Protein Toxins Research Articles

Diversity of biological activities of crude venom extracted from five species of South China Sea anemones

Developing novel, efficient, and safe peptide drugs from sea anemones has aroused great interest in countries around the world today. Sea anemones contain complex protein and peptide toxins, which determine the diversity of their biological activities. In this study, a variety of activities were assessed for crude venom extracted from five species of South China Sea anemones, including hemolytic, enzyme inhibition, anticancer, insecticidal, analgesic and lethal activities. The most toxic sea anemone was found to be Heteractis magnifica, which has high lethal activity in mice with an LD50 of 11.0 mg/kg. The crude venom of H. magnifica also exhibited a range of the most potent activities, including hemolytic, trypsin inhibitory, cytotoxic activity against U251 and A549 cells, insecticidal and analgesic activities. In addition, the crude venom of Stichodactyla haddoni was the most effective inhibitor of pepsin, and the crude venom of Heteractis crispa was extremely strong toxicity to HepG2 cells. These findings are of great significance for exploring the potential and application of South China Sea anemone resources, and are expected to provide new directions and possibilities for the development of novel anticancer drugs, analgesics and biopesticides.

Peptide Toxin Diversity and a Novel Antimicrobial Peptide from the Spider Oxyopes forcipiformis

Spider venoms are emerging as a rich source of bioactive peptide toxins with therapeutic potential. Lynx spiders of the genus Oxyopes are small, cursorial hunters that employ complex venom to subdue arthropod prey. However, extracting crude venom from these diminutive arachnids poses significant challenges. This study presents a transcriptome analysis of venom glands from an undescribed Oxyopes forcipiformis species, revealing 339 putative protein and peptide toxin sequences categorized into seven functional groups. The venom composition was dominated by membrane-active peptides (40.71%), venom auxiliary proteins (22.71%), neurotoxins (15.63%), channel active peptides (7.08%) and uncharacterized components (13.87%). Additionally, phylogenetic analysis of 65 disulfide-bond-rich peptides yielded six distinct families based on sequence homology and cysteine framework. Finally, a novel antimicrobial peptide, GK37, was identified using in silico and homology analyses. Our data suggested that GK37 presented significant antibacterial activity against Gram-positive bacteria Staphylococcus aureus with a minimum inhibitory concentration (MIC) of 1.552 µM by disrupting bacterial membranes. At 4× MICs, GK37 almost showed no hemolytic activity on blood cells or toxicity against Hek293T cells. Our findings provided a basis for targeted studies of the diversity and pharmacological effects of lynx spider peptide. We elucidated a valuable high-throughput approach for obtaining proteins and peptides from small-group spiders.

IGFBP6 Modulates Proteostasis by Activating ATF4 Targets and Reducing ER Retrotranslocon Expression.

Reduced expression of the IGFBP6 protein leads to an increase in the metastatic potential of breast cancer (BC) cells. The level of protein synthesis in tumor cells is increased, leading to a compensatory adjustment of proteostasis. One of the tools used to study proteostasis is protein toxins of the RIP-II family, which irreversibly inactivate ribosomes (particularly, viscumin). We investigated the effect of IGFBP6 gene knockdown on the proteostasis in the BC cell line MDA-MB-231. Ribosomes from MDA-MB-231IGFBP6 cells, knockdown for the IGFBP6 gene, are less efficiently modified by the toxin. This is probably due to the reduced transport of the viscumin catalytic subunit from the ER to the cytoplasm. MDA-MB-231IGFBP6 cells showed reduced expression of the retrotranslocon HRD1/Derlin subunit, which is a component of the ER-associated protein degradation system (ERAD). For ATF4 transcription factor, which is a part of the ER unfolded protein response (UPR) pathway, an increased expression of its targets was found.

Synthesis of Multivalent Glycoside-Immobilized Carboxymethyl Cellulose Nanohydrogel Particles with Superadsorption Ability for Lectins.

