Alpha-hemolysin promotes internalization of Staphylococcus aureus into human lung epithelial cells via caveolin-1- and cholesterol-rich lipid rafts.

Staphylococcus aureus is a pathogen associated with severe respiratory infections. The ability of S. aureus to internalize into lung epithelial cells complicates the treatment of respiratory infections caused by this bacterium. In the intracellular environment, S. aureus can avoid elimination by the immune system and the action of circulating antibiotics. Consequently, interfering with S. aureus internalization may represent a promising adjunctive therapeutic strategy to enhance the efficacy of conventional treatments. Here, we investigated the host-pathogen molecular interactions involved in S. aureus internalization into human lung epithelial cells. Lipid raft-mediated endocytosis was identified as the main entry mechanism. Thus, bacterial internalization was significantly reduced after the disruption of lipid rafts with methyl-ß-cyclodextrin. Confocal microscopy confirmed the colocalization of S. aureus with lipid raft markers such as ganglioside GM1 and caveolin-1. Adhesion of S. aureus to α5ß1 integrin on lung epithelial cells via fibronectin-binding proteins (FnBPs) was a prerequisite for bacterial internalization. A mutant S. aureus strain deficient in the expression of alpha-hemolysin (Hla) was significantly impaired in its capacity to enter lung epithelial cells despite retaining its capacity to adhere. This suggests a direct involvement of Hla in the bacterial internalization process. Among the receptors for Hla located in lipid rafts, caveolin-1 was essential for S. aureus internalization, whereas ADAM10 was dispensable for this process. In conclusion, this study supports a significant role of lipid rafts in S. aureus internalization into human lung epithelial cells and highlights the interaction between bacterial Hla and host caveolin-1 as crucial for the internalization process.

Inhibition of neutrophil swarming by type I interferon promotes intracellular bacterial evasion.

Listeria monocytogenes (LM) possesses the ability to breach multiple barriers and elicit intricate immune responses. However, there remains a lack of explicit understanding regarding how LM evades innate immune surveillance within the body. Here, we utilized liver intravital imaging to elucidate the dynamic process of LM during infection in the liver. We discovered that LM can rapidly escape from Kupffer cells (KCs) through listeriolysin O (LLO) and proliferate within hepatocytes. Upon LM exposure to the hepatic sinusoids, neutrophils rapidly aggregate at the site of infection. Subsequently, LM can induce type I interferon (IFN-I) production primarily in the spleen, which acts systemically on neutrophils to hamper their swarming by deactivating the ERK pathway, thus evading neutrophil-mediated eradication. Furthermore, our findings suggest that virus-induced IFN-I suppresses neutrophil swarming, and COVID-19 patients exhibit impaired neutrophil aggregation function. In conclusion, our findings provide compelling evidence demonstrating that intracellular bacteria represented by LM can hijack host defense mechanisms against viral infections to evade immune surveillance. Additionally, impaired neutrophil swarming caused by IFN-I is one of the significant factors contributing to the increased susceptibility to bacterial infections following viral infections.

Enhanced Staphylococcus aureus protection by uncoupling of the α-toxin-ADAM10 interaction during murine neonatal vaccination.

Staphylococcus aureus remains a leading global cause of bacterial infection-associated mortality and has eluded prior vaccine development efforts. S. aureus α-toxin (Hla) is an essential virulence factor in disease, impairing the T cell response to infection. The anti-Hla antibody response is a correlate of human protective immunity. Here we observe that this response is limited early in human life and design a vaccine strategy to elicit immune protection against Hla in a neonatal mice. By targeted disruption of the interaction of Hla with its receptor ADAM10, we identify a vaccine antigen (HlaH35L/R66C/E70C, HlaHRE) that elicits an ~100-fold increase in the neutralizing anti-Hla response. Immunization with HlaHRE enhances the T follicular helper (TFH) cell response to S. aureus infection, correlating with the magnitude of the neutralizing anti-toxin response and disease protection. Furthermore, maternal HlaHRE immunization confers protection to offspring. Together, these findings illuminate a path for S. aureus vaccine development at the maternal-infant interface.

