RESUMEN
Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen that can cause severe bacterial pneumonia. Amygdalin is the main active pharmaceutical ingredient of bitter almond, which has broad-spectrum antibacterial, anti-inflammatory, anti-oxidation and immunomodulatory effects. It is also the main ingredient of Yinhua Pinggan granule, which is commonly used to moisten the lung and relieve cough. However, little is known about the effects of amygdalin on MRSA. In this study, we found that amygdalin exhibited good antimicrobial activity in vitro against MRSA. Amygdalin has a protective effect on MRSA infected cells, and the effect is better when combined with levofloxacin. It also can reduce the adhesion and invasion of MRSA to cells. Amygdalin has anti-inflammatory and antioxidant effects, which can significantly reduce the increase of inflammatory factors and the production of ROS caused by infection. The protective mechanism of amygdalin on cells may be related to inhibiting the expression of NLRP3, ASC and IL-1ß pyroptosis pathways. Taken together, our study suggests that amygdalin exerts antibacterial effects by affecting biofilm formation, the expression of virulence factors, and drug resistance genes. Amygdalin combined with levofloxacin has a protective effect on A549 cells infected with MRSA, and the mechanism may be related to the inhibition of inflammatory response, oxidative damage and pyroptosis.
Asunto(s)
Amigdalina , Antibacterianos , Inflamación , Staphylococcus aureus Resistente a Meticilina , Estrés Oxidativo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Amigdalina/farmacología , Humanos , Estrés Oxidativo/efectos de los fármacos , Células A549 , Antibacterianos/farmacología , Inflamación/tratamiento farmacológico , Inflamación/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Células Epiteliales/metabolismo , Pulmón/microbiología , Pulmón/patología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Biopelículas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Levofloxacino/farmacología , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiologíaAsunto(s)
Células Epiteliales , Sindecano-1 , Factores de Virulencia , Humanos , Factores de Virulencia/metabolismo , Factores de Virulencia/genética , Células Epiteliales/microbiología , Células Epiteliales/metabolismo , Sindecano-1/metabolismo , Sindecano-1/genética , Bacillus anthracis/metabolismo , Bacillus anthracis/patogenicidad , Bacillus anthracis/genética , Carbunco/microbiologíaRESUMEN
Escherichia coli O157:H7 (E. coli O157) is known for causing severe foodborne illnesses such as hemorrhagic colitis and hemolytic uremic syndrome. Although E. coli O157 is typically regarded as an extracellular pathogen and a weak biofilm producer, some E. coli O157 strains, including a clinical strain ATCC 43895, exhibit a notable ability to invade bovine crypt cells and other epithelial cells, as well as to form robust biofilm. This invasive strain persists in the bovine host significantly longer than non-invasive strains. Various surface-associated factors, including lipopolysaccharides (LPS), flagella, and other adhesins, likely contribute to this enhanced invasiveness and biofilm formation. In this study, we constructed a series of LPS-core deletion mutations (waaI, waaG, waaF, and waaC) in E. coli O157 ATCC 43895, resulting in stepwise truncations of the LPS. This approach enabled us to investigate the effects on the biosynthesis of key surface factors, such as flagella and curli, and the ability of this invasive strain to invade host cells. We confirmed the LPS structure and found that all LPS-core mutants failed to form biofilms, highlighting the crucial role of core oligosaccharides in biofilm formation. Additionally, the LPS inner-core mutants ΔwaaF and ΔwaaC lost the ability to produce flagella and curli. Furthermore, these inner-core mutants exhibited a dramatic reduction in adherence to and invasion of epithelial cells (MAC-T), showing an approximately 100-fold decrease in cell invasion compared with the outer-core mutants (waaI and waaG) and the wild type. These findings underscore the critical role of LPS-core truncation in impairing flagella and curli biosynthesis, thereby reducing the invasion capability of E. coli O157 ATCC 43895.
Asunto(s)
Biopelículas , Escherichia coli O157 , Flagelos , Lipopolisacáridos , Flagelos/metabolismo , Flagelos/genética , Lipopolisacáridos/biosíntesis , Escherichia coli O157/genética , Escherichia coli O157/metabolismo , Escherichia coli O157/fisiología , Biopelículas/crecimiento & desarrollo , Animales , Bovinos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Adhesión Bacteriana , Células Epiteliales/microbiología , Células Epiteliales/metabolismoRESUMEN
Seminal plasma (SP) is the main vector of C. trachomatis (CT) during heterosexual transmission from male to female. It has immunomodulatory properties and impacts the susceptibility to HIV-1 infection, but its role has not been explored during CT infection. In the female reproductive tract (FRT), CT infection induces cytokine production and neutrophil recruitment. The role of neutrophils during CT infection is partially described, they could be at the origin of the pathology observed during CT infection. During this study, we developed an experimental in vitro model to characterize the impact of CT infection and SP on endocervical epithelial cell immune response in the FRT. We also studied the impact of the epithelial cell response on neutrophil phenotype and functions. We showed that the production by epithelial cells of pro-inflammatory cytokines increased during CT infection. Moreover, the pool of SP as well as individuals SP inhibited CT infection in a dose-dependent manner. The pool of SP inhibited cytokine production in a dose-dependent manner. The pool of SP altered gene expression profiles of infected cells. The culture supernatants of cells infected or not with CT, in presence or not of the pool of SP, had an impact on neutrophil phenotype and functions: they affected markers of neutrophil maturation, activation and adhesion capacity, as well as the survival, ROS production and phagocytosis ability. This study proposes a novel approach to study the impact of the environment on the phenotype and functions of neutrophils in the FRT. It highlights the impact of the factors of the FRT environment, in particular SP and CT infection, on the mucosal inflammation and the need to take into account the SP component while studying sexually transmitted infections during heterosexual transmission from male to female.