Carboxymethyl cellulose (CMC) is a water-soluble cellulose derivative that is nontoxic, biocompatible, biodegradable, and nonallergenic. As developing an adsorbent material for carbohydrate-binding proteins is challenging, we aimed to synthesize CMC nanohydrogel particles (CMCGPs) with an extremely high lectin adsorption tendency in this study. CMCGPs were used as the backbone of an adsorption carrier that was synthesized by cross-linking CMC with ethylene glycol diglycidyl ether. A series of glycoside-immobilized CMCGPs were synthesized by binding two types of glycans (LacNAc and lactose) to the polyvalent carboxymethyl groups that are present on the CMCGP surface and act as reaction sites. These immobilized glycosides function as molecular recognition sites. Glycan moieties were incorporated into the CMCGP backbone at degrees of immobilization (DI) ranging from 8.7 to 21.0% by altering the reaction composition. LacNAc-CMCGP (3b) showed a 19.9% DI of LacNAc glycoside to the CMCGP carboxymethyl group; on average, its particle size swelled to 418 nm in phosphate-buffered saline, which is approximately 1.4 times its dry-state size. Analyzing the adsorbent properties of glyco-CMCGPs using a lectin-binding assay showed the high structural specificity of glyco-CMCGPs to lectins. The equilibrium isotherm data was explained by the Langmuir adsorption model. Notably, compound 3b adsorbed 1.95 ± 0.05 μg of wheat germ agglutinin (WGA) lectin per 1.0 μg-dry of 3b particles at an adsorption equilibrium time of a few minutes. Furthermore, solid-state 13C nuclear magnetic resonance analysis showed that WGA lectin retained its natural structure without denaturation after binding to LacNAc-CMCGP. These results were also supported by affinity purification experiments of WGA from raw wheat germ extract using LacNAc-CMCGP, demonstrating that glyco-CMCGP is capable of adsorbing and desorbing lectin while maintaining its biological activity. Thus, multivalent glycoside-immobilized CMCGPs that use woody biomass derivatives as the backbone are expected to be applied as biorefinery materials, which specifically and abundantly adsorb not just plant lectins but also pathogenic viruses and toxin proteins.

Alpha-1 antitrypsin inhibits pertussis toxin

Pertussis (whooping cough) is a vaccine-preventable but re-emerging, highly infectious respiratory disease caused by Bordetella pertussis. There are currently no effective treatments for pertussis, complicating care for non-vaccinated individuals, especially newborns. Disease manifestations are predominantly caused by pertussis toxin (PT), a pivotal virulence factor classified as an ADP-ribosylating AB-type protein toxin. In this work, an unbiased approach using peptide libraries, bioassay-guided fractionation and mass spectrometry revealed α1-antitrypsin (α1AT) as a potent PT inhibitor. Biochemistry-, cell culture- and molecular modeling-based in vitro experimentation demonstrated that the α1AT mode of action is based on blocking PT-binding to the host target cell surface. In the infant mouse model of severe pertussis, α1AT expression was reduced upon infection. Further, systemic administration of α1AT significantly reduced B. pertussis-induced leukocytosis, which is a hallmark of infant infection and major risk factor for fatal pertussis. Taken together our data demonstrates that α1AT is a novel PT inhibitor and that further evaluation and development of α1AT as a therapeutic agent for pertussis is warranted. Importantly, purified α1AT is already in use clinically as an intravenous augmentation therapy for those with genetic α1AT deficiency and could be repurposed to clinical management of pertussis.

Phosphorylation of VapB antitoxins affects intermolecular interactions to regulate VapC toxin activity in Mycobacterium tuberculosis.