Encoding extracellular modification of artificial cell membranes using engineered self-translocating proteins.

The development of artificial cells has led to fundamental insights into the functional processes of living cells while simultaneously paving the way for transformative applications in biotechnology and medicine. A common method of generating artificial cells is to encapsulate protein expression systems within lipid vesicles. However, to communicate with the external environment, protein translocation across lipid membranes must take place. In living cells, protein transport across membranes is achieved with the aid of complex translocase systems which are difficult to reconstitute into artificial cells. Thus, there is need for simple mechanisms by which proteins can be encoded and expressed inside synthetic compartments yet still be externally displayed. Here we present a genetically encodable membrane functionalization system based on mutants of pore-forming proteins. We modify the membrane translocating loop of α-hemolysin to translocate functional peptides up to 52 amino acids across lipid membranes. Full membrane translocation occurs in the absence of any translocase machinery and the translocated peptides are recognized by specific peptide-binding ligands on the opposing membrane side. Engineered hemolysins can be used for genetically programming artificial cells to display interacting peptide pairs, enabling their assembly into artificial tissue-like structures.

Resistance to Cry14A family Bacillus thuringiensis crystal proteins in Caenornabditis elegans operates via the nhr-31 transcription factor and vacuolar-type ATPase pathway.

Bacillus thuringiensis (Bt) has been successfully used commercially for more than 60 years for biocontrol of insect pests. Since 1996, transgenic plants expressing Bt crystal (Cry) proteins have been used commercially to provide protection against insects that predate on corn and cotton. More recently, Bt Cry proteins that target nematodes have been discovered. One of these, Cry14Ab, has been expressed in transgenic soybean plants and found to provide significant protection against the soybean cyst nematode, Heterodera glycines. However, to date there has been no description of high-level resistance to any Cry14A family protein in nematodes. Here, we describe forward genetic screens to identify such mutants using the nematode Caenorhabditis elegans. Although non-conditional screens failed to identify highly resistant C. elegans, a conditional (temperature-sensitive) genetic screen identified one mutant, bre-6(ye123) (for Bt protein resistant), highly resistant to both Cry14Aa and Cry14Ab. The mutant comes at a high fitness cost, showing significant delays in growth and development and reduced fecundity. bre-6(ye123) hermaphrodites are only weakly resistant to copper intoxication, indicating that the mutant is not highly resistant to all insults. Backcrossing-whole genome sequencing was used to identify the gene mutated in ye123 as the nuclear hormone receptor nhr-31. RNAi, DNA rescue, and CRISPR analyses confirm that resistance to Cry14Aa intoxication in bre-6(ye123) is due to mutation of nhr-31 and was renamed nhr-31(ye123). As predicted for a mutation in this gene, nhr-31(ye123) animals showed significantly reduced expression of most of the subunits of the C. elegans vacuolar ATPase (vATPase). Mutants in the vATPase subunits unc-32 and vha-7 also show resistance to Cry14Aa and/or Cry14Ab. These data demonstrate that nhr-31 and the vATPase play a significant role in the intoxication of C. elegans by Cry14A family proteins, that reduction in vATPase levels result in high resistance to Cry14A family proteins, and that such resistance comes at a high fitness cost. Based on the relative difficulty of finding resistant mutants and the fitness cost associated with the vATPase pathway, our data suggest that transgenic Cry14Ab plants may hold up well to resistance by nematode parasites.

Transcriptomic Analysis of the Response of the Dioryctria abietella Larva Midgut to Bacillus thuringiensis 2913 Infection.