Asunto(s)
Infecciones por Chlamydia , Chlamydia trachomatis , Citocinas , Inmunidad Mucosa , Neutrófilos , Semen , Chlamydia trachomatis/inmunología , Chlamydia trachomatis/fisiología , Humanos , Femenino , Semen/inmunología , Semen/microbiología , Semen/metabolismo , Infecciones por Chlamydia/inmunología , Infecciones por Chlamydia/microbiología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Citocinas/metabolismo , Masculino , Células Epiteliales/microbiología , Células Epiteliales/metabolismo , Células Epiteliales/inmunología , Fagocitosis , Cuello del Útero/microbiología , Cuello del Útero/inmunologíaRESUMEN
The mucus serves as a protective barrier in the gastrointestinal tract against microbial attacks. While its role extends beyond merely being a physical barrier, the extent of its active bactericidal properties remains unclear, and the mechanisms regulating these properties are not yet understood. We propose that inflammation induces epithelial cells to secrete antimicrobial peptides, transforming mucus into an active bactericidal agent. To investigate the properties of mucus, we previously developed mucosoid culture models that mimic the healthy human stomach epithelium. Similar to organoids, mucosoids are stem cell-driven cultures; however, the cells are cultivated on transwells at air-liquid interface. The epithelial cells of mucosoids form a polarized monolayer, allowing differentiation into all stomach lineages, including mucus-secreting cells. This setup facilitates the secretion and accumulation of mucus on the apical side of the mucosoids, enabling analysis of its bactericidal effects and protein composition, including antimicrobial peptides. Our findings show that TNFα, IL1ß, and IFNγ induce the secretion of antimicrobials such as lactotransferrin, lipocalin2, complement component 3, and CXCL9 into the mucus. This antimicrobial-enriched mucus can partially eliminate Helicobacter pylori, a key stomach pathogen. The bactericidal activity depends on the concentration of each antimicrobial and their gene expression is higher in patients with inflammation and H.pylori-associated chronic gastritis. However, we also find that H. pylori infection can reduce the expression of antimicrobial encoding genes promoted by inflammation. These findings suggest that controlling antimicrobial secretion in the mucus is a critical component of epithelial immunity. However, pathogens like H. pylori can overcome these defenses and survive in the mucosa.
Asunto(s)
Péptidos Antimicrobianos , Mucosa Gástrica , Helicobacter pylori , Inflamación , Moco , Humanos , Moco/metabolismo , Moco/microbiología , Péptidos Antimicrobianos/metabolismo , Mucosa Gástrica/microbiología , Mucosa Gástrica/metabolismo , Mucosa Gástrica/inmunología , Inflamación/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/metabolismo , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/inmunología , Estómago/microbiología , Organoides/metabolismo , Organoides/microbiologíaRESUMEN
The collective involvement of virulence markers of Escherichia coli as an emerging pathogen associated with periodontitis remains unexplained. This study aimed to implement an in vitro model of infection using a human epithelial cell line to determine the virulome expression related to the antibiotic and disinfectant resistance genotype and pulse field gel electrophoresis (PFGE) type in E. coli strains isolated from patients with periodontal diseases. We studied 100 strains of E. coli isolated from patients with gingivitis (n = 12), moderate periodontitis (n = 59), and chronic periodontitis (n = 29). The identification of E. coli and antibiotic and disinfectant resistance genes was performed through PCR. To promote the expression of virulence genes in the strains, an in vitro infection model was used in the human epithelial cell line A549. RNA was extracted using the QIAcube robotic equipment and reverse transcription to cDNA was performed using the QuantiTect reverse transcription kit (Qiagen). The determination of virulence gene expression was performed through real-time PCR. Overall, the most frequently expressed adhesion genes among the isolated strains of gingivitis, moderate periodontitis, and chronic periodontitis were fimH (48%), iha (37%), and papA (18%); those for toxins were usp (33%); those for iron acquisition were feoB (84%), fyuA (62%), irp-2 (61%), and iroN (35%); those for protectins were traT (50%), KpsMT (35%), and ompT (28%); and those for pathogenicity islands were malX (45%). The most common antibiotic and disinfectant resistance genes among gingivitis, moderate periodontitis, and chronic periodontitis strains were sul-2 (43%), blaSHV (47%), blaTEM (45%), tet(A) (41%), dfrA1 (32%), marR-marO (57%), and qacEA1 (79%). The findings revealed the existence of a wide distribution of virulome expression profiles related to the antibiotic and disinfectant resistance genotype and PFGE type in periodontal strains of E. coli. These findings may contribute toward improving the prevention and treatment measures for periodontal diseases associated with E. coli.
