RESUMEN
Dysbiosis of the vaginal microbiome poses a serious risk for sexual human immunodeficiency virus type 1 (HIV-1) transmission. Prevotella spp are abundant during vaginal dysbiosis and associated with enhanced HIV-1 susceptibility; however, underlying mechanisms remain unclear. Here, we investigated the direct effect of vaginal bacteria on HIV-1 susceptibility of vaginal CD4+ T cells. Notably, pre-exposure to Prevotella timonensis enhanced HIV-1 uptake by vaginal T cells, leading to increased viral fusion and enhanced virus production. Pre-exposure to antiretroviral inhibitors abolished P timonensis-enhanced infection. Our study shows that the vaginal microbiome directly affects mucosal CD4+ T-cell susceptibility, emphasizing importance of vaginal dysbiosis diagnosis and treatment.
Asunto(s)
Linfocitos T CD4-Positivos , Disbiosis , Infecciones por VIH , VIH-1 , Prevotella , Vagina , Humanos , Femenino , Prevotella/aislamiento & purificación , Disbiosis/microbiología , Vagina/microbiología , Vagina/virología , Vagina/inmunología , Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/microbiología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Susceptibilidad a Enfermedades , Microbiota , Internalización del VirusRESUMEN
Mucin 1 (MUC1) is a transmembrane mucin expressed at the apical surface of epithelial cells at mucosal surfaces. MUC1 has a barrier function against bacterial invasion and is well known for its aberrant expression and glycosylation in adenocarcinomas. The MUC1 extracellular domain contains a variable number of tandem repeats (VNTR) of 20 amino acids, which are heavily O-linked glycosylated. Monoclonal antibodies against the MUC1 VNTR are powerful research tools with applications in the diagnosis and treatment of MUC1-expressing cancers. Here, we report direct mass spectrometry-based sequencing of anti-MUC1 hybridoma-derived 139H2 IgG, enabling reverse-engineering of the functional recombinant monoclonal antibody. The crystal structure of the 139H2 Fab fragment in complex with the MUC1 epitope was solved, revealing the molecular basis of 139H2 binding specificity to MUC1 and its tolerance to O-glycosylation of the VNTR. The available sequence of 139H2 will allow further development of MUC1-related diagnostic, targeting, and treatment strategies.
Asunto(s)
Mucina-1 , Neoplasias , Humanos , Secuencia de Aminoácidos , Mucina-1/genética , Mucina-1/química , Mucinas/genética , Mucinas/metabolismo , Glicosilación , Anticuerpos MonoclonalesRESUMEN
Glycosylated mucin proteins contribute to the essential barrier function of the intestinal epithelium. The transmembrane mucin MUC13 is an abundant intestinal glycoprotein with important functions for mucosal maintenance that are not yet completely understood. We demonstrate that in human intestinal epithelial monolayers, MUC13 localized to both the apical surface and the tight junction (TJ) region on the lateral membrane. MUC13 deletion resulted in increased transepithelial resistance (TEER) and reduced translocation of small solutes. TEER buildup in ΔMUC13 cells could be prevented by addition of MLCK, ROCK or protein kinase C (PKC) inhibitors. The levels of TJ proteins including claudins and occludin were highly increased in membrane fractions of MUC13 knockout cells. Removal of the MUC13 cytoplasmic tail (CT) also altered TJ composition but did not affect TEER. The increased buildup of TJ complexes in ΔMUC13 and MUC13-ΔCT cells was dependent on PKC. The responsible PKC member might be PKCδ (or PRKCD) based on elevated protein levels in the absence of full-length MUC13. Our results demonstrate for the first time that a mucin protein can negatively regulate TJ function and stimulate intestinal barrier permeability.