Dioryctria abietella Denis Schiffermuller (Lepidoptera: Pyralidae) is an oligophagous pest that mainly damages Pinaceae plants. Here, we investigated the effects of the Bacillus thuringiensis 2913 strain (Bt 2913), which carries the Cry1Ac, Cry2Ab, and Vip3Aa genes, on the D. abietella midgut transcriptome at 6, 12, and 24 h after infection. In total, 7497 differentially expressed genes (DEGs) were identified from the midgut transcriptome of D. abietella larvae infected with Bt 2913. Among these DEGs, we identified genes possibly involved in Bt 2913-induced perforation of the larval midgut. For example, the DEGs included 67 genes encoding midgut proteases involved in Cry/Vip toxin activation, 74 genes encoding potential receptor proteins that bind to insecticidal proteins, and 19 genes encoding receptor NADH dehydrogenases that may bind to Cry1Ac. Among the three transcriptomes, 88 genes related to metabolic detoxification and 98 genes related to immune defense against Bt 2913 infection were identified. Interestingly, 145 genes related to the 60S ribosomal protein were among the DEGs identified in the three transcriptomes. Furthermore, we performed bioinformatic analysis of zonadhesin, GST, CYP450, and CarE in the D. abietella midgut to determine their possible associations with Bt 2913. On the basis of the results of this analysis, we speculated that trypsin and other serine proteases in the D. abietella larval midgut began to activate Cry/Vip prototoxin at 6 h to 12 h after Bt 2913 ingestion. At 12 h after Bt 2913 ingestion, chymotrypsin was potentially involved in degrading the active core fragment of Vip3Aa toxin, and the detoxification enzymes in the larvae contributed to the metabolic detoxification of the Bt toxin. The ABC transporter and several other receptor-protein-related genes were also downregulated to increase resistance to Bt 2913. However, the upregulation of 60S ribosomal protein and heat shock protein expression weakened the resistance of larvae to Bt 2913, thereby enhancing the expression of NADH dehydrogenase and other receptor proteins that are highly expressed in the larval midgut and bind to activating toxins, including Cry1Ac. At 24 h after Bt 2913 ingestion, many activated toxins were bound to receptor proteins such as APN in the larval midgut, resulting in membrane perforation. Here, we clarified the mechanism of Bt 2913 infection in D. abietella larvae, as well as the larval immune defense response to Bt 2913, which provides a theoretical basis for the subsequent control of D. abietella using B. thuringiensis.

α-Hemolysin from Staphylococcus aureus Changes the Epigenetic Landscape of Th17 Cells.

The human body harbors a substantial population of bacteria, which may outnumber host cells. Thus, there are multiple interactions between both cell types. Given the common presence of Staphylococcus aureus in the human body and the role of Th17 cells in controlling this pathogen on mucous membranes, we sought to investigate the effect of α-hemolysin, which is produced by this bacterium, on differentiating Th17 cells. RNA sequencing analysis revealed that α-hemolysin influences the expression of signature genes for Th17 cells as well as genes involved in epigenetic regulation. We observed alterations in various histone marks and genome methylation levels via whole-genome bisulfite sequencing. Our findings underscore how bacterial proteins can significantly influence the transcriptome, epigenome, and phenotype of human Th17 cells, highlighting the intricate and complex nature of the interaction between immune cells and the microbiota.

YjbH contributes to Staphylococcus aureus skin pathology and immune response through Agr-mediated α-toxin regulation.

Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs) with Methicillin-Resistant S. aureus (MRSA) strains being a major contributor in both community and hospital settings. S. aureus relies on metabolic diversity and a large repertoire of virulence factors to cause disease. This includes α-hemolysin (Hla), an integral player in tissue damage found in various models, including SSTIs. Previously, we identified a role for the Spx adapter protein, YjbH, in the regulation of several virulence factors and as an inhibitor of pathogenesis in a sepsis model. In this study, we found that YjbH is critical for tissue damage during SSTI, and its absence leads to decreased proinflammatory chemokines and cytokines in the skin. We identified no contribution of YjbI, encoded on the same transcript as YjbH. Using a combination of reporters and quantitative hemolysis assays, we demonstrated that YjbH impacts Hla expression and activity both in vitro and in vivo. Additionally, expression of Hla from a non-native promoter reversed the tissue damage phenotype of the ΔyjbIH mutant. Lastly, we identified reduced Agr activity as the likely cause for reduced Hla production in the ΔyjbH mutant. This work continues to define the importance of YjbH in the pathogenesis of S. aureus infection as well as identify a new pathway important for Hla production.

