Successful colonization by the opportunistic pathogen Staphylococcus aureus depends on its ability to interact with other microorganisms. Staphylococcus aureus strains harbour a T7b subtype of type VII secretion system (T7SSb), a protein secretion system found in a wide variety of Bacillota, which functions in bacterial antagonism and virulence. Assessment of T7SSb activity in S. aureus has been hampered by low secretion activity under laboratory conditions and the lack of a sensitive assay to measure secretion. Here, we have utilized NanoLuc binary technology to develop a simple assay to monitor protein secretion via detection of bioluminescence. Fusion of the 11 amino acid NanoLuc fragment to the conserved substrate EsxA permits its extracellular detection upon supplementation with the large NanoLuc fragment and luciferase substrate. Following miniaturization of the assay to 384-well format, we use high-throughput analysis to demonstrate that T7SSb-dependent protein secretion differs across strains and growth temperature. We further show that the same assay can be used to monitor secretion of the surface-associated toxin substrate TspA. Using this approach, we identify three conserved accessory proteins required to mediate TspA secretion. Co-purification experiments confirm that all three proteins form a complex with TspA.

Current treatment of clostridial infections includes broad-spectrum antibiotics and antitoxins, yet antitoxins are ineffective against all Clostridiumspecies. Moreover, rising antimicrobial resistance (AMR) threatens treatment effectiveness and public health. This study therefore aimed to discover a common drug target for four pathogenic clostridial species, Clostridium botulinum, C. difficile, C. tetani, and C. perfringens through an in-silico core genomic approach. Using four reference genomes of C. botulinum, C. difficile, C. tetani, and C. perfringens, we identified 1484 core genomic proteins (371/genome) and screened them for potential drug targets. Through a subtractive approach, four core proteins were finally identified as drug targets, represented by type III pantothenate kinase (CoaX) and, selected for further analyses. Interestingly, the CoaX is involved in the phosphorylation of pantothenate (vitamin B5), which is a critical precursor for coenzyme A (CoA) biosynthesis. Investigation of druggability analysis on the identified drug target reinforces CoaX as a promising novel drug target for the selected Clostridium species. During the molecular screening of 1201 compounds, a known agonist drug compound (Vibegron) showed strong inhibitory activity against targeted clostridial CoaX. Additionally, we identified tazobactam, a beta-lactamase inhibitor, as effective against the newly proposed target, CoaX. Therefore, identifying CoaX as a single drug target effective against all four clostridial pathogens presents a valuable opportunity to develop a cost-effective treatment for multispecies clostridial infections.

Loxoscelism is the pathological condition triggered by a brown spider bite. The venom of these spiders is rich in phospholipases D (PLDs), which can induce virtually all local and systemic manifestations. Recombinant mutated PLDs from clinically relevant Loxosceles species in South America have been investigated as potential antigens to develop novel therapeutic strategies for loxoscelism. However, certain gaps need to be addressed before a clinical approach can be implemented. In this study, we examined the potential of these recombinant mutated PLDs as antigens by testing some variations in the immunization scheme. Furthermore, we evaluated the efficacy of the produced antibodies in neutralizing the nephrotoxicity and sphingomyelinase activity of brown spider venoms. Our findings indicate that the number of immunizations has a greater impact on the effectiveness of neutralization compared to the amount of antigen. Specifically, two or three doses were equally effective in reducing dermonecrosis and edema. Additionally, three immunizations proved to be more effective in neutralizing mice lethality than one or two. Moreover, immunizations mitigated the signs of kidney injury, a crucial aspect given that acute renal failure is a serious systemic complication. In vitro inhibition of the sphingomyelinase activity of Loxosceles venoms, a key factor in vivo toxicity, was nearly complete after incubation with antibodies raised against these antigens. These findings underscore the importance of implementing an effective immunization scheme with multiple immunizations, without the need for high antigen doses, and enhances the spectrum of neutralization exhibited by antibodies generated with these antigens. In summary, these results highlight the strong potential of these antigens for the development of new therapeutic strategies against cutaneous and systemic manifestations of loxoscelism.

