RESUMO
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.
Assuntos
Toxinas de Bacillus thuringiensis , Proteínas de Bactérias , Temperatura Baixa , Endotoxinas , Gossypium , Proteínas Hemolisinas , Nitrogênio , Sementes , Gossypium/genética , Gossypium/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Sementes/metabolismo , Nitrogênio/metabolismo , Plantas Geneticamente Modificadas , Bacillus thuringiensisRESUMO
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.
Assuntos
Toxinas de Bacillus thuringiensis , Proteínas de Bactérias , Endotoxinas , Proteínas Hemolisinas , Proteínas de Insetos , Inseticidas , Larva , Mariposas , Animais , Toxinas de Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis/toxicidade , Toxinas de Bacillus thuringiensis/química , Endotoxinas/metabolismo , Endotoxinas/genética , Endotoxinas/toxicidade , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacologia , Proteínas Hemolisinas/toxicidade , Proteínas Hemolisinas/genética , Mariposas/metabolismo , Mariposas/efeitos dos fármacos , Mariposas/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/toxicidade , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Larva/metabolismo , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/genética , Inseticidas/toxicidade , Inseticidas/farmacologia , Inseticidas/química , Bacillus thuringiensis/química , Bacillus thuringiensis/metabolismo , Bacillus thuringiensis/genética , Resistência a Inseticidas/genética , Controle Biológico de Vetores , Helicoverpa armigeraRESUMO
Staphylococcus aureus (S. aureus) is an opportunistic infectious pathogen, which causes a high mortality rate during bloodstream infections. The early detection of virulent strains in patients' blood samples is of medical interest for rapid diagnosis. The main virulent factors identified in patient isolates include leukocidins that bind to specific membrane receptors and lyse immune cells and erythrocytes. Duffy antigen receptor for chemokines (DARC) on the surface of specific cells is a main target of leukocidins such as gamma-hemolysin AB (HlgAB) and leukocidin ED (LukED). Among them, HlgAB is a conserved and critical leukocidin that binds to DARC and forms pores on the cell membranes, leading to cell lysis. Current methods are based on ELISA or bacterial culture, which takes hours to days. For detecting HlgAB with faster response and higher sensitivity, we developed a biosensor that combines single-walled carbon nanotube field effect transistors (swCNT-FETs) with immobilized DARC receptors as biosensing elements. DARC was purified from a bacterial expression system and successfully reconstituted into nanodiscs that preserve binding capability for HlgAB. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) showed an increase of the DARC-containing nanodisc size in the presence of HlgAB, indicating the formation of HlgAB prepore or pore complexes. We demonstrate that this sensor can specifically detect the leukocidins HlgA and HlgAB in a quantitative manner within the dynamic range of 1 fM to 100 pM with an LOD of 0.122 fM and an LOQ of 0.441 fM. The sensor was challenged with human serum spiked with HlgAB as simulated clinical samples. After dilution for decreasing nonspecific binding, it selectively detected the toxin with a similar detection range and apparent dissociation constant as in the buffer. This biosensor was demonstrated with remarkable sensitivity to detect HlgAB rapidly and has the potential as a tool for fundamental research and clinical applications, although this sensor cannot differentiate between HlgAB and LukED as both have the same receptor.
Assuntos
Técnicas Biossensoriais , Sistema do Grupo Sanguíneo Duffy , Leucocidinas , Staphylococcus aureus , Técnicas Biossensoriais/métodos , Sistema do Grupo Sanguíneo Duffy/química , Sistema do Grupo Sanguíneo Duffy/metabolismo , Leucocidinas/química , Leucocidinas/metabolismo , Humanos , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/química , Nanotubos de Carbono/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismoRESUMO
Cry toxins, produced by the bacterium Bacillus thuringiensis, are of significant agronomic value worldwide due to their potent and highly specific activity against various insect orders. However, some of these pore-forming toxins display specific activity against a range of human cancer cells whilst possessing no known insecticidal activity; Cry41Aa is one such toxin. Cry41Aa has similarities to its insecticidal counterparts in both its 3-domain toxic core structure and pore-forming abilities, but how it has evolved to target human cells is a mystery. This work shows that some insecticidal Cry toxins can enhance the toxicity of Cry41Aa against hepatocellular carcinoma cells, despite possessing no intrinsic toxicity themselves. This interesting crossover is not limited to human cancer cells, as Cry41Aa was found to inhibit some Aedes-active Cry toxins in mosquito larval assays. Here, we present findings that suggest that Cry41Aa shares a receptor with several insecticidal toxins, indicating a stronger evolutionary relationship than their divergent activities might suggest.