The role of male hormones in bacterial infections: enhancing Staphylococcus aureus virulence through testosterone-induced Agr activation.

Staphylococcus aureus is a notorious pathogen predominantly involved in skin and soft tissue infections, exhibiting a distinct innate sex bias. This study explores the influence of testosterone on the virulence of S. aureus and elucidates its underlying mechanisms. Utilizing a skin abscess model in intact and castrated male mice, we assessed the effects of testosterone on S. aureus pathogenicity. Compared to controls, castrated mice showed significantly reduced abscess sizes and decreased bacterial loads, highlighting the role of testosterone in modulating the severity of S. aureus infections. In vitro experiments revealed that testosterone enhances the hemolytic activity, cytotoxicity, and oxidative stress resistance of S. aureus. Real-time quantitative PCR analysis showed a significant upregulation of the genes encoding α-hemolysin (hla) and phenol-soluble modulin (psmα). Importantly, testosterone treatment significantly enhanced the expression of the accessory gene regulator (Agr) quorum-sensing system components (agrC, agrA, agrB, agrD), while the SaeRS system (saeR, saeS, and sbi) exhibited only slight changes. Gene knockout experiments revealed that deletion of agrC, rather than saeRS and agrBD, abolishes the testosterone-induced enhancement of hemolysis and gene expression, underscoring the key role of AgrC. Molecular docking simulations indicated a direct interaction between testosterone and AgrC protein, with a strong binding affinity at the active site residue SER201. This study provides new insights into the mechanistic basis of how testosterone enhances the pathogenicity of S. aureus, potentially contributing to increased male susceptibility to S. aureus infections and offering a targeted approach for therapeutic interventions.

The role of aquaporins in osmotic cell lysis induced by Bacillus thuringiensis Cry1Ac toxin in Helicoverpa armigera.

The insecticidal crystalline (Cry) and vegetative insecticidal (Vip) proteins derived from Bacillus thuringiensis (Bt) are used globally to manage insect pests, including the cotton bollworm, Helicoverpa armigera, one of the world's most damaging agricultural pests. Cry proteins bind to the ATP-binding cassette transporter C2 (ABCC2) receptor on the membrane surface of larval midgut cells, resulting in Cry toxin pores, and ultimately leading to cell swelling and/or lysis. Insect aquaporin (AQP) proteins within the membranes of larval midgut cells are proposed to allow the rapid influx of water into enterocytes following the osmotic imbalance triggered by the formation of Cry toxin pores. Here, we examined the involvement of H. armigera AQPs in Cry1Ac-induced osmotic cell swelling. We identified and characterized eight H. armigera AQPs and demonstrated that five are functional water channel proteins. Three of these (HaDrip1, HaPrip, and HaEglp1) were found to be expressed in the larval midgut. Xenopus laevis oocytes co-expressing the known Cry1Ac receptor HaABCC2 and each of the three HaAQPs displayed abnormal morphology and were lysed following exposure to Cry1Ac, suggesting a rapid influx of water was induced after Cry1Ac pore formation. In contrast, oocytes producing either HaABCC2 or HaAQP alone failed to swell or lyse after treatment with Cry1Ac, implying that both Cry1Ac pore formation and HaAQP function are needed for osmotic cell swelling. However, CRISPR/Cas9-mediated knockout of any one of the three HaAQP genes failed to cause significant changes in susceptibility to the Bt toxins Cry1Ac, Cry2Ab, or Vip3Aa. Our findings suggest that the multiple HaAQPs produced in larval midgut cells compensate for each other in allowing for the rapid influx of water in H. armigera midgut cells following Cry toxin pore formation, and that mutations affecting a single HaAQP are unlikely to confer resistance to Bt proteins.

Proven anti-virulence therapies in combating methicillin- and vancomycin-resistant Staphylococcus aureus infections.