Asunto(s)
Proteína Quinasa C , Proteínas de Uniones Estrechas , Humanos , Proteínas de Uniones Estrechas/metabolismo , Proteína Quinasa C/metabolismo , Intestinos , Mucosa Intestinal/metabolismo , Uniones Estrechas/metabolismo , Ocludina , Mucinas/metabolismo , Células Epiteliales/metabolismoRESUMEN
Clostridioides difficile infection is expected to become the most common healthcare-associated infection worldwide. C. difficile-induced pathogenicity is significantly attributed to its enterotoxin, TcdA, which primarily targets Rho-GTPases involved in regulating cytoskeletal and tight junction (TJ) dynamics, thus leading to cytoskeleton breakdown and ultimately increased intestinal permeability. This study investigated whether two non-digestible oligosaccharides (NDOs), alginate (AOS) and chitosan (COS) oligosaccharides, possess antipathogenic and barrier-protective properties against C. difficile bacteria and TcdA toxin, respectively. Both NDOs significantly reduced C. difficile growth, while cell cytotoxicity assays demonstrated that neither COS nor AOS significantly attenuated the TcdA-induced cell death 24 h post-exposure. The challenge of Caco-2 monolayers with increasing TcdA concentrations increased paracellular permeability, as measured by TEER and LY flux assays. In this experimental setup, COS completely abolished, and AOS mitigated, the deleterious effects of TcdA on the monolayer's integrity. These events were not accompanied by alterations in ZO-1 and occludin protein levels; however, immunofluorescence microscopy revealed that both AOS and COS prevented the TcdA-induced occludin mislocalization. Finally, both NDOs accelerated TJ reassembly upon a calcium-switch assay. Overall, this study established the antipathogenic and barrier-protective capacity of AOS and COS against C. difficile and its toxin, TcdA, while revealing their ability to promote TJ reassembly in Caco-2 cells.
Asunto(s)
Toxinas Bacterianas , Quitosano , Clostridioides difficile , Humanos , Toxinas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Células CACO-2 , Quitosano/farmacología , Clostridioides/metabolismo , Alginatos/farmacología , Ocludina , Enterotoxinas/toxicidad , Enterotoxinas/metabolismo , Oligosacáridos/farmacología , Oligosacáridos/metabolismoRESUMEN
Mucins play an essential role in protecting the respiratory tract against microbial infections while also acting as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance. Transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on epithelial entry of SARS-CoV-2. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of terminal mucin glycans sialic acid and fucose from the epithelial surface did not impact viral entry. In Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are located to the apical surface in close proximity and StcE treatment results in enhanced binding of purified spike protein. Both MUC1 and MUC16 are expressed on the surface of human organoid-derived air-liquid interface (ALI) differentiated airway cultures and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 replication. In these cultures, MUC1 was highly expressed in non-ciliated cells while MUC16 was enriched in goblet cells. In conclusion, the glycosylated extracellular domains of different transmembrane mucins might have similar protective functions in different respiratory cell types by restricting SARS-CoV-2 binding and entry.
Asunto(s)
COVID-19 , Mucinas , Humanos , Mucinas/metabolismo , Enzima Convertidora de Angiotensina 2 , SARS-CoV-2/metabolismo , Antígeno Ca-125/metabolismo , Pulmón/metabolismo , PolisacáridosRESUMEN
Dysbiosis of vaginal microbiota is associated with increased HIV-1 acquisition, but the underlying cellular mechanisms remain unclear. Vaginal Langerhans cells (LCs) protect against mucosal HIV-1 infection via autophagy-mediated degradation of HIV-1. As LCs are in continuous contact with bacterial members of the vaginal microbiome, we investigated the impact of commensal and dysbiosis-associated vaginal (an)aerobic bacterial species on the antiviral function of LCs. Most of the tested bacteria did not affect the HIV-1 restrictive function of LCs. However, Prevotella timonensis induced a vast uptake of HIV-1 by vaginal LCs. Internalized virus remained infectious for days and uptake was unaffected by antiretroviral drugs. P. timonensis-exposed LCs efficiently transmitted HIV-1 to target cells both in vitro and ex vivo. Additionally, P. timonensis exposure enhanced uptake and transmission of the HIV-1 variants that establish infection after sexual transmission, the so-called Transmitted Founder variants. Our findings, therefore, suggest that P. timonensis might set the stage for enhanced HIV-1 susceptibility during vaginal dysbiosis and advocate targeted treatment of P. timonensis during bacterial vaginosis to limit HIV-1 infection.