Assuntos
Toxinas de Bacillus thuringiensis , Bacillus thuringiensis , Proteínas de Bactérias , Endotoxinas , Proteínas Hemolisinas , Humanos , Endotoxinas/química , Endotoxinas/genética , Endotoxinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacologia , Animais , Inseticidas/química , Inseticidas/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Aedes/efeitos dos fármacos , Aedes/genética , Linhagem Celular TumoralRESUMO
Clostridium perfringens (C. perfringens), a Gram-positive bacterium, produces a variety of toxins and extracellular enzymes that can lead to disease in both humans and animals. Common symptoms include abdominal swelling, diarrhea, and intestinal inflammation. Severe cases can result in complications like intestinal hemorrhage, edema, and even death. The primary toxins contributing to morbidity in C. perfringens-infected intestines are CPA, CPB, CPB2, CPE, and PFO. Amongst these, CPB, CPB2, and CPE are implicated in apoptosis development, while CPA is associated with cell death, increased intracellular ROS levels, and the release of the inflammatory factor IL-18. However, the exact mechanism by which PFO toxins exert their effects in the infected gut is still unidentified. This study demonstrates that a C. perfringens PFO toxin infection disrupts the intestinal epithelial barrier function through in vitro and in vivo models. This study emphasizes the notable influence of PFO toxins on intestinal barrier integrity in the context of C. perfringens infections. It reveals that PFO toxins increase ROS production by causing mitochondrial damage, triggering pyroptosis in IPEC-J2 cells, and consequently resulting in compromised intestinal barrier function. These results offer a scientific foundation for developing preventive and therapeutic approaches against C. perfringens infections.
Assuntos
Toxinas Bacterianas , Clostridium perfringens , Células Epiteliais , Proteínas Hemolisinas , Mucosa Intestinal , Piroptose , Espécies Reativas de Oxigênio , Clostridium perfringens/patogenicidade , Toxinas Bacterianas/toxicidade , Toxinas Bacterianas/metabolismo , Piroptose/efeitos dos fármacos , Animais , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/toxicidade , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular , Camundongos , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacosRESUMO
Streptococcus suis type 2 (SS2) is a zoonotic pathogen capable of eliciting meningitis, presenting significant challenges to both the swine industry and public health. Suilysin (Sly), one of SS2 most potent virulence determinants, releases a surfeit of inflammatory agents following red blood cell lysis. Notably, while current research on Sly role in SS2-induced meningitis predominantly centers on its interaction with the blood-brain barrier (BBB), the repercussions of Sly hemolytic products on BBB function have largely been sidestepped. In this vein, our study delves into the ramifications of Sly-induced hemolysis on BBB integrity. We discern that Sly hemolytic derivatives exacerbate the permeability of Sly-induced in vitro BBB models. Within these Sly hemolytic products, Interleukin-33 (IL-33) disrupts the expression and distribution of Claudin-5 in brain microvascular endothelial cells, facilitating the release of Interleukin-6 (IL-6) and Interleukin-8 (IL-8), thereby amplifying BBB permeability. Preliminary mechanistic insights suggest that IL-33-driven expression of IL-6 and IL-8 is orchestrated by the p38-mitogen-activated protein kinase signaling, whereas matrix metalloproteinase 9 mediates IL-33-induced suppression of Claudin-5. To validate these in vitro findings, an SS2-infected mouse model was established, and upon intravenous administration of growth stimulation expressed gene 2 (ST2) antibodies, in vivo results further underscored the pivotal role of the IL-33/ST2 axis during SS2 cerebral invasion. In summation, this study pioneerly illuminates the involvement of Sly hemolytic products in SS2-mediated BBB compromise and spotlights the instrumental role and primary mechanism of IL-33 therein. These insights enrich our comprehension of SS2 meningitis pathogenesis, laying pivotal groundwork for therapeutic advancements against SS2-induced meningitis.IMPORTANCEThe treatment of meningitis caused by Streptococcus suis type 2 (SS2) has always been a clinical challenge. Elucidating the molecular mechanisms by which SS2 breaches the blood-brain barrier (BBB) is crucial for the development of meningitis therapeutics. Suilysin (Sly) is one of the most important virulence factors of SS2, which can quickly lyse red blood cells and release large amounts of damage-associated molecular patterns, such as hemoglobin, IL-33, cyclophilin A, and so on. However, the impact of these hemolytic products on the function of BBB is unknown and ignored. This study is the first to investigate the effect of Sly hemolytic products on BBB function. The data are crucial for the study of the pathogenesis of SS2 meningitis and can provide an important reference for the development of meningitis therapeutics.