Toxin-antitoxin modules are present in many bacterial pathogens. The VapBC family is particularly abundant in members of the Mycobacterium tuberculosis complex, with 50 modules present in the M. tuberculosis genome. In type IIA modules, the VapB antitoxin protein binds to and inhibits the activity of the co-expressed cognate VapC toxin protein. VapB proteins may also bind to promoter region sequences and repress the expression of the vapB-vapC operon. Though VapB-VapC interactions can control the amount of free VapC toxin in the bacterial cell, the mechanisms that affect this interaction are poorly understood. Based on our recent finding of Ser/Thr phosphorylation of VapB proteins in M. tuberculosis, we substituted phosphomimetic or phosphoablative amino acids at the phosphorylation sites of two VapB proteins. We found that phosphomimetic substitution of VapB27 and VapB46 resulted in decreased interaction with their respective cognate VapC proteins, whereas phosphoablative substitution did not alter binding. Similarly, we determined that phosphomimetic substitution interfered with VapB binding to promoter region DNA sequences. Both decreased VapB-VapC interaction and decreased VapB repression of vapB-vapC operon transcription would result in increased free VapC in the M. tuberculosis cell. In growth inhibition experiments, M. tuberculosis strains expressing vapB46-vapC46 constructs containing a phosphoablative vapB mutation resulted in lower toxicity compared to a strain expressing native vapB46, whereas similar or greater toxicity was observed in the strain expressing the phosphomimetic vapB mutation. These results identify a novel mechanism by which VapC toxicity activity can be regulated by VapB phosphorylation.IMPORTANCEIntracellular bacterial toxins are present in many bacterial pathogens and have been linked to bacterial survival in response to stresses encountered during infection. The activity of many toxins is regulated by a co-expressed antitoxin protein that binds to and sequesters the toxin protein. The mechanisms by which an antitoxin may respond to stresses to alter toxin activity are poorly understood. Here, we show that antitoxin interactions with its cognate toxin and with promoter DNA required for antitoxin and toxin expression can be altered by Ser/Thr phosphorylation of the antitoxin and, thus, affect toxin activity. This reversible modification may play an important role in regulating toxin activity within the bacterial cell in response to signals generated during infection.

Host Tropism and Structural Biology of ABC Toxin Complexes.

ABC toxin complexes are a class of protein toxin translocases comprised of a multimeric assembly of protein subunits. Each subunit displays a unique composition, contributing to the formation of a syringe-like nano-machine with natural cargo carrying, targeting, and translocation capabilities. Many of these toxins are insecticidal, drawing increasing interest in agriculture for use as biological pesticides. The A subunit (TcA) is the largest subunit of the complex and contains domains associated with membrane permeation and targeting. The B and C subunits, TcB and TcC, respectively, package into a cocoon-like structure that contains a toxic peptide and are coupled to TcA to form a continuous channel upon final assembly. In this review, we outline the current understanding and gaps in the knowledge pertaining to ABC toxins, highlighting seven published structures of TcAs and how these structures have led to a better understanding of the mechanism of host tropism and toxin translocation. We also highlight similarities and differences between homologues that contribute to variations in host specificity and conformational change. Lastly, we review the biotechnological potential of ABC toxins as both pesticides and cargo-carrying shuttles that enable the transport of peptides into cells.

A Comparative Analysis of Different Xenorhabdus Strains Reveals a Virulent Factor, Cyclic Pro-Phe, Using a Differential Expression Profile Analysis of Non-Ribosomal Peptide Synthetases.

Entomopathogenic bacteria, classified into the genus Xenorhabdus, exhibit a dual lifestyle as mutualistic symbionts to Steinernema nematodes and as pathogens to a broad range of insects. Bacterial virulence depends on toxin proteins that induce toxemia and various immunosuppressive secondary metabolites that cause septicemia. Particularly, the immunosuppressive properties of Xenorhabdus bacteria determine the variability of their insecticidal activities. This study explored the role of peptide metabolites in virulence and its variation among six bacterial strains across three species: X. nematophila, X. bovienii, and X. hominickii. Initially, their virulence significantly varied against a susceptible lepidopteran host, Maruca vitrata, but showed less variation against a tolerant coleopteran host, Tenebrio molitor, with high median lethal bacterial doses. In M. vitrata, virulence was strongly correlated with bacterial growth rate and inhibitory activity against phospholipase A2. Secondly, the six strains differed in the compositions of their secreted secondary metabolites, analyzed by GC-MS following ethyl acetate extraction. Notably, there was significant variation in the production of di- or tetra-peptides. Highly virulent strains commonly produced the cyclic Pro-Phe (cPF). Thirdly, the expression of non-ribosomal peptide synthetase (NRPS) genes varied greatly among the strains. NRPS genes were minimally expressed in the tolerant T. molitor and highly expressed in the susceptible M. vitrata. In M. vitrata, specific NRPS genes were markedly expressed in the virulent strains. Finally, cPF demonstrated potent immunosuppressive activity against the cellular and humoral responses of M. vitrata. The addition of cPF significantly enhanced the virulence against the tolerant T. molitor. These findings suggest that immunosuppression is necessary for the pathogenicity of Xenorhabdus bacteria, wherein NRPS products play a critical role in suppressing immune-associated factors in target insects.

Conjugation of anti-HIV gp41 monoclonal antibody to a drug capable of targeting resting lymphocytes produces an effective cytotoxic anti-HIV immunoconjugate.