Asunto(s)
Infecciones por VIH , VIH-1 , Antivirales , Disbiosis , Femenino , Humanos , Células de Langerhans , PrevotellaRESUMEN
Fucosidases are associated with several pathological conditions and play an important role in the health of the human gut. For example, fucosidases have been shown to be indicators and/or involved in hepatocellular carcinoma, breast cancer, and helicobacter pylori infections. A prerequisite for the detection and profiling of fucosidases is the formation of a specific covalent linkage between the enzyme of interest and the activity-based probe (ABP). The most commonly used fucosidase ABPs are limited to only one of the classes of fucosidases, the retaining fucosidases. New approaches are needed that allow for the detection of the second class of fucosidases, the inverting type. Here, we report an ortho-quinone methide-based probe with an azide mini-tag that selectively labels both retaining and inverting bacterial α-l-fucosidases. Mass spectrometry-based intact protein and sequence analysis of a probe-labeled bacterial fucosidase revealed almost exclusive single labeling at two specific tryptophan residues outside of the active site. Furthermore, the probe could detect and image extracellular fucosidase activity on the surface of live bacteria.
Asunto(s)
Infecciones por Helicobacter , Helicobacter pylori , Indolquinonas , Helicobacter pylori/metabolismo , Humanos , alfa-L-Fucosidasa/metabolismoRESUMEN
At the intestinal host-microbe interface, the transmembrane mucin MUC1 can function as a physical barrier as well as a receptor for bacteria. MUC1 also influences epithelial cell morphology and receptor function. Various bacterial pathogens can exploit integrins to infect eukaryotic cells. It is yet unclear whether MUC1 influences the interaction of bacteria with integrins. We used Escherichia coli expressing the invasin (inv) protein of Yersinia pseudotuberculosis (E. coli inv) to assess the effects of MUC1 on ß1 integrin (ITGB1)-mediated bacterial invasion. Our results show that expression of full-length MUC1 does not yield a physical barrier but slightly enhances E. coli inv uptake. Enzymatic removal of the MUC1 extracellular domain (ED) using a secreted protease of C1 esterase inhibitor (StcE) of pathogenic Escherichia coli had no additional effect on E. coli inv invasion. In contrast, expression of a truncated MUC1 that lacks the cytoplasmic tail (CT) reduced bacterial entry substantially. Substitution of tyrosine residues in the MUC1 CT also reduced bacterial uptake, while deletion of the C-terminal half of the cytoplasmic tail only had a minor effect, pointing to a regulatory role of tyrosine phosphorylation and the N-terminal region of the MUC1 CT in integrin-mediated uptake process. Unexpectedly, StcE removal of the ED in MUC1-ΔCT cells reversed the block in bacterial invasion. Together, these findings indicate that MUC1 can facilitate ß1-integrin-mediated bacterial invasion by a concerted action of the large glycosylated extracellular domain and the membrane-juxtaposed cytoplasmic tail region.IMPORTANCE Bacteria can exploit membrane receptor integrins for cellular invasion, either by direct binding of bacterial adhesins or utilizing extracellular matrix components. MUC1 is a large transmembrane glycoprotein expressed by most epithelial cells that can have direct defensive or receptor functions at the host-microbe interface and is involved in facilitating integrin clustering. We investigated the role of epithelial MUC1 on ß1 integrin-mediated bacterial invasion. We discovered that MUC1 does not act as a barrier but facilitates bacterial entry through ß1 integrins. This process involves a concerted action of the MUC1 O-glycosylated extracellular domain and cytoplasmic tail. Our findings add a new dimension to the complexity of bacterial invasion mechanisms and provide novel insights into the distinct functions of MUC1 domains at the host-microbe interface.