Assuntos
Barreira Hematoencefálica , Células Endoteliais , Proteínas Hemolisinas , Hemólise , Interleucina-33 , Streptococcus suis , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/microbiologia , Animais , Camundongos , Interleucina-33/metabolismo , Humanos , Proteínas Hemolisinas/metabolismo , Streptococcus suis/patogenicidade , Células Endoteliais/microbiologia , Células Endoteliais/metabolismo , Infecções Estreptocócicas/microbiologia , Interleucina-6/metabolismo , Interleucina-6/genética , Interleucina-8/metabolismo , Suínos , Metaloproteinase 9 da Matriz/metabolismoRESUMO
The increasing prevalence of infections related to methicillin-resistant Staphylococcus aureus (MRSA) necessitates the exploration of innovative therapeutic strategies that diverge from conventional antibiotic treatments. This is imperative to effectively combat resistance and manage these infections. The adoption of antivirulence strategies has emerged as a particularly promising avenue. This approach applies a heightened selective pressure on pathogens, thereby diminishing the likelihood of bacteria evolving resistance to antibiotics. In our pursuit of novel therapeutics for treating MRSA infections, we have focused on agents that inhibit the virulence of S. aureus without impeding its growth, aiming to minimize the development of drug resistance. α-Hemolysin, a critical virulence factor encoded by the hla gene, is a cytotoxin that forms pores in host cell membranes and plays a pivotal role in the progression of disease during bacterial infections. Herein, we identified that norwogonin could effectively inhibit Hla production via targeting agrAC, a crucial protein in quorum sensing, resulting in dose-dependent inhibition of hemolytic activity without suppressing S. aureus growth. In vitro assays illustrated that norwogonin decreased the thermal stability of agrAC, providing evidence of interaction between norwogonin and agrAC. Meanwhile, norwogonin alleviated Hla-mediated A549 cell damage and reduced lactate dehydrogenase release. In vivo studies suggested that norwogonin treatment blocked the establishment of a mouse model of pneumonia caused by S. aureus USA300. Notably, norwogonin enhanced the antibacterial potency of oxacillin. In conclusion, norwogonin is a promising candidate for treating S. aureus infections, offering a novel alternative to traditional antibiotics by targeting virulence factors and enhancing the efficacy of existing treatments.
Assuntos
Antibacterianos , Proteínas de Bactérias , Proteínas Hemolisinas , Staphylococcus aureus Resistente à Meticilina , Fatores de Virulência , Animais , Feminino , Humanos , Camundongos , Células A549 , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Toxinas Bacterianas/metabolismo , Modelos Animais de Doenças , Proteínas Hemolisinas/metabolismo , Hemólise/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Percepção de Quorum/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Virulência/efeitos dos fármacos , Fatores de Virulência/metabolismoRESUMO
Staphylococcus aureus is a gram-positive bacteria, and its virulence factors can cause many kinds of infections, such as pneumonia, sepsis, enteritis and osteomyelitis. Traditional antibiotics can not only kill bacteria, but also easily lead to bacterial resistance. Jingfang Mixture (JFM) has the effects of inducing sweating and relieving the exterior, dispelling wind and eliminating dampness, and is commonly used in clinic to prevent and treat epidemic diseases and infectious diseases. The main purpose of this study is to explore the inhibitory effect of JFM on alpha-hemolysin (Hla) of S. aureus and to alleviate the damage caused by Hla. We found that JFM could inhibit the hemolytic activity, transcription level and neutralizing activity of Hla in a dose-dependent manner at the concentrations of 125, 250 and 500 µg/mL, without affecting the growth of bacteria. In addition, JFM reduced the damage of Hla to A549 cells and the release of lactate dehydrogenase (LDH). We also observed that in the S. aureus - induced pneumonia mouse model, JFM could significantly prolong the life of mice, reduce the bacterial load in the lungs, significantly improve the pathological state of the lungs and alleviate the damage caused by inflammatory factors, and the pathogenicity of gene deletion strain DU 1090 of S. aureus to pneumonia mice was also significantly reduced. In conclusion, this study proved that JFM is a potential drug against S. aureus infection, and this study provided a preliminary study for better guidance of clinical drug use.