HIV-infected cells persisting in the face of suppressive antiretroviral therapy are the barrier to curing infection. Cytotoxic immunoconjugates targeted to HIV antigens on the cell surface may clear these cells. We showed efficacy in mouse and macaque models using immunotoxins, but immunogenicity blunted the effect. As an alternative, we propose antibody drug conjugates (ADCs), as used in cancer immunotherapy. In cancer, the target is a dividing cell, whereas it may not be in HIV. We screened cytotoxic drugs on human primary cells and cell lines. An anthracycline derivative, PNU-159682 (PNU), was highly cytotoxic to both proliferating and resting cells. Human anti-gp41 mAb 7B2 was conjugated to ricin A chain or PNU. The conjugates were tested in vitro for cytotoxic efficacy and anti-viral effect, and in vivo for tolerability. The specificity of killing for both conjugates was demonstrated on Env+ and Env- cells. The toxin conjugate was more potent and killed more rapidly, but 7B2-PNU was effective at levels achievable in patients. The ricin conjugate was well tolerated in mice; 7B2-PNU was toxic when administered intraperitoneally but was tolerated intravenously. We have produced an ADC with potential to target the persistent HIV reservoir in both dividing and non-dividing cells while avoiding immunogenicity. Cytotoxic anti-HIV immunoconjugates may have greatest utility as part of an "activate and purge" regimen, involving viral activation in the reservoir. This is a unique comparison of an immunotoxin and ADC targeted by the same antibody and tested in the same systems.IMPORTANCEHIV infection can be controlled with anti-retroviral therapy, but it cannot be cured. Despite years of therapy that suppresses HIV, patients again become viremic shortly after discontinuing treatment. A long-lived population of memory T cells retain the genes encoding HIV, and these cells secrete infectious HIV when no longer suppressed by therapy. This is the persistent reservoir of HIV infection. The therapies described here use anti-HIV antibodies conjugated to poisons to kill the cells in this reservoir. These poisons may be of several types, including protein toxins (immunotoxins) or anti-cancer drugs (antibody drug conjugates, ADCs). We have previously shown that an anti-HIV immunotoxin had therapeutic effects in animal models, but it elicited an anti-drug immune response. Here, we have prepared an anti-HIV ADC, which would be less likely to provoke an immune response, and show its potential for use in eliminating the persistent reservoir of HIV infection.

In Vitro Evaluation of Antipseudomonal Activity and Safety Profile of Peptidomimetic Furin Inhibitors.

Inhibitors of the serine protease furin have been widely studied as antimicrobial agents due to their ability to block the cleavage and activation of certain viral surface proteins and bacterial toxins. In this study, the antipseudomonal effects and safety profiles of the furin inhibitors MI-1851 and MI-2415 were assessed. Fluorescence quenching studies suggested no relevant binding of the compounds to human serum albumin and α1-acid glycoprotein. Both inhibitors demonstrated significant antipseudomonal activity in Madin-Darby canine kidney cells, especially compound MI-1851 at very low concentrations (0.5 µM). Using non-tumorigenic porcine IPEC-J2 cells, neither of the two furin inhibitors induced cytotoxicity (CCK-8 assay) or altered significantly the intracellular (Amplex Red assay) or extracellular (DCFH-DA assay) redox status even at a concentration of 100 µM. The same assays with MI-2415 conducted on primary human hepatocytes also resulted in no changes in cell viability and oxidative stress at up to 100 µM. Microsomal and hepatocyte-based CYP3A4 activity assays showed that both inhibitors exhibited a concentration-dependent inhibition of the isoenzyme at high concentrations. In conclusion, this study indicates a good safety profile of the furin inhibitors MI-1851 and MI-2415, suggesting their applicability as antimicrobials for further in vivo investigations, despite some inhibitory effects on CYP3A4.

Nanofractionation Analytics for Comparing MALDI-MS and ESI-MS Data of Viperidae Snake Venom Toxins.