Asunto(s)
Antibacterianos , Desinfectantes , Farmacorresistencia Bacteriana , Infecciones por Escherichia coli , Escherichia coli , Factores de Virulencia , Humanos , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/patogenicidad , Factores de Virulencia/genética , Antibacterianos/farmacología , Infecciones por Escherichia coli/microbiología , Farmacorresistencia Bacteriana/genética , Desinfectantes/farmacología , Periodontitis/microbiología , Virulencia/genética , Células A549 , Células Epiteliales/microbiología , Genotipo , Adulto , Femenino , Masculino , Persona de Mediana Edad , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Electroforesis en Gel de Campo PulsadoRESUMEN
The conjunctiva has immune-responsive properties to protect the eye from infections. Its innate immune system reacts against external pathogens, such as fungi. The complement factor C5a is an important contributor to the initial immune response. It is known that activation of transient-receptor-potential-vanilloid 1 (TRPV1) and TRP-melastatin 8 (TRPM8) channels is involved in different immune reactions and inflammation in the human body. The aim of this study was to determine if C5a and mucor racemosus e voluminae cellulae (MR) modulate Ca2+-signaling through changes in TRPs activity in human conjunctival epithelial cells (HCjECs). Furthermore, crosstalk was examined between C5a and MR in mediating calcium regulation. Intracellular Ca2+-concentration ([Ca2+]i) was measured by fluorescence calcium imaging, and whole-cell currents were recorded using the planar-patch-clamp technique. MR was used as a purified extract. Application of C5a (0.05-50 ng/mL) increased both [Ca2+]i and whole-cell currents, which were suppressed by either the TRPV1-blocker AMG 9810 or the TRPM8-blocker AMTB (both 20 µM). The N-terminal peptide C5L2p (20-50 ng/mL) blocked rises in [Ca2+]i induced by C5a. Moreover, the MR-induced rise in Ca2+-influx was suppressed by AMG 9810 and AMTB, as well as 0.05 ng/mL C5a. In conclusion, crosstalk between C5a and MR controls human conjunctival cell function through modulating interactions between TRPV1 and TRPM8 channel activity.
Asunto(s)
Calcio , Complemento C5a , Conjuntiva , Células Epiteliales , Humanos , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Conjuntiva/metabolismo , Conjuntiva/microbiología , Calcio/metabolismo , Complemento C5a/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Señalización del Calcio , Canales Catiónicos TRPV/metabolismoRESUMEN
Shiga toxin (Stx) is produced by some pathogenic strains of Escherichia coli. To study the impact of Stx on the human intestine, we utilized human intestinal organoids and human intestinal enteroids grown as human intestinal enteroid monolayers (HIEMs) in transwells. To establish optimal experimental conditions, HIEMs were grown with or without mesenchymal cells added to the basolateral wells to recapitulate the interactions between the intestinal epithelium and the underlying mesenchyme. Monolayer barrier integrity was determined through transepithelial electrical resistance (TEER) readings. Apical saline was used on the apical surface since growth medium caused uneven development of the TEER. The medium used for epithelial cells contains added growth factors, while the mesenchymal medium lacks these growth factors. We have shown that mesenchymal cells can maintain the epithelial monolayer in the medium lacking growth factors, suggesting they produce these factors. Furthermore, growth factors produced by mesenchymal cells need to build up in the medium over time, since daily medium changes were not as effective as medium changes performed every 3 days. We have also shown that addition of growth factors is toxic to mesenchymal cells. Epithelial cells were more resistant to Stx2 than the mesenchymal cells, and mesenchymal cells contributed to epithelial cell death. Epithelial cells tolerated luminal exposure better than basolateral exposure. These studies demonstrate the importance of understanding tissue interactions in a disease state when using in vitro and in vitro models. IMPORTANCE: These studies have cemented the need for complex cell culture models when studying host-pathogen interactions. Common animal models such as mice are resistant to E. coli O157:H7 infections and intestinal delivery of Stx2, while humans appear to be sensitive to both. It has been proposed that in humans, shiga toxin-producing E. coli-mediated intestinal damage destroys the intestinal barrier and allows basolateral access to Stx2. In mice, there is no epithelial damage; therefore, they are resistant to epithelial delivery of Stx2 while remaining sensitive to Stx2 injection. Our studies show that like mice, the human epithelial layer is quite resistant to Stx2, and it is the sensitivity of the mesenchymal cells that kills the epithelial cells. We have shown that Stx2 is transported through the intact epithelium without causing damage to the resistant epithelial layer. Understanding tissue interactions during infections is therefore critical in determining the effects of pathogens on human tissues.
