacteria (Vibrio parahaemolyticus, Vp; V. campbellii, Vc). Also, we used the apoptosis inducers, staurosporine (STP), and camptothecin (CPT). STP, CPT, STX, and GTX 2/3, provoked high hemocyte mortality characterized by apoptosis hallmarks such as phosphatidylserine translocation into the outer leaflet of the cell membrane, exacerbated chromatin condensation, DNA oligonucleosomal fragments, and variation in gene expression levels of apoptotic caspases 2, 3, 7, and 8. The mixture of PbTx-2,-3 also showed many apoptosis features; however, they did not show apoptotic DNA oligonucleosomal fragments. Likewise, PbTx-2, OA/DTX-1, and proteinaceous extracts from bacteria Vp, and Vc, induced a minor degree of cell death with high gene expression of the pro-inflammatory initiator caspase-1, which could indicate a process of pyroptosis-like PCD. Hemocytes could carry out both PCD types simultaneously. Therefore, marine toxins trigger PCD's signaling pathways in C. gigas hemocytes, depending on the toxin's nature, which appears to be highly conserved both structurally and functionally.

Hunting the eagle killer: A cyanobacterial neurotoxin causes vacuolar myelinopathy.

Vacuolar myelinopathy is a fatal neurological disease that was initially discovered during a mysterious mass mortality of bald eagles in Arkansas in the United States. The cause of this wildlife disease has eluded scientists for decades while its occurrence has continued to spread throughout freshwater reservoirs in the southeastern United States. Recent studies have demonstrated that vacuolar myelinopathy is induced by consumption of the epiphytic cyanobacterial species Aetokthonos hydrillicola growing on aquatic vegetation, primarily the invasive Hydrilla verticillata Here, we describe the identification, biosynthetic gene cluster, and biological activity of aetokthonotoxin, a pentabrominated biindole alkaloid that is produced by the cyanobacterium A. hydrillicola We identify this cyanobacterial neurotoxin as the causal agent of vacuolar myelinopathy and discuss environmental factors-especially bromide availability-that promote toxin production.

Production and purification of Clostridium perfringens type D epsilon toxin and IgY antitoxin.

The aim of this study was to purify Clostridium perfringens type D epsilon toxin and produce and purify anti-epsilon chicken immunoglobulin Y (IgY). A single-step ion exchange chromatography resulted in a high-yield and high-purity toxin, while ion exchange chromatography followed by gel filtration resulted in the highest purity of the toxin, but at a lower yield. Purified and inactivated epsilon toxin were then administered in chickens via four inoculations and IgY was obtained at a high purity and yield, with an antibody titer of 50 IU/mL and high levels of avidity (73.2%). In summary, C. perfringens type D epsilon toxin and chicken anti-epsilon IgY were successfully produced and purified, and may be used for the diagnosis of enterotoxemia caused by the epsilon toxin, as well as in potency tests of existing and future vaccines against enterotoxemia.

Multicenter evaluation of Verigene Enteric Pathogens Nucleic Acid Test for detection of gastrointestinal pathogens.

We investigated the efficiency of the Verigene Enteric Pathogens Nucleic Acid Test (Verigene EP test), which is an automated microarray-based assay system that enables rapid and simultaneous genetic detection of gastrointestinal pathogens and toxins, including those in the Campylobacter Group, Salmonella species, Shigella species, the Vibrio Group, Yersinia enterocolitica, Shiga toxin 1 and 2, norovirus GI/GII, and rotavirus A. Three clinical laboratories evaluated the Verigene EP test, using 268 stool samples for bacterial and toxin genes and 167 samples for viral genes. Culture-based reference methods were used for the detection of bacteria and toxins, while a different molecular assay was used for viral detection. The overall concordance rate between the Verigene EP test and the reference methods for the 1940 assays was 99.0%. The overall sensitivity and specificity of the Verigene EP test were 97.0% and 99.3%, respectively. Of the 19 samples with discordant results, 13 samples were false positives and six were false negatives. The Verigene EP test simultaneously detected two targets in 11 samples; overall, the test demonstrated high efficiency in detecting crucial diarrheagenic pathogens, indicating its suitability for clinical practice.