Asunto(s)
Células Epiteliales/microbiología , Escherichia coli/metabolismo , Integrina beta1/metabolismo , Mucina-1/metabolismo , Yersinia pseudotuberculosis/genética , Adhesinas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/patogenicidad , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Células HeLa , Humanos , Integrina beta1/genética , Mucina-1/genéticaRESUMEN
GH29 α-l-fucosidases catalyze hydrolysis of terminal α-l-fucosyl linkages with varying specificity and are expressed by prominent members of the human gut microbiota. Both homeostasis and dysbiosis at the human intestinal microbiota interface have been correlated with altered fucosidase activity. Herein we describe the development of a 2-deoxy-2-fluoro fucosyl fluoride derivative with an azide mini-tag as an activity-based probe (ABP) for selective in vitro labelling of GH29 α-l-fucosidases. Only catalytically active fucosidases are inactivated by this ABP, allowing their functionalization with a biotin reporter group via the CuAAC reaction and subsequent in-gel detection at nanogram levels. The ABP we present here is shown to be active against a GH29 α-l-fucosidase from Bacteroides fragilis and capable of labeling two other GH29 α-l-fucosidases with different linkage specificity, illustrating its broader utility. This novel ABP is a valuable addition to the toolbox of fucosidase probes by allowing identification and functional studies of the wide variety of GH29 fucosidases, including those in the gut microbiota.
Asunto(s)
Fucosa/química , Sondas Moleculares/química , alfa-L-Fucosidasa/análisis , Bacteroides fragilis/enzimología , Fucosa/análogos & derivados , Fucosa/farmacología , Microbioma Gastrointestinal , Humanos , Sondas Moleculares/síntesis química , Sondas Moleculares/farmacología , Estructura Molecular , alfa-L-Fucosidasa/antagonistas & inhibidores , alfa-L-Fucosidasa/metabolismoRESUMEN
Mucus plays a pivotal role in protecting the respiratory tract against microbial infections. It acts as a primary contact site to entrap microbes and facilitates their removal from the respiratory tract via the coordinated beating of motile cilia. The major components of airway mucus are heavily O-glycosylated mucin glycoproteins, divided into gel-forming mucins and transmembrane mucins. The gel-forming mucins MUC5AC and MUC5B are the primary structural components of airway mucus, and they enable efficient clearance of pathogens by mucociliary clearance. MUC5B is constitutively expressed in the healthy airway, whereas MUC5AC is upregulated in response to inflammatory challenge. MUC1, MUC4, and MUC16 are the three major transmembrane mucins of the respiratory tracts which prevent microbial invasion, can act as releasable decoy receptors, and activate intracellular signal transduction pathways. Pathogens have evolved virulence factors such as adhesins that facilitate interaction with specific mucins and mucin glycans, for example, terminal sialic acids. Mucin expression and glycosylation are dependent on the inflammatory state of the respiratory tract and are directly regulated by proinflammatory cytokines and microbial ligands. Gender and age also impact mucin glycosylation and expression through the female sex hormone estradiol and age-related downregulation of mucin production. Here, we discuss what is currently known about the role of respiratory mucins and their glycans during bacterial and viral infections of the airways and their relevance for the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Understanding the impact of microbe-mucin interaction in the respiratory tract could inspire the development of novel therapies to boost mucosal defense and combat respiratory infections.