Worldwide, it is estimated that there are 1.8 to 2.7 million cases of envenoming caused by snakebites. Snake venom is a complex mixture of protein toxins, lipids, small molecules, and salts, with the proteins typically responsible for causing pathology in snakebite victims. For their chemical characterization and identification, analytical methods are required. Reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (RP-LC-ESI-MS) is a widely used technique due to its ease of use, sensitivity, and ability to be directly coupled after LC separation. This method allows for the efficient separation of complex mixtures and sensitive detection of analytes. On the other hand, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is also sometimes used, and though it typically requires additional sample preparation steps, it offers desirable suitability for the analysis of larger biomolecules. In this study, seven medically important viperid snake venoms were separated into their respective venom toxins and measured by ESI-MS. In parallel, using nanofractionation analytics, post-column high-resolution fractionation was used to collect the eluting toxins for further processing for MALDI-MS analysis. Our comparative results showed that the deconvoluted snake venom toxin masses were observed with good sensitivity from both ESI-MS and MALDI-MS approaches and presented overlap in the toxin masses recovered (between 25% and 57%, depending on the venom analyzed). The mass range of the toxins detected in high abundance was between 4 and 28 kDa. In total, 39 masses were found in both the ESI-MS and/or MALDI-MS analyses, with most being between 5 and 9 kDa (46%), 13 and 15 kDa (38%), and 24 and 28 kDa (13%) in size. Next to the post-column MS analyses, additional coagulation bioassaying was performed to demonstrate the parallel post-column assessment of venom activity in the workflow. Most nanofractionated venoms exhibited anticoagulant activity, with three venoms additionally exhibiting toxins with clear procoagulant activity (Bothrops asper, Crotalus atrox, and Daboia russelii) observed post-column. The results of this study highlight the complementarity of ESI-MS and MALDI-MS approaches for characterizing snake venom toxins and provide a complementary overview of defined toxin masses found in a diversity of viper snake venoms.

Enhancing the cytotoxicity of immunotoxins by facilitating their dissociation from target receptors under the reducing conditions of the endocytic pathway

Immunotoxins (ITs) are recombinant chimeric proteins that combine a protein toxin with a targeting moiety to facilitate the selective delivery of the toxin to cancer cells. Here, we present a novel strategy to enhance the cytosolic access of ITs by promoting their dissociation from target receptors under the reducing conditions of the endocytic pathway. We engineered monobodySS, a human fibronectin type III domain-based monobody with disulfide bond (SS)-containing paratopes, targeting receptors such as EGFR, EpCAM, Her2, and FAP. MonobodySS exhibited SS-dependent target receptor binding with a significant reduction in binding under reducing conditions. We then created monobodySS-based ITs carrying a 25 kDa fragment of Pseudomonas exotoxin A (PE25), termed monobodySS-PE25. These ITs showed dose-dependent cytotoxicity against target receptor-expressing cancer cells and a wider therapeutic window due to higher efficacy at lower doses compared to controls with SS reduction inhibited. ERSS/28-PE25, with a KD of 28 nM for EGFR, demonstrated superior tumor-killing potency compared to ER/21-PE25, which lacks an SS bond, at equivalent and lower doses. In vivo, ERSS/28-PE25 outperformed ER/21-PE25 in suppressing tumor growth in EGFR-overexpressing xenograft mouse models. This study presents a strategy for developing solid tumor-targeting ITs using SS-containing paratopes to enhance cytosolic delivery and antitumor efficacy.

Comparative Analysis of Tentacle Extract and Nematocyst Venom: Toxicity, Mechanism, and Potential Intervention in the Giant Jellyfish Nemopilema nomurai.

The giant jellyfish Nemopilema nomurai sting can cause local and systemic reactions; however, comparative analysis of the tentacle extract (TE) and nematocyst venom extract (NV), and its toxicity, mechanism, and potential intervention are still limited. This study compared venom from TE and NV for their composition, toxicity, and efficacy in vitro and in vivo used RAW264.7 cells and ICR mice. A total of 239 and 225 toxin proteins were identified in TE and NV by proteomics, respectively. Pathological analysis revealed that TE and NV caused heart and liver damage through apoptosis, necrosis, and inflammation, while TE exhibited higher toxicity ex vivo and in vivo. Biochemical markers indicated TE and NV elevated creatine kinase, lactatedehydrogenase, and aspartate aminotransferase, with the TE group showing a more significant increase. Transcriptomics and Western blotting indicated both venoms increased cytokines expression and MAPK signaling pathways. Additionally, 1 mg/kg PACOCF3 (the phospholipase A2 inhibitor) improved survival from 16.7% to 75% in mice. Our results indicate that different extraction methods impact venom activities, tentacle autolysis preserves toxin proteins and their toxicity, and PACOCF3 is a potential antidote, which establishes a good extraction method of jellyfish venom, expands our understanding of jellyfish toxicity, mechanism, and provides a promising intervention.