Identification of Galtox, a new protein toxin from Photorhabdus bacterial symbionts of Heterorhabditis nematodes.

The Gram-negative bacteria Photorhabdus lives in a symbiotic relationship with the insect-pathogenic Heterorhabditis nematodes and produces numerous hydrolytic enzymes, secondary metabolites and protein toxins. Seven Photorhabdus strains were previously isolated from the Heterorhabditis nematodes collected from different geographical regions of India. The strains IARI-SGMG3, IARI-SGHR2, IARI-SGHR4, IARI-SGMS1 and IARI-SGGJ2 were identified as P. akhurstii, whereas IARI-SGLDK1 and IARI-SGHP1 were identified as P. laumondii subsp. laumondii and P. laumondii subsp. clarkeii, respectively. A new and previously unreported 35 kDa molecular weight protein toxin 'Galtox' was identified from these Photorhabdus strains. The nucleotide sequences of the toxin gene from seven Photorhabdus strains were PCR amplified, sequenced, cloned into pET protein expression vector, and the protein toxin was expressed and purified. The Galtox sequence from various strains showed variations in sequence and toxicity against Galleria mellonella. The injection of purified Galtox protein into the 4th instar larvae showed median lethal dose (LD50) values of 2.39-26.08 ng toxin/g G. mellonella bodyweight after 48 h. The protein injection killed the insects quickly and exhibited a median lethal time (LT50) of 12-60 h when injected at the rate of 3.1-31.2 ng toxin/g G. mellonella bodyweight. Galtox protein sequence analysis indicated similarity to several bacterial toxin-related protein domains, such as 6rgnA domain of Bordetella membrane targeting toxin BteA, 6gy6 domain of Xenorhabdus α-Xenorhabdolysins, 4mu6A and 4xa9a domains similar to effector protein LegC3 from Legionella pneumophila and 1cv8.1 domain of staphylococcal cysteine proteinase staphopain B. The mode of action of Galtox needs to be understood to enable its use for the management of agricultural insect-pests.

Contact-independent killing mediated by a T6SS effector with intrinsic cell-entry properties.

Bacterial type VI secretion systems (T6SSs) inject toxic effectors into adjacent eukaryotic and prokaryotic cells. It is generally thought that this process requires physical contact between the two cells. Here, we provide evidence of contact-independent killing by a T6SS-secreted effector. We show that the pathogen Yersinia pseudotuberculosis uses a T6SS (T6SS-3) to secrete a nuclease effector that kills other bacteria in vitro and facilitates gut colonization in mice. The effector (Tce1) is a small protein that acts as a Ca2+- and Mg2+-dependent DNase, and its toxicity is inhibited by a cognate immunity protein, Tci1. As expected, T6SS-3 mediates canonical, contact-dependent killing by directly injecting Tce1 into adjacent cells. In addition, T6SS-3 also mediates killing of neighboring cells in the absence of cell-to-cell contact, by secreting Tce1 into the extracellular milieu. Efficient contact-independent entry of Tce1 into target cells requires proteins OmpF and BtuB in the outer membrane of target cells. The discovery of a contact-independent, long-range T6SS toxin delivery provides a new perspective for understanding the physiological roles of T6SS in competition. However, the mechanisms mediating contact-independent uptake of Tce1 by target cells remain unclear.

Sensitive and rapid detection of cytolethal distending toxin encoding genes in Providencia alcalifaciens.

Cytolethal distending toxins (CDTs) produced by P. alcalifaciens are considered as potential virulence factors. A loop-mediated isothermal amplification (LAMP) method for the detection of cdtA, cdtB, and cdtC genes was established which showed high specificity and strong sensitivity. The LAMP assay showed a detection threshold was 3.13 pg/µl within 40 min.