Asunto(s)
Glicoproteínas/metabolismo , Mucinas/metabolismo , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/virología , Infecciones Bacterianas/metabolismo , COVID-19/virología , Glicosilación , Humanos , Mucina 5AC/metabolismo , Mucina-1/metabolismo , Mucina 5B/metabolismo , Infecciones del Sistema Respiratorio/prevención & control , SARS-CoV-2/patogenicidad , Virosis/metabolismoRESUMEN
The enteropathogenic bacterium, Campylobacter jejuni, was considered to be non-saccharolytic, but recently it emerged that l-fucose plays a central role in C. jejuni virulence. Half of C. jejuni clinical isolates possess an operon for l-fucose utilisation. In the intestinal tract, l-fucose is abundantly available in mucin O-linked glycan structures, but C. jejuni lacks a fucosidase enzyme essential to release the l-fucose. We set out to determine how C. jejuni can gain access to these intestinal l-fucosides. Growth of the fuc + C. jejuni strains, 129,108 and NCTC 11168, increased in the presence of l-fucose while fucose permease knockout strains did not benefit from additional l-fucose. With fucosidase assays and an activity-based probe, we confirmed that Bacteriodes fragilis, an abundant member of the intestinal microbiota, secretes active fucosidases. In the presence of mucins, C. jejuni was dependent on B. fragilis fucosidase activity for increased growth. Campylobacter jejuni invaded Caco-2 intestinal cells that express complex O-linked glycan structures that contain l-fucose. In infection experiments, C. jejuni was more invasive in the presence of B. fragilis and this increase is due to fucosidase activity. We conclude that C. jejuni fuc + strains are dependent on exogenous fucosidases for increased growth and invasion.
Asunto(s)
Bacteroides fragilis/enzimología , Campylobacter jejuni/crecimiento & desarrollo , Campylobacter jejuni/patogenicidad , Fucosa/metabolismo , Mucinas/metabolismo , alfa-L-Fucosidasa/metabolismo , Células CACO-2 , Campylobacter jejuni/genética , Humanos , Interacciones Microbianas/fisiología , Virulencia , alfa-L-Fucosidasa/biosíntesisRESUMEN
Dysbiosis of the vaginal microbiome as a result of overgrowth of anaerobic bacteria leads to bacterial vaginosis (BV) which is associated with increased inflammation in the genital mucosa. Moreover, BV increases susceptibility to sexual transmitted infections (STIs) and is associated with adverse pregnancy outcomes. It remains unclear how specific vaginal aerobic and anaerobic bacteria affect health and disease. We selected different vaginal bacteria ranging from true commensals to species associated with dysbiosis and investigated their effects on activation of dendritic cells (DCs). Commensal Lactobacilli crispatus did not induce DC maturation nor led to production of pro-inflammatory cytokines. In contrast, BV-associated bacteria Megasphaera elsdenii and Prevotella timonensis induced DC maturation and increased levels of pro-inflammatory cytokines. Notably, DCs stimulated with Prevotella timonensis suppressed Th2 responses and induced Th1 skewing, typically associated with preterm birth. In contrast, Lactobacillus crispatus and Megasphaera elsdenii did not affect Th cell polarization. These results strongly indicate that the interaction of vaginal bacteria with mucosal DCs determines mucosal inflammation and we have identified the anaerobic bacterium Prevotella timonensis as a strong inducer of inflammatory responses. Specifically targeting these inflammation-inducing bacteria might be a therapeutic strategy to prevent BV and associated risks in STI susceptibility and preterm birth.