Assuntos
Antibacterianos , Proteínas Hemolisinas , Infecções Estafilocócicas , Staphylococcus aureus , Animais , Feminino , Humanos , Camundongos , Células A549 , Antibacterianos/farmacologia , Carga Bacteriana/efeitos dos fármacos , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/farmacologia , Proteínas Hemolisinas/metabolismo , Hemólise/efeitos dos fármacos , Pulmão/microbiologia , Pulmão/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Pneumonia Estafilocócica/tratamento farmacológico , Pneumonia Estafilocócica/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Fatores de Virulência/genéticaRESUMO
Protein nanopores have proven to be effective for single-molecule studies, particularly for single-stranded DNA (ssDNA) translocation. Previous experiments demonstrated their ability to distinguish differences in purine and pyrimidine bases and in the orientation of the ssDNA molecule inside nanopores. Unfortunately, the microscopic details of ssDNA translocation over experimental time scales, which are not accessible through all-atom molecular dynamics (MD), have yet to be examined. However, coarse-grained (CG) MD simulations enable systems to be simulated over longer characteristic times closer to experiments than all-atom MD. This paper studies ssDNA translocation through α-hemolysin nanopores exploiting steered MD using the MARTINI CG force field. The impacts of the sequence length, orientation inside the nanopore and DNA charges on translocation dynamics as well as the conformational dynamics of ssDNA during the translocation are explored. Our results highlight the efficacy of CG molecular dynamics in capturing the experimental properties of ssDNA translocation, including a wide distribution in translocation times per base. In particular, the phosphate charges of the DNA molecule are crucial in the translocation dynamics and impact the translocation rate. Additionally, the influence of the ssDNA molecule orientation on the translocation rate is explained by the conformational differences of ssDNA inside the nanopore during its translocation. Our study emphasizes the significance of obtaining sufficient statistics via CG MD, which can elucidate the great variety of translocation processes.
Assuntos
DNA de Cadeia Simples , Proteínas Hemolisinas , Simulação de Dinâmica Molecular , Nanoporos , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Conformação de Ácido NucleicoRESUMO
Vibrio cholerae (V. cholerae), the etiological agent of cholera, employs various virulence factors to adapt and thrive within both aquatic and human host environments. Among these factors, the type VI secretion system (T6SS) stands out as one of the crucial determinants of its pathogenicity. Valine glycine repeat protein G1 (VgrG1) and hemolysin coregulated protein (HCP) are considered major effector molecules of T6SS. Previous studies have highlighted that VgrG1 interacts with HCP proteins. Additionally, it has been shown that VgrG1 possesses an actin cross-linking domain (ACD) with actin-binding activity. Interestingly, it was reported that purified HCP protein treatment increased the stress fibers within cells. Therefore, we hypothesize that HCP may interact with host cell actin, potentially playing a role in the cytoskeletal rearrangement during V. cholerae infection. To test this hypothesis, we characterized HCP from the V. cholerae O139 serotype and demonstrated its interaction with actin monomers. In silico analysis and experimental validation revealed the presence of an actin-binding site within HCP. Furthermore, overexpression of HCP resulted in its colocalization with actin stress fibers in host cells. Our findings establish HCP as an effector molecule for potent host cell actin cytoskeleton remodeling during V. cholerae infection, providing new insights into bacterial pathogenicity mechanisms. Understanding the interplay between bacterial effectors and host cell components is crucial for developing targeted therapeutic interventions against cholera and related infectious diseases.
Assuntos
Citoesqueleto de Actina , Proteínas de Bactérias , Vibrio cholerae , Vibrio cholerae/patogenicidade , Vibrio cholerae/metabolismo , Vibrio cholerae/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Humanos , Citoesqueleto de Actina/metabolismo , Interações Hospedeiro-Patógeno , Ligação Proteica , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Actinas/metabolismo , Cólera/microbiologia , Proteínas Hemolisinas/metabolismoRESUMO
Staphylococcus aureus α-hemolysin (Hla) is a pore-forming toxin critical for the pathogenesis of skin and soft tissue infections, which causes the pathognomonic lesion of cutaneous necrosis (dermonecrosis) in mouse models. To determine the mechanism by which dermonecrosis develops during S. aureus skin infection, mice were given control serum, Hla-neutralizing antiserum, or an inhibitor of Hla receptor [A-disintegrin and metalloprotease 10 (ADAM10) inhibitor] followed by subcutaneous infection by S. aureus, and the lesions were evaluated using immunohistochemistry and immunofluorescence. Hla induced apoptosis in the vascular endothelium at 6 hours post-infection (hpi), followed by apoptosis in keratinocytes at 24 hpi. The loss of vascular endothelial (VE)-cadherin expression preceded the loss of epithelial-cadherin expression. Hla also induced hypoxia in the keratinocytes at 24 hpi following vascular injury. Treatment with Hla-neutralizing antibody or ADAM10 inhibitor attenuated early cleavage of VE-cadherin, cutaneous hypoxia, and dermonecrosis. These findings suggest that Hla-mediated vascular injury with cutaneous hypoxia underlies the pathogenesis of S. aureus-induced dermonecrosis.