Calcium leakage involved in nematotoxic effects of the Conidiobolus obscurus CytCo protein on the pine wood nematode, Bursaphelenchus xylophilus.

The pine wood nematode Bursaphelenchus xylophilus, a severe invasive species, is responsible for causing widespread pine wilt disease. The CytCo protein, a pore-forming toxin derived from Conidiobolus obscurus, exhibits nematotoxicity towards B. xylophilus. Our present study reveals the expression variation of a range of gene products in B. xylophilus that respond to the effects of CytCo using the isobaric tags for relative and absolute quantification proteomics technology. Functional enrichment analysis indicates that many differentially expressed proteins are linked to calcium signaling system, proteasome, energy production and conversion, and the determination of adult lifespan. It suggests that the dysregulation of calcium homeostasis, energy metabolism, and apoptosis contribute to the CytCo nematotoxicity. Using the calcium ion (Ca2+)-indicator calcein, we detected changes in Ca2+ levels in B. xylophilus, with a significantly increase in fluorescence in the nematode's intestine and pseudocoelom following CytCo treatments. Meanwhile, the apoptosis and reactive oxygen species (ROS) assays showed an enhancement of fluorescence in B. xylophilus cells, with increased CytCo concentrations. The protein toxin CytCo triggers Ca2+ leakage, disrupts Ca2+ balance in B. xylophilus, and induces apoptosis and ROS outburst, thereby intensifying its nematotoxic effects. This finding facilitates our understanding of the modes of action of nematotoxic proteins, and contributes to the development of innovative nematode control strategies. © 2024 Society of Chemical Industry.

Comparative transcriptomic analysis of hepatopancreas reveals that more genes are involved in the exposure response of Vibrio parahaemolyticus PirAvp compared to PirBvp

Vibrio parahaemolyticus (VP-AHPND) is regarded as one of the main pathogens that caused acute hepatopancreatic necrosis disease (AHPND) in the Pacific white shrimp Litopenaeus vannamei. PirAvp and PirBvp toxin proteins are the main pathogenic proteins of AHPND in shrimp. Knowledge about the mechanism of shrimp response to PirAvp or PirBvp toxin is very helpful for developing new prevention and control strategy of AHPND in shrimp. In this study, the pathological sections showed that after 4 h treatment, significant pathological changes were observed in the PirBvp treated group, and no obvious pathological changes was found in PirAvp treated group. In order to learn the mechanism of shrimp response to PirAvp and PirBvp, comparative transcriptome was applied to analyze the different expressions of genes in the hepatopancreas of shrimp after treatment with PirAvp or PirBvp. A total of 9978 differentially expressed genes (DEGs) were identified between PirAvp or PirBvp-treated and PBS control shrimp, including 6616 DEGs in the PirAvp treated group and 3362 DEGs in the PirBvp treated group. There were 2263 DEGs that were commonly expressed, 4353 DEGs were only expressed in PirAvp VS PBS group and 1099 DEGs were uniquely expressed in PirBvp VS PBS group. Among these DEGs, the anti-apoptosis related pathways and immune response related genes significantly expressed in the commonly expressed DEGs of PirAvp VS PBS group and PirBvp VS PBS group, and small GTPase-mediated signaling and DNA metabolic process might relate to the host special reaction towards PirAvp and PirBvp exposure. The data suggested that the differential expression of these immune and metabolic-related genes in hepatopancreas might contribute to the pathogenicity variations of shrimp to VP-AHPND. The identified genes in this study will be useful for clarifying the response mechanism of shrimp toward different toxins of VP-AHPND and will further provide molecular basis for understanding the pathogenic mechanism of VP-AHPND.

CAMP signaling of Bordetella adenylate cyclase toxin blocks M-CSF triggered upregulation of iron acquisition receptors on differentiating CD14+ monocytes.