Introduction: Despite years of efforts to develop new antibiotics for eradicating multidrug-resistant (MDR) and multi-virulent Methicillin-Resistant Staphylococcus aureus (MRSA) and Vancomycin-Resistant Staphylococcus aureus (VRSA) infections, treatment failures and poor prognoses in most cases have been common. Therefore, there is an urgent need for new therapeutic approaches targeting virulence arrays. Our aim is to discover new anti-virulence therapies targeting MRSA and VRSA virulence arrays. Methodology: We employed phenotypic, molecular docking, and genetic studies to screen for anti-virulence activities among selected promising compounds: Coumarin, Simvastatin, and Ibuprofen. Results: We found that nearly all detected MRSA and VRSA strains exhibited MDR and multi-virulent profiles. The molecular docking results aligned with the phenotypic and genetic assessments of virulence production. Biofilm and hemolysin productions were inhibited, and all virulence genes were downregulated upon treatment with sub-minimum inhibitory concentration (sub-MIC) of these promising compounds. Ibuprofen was the most active compound, exhibiting the highest inhibition and downregulation of virulence gene products. Moreover, in vivo and histopathological studies confirmed these results. Interestingly, we observed a significant decrease in wound area and improvements in re-epithelialization and tissue organization in the Ibuprofen and antimicrobial treated group compared with the group treated with antimicrobial alone. These findings support the idea that a combination of Ibuprofen and antimicrobial drugs may offer a promising new therapy for MRSA and VRSA infections. Conclusion: We hope that our findings can be implemented in clinical practice to assist physicians in making the most suitable treatment decisions.

Entangling roles of cholesterol-dependent interaction and cholesterol-mediated lipid phase heterogeneity in regulating listeriolysin O pore-formation.

Cholesterol-dependent cytolysins (CDCs) are the distinct class of ß-barrel pore-forming toxins (ß-PFTs) that attack eukaryotic cell membranes, and form large, oligomeric, transmembrane ß-barrel pores. Listeriolysin O (LLO) is a prominent member in the CDC family. As documented for the other CDCs, membrane cholesterol is essential for the pore-forming functionality of LLO. However, it remains obscure how exactly cholesterol facilitates its pore formation. Here, we show that cholesterol promotes both membrane-binding and oligomerization of LLO. We demonstrate cholesterol not only facilitates membrane-binding, it also enhances the saturation threshold of LLO-membrane association, and alteration of the cholesterol-recognition motif in the LLO mutant (LLOT515G-L516G) compromises its pore-forming efficacy. Interestingly, such defect of LLOT515G-L516G could be rescued in the presence of higher membrane cholesterol levels, suggesting cholesterol can augment the pore-forming efficacy of LLO even in the absence of a direct toxin-cholesterol interaction. Furthermore, we find the membrane-binding and pore-forming abilities of LLOT515G-L516G, but not those of LLO, correlate with the cholesterol-dependent rigidity/ordering of the membrane lipid bilayer. Our data further suggest that the line tension derived from the lipid phase heterogeneity of the cholesterol-containing membranes could play a pivotal role in LLO function, particularly in the absence of cholesterol binding. Therefore, in addition to its receptor-like role, we conclude cholesterol can further facilitate the pore-forming, membrane-damaging functionality of LLO by asserting the optimal physicochemical environment in membranes. To the best of our knowledge, this aspect of the cholesterol-mediated regulation of the CDC mode of action has not been appreciated thus far.

Downregulation of APN1 and ABCC2 mutation in Bt Cry1Ac-resistant Trichoplusia ni are genetically independent.