Anabaenopeptins from Cyanobacteria in Freshwater Bodies of Greece.

Cyanobacteria are photosynthetic microorganisms that are able to produce a large number of secondary metabolites. In freshwaters, under favorable conditions, they can rapidly multiply, forming blooms, and can release their toxic/bioactive metabolites in water. Among them, anabaenopeptins (APs) are a less studied class of cyclic bioactive cyanopeptides. The occurrence and structural variety of APs in cyanobacterial blooms and cultured strains from Greek freshwaters were investigated. Cyanobacterial extracts were analyzed with LC-qTRAP MS/MS using information-dependent acquisition in enhanced ion product mode in order to obtain the fragmentation mass spectra of APs. Thirteen APs were detected, and their possible structures were annotated based on the elucidation of fragmentation spectra, including three novel ones. APs were present in the majority of bloom samples (91%) collected from nine Greek lakes during different time periods. A large variety of APs was observed, with up to eight congeners co-occurring in the same sample. AP F (87%), Oscillamide Y (87%) and AP B (65%) were the most frequently detected congeners. Thirty cyanobacterial strain cultures were also analyzed. APs were only detected in one strain (Microcystis ichtyoblabe). The results contribute to a better understanding of APs produced by freshwater cyanobacteria and expand the range of structurally characterized APs.

Several New Putative Bacterial ADP-Ribosyltransferase Toxins Are Revealed from In Silico Data Mining, Including the Novel Toxin Vorin, Encoded by the Fire Blight Pathogen Erwinia amylovora.

Mono-ADP-ribosyltransferase (mART) toxins are secreted by several pathogenic bacteria that disrupt vital host cell processes in deadly diseases like cholera and whooping cough. In the last two decades, the discovery of mART toxins has helped uncover the mechanisms of disease employed by pathogens impacting agriculture, aquaculture, and human health. Due to the current abundance of mARTs in bacterial genomes, and an unprecedented availability of genomic sequence data, mART toxins are amenable to discovery using an in silico strategy involving a series of sequence pattern filters and structural predictions. In this work, a bioinformatics approach was used to discover six bacterial mART sequences, one of which was a functional mART toxin encoded by the plant pathogen, Erwinia amylovora, called Vorin. Using a yeast growth-deficiency assay, we show that wild-type Vorin inhibited yeast cell growth, while catalytic variants reversed the growth-defective phenotype. Quantitative mass spectrometry analysis revealed that Vorin may cause eukaryotic host cell death by suppressing the initiation of autophagic processes. The genomic neighbourhood of Vorin indicated that it is a Type-VI-secreted effector, and co-expression experiments showed that Vorin is neutralized by binding of a cognate immunity protein, VorinI. We demonstrate that Vorin may also act as an antibacterial effector, since bacterial expression of Vorin was not achieved in the absence of VorinI. Vorin is the newest member of the mART family; further characterization of the Vorin/VorinI complex may help refine inhibitor design for mART toxins from other deadly pathogens.

Detection of Necrotic Enteritis B-like Toxin Secreted by Clostridium perfringens Using Capture Enzyme-Linked Immunosorbent Assay.