Asunto(s)
Células Epiteliales , Mucosa Intestinal , Organoides , Humanos , Células Epiteliales/microbiología , Células Epiteliales/efectos de los fármacos , Mucosa Intestinal/microbiología , Animales , Ratones , Toxina Shiga/metabolismo , Intestinos/microbiologíaRESUMEN
The bacterial pathogen Staphylococcus aureus employs a thick cell wall for protection against physical and chemical insults. This wall requires continuous maintenance to ensure strength and barrier integrity, but also to permit bacterial growth and division. The main cell wall component is peptidoglycan. Accordingly, the bacteria produce so-called peptidoglycan hydrolases (PGHs) that cleave glycan strands to facilitate growth, cell wall remodelling, separation of divided cells and release of exported proteins into the extracellular milieu. A special class of PGHs contains so-called 'cysteine, histidine-dependent amidohydrolase/peptidase' (CHAP) domains. In the present study, we profiled the roles of 11 CHAP PGHs encoded by the core genome of S. aureus USA300 LAC. Mutant strains lacking individual CHAP PGHs were analysed for growth, cell morphology, autolysis, and invasion and replication inside human lung epithelial cells. The results show that several investigated CHAP PGHs contribute to different extents to extracellular and intracellular growth and replication of S. aureus, septation of dividing cells, daughter cell separation once the division process is completed, autolysis and biofilm formation. In particular, the CHAP PGHs Sle1 and SAUSA300_2253 control intracellular staphylococcal replication and the resistance to ß-lactam antibiotics like oxacillin. This makes the S. aureus PGHs in general, and the Sle1 and SAUSA300_2253 proteins in particular, attractive targets for future prophylactic or therapeutic anti-staphylococcal interventions. Alternatively, these cell surface-exposed enzymes, or particular domains of these enzymes, could be applied in innovative anti-staphylococcal therapies.
Asunto(s)
Proteínas Bacterianas , Pared Celular , N-Acetil Muramoil-L-Alanina Amidasa , Staphylococcus aureus , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/genética , Humanos , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Staphylococcus aureus/fisiología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Pared Celular/metabolismo , Peptidoglicano/metabolismo , Antibacterianos/farmacología , Biopelículas/crecimiento & desarrollo , Biopelículas/efectos de los fármacos , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Células Epiteliales/microbiologíaRESUMEN
Membrane lipoproteins serve as primary pro-inflammatory virulence factors in Mycoplasma genitalium. Membrane lipoproteins primarily induce inflammatory responses by activating Toll-like Receptor 2 (TLR2); however, the role of the metabolic status of urethral epithelial cells in inflammatory response remains unclear. In this study, we found that treatment of uroepithelial cell lines with M. genitalium membrane lipoprotein induced metabolic reprogramming, characterized by increased aerobic glycolysis, decreased oxidative phosphorylation, and increased production of the metabolic intermediates acetyl-CoA and malonyl-CoA. The metabolic shift induced by membrane lipoproteins is reversible upon blocking MyD88 and TRAM. Malonyl-CoA induces malonylation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and malonylated GAPDH could dissociate from the 3' untranslated region of TNF-α and IFN-γ mRNA. This dissociation greatly reduces the inhibitory effect on the translation of TNF-α and IFN-γ mRNA, thus achieving fine-tuning control over cytokine secretion. These findings suggest that GAPDH malonylation following M. genitalium infection is an important inflammatory signal that plays a crucial role in urogenital inflammatory diseases.
Asunto(s)
Citocinas , Células Epiteliales , Interferón gamma , Mycoplasma genitalium , Factor de Necrosis Tumoral alfa , Mycoplasma genitalium/metabolismo , Mycoplasma genitalium/genética , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Humanos , Citocinas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Interferón gamma/metabolismo , Línea Celular , Lipoproteínas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Uretra/microbiología , Uretra/metabolismo , Infecciones por Mycoplasma/metabolismo , Infecciones por Mycoplasma/microbiología , Factores de Virulencia/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Glucólisis , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 2/genéticaRESUMEN
Despite Staphylococcus aureus (S. aureus) being a highly studied zoontic bacterium, its enteropathogenicity remains elusive. Herein, our findings demonstrated that S. aureus infection led to the accumulation of lipid droplets (LDs) in intestinal epithelial cells, accompanied by marked elevation inflammatory response that ultimately decreases intracellular bacterial load. The aforestated phenomenon may be partly attributed to the up-regulation of hypoxia-inducible lipid droplet-associated protein (HILPDA) and the concomitant down-regulation of cystathionine ß-synthase (CBS) protein. Moreover, S. aureus infection up-regulated the expression of HILPDA, thereby promoting LDs accumulation, and down-regulated that of CBS, consequently inhibiting microsomal triglyceride transfer protein (MTTP) expression. This process may suppress the transport of LDs to the extracellular environment, further contributing to the formation of intracellular LDs. In summary, the results of this study provide significant insights into the intricate mechanisms through which the host organism combats pathogens and maintains the balance of sulfur and lipid metabolism. These findings not only enhance our understanding of the host's defense mechanisms but also offer promising avenues for the development of novel strategies to combat intestinal infectious diseases.