Assuntos
Proteína ADAM10 , Toxinas Bacterianas , Caderinas , Proteínas Hemolisinas , Queratinócitos , Necrose , Staphylococcus aureus , Animais , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/toxicidade , Camundongos , Toxinas Bacterianas/toxicidade , Toxinas Bacterianas/metabolismo , Staphylococcus aureus/patogenicidade , Queratinócitos/microbiologia , Queratinócitos/metabolismo , Proteína ADAM10/metabolismo , Caderinas/metabolismo , Apoptose , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Antígenos CD/metabolismo , Proteínas de Membrana/metabolismo , Infecções Cutâneas Estafilocócicas/microbiologia , Infecções Cutâneas Estafilocócicas/patologia , Infecções Cutâneas Estafilocócicas/imunologia , Pele/patologia , Pele/microbiologia , Feminino , Endotélio Vascular/patologia , Endotélio Vascular/microbiologia , Endotélio Vascular/metabolismo , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/patologia , Modelos Animais de DoençasRESUMO
The flagellar MS-ring, uniquely constituted by FliF, is essential for flagellar biogenesis and functionality in several bacteria. The aim of this study was to dissect the role of FliF in the Gram-positive and peritrichously flagellated Bacillus cereus. We demonstrate that fliF forms an operon with the upstream gene fliE. In silico analysis of B. cereus ATCC 14579 FliF identifies functional domains and amino acid residues that are essential for protein functioning. The analysis of a ΔfliF mutant of B. cereus, constructed in this study using an in frame markerless gene replacement method, reveals that the mutant is unexpectedly able to assemble flagella, although in reduced amounts compared to the parental strain. Nevertheless, motility is completely abolished by fliF deletion. FliF deprivation causes the production of submerged biofilms and affects the ability of B. cereus to adhere to gastrointestinal mucins. We additionally show that the fliF deletion does not compromise the secretion of the three components of hemolysin BL, a toxin secreted through the flagellar type III secretion system. Overall, our findings highlight the important role of B. cereus FliF in flagella-related functions, being the protein required for complete flagellation, motility, mucin adhesion, and pellicle biofilms.
Assuntos
Bacillus cereus , Proteínas de Bactérias , Biofilmes , Flagelos , Óperon , Bacillus cereus/metabolismo , Bacillus cereus/genética , Flagelos/metabolismo , Flagelos/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/genética , Aderência Bacteriana , Regulação Bacteriana da Expressão Gênica , Deleção de Genes , Proteínas de MembranaRESUMO
Trueperella pyogenes is an important opportunistic pathogenic bacterium widely distributed in the environment. Pyolysin (PLO) is a primary virulence factor of T. pyogenes and capable of lysing many different cells. PLO is a member of the cholesterol-dependent cytolysin (CDC) family of which the primary structure only presents a low level of homology with other members from 31% to 45%. By deeply studying PLO, we can understand the overall pathogenic mechanism of CDC family proteins. This study established a mouse muscle tissue model infected with recombinant PLO (rPLO) and its single-point mutations, rPLO N139K and rPLO F240A, and explored its mechanism of causing inflammatory damage. The inflammatory injury abilities of rPLO N139K and rPLO F240A are significantly reduced compared to rPLO. This study elaborated on the inflammatory mechanism of PLO by examining its unit point mutations in detail. Our data also provide a theoretical basis and practical significance for future research on toxins and bacteria.