Spider venom is a natural source of diverse biomolecules, but due to technical limitations, only a small fraction has been studied. With the advancement of omics technologies, research on spider venom has broadened, greatly promoting systematic studies of spider venom. Agelena limbata is a common spider found in vegetation, known for constructing funnel-shaped webs, and feeding on insects such as Diptera and Homoptera. However, due to its small size and the difficulty in obtaining venom, the composition of Agelena limbata venom has never been studied. In this study, a transcriptomics approach was used to analyze the toxin components in the venom of Agelena limbata, resulting in the identification of 28 novel toxin-like sequences and 24 peptidases. Based on sequence similarity and differences in cysteine motifs, the 28-novel toxin-like sequences were classified into 10 superfamilies. According to the results annotated in the database, the 24 peptidases were divided into six distinct families, with the serine protease family being the most common. A phylogenetic tree was constructed using the toxin-like sequences of Agelena limbata along with Psechrus triangulus and Hippasa lycosina. An analysis of the structural domains and motifs of Agelena limbata was also conducted. The results indicated that Agelena limbata is more distantly related to the other two species of funnel-web spiders, and that the toxin superfamily IX has a unique function compared to the other superfamilies. This study reveals the components of the Agelena limbata venom, deepening our understanding of it, and through bioinformatics analysis, has identified unique functions of the toxin superfamilies, providing a scientific basis for the development of bioactive drugs in the future.

UNASSIGNED: Klebsiella oxytoca (K. oxytoca) is the second bacterial cause of nosocomial infections in the general population after K. pneumoniae. This study surveyed the frequency of cytotoxin-producing strains of K. oxytoca and their antibiotic susceptibility profile in a cohort of children admitted to a referral hospital with different malignancies.
UNASSIGNED: The Stool samples of children admitted to the Cancer Chemotherapy Unit of the Mofid Children\'s Hospital, Tehran, Iran were analyzed using conventional biochemical tests and polymerase chain reaction targeting the pehX gene to identify K. oxytoca. The antibiotic susceptibility profile of isolated K. oxytoca against commonly prescribed antibiotics used in treating infection at the facility was determined using the Kirby-Bauer disk diffusion technique. Also, the prevalence of genes encoding toxins among K. oxytoca was identified by PCR assay.
UNASSIGNED: The Stool samples of 280 participants were taken for the study of which 38 samples [(55.3% (21/38) 42 males and 44.7% (17/38) females)] tested positive for various Klebsiella spp. Out of this, K. oxytoca was identified in 2.5% (7/280) stools using cultures and conventional biochemical tests. Also, the stools of 2.9% (8/280) of the participants tested positive for K. oxytoca using PCR assay. Using PCR, (2/7) of the K. oxytoca isolates tested positive for the npsA and npsB genes and were identified as toxigenic K. oxytoca strains.
UNASSIGNED: The prevalence of toxin-producing K. oxytoca strains in stool samples of children diagnosed with cancer in Iran is relatively low. Most of the K. oxytoca isolates were susceptible to tested antibiotics. Globally, active surveillance of toxigenic K. oxytoca strains in patients with different malignancies or immunocompromised patients is recommended in healthcare settings.