Asunto(s)
Cistationina betasintasa , Células Epiteliales , Gotas Lipídicas , Staphylococcus aureus , Staphylococcus aureus/metabolismo , Gotas Lipídicas/metabolismo , Cistationina betasintasa/metabolismo , Cistationina betasintasa/genética , Humanos , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Animales , Metabolismo de los Lípidos , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/patología , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Células CACO-2 , RatonesRESUMEN
Helicobacter pylori infection has been thought to be associated with liver diseases, although the exact mechanisms remain elusive. This study identified H. pylori-induced liver inflammation and tissue damage in infected mice and examined the exosome-mediated mechanism underlying H. pylori infection's impact on liver injury. Exosomes were isolated from H. pylori-infected gastric epithelial GES-1 cells (Hp-GES-EVs), and the crucial virulence factor CagA was identified within these exosomes. Fluorescent labeling demonstrated that Hp-GES-EVs can be absorbed by liver cells. Treatment with Hp-GES-EVs enhanced the proliferation, migration, and invasion of Hep G2 and Hep 3B cells. Additionally, exposure to Hp-GES-EVs activated NF-κB and PI3K/AKT signaling pathways, which provides a reasonable explanation for the liver inflammation and neoplastic traits. Using a mouse model established via tail vein injection of Hp-GES-EVs, exosome-driven liver injury was evidenced by slight hepatocellular erosion around the central hepatic vein and elevated serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and IL-6. Administering the exosome inhibitor GW4869 via intraperitoneal injection in mice resulted in a reduction of liver damage caused by H. pylori infection. These findings illuminate the exosome-mediated pathogenesis of H. pylori-induced liver injury and offer valuable insights into the extra-gastrointestinal manifestations of H. pylori infection.
Asunto(s)
Antígenos Bacterianos , Proteínas Bacterianas , Modelos Animales de Enfermedad , Exosomas , Infecciones por Helicobacter , Helicobacter pylori , Hígado , Transducción de Señal , Exosomas/metabolismo , Animales , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/patogenicidad , Ratones , Humanos , Proteínas Bacterianas/metabolismo , Hígado/patología , Hígado/metabolismo , Hígado/microbiología , Antígenos Bacterianos/metabolismo , Compuestos de Bencilideno/farmacología , Compuestos de Anilina/farmacología , FN-kappa B/metabolismo , Células Hep G2 , Aspartato Aminotransferasas/sangre , Interleucina-6/metabolismo , Alanina Transaminasa/sangre , Proliferación Celular , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/metabolismo , Movimiento Celular , Línea Celular , Masculino , Factores de Virulencia/metabolismoRESUMEN
Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a systemic infection that affects millions of people worldwide. S. Typhi can invade and survive within host cells, such as intestinal epithelial cells and macrophages, by modulating their immune responses. However, the immunomodulatory capability of S. Typhi in relation to TolC-facilitated efflux pump function remains unclear. The role of TolC, an outer membrane protein that facilitates efflux pump function, in the invasion and immunomodulation of S. Typhi, was studied in human intestinal epithelial cells and macrophages. The tolC deletion mutant of S. Typhi was compared with the wild-type and its complemented strain in terms of their ability to invade epithelial cells, survive and induce cytotoxicity in macrophages, and elicit proinflammatory cytokine production in macrophages. The tolC mutant, which has a defective outer membrane, was impaired in invading epithelial cells compared to the wild-type strain, but the intracellular presence of the tolC mutant exhibited greater cytotoxicity and induced higher levels of proinflammatory cytokines (IL-1ß and IL-8) in macrophages compared to the wild-type strain. These effects were reversed by complementing the tolC mutant with a functional tolC gene. Our results suggest that TolC plays a role in S. Typhi to efficiently invade epithelial cells and suppress host immune responses during infection. TolC may be a potential target for the development of novel therapeutics against typhoid fever.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Células Epiteliales , Macrófagos , Salmonella typhi , Fiebre Tifoidea , Salmonella typhi/patogenicidad , Salmonella typhi/inmunología , Salmonella typhi/genética , Humanos , Macrófagos/microbiología , Macrófagos/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/inmunología , Células Epiteliales/microbiología , Células Epiteliales/inmunología , Fiebre Tifoidea/inmunología , Fiebre Tifoidea/microbiología , Inmunomodulación , Citocinas/metabolismo , Citocinas/inmunología , Viabilidad Microbiana , Interleucina-8/metabolismo , Interleucina-1beta/metabolismo , Interleucina-1beta/inmunología , Línea CelularRESUMEN