Assuntos
Proteínas de Bactérias , Proteínas Hemolisinas , Proteína 3 que Contém Domínio de Pirina da Família NLR , Mutação Puntual , Animais , Camundongos , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Inflamação/metabolismo , Inflamação/genética , Potássio/metabolismo , Transdução de Sinais , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Inflamassomos/metabolismo , HumanosRESUMO
The G protein-coupled estrogen receptor, also known as GPER1 or originally GPR30, is found in various tissues, indicating its diverse functions. It is typically present in immune cells, suggesting its role in regulating immune responses to infectious diseases. Our previous studies have shown that G-1, a selective GPER agonist, can limit the pathogenesis mediated by Staphylococcus aureus alpha-hemolysin (Hla). It aids in clearing bacteria in a mouse skin infection model and restricts the surface display of the Hla receptor, ADAM10 (a disintegrin and metalloprotease 10) in HaCaT keratinocytes. In this report, we delve into the modulation of GPER in human immune cells in relation to the NLRP3 inflammasome. We used macrophage-like differentiated THP-1 cells for our study. We found that treating these cells with G-1 reduces ATP release, decreases the activity of the caspase-1 enzyme, and lessens cell death following Hla intoxication. This is likely due to the reduced levels of ADAM10 and NLRP3 proteins, as well as the decreased display of the ADAM10 receptor in the G-1-treated THP-1 cells. Our studies, along with our previous work, suggest the potential therapeutic use of G-1 in reducing Hla susceptibility in humans. This highlights the importance of GPER in immune regulation and its potential as a therapeutic target.
Assuntos
Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide , Toxinas Bacterianas , Proteínas Hemolisinas , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Receptores de Estrogênio , Receptores Acoplados a Proteínas G , Staphylococcus aureus , Proteína ADAM10/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Humanos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Hemolisinas/metabolismo , Inflamassomos/metabolismo , Toxinas Bacterianas/metabolismo , Células THP-1 , Receptores de Estrogênio/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/agonistas , Caspase 1/metabolismo , Trifosfato de Adenosina/metabolismo , Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/microbiologia , Dipeptídeos , Ácidos HidroxâmicosRESUMO
The Aedes aegypti cadherin-like protein (Aae-Cad) and the membrane-bound alkaline phosphatase (Aae-mALP) are membrane proteins identified as putative receptors for the larvicidal Cry toxins produced by Bacillus thuringiensis subsp. israelensis bacteria. Cry toxins are the most used toxins in the control of different agricultural pest and mosquitos. Despite the relevance of Aae-Cad and Aae-mALP as possible toxin-receptors in mosquitoes, previous efforts to establish a clear functional connection among them and Cry toxins activity have been relatively limited. In this study, we used CRISPR-Cas9 to generate knockout (KO) mutations of Aae-Cad and Aae-mALP. The Aae-mALP KO was successfully generated, in contrast to the Aae-Cad KO which was obtained only in females. The female-linked genotype was due to the proximity of aae-cad gene to the sex-determining loci (M:m). Both A. aegypti KO mutant populations were viable and their insect-development was not affected, although a tendency on lower egg hatching rate was observed. Bioassays were performed to assess the effects of these KO mutations on the susceptibility of A. aegypti to Cry toxins, showing that the Aae-Cad female KO or Aae-mALP KO mutations did not significantly alter the susceptibility of A. aegypti larvae to the mosquitocidal Cry toxins, including Cry11Aa, Cry11Ba, Cry4Ba, and Cry4Aa. These findings suggest that besides the potential participation of Aae-Cad and Aae-mALP as Cry toxin receptors in A. aegypti, additional midgut membrane proteins are involved in the mode of action of these insecticidal toxins.
Assuntos
Aedes , Fosfatase Alcalina , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias , Sistemas CRISPR-Cas , Caderinas , Endotoxinas , Proteínas Hemolisinas , Animais , Aedes/genética , Aedes/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Endotoxinas/genética , Endotoxinas/metabolismo , Feminino , Caderinas/genética , Caderinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Resistência a Inseticidas/genética , Técnicas de Inativação de Genes , Larva/genética , Larva/crescimento & desenvolvimento , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Masculino , Inseticidas/farmacologiaRESUMO
The global landscape of Candida infections has seen a significant shift. Previously, Candida albicans was the predominant species. However, there has been an emergence of non-albicans Candida species, which are often less susceptible to antifungal treatment. Candida kefyr, in particular, has been increasingly associated with infections. This study aimed to investigate the profiles of enzymatic activity and biofilm formation in both clinical and non-clinical isolates of C. kefyr. A total of 66 C. kefyr isolates were analysed. The activities of proteinase and phospholipase were assessed using bovine serum albumin and egg yolk agar, respectively. Haemolysin, caseinolytic and esterase activities were evaluated using specific methods. Biofilm formation was investigated using crystal violet staining. The findings indicated that biofilm and proteinase activity were detected in 81.8% and 93.9% of all the isolates, respectively. Haemolysin activity was observed with the highest occurrence (95.5%) among normal microbiota isolates. Esterase activity was predominantly identified in dairy samples and was absent in hospital samples. Caseinase production was found with the highest occurrence (18.2%) in normal microbiota and hospital samples. Phospholipase activity was limited, found in only 3% of all the isolates. These findings reveal variations in enzyme activity between clinical and non-clinical C. kefyr isolates. This sheds light on their pathogenic potential and has implications for therapeutic strategies.