The resistance to the insecticidal protein Cry1Ac from the bacterium Bacillus thuringiensis (Bt) in the cabbage looper, Trichoplusia ni, has previously been identified to be associated with a frameshift mutation in the ABC transporter ABCC2 gene and with altered expression of the aminopeptidase N (APN) genes APN1 and APN6, shown as missing of the 110-kDa APN1 (phenotype APN1¯) in larval midgut brush border membrane vesicles (BBMV). In this study, genetic linkage analysis identified that the APN1¯ phenotype and the ABCC2 mutation in Cry1Ac-resistant T. ni segregated independently, although they were always associated under Cry1Ac selection. The ABCC2 mutation and APN1¯ phenotype were separated into two T. ni strains respectively. Bioassays of the T. ni strains with Cry1Ac determined that the T. ni with the APN1¯ phenotype showed a low level resistance to Cry1Ac (3.5-fold), and the associated resistance is incompletely dominant in the background of the ABCC2 mutation. Whereas the ABCC2 mutation-associated resistance to Cry1Ac is at a moderate level, and the resistance is incompletely recessive in the genetic background of downregulated APN1. Analysis of Cry1Ac binding to larval midgut BBMV indicated that the midgut in larvae with the APN1¯ phenotype had reduced binding affinity for Cry1Ac, but the number of binding sites remained unchanged, and the midgut in larvae with the ABCC2 mutation had both reduced binding affinity and reduced number of binding sites for Cry1Ac. The reduced Cry1Ac binding to BBMV from larvae with the ABCC2 mutation or APN1¯ phenotype correlated with the lower levels of resistance.IMPORTANCEThe soil bacterium Bacillus thuringiensis (Bt) is an important insect pathogen used as a bioinsecticide for pest control. Bt genes coding for insecticidal proteins are the primary transgenes engineered into transgenic crops (Bt crops) to confer insect resistance. However, the evolution of resistance to Bt proteins in insect populations in response to exposure to Bt threatens the sustainable application of Bt biotechnology. Cry1Ac is a major insecticidal toxin utilized for insect control. Genetic mechanisms of insect resistance to Cry1Ac are complex and require to be better understood. The resistance to Cry1Ac in Trichoplusia ni is associated with a mutation in the ABCC2 gene and also associated with the APN expression phenotype APN1¯. This study identified the genetic independence of the APN1¯ phenotype from the ABCC2 mutation and isolated and analyzed the ABCC2 mutation-associated and APN1¯ phenotype-associated resistance traits in T. ni to provide new insights into the genetic mechanisms of Cry1Ac resistance in insects.

In vitro evolution driven by epistasis reveals alternative cholesterol-specific binding motifs of perfringolysin O.

The crucial molecular factors that shape the interfaces of lipid-binding proteins with their target ligands and surfaces remain unknown due to the complex makeup of biological membranes. Cholesterol, the major modulator of bilayer structure in mammalian cell membranes, is recognized by various proteins, including the well-studied cholesterol-dependent cytolysins. Here, we use in vitro evolution to investigate the molecular adaptations that preserve the cholesterol specificity of perfringolysin O, the prototypical cholesterol-dependent cytolysin from Clostridium perfringens. We identify variants with altered membrane-binding interfaces whose cholesterol-specific activity exceeds that of the wild-type perfringolysin O. These novel variants represent alternative evolutionary outcomes and have mutations at conserved positions that can only accumulate when epistatic constraints are alleviated. Our results improve the current understanding of the biochemical malleability of the surface of a lipid-binding protein.

Efficient control of root-knot nematodes by expressing Bt nematicidal proteins in root leucoplasts.

Root-knot nematodes (RKNs) are plant pests that infect the roots of host plants. Bacillus thuringiensis (Bt) nematicidal proteins exhibited toxicity to nematodes. However, the application of nematicidal proteins for plant protection is hampered by the lack of effective delivery systems in transgenic plants. In this study, we discovered the accumulation of leucoplasts (root plastids) in galls and RKN-induced giant cells. RKN infection causes the degradation of leucoplasts into small vesicle-like structures, which are responsible for delivering proteins to RKNs, as observed through confocal microscopy and immunoelectron microscopy. We showed that different-sized proteins from leucoplasts could be taken up by Meloidogyne incognita female. To further explore the potential applications of leucoplasts, we introduced the Bt crystal protein Cry5Ba2 into tobacco and tomato leucoplasts by fusing it with a transit peptide. The transgenic plants showed significant resistance to RKNs. Intriguingly, RKN females preferentially took up Cry5Ba2 protein when delivered through plastids rather than the cytosol. The decrease in progeny was positively correlated with the delivery efficiency of the nematicidal protein. In conclusion, this study offers new insights into the feeding behavior of RKNs and their ability to ingest leucoplast proteins, and demonstrates that root leucoplasts can be used for delivering nematicidal proteins, thereby offering a promising approach for nematode control.