Bacterial vaginosis (BV) is a polymicrobial infection of the female reproductive tract. BV is characterized by replacement of health-associated Lactobacillus species by diverse anerobic bacteria, including the well-known Gardnerella vaginalis. Prevotella timonensis, and Prevotella bivia are anerobes that are found in a significant number of BV patients, but their contributions to the disease process remain to be determined. Defining characteristics of anerobic overgrowth in BV are adherence to the mucosal surface and the increased activity of mucin-degrading enzymes such as sialidases in vaginal secretions. We demonstrate that P. timonensis, but not P. bivia, strongly adheres to vaginal and endocervical cells to a similar level as G. vaginalis but did not elicit a comparable proinflammatory epithelial response. The P. timonensis genome uniquely encodes a large set of mucus-degrading enzymes, including four putative fucosidases and two putative sialidases, PtNanH1 and PtNanH2. Enzyme assays demonstrated that fucosidase and sialidase activities in P. timonensis cell-bound and secreted fractions were significantly higher than for other vaginal anerobes. In infection assays, P. timonensis efficiently removed fucose and α2,3- and α2,6-linked sialic acid moieties from the epithelial glycocalyx. Recombinantly expressed P. timonensis NanH1 and NanH2 cleaved α2,3 and α2,6-linked sialic acids from the epithelial surface, and sialic acid removal by P. timonensis could be blocked using inhibitors. This study demonstrates that P. timonensis has distinct virulence-related properties that include initial adhesion and a high capacity for mucin degradation at the vaginal epithelial mucosal surface. Our results underline the importance of understanding the role of different anerobic bacteria in BV. IMPORTANCE: Bacterial vaginosis (BV) is a common vaginal infection that affects a significant proportion of women and is associated with reduced fertility and increased risk of secondary infections. Gardnerella vaginalis is the most well-known BV-associated bacterium, but Prevotella species including P. timonensis and P. bivia may also play an important role. We showed that, similar to G. vaginalis, P. timonensis adhered well to the vaginal epithelium, suggesting that both bacteria could be important in the first stage of infection. Compared to the other bacteria, P. timonensis was unique in efficiently removing the protective mucin sugars that cover the vaginal epithelium. These results underscore that vaginal bacteria play different roles in the initiation and development of BV.
Asunto(s)
Glicocálix , Neuraminidasa , Prevotella , Vagina , Vaginosis Bacteriana , alfa-L-Fucosidasa , Femenino , Neuraminidasa/metabolismo , Neuraminidasa/genética , Prevotella/enzimología , Prevotella/genética , Prevotella/patogenicidad , Prevotella/metabolismo , Humanos , Vagina/microbiología , alfa-L-Fucosidasa/metabolismo , alfa-L-Fucosidasa/genética , Vaginosis Bacteriana/microbiología , Glicocálix/metabolismo , Adhesión Bacteriana , Células Epiteliales/microbiologíaRESUMEN
Cytotoxic chemotherapies have devastating side effects, particularly within the gastrointestinal tract. Gastrointestinal toxicity includes the death and damage of the epithelium and an imbalance in the intestinal microbiota, otherwise known as dysbiosis. Whether dysbiosis is a direct contributor to tissue toxicity is a key area of focus. Here, from both mammalian and bacterial perspectives, we uncover an intestinal epithelial cell death-Enterobacteriaceae signaling axis that fuels dysbiosis. Specifically, our data demonstrate that chemotherapy-induced epithelial cell apoptosis and the purine-containing metabolites released from dying cells drive the inter-kingdom transcriptional re-wiring of the Enterobacteriaceae, including fundamental shifts in bacterial respiration and promotion of purine utilization-dependent expansion, which in turn delays the recovery of the intestinal tract. Inhibition of epithelial cell death or restriction of the Enterobacteriaceae to homeostatic levels reverses dysbiosis and improves intestinal recovery. These findings suggest that supportive therapies that maintain homeostatic levels of Enterobacteriaceae may be useful in resolving intestinal disease.