Assuntos
Biofilmes , Candida , Candidíase , Fosfolipases , Biofilmes/crescimento & desenvolvimento , Candida/isolamento & purificação , Candida/enzimologia , Candida/fisiologia , Candida/classificação , Humanos , Candidíase/microbiologia , Fosfolipases/metabolismo , Esterases/metabolismo , Proteínas Hemolisinas/metabolismo , Peptídeo Hidrolases/metabolismo , Microbiologia AmbientalRESUMO
Vibrio vulnificus (V. vulnificus) is a halophilic gram-negative bacterium that inhabits coastal warm water and induce severe diseases such as primary septicemia. To investigate the mechanisms of rapid bacterial translocation on intestinal infection, we focused on outer membrane vesicles (OMVs), which are extracellular vesicles produced by Gram-negative bacteria and deliver virulence factors. However, there are very few studies on the pathogenicity or contents of V. vulnificus OMVs (Vv-OMVs). In this study, we investigated the effects of Vv-OMVs on host cells. Epithelial cells INT407 were stimulated with purified OMVs and morphological alterations and levels of lactate dehydrogenase (LDH) release were observed. In cells treated with OMVs, cell detachment without LDH release was observed, which exhibited different characteristics from cytotoxic cell detachment observed in V. vulnificus infection. Interestingly, OMVs from a Vibrio Vulnificus Hemolysin (VVH) and Multifunctional-autoprocessing repeats-in -toxin (MARTX) double-deletion mutant strain also caused cell detachment without LDH release. Our results suggested that the proteolytic function of a serine protease contained in Vv-OMVs may contribute to pathogenicity of V. vulnificus by assisting bacterial translocation. This study reveals a new pathogenic mechanism during V. vulnificus infections. J. Med. Invest. 71 : 102-112, February, 2024.
Assuntos
Vesículas Extracelulares , Vibrio vulnificus , Vibrio vulnificus/patogenicidade , Vibrio vulnificus/metabolismo , Humanos , Vesículas Extracelulares/metabolismo , Proteínas Hemolisinas/metabolismo , L-Lactato Desidrogenase/metabolismo , Membrana Externa Bacteriana/metabolismo , Células Epiteliais/microbiologiaRESUMO
The pathogenicity of many bacteria, including Bacillus cereus and Staphylococcus aureus, depends on pore-forming toxins (PFTs), which cause the lysis of host cells by forming pores in the membranes of eukaryotic cells. Bioinformatic analysis revealed a region homologous to the Lys171-Gly250 sequence in hemolysin II (HlyII) from B. cereus in over 600 PFTs, which we designated as a "homologous peptide". Three ß-barrel PFTs were used for a detailed comparative analysis. Two of them-HlyII and cytotoxin K2 (CytK2)-are synthesized in Bacillus cereus sensu lato; the third, S. aureus α-toxin (Hla), is the most investigated representative of the family. Protein modeling showed certain amino acids of the homologous peptide to be located on the surface of the monomeric forms of these ß-barrel PFTs. We obtained monoclonal antibodies against both a cloned homologous peptide and a 14-membered synthetic peptide, DSFNTFYGNQLFMK, as part of the homologous peptide. The HlyII, CytK2, and Hla regions recognized by the obtained antibodies, as well as an antibody capable of suppressing the hemolytic activity of CytK2, were identified in the course of this work. Antibodies capable of recognizing PFTs of various origins can be useful tools for both identification and suppression of the cytolytic activity of PFTs.