Immunolocalization and Ultrastructure Show Ingestion of Cry Protein Expressed in Glycine max by Heterodera glycines and Its Mode of Action.

Great interest exists in developing a transgenic trait that controls the economically important soybean (Glycine max) pest, soybean cyst nematode (SCN, Heterodera glycines), due to its adaptation to native resistance. Soybean plants expressing the Bacillus thuringiensis delta-endotoxin, Cry14Ab, were recently demonstrated to control SCN in both growth chamber and field testing. In that communication, ingestion of the Cry14Ab toxin by SCN second stage juveniles (J2s) was demonstrated using fluorescently labeled Cry14Ab in an in vitro assay. Here, we show that consistent with expectations for a Cry toxin, Cry14Ab has a mode of action unique from the native resistance sources Peking and PI 88788. Further, we demonstrate in planta the ingestion and localization of the Cry14Ab toxin in the midgut of nematodes feeding on roots expressing Cry14Ab using immunogold labeling and transmission electron microscopy. We observed immunolocalization of the toxin and resulting intestinal damage primarily in the microvillus-like structure (MvL)-containing region of the midgut intestine but not in nematodes feeding on roots lacking toxin. This demonstrated that Cry14Ab was taken up by the J2 SCN, presumably through the feeding tube within the plant root cell that serves as its feeding site. This suggests that relatively large proteins can be taken up through the feeding tube. Electron microscopy showed that Cry14Ab caused lysis of the midgut MvL membrane and eventual degradation of the MvL and the lysate, forming particulate aggregates. The accumulated electron-dense aggregate in the posterior midgut intestine was not observed in SCN in nonCry14Ab-expressing plants. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Low temperature exposure influences nitrogen metabolism resulting in decreased Cry1Ac insecticidal endotoxin content in cotton seeds.

BACKGROUND: Sudden temperature drops, resulting from extreme weather events, often occur during the boll-setting period of cotton in Xinjiang, China, causing decreased expression of Bacillus thuringiensis (Bt) insecticidal proteins in cotton bolls. The precise threshold temperatures and durations that lead to significant changes in Cry1Ac endotoxin levels under low temperatures remain unclear. To address this, we investigated the effects of different temperatures and stress durations on Cry1Ac endotoxin levels in cotton bolls. In 2020-2021, two Bt transgenic cotton varieties, conventional Sikang1 and hybrid Sikang3, were selected as experimental materials. Various low temperatures (ranging from 16 to 20 °C) with different durations (12 h, 24 h and 48 h) were applied during the peak boll-setting period. RESULTS: As the temperature decreased, the Cry1Ac endotoxin content in the boll shell, fiber, and seed exhibited a declining trend. Moreover, the threshold temperature which caused a significant reduction in Cry1Ac endotoxin content increased with the prolonged duration of low-temperature stress. Among the components of cotton bolls, seeds were most affected by low-temperature stress, with the threshold temperature for a significant reduction in Cry1Ac endotoxin content ranging from 17 °C to 19 °C. Correlation analysis indicated that low temperatures led to a decrease in protein synthesis capacity and an increase in degradation ability, resulting in reduced Cry1Ac endotoxin content. Pathway analysis revealed that both free amino acid and peptidase had significant negative effects on Cry1Ac endotoxin content. CONCLUSION: In summary, when the daily average temperature was ≤ 19 °C, implementing cultural practices to reduce free amino acid content and peptidase activity could serve as effective cold defense strategies for Bt cotton production.