Asunto(s)
Células Dendríticas/inmunología , Disbiosis/complicaciones , Megasphaera elsdenii/inmunología , Prevotella/inmunología , Vaginosis Bacteriana/inmunología , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/metabolismo , Disbiosis/inmunología , Disbiosis/microbiología , Femenino , Humanos , Mediadores de Inflamación/metabolismo , Leucocitos Mononucleares , Prevotella/aislamiento & purificación , Cultivo Primario de Células , Vagina/citología , Vagina/inmunología , Vagina/microbiología , Vaginosis Bacteriana/microbiologíaRESUMEN
The cellular invasion machinery of the enteric pathogen Salmonella consists of a type III secretion system (T3SS) with injectable virulence factors that induce uptake by macropinocytosis. Salmonella invasion at the apical surface of intestinal epithelial cells is inefficient, presumably because of a glycosylated barrier formed by transmembrane mucins that prevents T3SS contact with host cells. We observed that Salmonella is capable of apical invasion of intestinal epithelial cells that express the transmembrane mucin MUC1. Knockout of MUC1 in HT29-MTX cells or removal of MUC1 sialic acids by neuraminidase treatment reduced Salmonella apical invasion but did not affect lateral invasion that is not hampered by a defensive barrier. A Salmonella deletion strain lacking the SiiE giant adhesin was unable to invade intestinal epithelial cells through MUC1. SiiE-positive Salmonella closely associated with the MUC1 layer at the apical surface, but invaded Salmonella were negative for the adhesin. Our findings uncover that the transmembrane mucin MUC1 is required for Salmonella SiiE-mediated entry of enterocytes via the apical route.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Mucina-1/fisiología , Infecciones por Salmonella/metabolismo , Proteínas Bacterianas , Línea Celular , Elonguina/metabolismo , Enterocitos , Células Epiteliales , Humanos , Mucina-1/genética , Mucina-1/metabolismo , Salmonella enterica/patogenicidad , Salmonella typhimurium/patogenicidad , Factores de VirulenciaRESUMEN
Mucosal surfaces line our body cavities and provide the interaction surface between commensal and pathogenic microbiota and the host. The barrier function of the mucosal layer is largely maintained by gel-forming mucin proteins that are secreted by goblet cells. In addition, mucosal epithelial cells express cell-bound mucins that have both barrier and signaling functions. The family of transmembrane mucins consists of diverse members that share a few characteristics. The highly glycosylated extracellular mucin domains inhibit invasion by pathogenic bacteria and can form a tight mesh structure that protects cells in harmful conditions. The intracellular tails of transmembrane mucins can be phosphorylated and connect to signaling pathways that regulate inflammation, cell-cell interactions, differentiation, and apoptosis. Transmembrane mucins play important roles in preventing infection at mucosal surfaces, but are also renowned for their contributions to the development, progression, and metastasis of adenocarcinomas. In general, transmembrane mucins seem to have evolved to monitor and repair damaged epithelia, but these functions can be highjacked by cancer cells to yield a survival advantage. This review presents an overview of the current knowledge of the functions of transmembrane mucins in inflammatory processes and carcinogenesis in order to better understand the diverse functions of these multifunctional proteins.
Asunto(s)
Células Epiteliales/metabolismo , Inflamación/inmunología , Proteínas de la Membrana/metabolismo , Mucinas/metabolismo , Membrana Mucosa/metabolismo , Neoplasias/inmunología , Animales , Células Epiteliales/inmunología , HumanosRESUMEN
Flagella are nanofibers that drive bacterial movement. The filaments are generally composed of thousands of tightly packed flagellin subunits with a terminal cap protein, named FliD. Here, we report that the FliD protein of the bacterial pathogen Campylobacter jejuni binds to host cells. Live-cell imaging and confocal microscopy showed initial contact of the bacteria with epithelial cells via the flagella tip. Recombinant FliD protein bound to the surface of intestinal epithelial cells in a dose-dependent fashion. Search for the FliD binding site on the host cell using cells with defined glycosylation defects indicated glycosaminoglycans as a putative target. Heparinase treatment of wild type cells and an excess of soluble heparin abolished FliD binding. Binding assays showed direct and specific binding of FliD to heparin. Addition of an excess of purified FliD or heparin reduced the attachment of viable C. jejuni to the host cells. The host cell binding domain of FliD was mapped to the central region of the protein. Overall, our results indicate that the C. jejuni flagellar tip protein FliD acts as an attachment factor that interacts with cell surface heparan sulfate glycosaminoglycan receptors.