Assuntos
Bacillus cereus , Toxinas Bacterianas , Proteínas Hemolisinas , Staphylococcus aureus , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Bacillus cereus/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Staphylococcus aureus/metabolismo , Sequência de Aminoácidos , Hemólise , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Modelos Moleculares , Animais , Anticorpos Monoclonais/química , Humanos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismoRESUMO
The entomopathogenic fungus Beauveria bassiana provides an eco-friendly substitute to chemical insecticides for mosquito control. Nevertheless, its widespread application has been hindered by its comparatively slow efficacy in eliminating mosquitoes. To augment the potency of B. bassiana against Aedes mosquitoes, a novel recombinant strain, Bb-Cyt1Aa, was developed by incorporating the Bacillus thuringiensis toxin gene Cyt1Aa into B. bassiana. The virulence of Bb-Cyt1Aa was evaluated against Aedes aegypti and Aedes albopictus using insect bioassays. Compared to the wild-type (WT) strain, the median lethal time (LT50) for A. aegypti larvae infected with Bb-Cyt1Aa decreased by 33.3% at a concentration of 1 × 108 conidia/mL and by 22.2% at 1 × 107 conidia/mL. The LT50 for A. aegypti adults infected with Bb-Cyt1Aa through conidia ingestion was reduced by 37.5% at 1 × 108 conidia/mL and by 33.3% at 1 × 107 conidia/mL. Likewise, the LT50 for A. aegypti adults infected with Bb-Cyt1Aa through cuticle contact decreased by 33.3% and 30.8% at the same concentrations, respectively. Furthermore, the Bb-Cyt1Aa strain also demonstrated increased toxicity against both larval and adult A. albopictus, when compared to the WT strain. In conclusion, our study demonstrated that the expression of B. thuringiensis toxin Cyt1Aa in B. bassiana enhanced its virulence against Aedes mosquitoes. This suggests that B. bassiana expressing Cyt1Aa has potential value for use in mosquito control. IMPORTANCE: Beauveria bassiana is a naturally occurring fungus that can be utilized as a bioinsecticide against mosquitoes. Cyt1Aa is a delta-endotoxin protein produced by Bacillus thuringiensis that exhibits specific and potent insecticidal activity against mosquitoes. In our study, the expression of this toxin Cyt1Aa in B. bassiana enhances the virulence of B. bassiana against Aedes aegypti and Aedes albopictus, thereby increasing their effectiveness in killing mosquitoes. This novel strain can be used alongside chemical insecticides to reduce dependence on harmful chemicals, thereby minimizing negative impacts on the environment and human health. Additionally, the potential resistance of B. bassiana against mosquitoes in the future could be overcome by acquiring novel combinations of exogenous toxin genes. The presence of B. bassiana that expresses Cyt1Aa is of significant importance in mosquito control as it enhances genetic diversity, creates novel virulent strains, and contributes to the development of safer and more sustainable methods of mosquito control.
Assuntos
Aedes , Toxinas de Bacillus thuringiensis , Bacillus thuringiensis , Beauveria , Endotoxinas , Proteínas Hemolisinas , Larva , Controle de Mosquitos , Controle Biológico de Vetores , Animais , Beauveria/genética , Beauveria/patogenicidade , Beauveria/metabolismo , Aedes/microbiologia , Controle de Mosquitos/métodos , Toxinas de Bacillus thuringiensis/genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Endotoxinas/genética , Endotoxinas/metabolismo , Controle Biológico de Vetores/métodos , Larva/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Virulência/genética , Esporos Fúngicos/genética , Inseticidas/farmacologia , Inseticidas/metabolismoRESUMO
A novel group of biocidal compounds are the Crystal 3D (Cry) and Cytolytic (Cyt) proteins produced by Bacillus thuringiensis (Bt). Some Bt Cry proteins have a selective nematocidal activity, with Cry5B being the most studied. Cry5B kills nematode parasites by binding selectively to membrane glycosphingolipids, then forming pores in the cell membranes of the intestine leading to damage. Cry5B selectively targets multiple species of nematodes from different clades and has no effect against mammalian hosts. Levamisole is a cholinergic anthelmintic that acts by selectively opening L-subtype nicotinic acetylcholine receptor ion-channels (L-AChRs) that have been found on muscles of nematodes. A synergistic nematocidal interaction between levamisole and Cry5B at the whole-worm level has been described previously, but the location, mechanism and time-course of this synergism is not known. In this study we follow the timeline of the effects of levamisole and Cry5B on the Ca2+ levels in enterocyte cells in the intestine of Ascaris suum using fluorescence imaging. The peak Ca2+ responses to levamisole were observed after approximately 10 minutes while the peak responses to activated Cry5B were observed after approximately 80 minutes. When levamisole and Cry5B were applied simultaneously, we observed that the responses to Cry5B were bigger and occurred sooner than when it was applied by itself. It is proposed that the synergism is due to the cytoplasmic Ca2+ overload that is induced by the combination of levamisole opening Ca2+ permeable L-subtype nAChRs and the Ca2+ permeable Cry5B toxin pores produced in the enterocyte plasma membranes. The effect of levamisole potentiates and speeds the actions of Cry5B that gives rise to bigger Ca2+ overloads that accelerates cell-death of the enterocytes.