Bordetella pertussis infects the upper airways of humans and disarms host defense by the potent immuno-subversive activities of its pertussis (PT) and adenylate cyclase (CyaA) toxins. CyaA action near-instantly ablates the bactericidal activities of sentinel CR3-expressing myeloid phagocytes by hijacking cellular signaling pathways through the unregulated production of cAMP. Moreover, CyaA-elicited cAMP signaling also inhibits the macrophage colony-stimulating factor (M-CSF)-induced differentiation of incoming inflammatory monocytes into bactericidal macrophages. We show that CyaA/cAMP signaling via protein kinase A (PKA) downregulates the M-CSF-elicited expression of monocyte receptors for transferrin (CD71) and hemoglobin-haptoglobin (CD163), as well as the expression of heme oxygenase-1 (HO-1) involved in iron liberation from internalized heme. The impact of CyaA action on CD71 and CD163 levels in differentiating monocytes is largely alleviated by the histone deacetylase inhibitor trichostatin A (TSA), indicating that CyaA/cAMP signaling triggers epigenetic silencing of genes for micronutrient acquisition receptors. These results suggest a new mechanism by which B. pertussis evades host sentinel phagocytes to achieve proliferation on airway mucosa.IMPORTANCETo establish a productive infection of the nasopharyngeal mucosa and proliferate to sufficiently high numbers that trigger rhinitis and aerosol-mediated transmission, the pertussis agent Bordetella pertussis deploys several immunosuppressive protein toxins that compromise the sentinel functions of mucosa patrolling phagocytes. We show that cAMP signaling elicited by very low concentrations (22 pM) of Bordetella adenylate cyclase toxin downregulates the iron acquisition systems of CD14+ monocytes. The resulting iron deprivation of iron, a key micronutrient, then represents an additional aspect of CyaA toxin action involved in the inhibition of differentiation of monocytes into the enlarged bactericidal macrophage cells. This corroborates the newly discovered paradigm of host defense evasion mechanisms employed by bacterial pathogens, where manipulation of cellular cAMP levels blocks monocyte to macrophage transition and replenishment of exhausted phagocytes, thereby contributing to the formation of a safe niche for pathogen proliferation and dissemination.

Identification of novel toxins associated with the extracellular contractile injection system using machine learning

Secretion systems play a crucial role in microbe-microbe or host-microbe interactions. Among these systems, the extracellular contractile injection system (eCIS) is a unique bacterial and archaeal extracellular secretion system that injects protein toxins into target organisms. However, the specific proteins that eCISs inject into target cells and their functions remain largely unknown. Here, we developed a machine learning classifier to identify eCIS-associated toxins (EATs). The classifier combines genetic and biochemical features to identify EATs. We also developed a score for the eCIS N-terminal signal peptide to predict EAT loading. Using the classifier we classified 2,194 genes from 950 genomes as putative EATs. We validated four new EATs, EAT14-17, showing toxicity in bacterial and eukaryotic cells, and identified residues of their respective active sites that are critical for toxicity. Finally, we show that EAT14 inhibits mitogenic signaling in human cells. Our study provides insights into the diversity and functions of EATs and demonstrates machine learning capability of identifying novel toxins. The toxins can be employed in various applications dependently or independently of eCIS.

Alcohol in Baijiu Contributes to the Increased Probability of Host Infection by Clostridioides difficile Spores.

Clostridioides difficile and its endospores possess the characteristics of a foodborne pathogen and have been detected at several stages in the food chain. In the presence of an imbalance in host intestinal ecology, C. difficile can proliferate and cause intestinal infections. Multiple food source factors can substantially alter the host's gut ecosystem, including the consumption of baijiu. However, it remains to be known whether the gut ecological changes induced by the consumption of baijiu increase the risk of C. difficile invasion and infection. In this study, C. difficile cells were exposed to two commercially available baijiu to evaluate the effect of baijiu on C. difficile cells and to verify through a mouse model. The results showed that baijiu effectively inhibited the growth and biofilm production of C. difficile, downregulated the expression levels of tcdA and tcdB virulence genes but upregulated the expression level of spore-producing genes Spo0A, enhanced the spore production, as well as increased C. difficile cell adhesion to Caco-2 cells. The mouse model showed that the intake of baijiu promoted the invasion and infection of C. difficile spores, causing damage to the cecum tissue, accompanied by an increase in the gut lipid carrier protein-2 (Lcn-2) and TcdA toxin protein levels. Simultaneously, cholic acid was elevated, whereas deoxycholic acid was decreased. This study is the first to find a possible link between baijiu intake and C. difficile spore invasion and infection.