Asunto(s)
Adhesión Bacteriana/fisiología , Proteínas Bacterianas/metabolismo , Campylobacter jejuni/metabolismo , Flagelos/metabolismo , Glicosaminoglicanos/metabolismo , Mucosa Intestinal/parasitología , Animales , Adhesión Bacteriana/efectos de los fármacos , Proteínas Bacterianas/genética , Sitios de Unión/fisiología , Células CHO , Infecciones por Campylobacter/microbiología , Infecciones por Campylobacter/patología , Línea Celular Tumoral , Cricetulus , Células Epiteliales/citología , Células Epiteliales/parasitología , Flagelina/metabolismo , Células HT29 , Liasa de Heparina/farmacología , Humanos , Mucosa Intestinal/citologíaRESUMEN
Short-chain fatty acids (SCFAs) are products of microbial fermentation that are important for intestinal epithelial health. Here, we describe that SCFAs have rapid and reversible effects on toll-like receptor (TLR) responses in epithelial cells. Incubation of HEK293 or HeLa epithelial cells with the SCFAs butyrate or propionate at physiological concentrations enhanced NF-κB activation induced by TLR5, TLR2/1, TLR4, and TLR9 agonists. NF-κB activation in response to tumor necrosis factor α (TNFα) was also increased by SCFAs. Comparative transcript analysis of HT-29 colon epithelial cells revealed that SCFAs enhanced TLR5-induced transcription of TNFα but dampened or even abolished the TLR5-mediated induction of IL-8 and monocyte chemotactic protein 1. SCFAs are known inhibitors of histone deacetylases (HDACs). Butyrate or propionate caused a rapid increase in histone acetylation in epithelial cells, similar to the small molecule HDAC inhibitor trichostatin A (TSA). TSA also mimicked the effects of SCFAs on TLR-NF-κB responses. This study shows that bacterial SCFAs rapidly alter the epigenetic state of host cells resulting in redirection of the innate immune response and selective reprograming of cytokine/chemokine expression.
RESUMEN
The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.
Asunto(s)
Candida albicans , Candidiasis/inmunología , Interacciones Huésped-Patógeno/inmunología , Fagocitosis/fisiología , Esfingolípidos/biosíntesis , Animales , Candida albicans/inmunología , Línea Celular , Cromatografía en Capa Delgada , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Modelos Animales de Enfermedad , Citometría de Flujo , Técnicas de Inactivación de Genes , Humanos , Espectrometría de Masas , RatonesRESUMEN
The major fungal pathogen of humans, Candida albicans, is exposed to reactive nitrogen and oxygen species following phagocytosis by host immune cells. In response to these toxins, this fungus activates potent anti-stress responses that include scavenging of reactive nitrosative and oxidative species via the glutathione system. Here we examine the differential roles of two glutathione recycling enzymes in redox homeostasis, stress adaptation and virulence in C. albicans: glutathione reductase (Glr1) and the S-nitrosoglutathione reductase (GSNOR), Fdh3. We show that the NADPH-dependent Glr1 recycles GSSG to GSH, is induced in response to oxidative stress and is required for resistance to macrophage killing. GLR1 deletion increases the sensitivity of C. albicans cells to H2O2, but not to formaldehyde or NO. In contrast, Fdh3 detoxifies GSNO to GSSG and NH3, and FDH3 inactivation delays NO adaptation and increases NO sensitivity. C. albicans fdh3â cells are also sensitive to formaldehyde, suggesting that Fdh3 also contributes to formaldehyde detoxification. FDH3 is induced in response to nitrosative, oxidative and formaldehyde stress, and fdh3Δ cells are more sensitive to killing by macrophages. Both Glr1 and Fdh3 contribute to virulence in the Galleria mellonella and mouse models of systemic infection. We conclude that Glr1 and Fdh3 play differential roles during the adaptation of C. albicans cells to oxidative, nitrosative and formaldehyde stress, and hence during the colonisation of the host. Our findings emphasise the importance of the glutathione system and the maintenance of intracellular redox homeostasis in this major pathogen.