Asunto(s)
Disbiosis , Enterobacteriaceae , Microbioma Gastrointestinal , Mucosa Intestinal , Disbiosis/inducido químicamente , Animales , Microbioma Gastrointestinal/efectos de los fármacos , Ratones , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Ratones Endogámicos C57BL , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Intestinos/efectos de los fármacos , Intestinos/microbiología , Transducción de Señal , Purinas/metabolismo , Purinas/farmacologíaRESUMEN
Streptococcus pneumoniae (Sp), a leading cause of community-acquired pneumonia, can spread from the lung into the bloodstream to cause septicemia and meningitis, with a concomitant threefold increase in mortality. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that target pathogenic immune processes. Polymorphonuclear leukocytes (PMNs) are essential for infection control but can also promote tissue damage and pathogen spread. The major Sp virulence factor, pneumolysin, triggers acute inflammation by stimulating the 12-lipoxygenase (12-LOX) eicosanoid synthesis pathway in epithelial cells. This pathway is required for systemic spread in a mouse pneumonia model and produces a number of bioactive lipids, including hepoxilin A3 (HXA3), a hydroxy epoxide PMN chemoattractant that has been hypothesized to facilitate breach of mucosal barriers. To understand how 12-LOX-dependent inflammation promotes dissemination during Sp lung infection and dissemination, we utilized bronchial stem cell-derived air-liquid interface cultures that lack this enzyme to show that HXA3 methyl ester (HXA3-ME) is sufficient to promote basolateral-to-apical PMN transmigration, monolayer disruption, and concomitant Sp barrier breach. In contrast, PMN transmigration in response to the non-eicosanoid chemoattractant N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP) did not lead to epithelial disruption or bacterial translocation. Correspondingly, HXA3-ME but not fMLP increased the release of neutrophil elastase (NE) from Sp-infected PMNs. Pharmacologic blockade of NE secretion or activity diminished epithelial barrier disruption and bacteremia after pulmonary challenge of mice. Thus, HXA3 promotes barrier-disrupting PMN transmigration and NE release, pathological events that can be targeted to curtail systemic disease following pneumococcal pneumonia.IMPORTANCEStreptococcus pneumoniae (Sp), a leading cause of pneumonia, can spread from the lung into the bloodstream to cause systemic disease. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that limit pathologic host immune responses to Sp. Excessive polymorphonuclear leukocyte (PMN) infiltration into Sp-infected airways promotes systemic disease. Using stem cell-derived respiratory cultures that reflect bona fide lung epithelium, we identified eicosanoid hepoxilin A3 as a critical pulmonary PMN chemoattractant that is sufficient to drive PMN-mediated epithelial damage by inducing the release of neutrophil elastase. Inhibition of the release or activity of this protease in mice limited epithelial barrier disruption and bacterial dissemination, suggesting a new host-directed treatment for Sp lung infection.
Asunto(s)
Bacteriemia , Elastasa de Leucocito , Neutrófilos , Streptococcus pneumoniae , Animales , Ratones , Streptococcus pneumoniae/inmunología , Elastasa de Leucocito/metabolismo , Bacteriemia/microbiología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Pulmón/microbiología , Pulmón/inmunología , Humanos , Células Epiteliales/microbiología , Células Epiteliales/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Neumonía Neumocócica/inmunología , Neumonía Neumocócica/microbiología , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Estreptolisinas/metabolismo , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 12-Lipooxigenasa/genéticaRESUMEN
Helicobacter pylori (H. pylori) infection is the primary risk factor for the progress of gastric diseases. The persistent stomach colonization of H. pylori is closely associated with the development of gastritis and malignancies. Although the involvement of progranulin (PGRN) in various cancer types has been well-documented, its functional role and underlying mechanisms in gastric cancer (GC) associated with H. pylori infection remain largely unknown. This report demonstrated that PGRN was up-regulated in GC and associated with poor prognosis, as determined through local and public database analysis. Additionally, H. pylori induced the up-regulation of PGRN in gastric epithelial cells both in vitro and in vivo. Functional studies have shown that PGRN promoted the intracellular colonization of H. pylori. Mechanistically, H. pylori infection induced autophagy, while PGRN inhibited autophagy to promote the intracellular colonization of H. pylori. Furthermore, PGRN suppressed H. pylori-induced autophagy by down-regulating decorin (DCN) through the mTOR pathway. In general, PGRN inhibited autophagy to facilitate intracellular colonization of H. pylori via the PGRN/mTOR/DCN axis. This study provides new insights into the molecular mechanisms underlying the progression of gastric diseases, suggesting PGRN as a potential therapeutic target and prognostic predictor for these disorders.