Necrotic enteritis (NE) is a devastating enteric disease caused by Clostridium perfringens type A/G, which affects global poultry industry by compromising the performance, health, and welfare of chickens. The causative main virulent factor responsible for NE pathogenesis has been shifted from a phospholipase C portion of an α-toxin, to an NE B-like (NetB) toxin, a plasmid-encoded pore-forming heptameric protein, in NE development. Therefore, the ability to detect NetB toxin will enable early diagnosis of field NE. Because the NetB protein can only be detected by western blot analysis with polyclonal anti-NetB antiserum, we developed a NetB-specific monoclonal antibody (mAb)-based capture enzyme-linked immunosorbent assay (ELISA). Twenty mAbs reacting with Escherichia coli-expressed NetB protein were selected, isotyped, and conjugated with horseradish peroxidase for antibody pair tests. Multiple mAb pairs were found to detect E. coli NetB protein and native NetB protein secreted by netB-positive C. perfringens isolates. The developed capture (sandwich) ELISA could be useful to identify in vitro production of native NetB protein secreted from netB-positive field C. perfringens isolates and to conduct a large field test of commercial chickens undergoing NE infection. Here, we first report that native NetB toxin can be detected in C. perfringens NetB-specific mAb-based capture ELISA.

The A component (SmhA) of a tripartite pore-forming toxin from Serratia marcescens: expression, purification and crystallographic analysis.

Tripartite α-pore-forming toxins are constructed of three proteins (A, B and C) and are found in many bacterial pathogens. While structures of the B and C components from Gram-negative bacteria have been described, the structure of the A component of a Gram-negative α-pore-forming toxin has so far proved elusive. SmhA, the A component from the opportunistic human pathogen Serratia marcescens, has been cloned, overexpressed and purified. Crystals were grown of selenomethionine-derivatized protein and anomalous data were collected. Phases were calculated and an initial electron-density map was produced.

Bacteroides fragilis Enterotoxin Induces Sulfiredoxin-1 Expression in Intestinal Epithelial Cell Lines Through a Mitogen-Activated Protein Kinases- and Nrf2-Dependent Pathway, Leading to the Suppression of Apoptosis.

Enterotoxigenic Bacteroides fragilis is a causative agent of colitis and secrets enterotoxin (BFT), leading to the disease. Sulfiredoxin (Srx)-1 serves to protect from oxidative damages. Although BFT can generate reactive oxygen species in intestinal epithelial cells (IECs), no Srx-1 expression has been reported in ETBF infection. In this study, we explored the effects of ETBF-produced BFT on Srx-1 induction in IECs. Treatment of IECs with BFT resulted in increased expression of Srx-1 in a time-dependent manner. BFT treatment also activated transcriptional signals including Nrf2, AP-1 and NF-κB, and the Srx-1 induction was dependent on the activation of Nrf2 signals. Nrf2 activation was assessed using immunoblot and Nrf2-DNA binding activity and the specificity was confirmed by supershift and competition assays. Suppression of NF-κB or AP-1 signals did not affect the upregulation of Srx-1 expression. Nrf2-dependent Srx-1 expression was associated with the activation of p38 mitogen-activated protein kinases (MAPKs) in IECs. Furthermore, suppression of Srx-1 significantly enhanced apoptosis while overexpression of Srx-1 significantly attenuated apoptosis during exposure to BFT. These results imply that a signaling cascade involving p38 and Nrf2 is essential for Srx-1 upregulation in IECs stimulated with BFT. Following this upregulation, Srx-1 may control the apoptosis in BFT-exposed IECs.

Optimized high-purity protein preparation of biologically active recombinant VacA cytotoxin variants from Helicobacter pylori.

Vacuolating cytotoxin A (VacA) is a highly polymorphic virulence protein produced by the human gastric pathogen Helicobacter pylori which can cause gastritis, peptic ulcer and gastric cancer. Here, we present an optimized protein preparation of the mature full-length VacA variants (m1-and m2-types) and their 33-kDa N-terminal and 55/59-kDa C-terminal domains as biologically active recombinant proteins fused with an N-terminal His(6) tag. All recombinant VacA constructs were over-expressed in Escherichia coli as insoluble inclusions which were soluble when phosphate buffer (pH 7.4) was supplemented with 5-6 M urea. Upon immobilized-Ni2+ affinity purification under 5-M urea denaturing conditions, homogenous products (>95% purity) of 55/59-kDa domains were consistently obtained while only ~80% purity of both mature VacA variants and the 33-kDa truncate was achieved, thus requiring additional purification by size-exclusion chromatography. After successive refolding via optimized stepwise dialysis, all refolded VacA proteins were proven to possess both cytotoxic and vacuolating activity against cultured human gastric epithelial cells albeit the activity observed for VacA-m2 was lower than the m1-type variant. Such an optimized protocol described herein was effective for production of high-purity recombinant VacA proteins in large amounts (~30-40 mg per liter culture) that would pave the way for further studies on sequence-structure and function relationships of different VacA variants.