Asunto(s)
Adaptación Fisiológica , Aldehído Oxidorreductasas , Candida albicans , Proteínas Fúngicas , Glutatión Reductasa , Estrés Oxidativo , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Animales , Candida albicans/enzimología , Candida albicans/genética , Candida albicans/patogenicidad , Candidiasis/enzimología , Candidiasis/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glutatión Reductasa/genética , Glutatión Reductasa/metabolismo , Humanos , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Óxido Nítrico/metabolismoRESUMEN
Membrane reshaping resides at the core of many important cellular processes, and among its mediators are the BAR (Bin, Amphiphysin, Rvs) domain-containing proteins. We have explored the diversity and function of the Rvs BAR proteins in Candida albicans and identified a novel family member, Rvs167-3 (orf19.1861). We show that Rvs167-3 specifically interacts with Rvs162 to form a stable BAR heterodimer able to bind liposomes in vitro. A second, distinct heterodimer is formed by the canonical BAR proteins Rvs161 and Rvs167. Purified Rvs161/Rvs167 complex also binds liposomes, indicating that C. albicans expresses two functional BAR heterodimers. We used live-cell imaging to localize green fluorescent protein (GFP)-tagged Rvs167-3 and Rvs167 and show that both proteins concentrate in small cortical spots. However, while Rvs167 strictly colocalizes with the endocytic marker protein Abp1, we do not observe any colocalization of Rvs167-3 with sites of endocytosis marked by Abp1. Furthermore, the rvs167-3Δ/Δ mutant is not defective in endocytosis and strains lacking Rvs167-3 or its partner Rvs162 do not display increased sensitivity to high salt concentrations or decreased cell wall integrity, phenotypes which have been observed for rvs167Δ/Δ and rvs161Δ/Δ strains and which are linked to endocytosis defects. Taken together, our results indicate different roles for the two BAR heterodimers in C. albicans: the canonical Rvs161/Rvs167 heterodimer functions in endocytosis, whereas the novel Rvs162/Rvs167-3 heterodimer seems not to be involved in this process. Nevertheless, despite their different roles, our phenotypic analysis revealed a genetic interaction between the two BAR heterodimers, suggesting that they may have related but distinct membrane-associated functions.
Asunto(s)
Candida albicans/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas Fúngicas/metabolismo , Candida albicans/metabolismo , Membrana Celular/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Endocitosis , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Estructura Terciaria de Proteína , Transporte de ProteínasRESUMEN
The commensal yeast Candida albicans is part of the human intestinal microflora and is considered a "pathobiont", a resident microbe with pathogenic potential yet harmless under normal conditions. The aim of this study was to investigate the effect of C. albicans on inflammation of the intestinal tract and the role of Bruton's tyrosine kinase (Btk). Btk is an enzyme that modulates downstream signaling of multiple receptors involved in innate and adaptive immunity, including the major anti-fungal receptor Dectin-1. Colitis was induced in wild type and Btk-/- mice by treatment with dextran sodium sulfate (DSS) and the gastrointestinal tract of selected treatment groups were then colonized with C. albicans. Colonization by C. albicans neither dampened nor exacerbated inflammation in wild type mice, but colon length and spleen weight were improved in Btk-deficient mice colonized with C. albicans. Neutrophil infiltration was comparable between wild type and Btk-/- mice, but the knockout mice displayed severely reduced numbers of macrophages in the colon during both DSS and DSS/Candida treatment. Smaller numbers and reduced responsiveness of Btk-/- macrophages might partially explain the improved colon length of Btk-/- mice as a result of Candida colonization. Surprisingly, DSS/Candida-treated Btk-/- animals had higher levels of certain pro-inflammatory cytokines and levels of the anti-inflammatory cytokine TGF-ß were reduced compared to wild type. A clustering and correlation analysis showed that for wild type animals, spleen TGF-ß and colon IL-10 and for Btk-/- spleen and colon levels of IL-17A best correlated with the inflammatory parameters. We conclude that in Btk-/- immunocompromised animals, colonization of the gastrointestinal tract by the commensal yeast C. albicans alters inflammatory symptoms associated with colitis.