Jellyfish Venom Peptides Targeting Human Potassium Channels Identified through Ligand Screening: Morphometric and Molecular Identification of the Species and Antibiotic Potential.

The relative lack of marine venom could be attributed to the difficulty in dealing with venomous marine animals. Moreover, the venom of marine animals consists of various bioactive molecules, many of which are proteins with unique properties. In this study, we investigated the potential toxic proteins of jellyfish collected for ligand screening to understand the mechanism of the toxic effects of jellyfish. Since taxonomic identification is problematic due to the lack of proper keys, we conducted morphological and molecular mitochondrial DNA sequencing from COI and ITS regions. The venom extract from nematocysts found along the bell margins was used for protein characterization using SDS-gel electrophoresis and nano-liquid chromatography-tandem mass spectrometry. Ligand screening for the most potent toxin and antibacterial and cytotoxicity assays were carried out. The phylogenetic tree showed distinct clustering from other Catostylus sp. The proteomic analysis revealed venom with many bioactive proteins. Only 13 venom proteins were identified with molecular weights ranging from 4318 to 184,923 Da, exhibiting the venom's complexity. The overall toxin protein composition of Catostylus sp. venom was dominated by potassium channel toxin alpha-KTx. Molecular docking of toxin alpha-KTx 1.13 revealed high specificity towards the human voltage-gated potassium channel Kv3 with a high fitness score and a minimum energy barrier of -17.9 kcal/mol. Disc diffusion and cytotoxicity assays revealed potent antibacterial activity against Klebsiella pneumoniae with no cytotoxicity. Further studies on detailed characterization and therapeutic potentials are warranted.

Hierarchical determinants in cytotoxic necrotizing factor (CNF) toxins driving Rho G-protein deamidation versus transglutamination.

The cytotoxic necrotizing factor (CNF) family of AB-type bacterial protein toxins catalyze two types of modification on their Rho GTPase substrates: deamidation and transglutamination. It has been established that E. coli CNF1 and its close homolog proteins catalyze primarily deamidation and Bordetella dermonecrotic toxin (DNT) catalyzes primarily transglutamination. The rapidly expanding microbial genome sequencing data have revealed that there are at least 13 full-length variants of CNF1 homologs. CNFx from E. coli strain GN02091 is the most distant from all other members of the CNF family with 50%-55% sequence identity at the protein level and 0.45-0.52 nucleotide substitutions per site at the DNA level. CNFx modifies RhoA, Rac1, and Cdc42, and like CNF1, activates downstream SRE-dependent mitogenic signaling pathways in human HEK293T cells, but at a 1,000-fold higher EC50 value. Unlike other previously characterized CNF toxins, CNFx modifies Rho proteins primarily through transglutamination, as evidenced by gel-shift assay and confirmed by MALDI mass spectral analysis, when coexpressed with Rho-protein substrates in E. coli BL21 cells or through direct treatment of HEK293T cells. A comparison of CNF1 and CNFx sequences identified two critical active-site residues corresponding to positions 832 and 862 in CNF1. Reciprocal site-specific mutations at these residues in each toxin revealed hierarchical rules that define the preference for deamidase versus a transglutaminase activity in CNFs. An additional unique Cys residue at the C-terminus of CNFx was also discovered to be critical for retarding cargo delivery.IMPORTANCECytotoxic necrotizing factor (CNF) toxins not only play important virulence roles in pathogenic E. coli and other bacterial pathogens, but CNF-like genes have also been found in an expanding number of genomes from clinical isolates. Harnessing the power of evolutionary relationships among the CNF toxins enabled the deciphering of the hierarchical active-site determinants that define whether they modify their Rho GTPase substrates through deamidation or transglutamination. With our finding that a distant CNF variant (CNFx) unlike other known CNFs predominantly transglutaminates its Rho GTPase substrates, the paradigm of "CNFs deamidate and DNTs transglutaminate" could finally be attributed to two critical amino acid residues within the active site other than the previously identified catalytic Cys-His dyad residues. The significance of our approach and research findings is that they can be applied to deciphering enzyme reaction determinants and substrate specificities for other bacterial proteins in the development of precision therapeutic strategies.