Asunto(s)
Autofagia , Células Epiteliales , Mucosa Gástrica , Infecciones por Helicobacter , Helicobacter pylori , Progranulinas , Neoplasias Gástricas , Serina-Treonina Quinasas TOR , Progranulinas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Humanos , Células Epiteliales/microbiología , Células Epiteliales/metabolismo , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/metabolismo , Animales , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Mucosa Gástrica/microbiología , Mucosa Gástrica/metabolismo , Ratones , Transducción de SeñalRESUMEN
Mucus and its movements are essential to epithelial tissue immune defenses against pathogens, including fungal pathogens, which can infect respiratory, gastrointestinal or the genito-urinary tracts. Several epithelial cell types contribute to their immune defense. This review focuses on the respiratory tract because of its paramount importance, but the observations will apply to epithelial cell defenses of other mucosal tissue, including the gastrointestinal and genito-urinary tracts. Mucus and its movements can enhance or degrade the immune defenses of the respiratory tract, particularly the lungs. The enhancements include inhaled pathogen entrapments, including fungal pathogens, pollutants and particulates, for their removal. The detriments include smaller lung airway obstructions by mucus, impairing the physical removal of pathogens and impairing vital transfers of oxygen and carbon dioxide between the alveolar circulatory system and the pulmonary air. Inflammation, edema and/or alveolar cellular damage can also reduce vital transfers of oxygen and carbon dioxide between the lung alveolar circulatory system and the pulmonary air. Furthermore, respiratory tract defenses are affected by several fatty acid mediators which activate cellular receptors to manipulate neutrophils, macrophages, dendritic cells, various innate lymphoid cells including the natural killer cells, T cells, γδ T cells, mucosal-associated invariant T cells, NKT cells and mast cells. These mediators include the inflammatory and frequently immunosuppressive prostaglandins and leukotrienes, and the special pro-resolving mediators, which normally resolve inflammation and immunosuppression. The total effects on the various epithelial cell and immune cell types, after exposures to pathogens, pollutants or particulates, will determine respiratory tract health or disease.
Asunto(s)
Células Epiteliales , Humanos , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Hongos , AnimalesRESUMEN
In the current study, two Salmonella Typhimurium strains, JOL 912 and JOL 1800, were engineered from the wild-type JOL 401 strain through in-frame deletions of the lon and cpxR genes, with JOL 1800 also lacking rfaL. These deletions significantly attenuated the strains, impairing their intracellular survival and creating unique immunological profiles. This study investigates the response of these strains to various abiotic stress conditions commonly experienced in vivo, including temperature, acidity, osmotic, and oxidative stress. Notably, cold stress induced a non-significant trend towards increased invasion by Salmonella compared to other stressors. Despite the observed attenuation, no significant alterations in entry mechanisms (trigger vs. zipper) were noted between these strains, although variations were evident depending on the host cell type. Both strains effectively localized within the cytoplasm, demonstrating their ability to invade and interact with the intracellular environment. Immunologically, JOL 912 elicited a robust response, marked by substantial activation of nuclear factor kappa B (NF-kB), and chemokines, interleukin 8 (CXCL 8) and interleukin 10 (CXCL 10), comparable to the wild-type JOL 401 (over a fourfold increase compared to JOL 1800). In contrast, JOL 1800 exhibited a minimal immune response. Additionally, these attenuations influenced the expression of cyclins D1 and B1 and caspases 3 and 7, indicating cell cycle arrest at the G2/M phase and promotion of the G0/G1 to S phase transition, alongside apoptosis in infected cells. These findings provide valuable insights into the mechanisms governing the association, internalization, and survival of Salmonella mutants, enhancing our understanding of their regulatory effects on host cell physiology.
Asunto(s)
Proteínas Bacterianas , Salmonella typhimurium , Estrés Fisiológico , Salmonella typhimurium/patogenicidad , Salmonella typhimurium/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Estrés Fisiológico/genética , Humanos , Virulencia/genética , Células Epiteliales/microbiología , Células Epiteliales/metabolismo , Proteasa La/metabolismo , Proteasa La/genética , Mutación , Infecciones por Salmonella/microbiología , Infecciones por Salmonella/genética , FN-kappa B/metabolismoRESUMEN
Numerous Aspergillus fumigatus (Af) airborne spores are inhaled daily by humans and animals due to their ubiquitous presence. The interaction between the spores and the respiratory epithelium, as well as its impact on the epithelial barrier function, remains largely unknown. The epithelial barrier protects the respiratory epithelium against viral infections. However, it can be compromised by environmental contaminants such as pollen, thereby increasing susceptibility to respiratory viral infections, including alphaherpesvirus equine herpesvirus type 1 (EHV-1). To determine whether Af spores disrupt the epithelial integrity and enhance susceptibility to viral infections, equine respiratory mucosal ex vivo explants were pretreated with Af spore diffusate, followed by EHV-1 inoculation. Spore proteases were characterized by zymography and identified using mass spectrometry-based proteomics. Proteases of the serine protease, metalloprotease, and aspartic protease groups were identified. Morphological analysis of hematoxylin-eosin (HE)-stained sections of the explants revealed that Af spores induced the desquamation of epithelial cells and a significant increase in intercellular space at high and low concentrations, respectively. The increase in intercellular space in the epithelium caused by Af spore proteases correlated with an increase in EHV-1 infection. Together, our findings demonstrate that Af spore proteases disrupt epithelial integrity, potentially leading to increased viral infection of the respiratory epithelium.