Toxin-antitoxin (TA) modules are widely found in the genomes of pathogenic bacteria. They regulate vital cellular functions like transcription, translation, and DNA replication, and are therefore essential to the survival of bacteria under stress. With a focus on the type II parDE modules, this study thoroughly examines TAome in Pseudomonas aeruginosa, a bacterium well-known for its adaptability and antibiotic resistance. We explored the TAome in three P. aeruginosa strains: ATCC 27,853, PAO1, and PA14, and found 15 type II TAs in ATCC 27,853, 12 in PAO1, and 13 in PA14, with significant variation in the associated mobile genetic elements. Five different parDE homologs were found by further TAome analysis in ATCC 27,853, and their relationships were confirmed by sequence alignments and precise genomic positions. After comparing these ParDE modules\' sequences to those of other pathogenic bacteria, it was discovered that they were conserved throughout many taxa, especially Proteobacteria. Nucleic acids were predicted as potential ligands for ParD antitoxins, whereas ParE toxins interacted with a wide range of small molecules, indicating a diverse functional repertoire. The interaction interfaces between ParDE TAs were clarified by protein-protein interaction networks and docking studies, which also highlighted important residues involved in binding. This thorough examination improves our understanding of the diversity, evolutionary dynamics, and functional significance of TA systems in P. aeruginosa, providing insights into their roles in bacterial physiology and pathogenicity.

Knowledge of anatomy not only allows optimal treatment and therefore full satisfaction of our patients, but is also fundamental in the prevention of complications. A thorough understanding of aging allows for effective treatments, as most patients demand a natural result, removing the inevitable signs of aging, which can only be understood by considering their aging. Facial aging is a natural but complex multifactorial process, particularly for the forehead. In this article, we will focus on botulinum toxin as well as fillers in aging.

Ricin is a toxic protein regarded as a potential chemical weapon for bioterrorism or criminal use. In the event of a ricin incident, rapid analytical methods are essential for ricin confirmation in a diversity of matrices, from environmental to human or food samples. Mass spectrometry-based methods provide specific toxin identification but require prior enrichment by antibodies to reach trace-level detection in matrices. Here, we describe a novel assay using the glycoprotein asialofetuin as an alternative to antibodies for ricin enrichment, combined with the specific detection of signature peptides by high-resolution mass spectrometry. Additionally, optimizations made to the assay reduced the sample preparation time from 5 h to 80 min only. Method evaluation confirmed the detection of ricin at trace levels over a wide range of pH and in protein-rich samples, illustrating challenging matrices. This new method constitutes a relevant antibody-free solution for the fast and specific mass spectrometry detection of ricin in the situation of a suspected toxin incident, complementary to active ricin determination by adenine release assays.

Equine atypical myopathy (AM) is a severe rhabdomyolysis syndrome primarily caused by hypoglycin A (HGA) and methylenecyclopropylglycine protoxins. This study aimed to refine diagnostic and prognostic criteria for AM while exploring apparently healthy cograzers. Blood samples from 263 horses, including AM cases (n= 95), cograzers (n= 73), colic horses (n= 19), and controls (n= 76), were analyzed for HGA, its toxic metabolite, and acylcarnitines profile. Diseased horses exhibited alterations in acylcarnitines that strongly distinguished them from controls and colic horses. Regression analyses identified distinct acylcarnitines profiles among groups, with cograzers showing intermediate alterations. Age and gelding status emerged as protective factors against AM. Furthermore, serum acylcarnitines profiling was valuable in predicting AM survival, with isovaleryl-/2-methylbutyrylcarnitine (i.e., C5 acylcarnitine) showing promise as both a diagnostic and prognostic marker. Subclinical alterations in cograzers underscore a novel aspect: the presence of subclinical cases of AM.

Expression of camelid-derived, single-domain antibodies (VHHs) within the cytoplasm of mammalian cells as \"intrabodies\" has opened-up novel avenues for medical countermeasures against fast-acting biothreat agents. In this report, we describe a heterodimeric intrabody that renders Vero cells virtually impervious to ricin toxin (RT), a potent Category B ribosome-inactivating protein (RIP). The intrabody consists of two structurally defined VHHs that target distinct epitopes on RT\'s enzymatic subunit (RTA): V9E1 targets RTA\'s P-stalk recruitment site, and V2A11 targets RTA\'s active site. Resistance to RT conferred by the biparatopic VHH construct far exceeded that of either of the VHHs alone and effectively inhibited all measurable RT-induced cytotoxicty in vitro. We propose that targeted delivery of bispecific intrabodies to lung tissues may represent a novel means to shield the airways from the effects of inhalational RT exposure.