Development of an indirect Enzyme Linked Immunoassay (iELISA) using monoclonal antibodies against Photorhabdus insect related toxins, PirAVp and PirBVp released from Vibrio spp.

An acute hepatopancreatic necrosis disease (AHPND) causes serious losses to the global shrimp industry. The etiologic agent of AHPND is Vibrio spp. carrying a large plasmid which encodes a binary toxin, PirAB. Currently, AHPND is diagnosed by PCR based methods that detect the presences of both pirA and pirB genes. However, the bacterial strains containing the pirA and pirB genes do not always express the binary toxin, resulting in mis-estimation of the virulence of bacterial strains containing pirA and pirB genes. Thus, the immuno based assay (i.e. ELISA) is a promising approach to detect PirAVp and PirBVp. In the present study, a total of forty monoclonal antibodies clones (mAb) against PirAVp (20 mAbs) and PirBVp (20 mAbs) were screened by western blot analysis to select four mAb clones that show the strongest immunoreactivity in indirect ELISA (iELISA). The four selected mAbs (i.e. 1B9 and 5E9 against PirAVp; 7B7 and 7B9 against PirBVp) detected specifically Vibrio spp. causing AHPND. In addition, four selected mAbs were able to detect either PirAVp or PirBVp down to 0.008 ng/µl. A double blind assay using thirty AHPND-infected and six SPF shrimp Penaeus vannamei were analyzed by iELISA to determine the detection sensitivity of the assay. The results showed that iELISA was able to accurately detect 29 out of 30 AHPND infected shrimp. These finding indicated that iELISA is a reliable method to detect PirAVp and PirBVp toxins in infected shrimp and will be a useful tool in AHPND diagnosis and in studying the role of binary toxins in AHPND pathogenesis.

Crystal structure of the YoeBSa1-YefMSa1 complex from Staphylococcus aureus.

Toxin-antitoxin (TA) systems are ubiquitously found in bacteria and are related to cell maintenance and survival under environmental stresses such as heat shock, nutrient starvation, and antibiotic treatment. Here, we report for the first time the crystal structure of the Staphylococcus aureus TA complex YoeBSa1-YefMSa1 at a resolution of 1.7 Å. This structure reveals a heterotetramer with a 2:2 stoichiometry between YoeBSa1 and YefMSa1. The N-terminal regions of the YefMSa1 antitoxin form a homodimer characteristic of a hydrophobic core, and the C-terminal extended region of each YefMSa1 protomer makes contact with each YoeBSa1 monomer. The binding stoichiometry of YoeBSa1 and YefMSa1 is different from that of YoeB and YefM of E. coli (YoeBEc and YefMEc), which is the only structural homologue among YoeB-YefM families; however, the structures of individual YoeBSa1 and YefMSa1 subunits in the complex are highly similar to the corresponding structures in E. coli. In addition, docking simulation with a minimal RNA substrate provides structural insight into the guanosine specificity of YoeBSa1 for cleavage in the active site, which is distinct from the specificity of YoeBEc for adenosine rather than guanosine. Given the previous finding that YoeBSa1 exhibits fatal toxicity without inducing persister cells, the structure of the YoeBSa1-YefMSa1 complex will contribute to the design of a new category of anti-staphylococcal agents that disrupt the YoeBSa1-YefMSa1 complex and increase YoeBSa1 toxicity.