Death-Associated LIM-Only Protein Reduces Cry1Ac Toxicity by Sequestration of Cry1Ac Protoxin and Activated Toxin in Helicoverpa armigera.

The extensive use of Bacillus thuringiensis (Bt) in pest management has driven the evolution of pest resistance to Bt toxins, particularly Cry1Ac. Effective management of Bt resistance necessitates a good understanding of which pest proteins interact with Bt toxins. In this study, we screened a Helicoverpa armigera larval midgut cDNA library and captured 208 potential Cry1Ac-interacting proteins. Among these, we further examined the interaction between Cry1Ac and a previously unknown Cry1Ac-interacting protein, HaDALP (H. armigera death-associated LIM-only protein), as well as its role in toxicology. The results revealed that HaDALP specifically binds to both the Cry1Ac protoxin and activated toxin, significantly enhancing cell and larval tolerance to Cry1Ac. Additionally, HaDALP was overexpressed in a Cry1Ac-resistant H. armigera strain. These findings reveal a greater number of Cry1Ac-interacting proteins than previously known and demonstrate, for the first time, that HaDALP reduces Cry1Ac toxicity by sequestering both the protoxin and activated toxin.

The presence of differentiated C2C12 muscle cells enhances toxin production and growth by Clostridium perfringens type A strain ATCC3624.

Clostridium perfringens type A causes gas gangrene, which involves muscle infection. Both alpha toxin (PLC), encoded by the plc gene, and perfringolysin O (PFO), encoded by the pfoA gene, are important when type A strains cause gas gangrene in a mouse model. This study used the differentiated C2C12 muscle cell line to test the hypothesis that one or both of those toxins contributes to gas gangrene pathogenesis by releasing growth nutrients from muscle cells. RT-qPCR analyses showed that the presence of differentiated C2C12 cells induces C. perfringens type A strain ATCC3624 to upregulate plc and pfoA expression, as well as increase expression of several regulatory genes, including virS/R, agrB/D, and eutV/W. The VirS/R two component regulatory system (TCRS) and its coupled Agr-like quorum sensing system, along with the EutV/W TCRS (which regulates expression of genes involved in ethanolamine [EA] utilization), were shown to mediate the C2C12 cell-induced increase in plc and pfoA expression. EA was demonstrated to increase toxin gene expression. ATCC3624 growth increased in the presence of differentiated C2C12 muscle cells and this effect was shown to involve both PFO and PLC. Those membrane-active toxins were each cytotoxic for differentiated C2C12 cells, suggesting they support ATCC3624 growth by releasing nutrients from differentiated C2C12 cells. These findings support a model where, during gas gangrene, increased production of PFO and PLC in the presence of muscle cells causes more damage to those host cells, which release nutrients like EA that are then used to support C. perfringens growth in muscle.

Enterococcus faecalis-derived adenine enhances enterohaemorrhagic Escherichia coli Type 3 Secretion System-dependent virulence.

Interactions between microbiota and enteric pathogens can promote colonization resistance or enhance pathogenesis. The pathobiont Enterococcus faecalis increases enterohaemorrhagic E. coli (EHEC) virulence by upregulating Type 3 Secretion System (T3SS) expression, effector translocation, and attaching and effacing (AE) lesion formation on enterocytes, but the mechanisms underlying this remain unknown. Using co-infection of organoids, metabolomics, supplementation experiments and bacterial genetics, here we show that co-culture of EHEC with E. faecalis increases the xanthine-hypoxanthine pathway activity and adenine biosynthesis. Adenine or E. faecalis promoted T3SS gene expression, while transcriptomics showed upregulation of adeP expression, which encodes an adenine importer. Mechanistically, adenine relieved High hemolysin activity (Hha)-dependent repression of T3SS gene expression in EHEC and promoted AE lesion formation in an AdeP-dependent manner. Microbiota-derived purines, such as adenine, support multiple beneficial host responses; however, our data show that this metabolite also increases EHEC virulence, highlighting the complexity of pathogen-microbiota-host interactions in the gut.