RESUMO
BACKGROUND: The cell envelope of Staphylococcus aureus contains two major secondary cell wall glycopolymers: capsular polysaccharide (CP) and wall teichoic acid (WTA). Both the CP and the WTA are attached to the cell wall and play distinct roles in S. aureus colonization, pathogenesis, and bacterial evasion of host immune defenses. OBJECTIVE: We aimed to investigate whether CP interferes with WTA-mediated properties. METHODS: Strains with natural heterogeneous expression of CP, strains with homogeneous high CP expression and CP-deficient strains were compared to WTA deficient controls regarding WTA dependent phage binding, cell adhesion, IgG deposition, and virulence in vivo. RESULTS: WTA-mediated phage adsorption, specific antibody deposition and cell adhesion were negatively correlated with CP expression. WTA, but not CP, enhanced the bacterial burden in a mouse abscess model, while CP overexpression resulted in intermediate virulence in vivo. CONCLUSIONS: CP protects the bacteria from WTA-dependent opsonization and phage binding. This protection comes at the cost of diminished adhesion to host cells. The highly complex regulation and mostly heterogeneous expression of CP has probably evolved to ensure the survival and optimal physiological adaptation of the bacterial population as a whole.
RESUMO
Wall teichoic acids (WTAs) are important components of the cell wall of the opportunistic Gram-positive bacterium Staphylococcus aureus. WTAs are composed of repeating ribitol phosphate (RboP) residues that are decorated with d-alanine and N-acetyl-d-glucosamine (GlcNAc) modifications, in a seemingly random manner. These WTA-modifications play an important role in shaping the interactions of WTA with the host immune system. Due to the structural heterogeneity of WTAs, it is impossible to isolate pure and well-defined WTA molecules from bacterial sources. Therefore, here synthetic chemistry to assemble a broad library of WTA-fragments, incorporating all possible glycosylation modifications (α-GlcNAc at the RboP C4; ß-GlcNAc at the RboP C4; ß-GlcNAc at the RboP C3) described for S. aureus WTAs, is reported. DNA-type chemistry, employing ribitol phosphoramidite building blocks, protected with a dimethoxy trityl group, was used to efficiently generate a library of WTA-hexamers. Automated solid phase syntheses were used to assemble a WTA-dodecamer and glycosylated WTA-hexamer. The synthetic fragments have been fully characterized and diagnostic signals were identified to discriminate the different glycosylation patterns. The different glycosylated WTA-fragments were used to probe binding of monoclonal antibodies using WTA-functionalized magnetic beads, revealing the binding specificity of these WTA-specific antibodies and the importance of the specific location of the GlcNAc modifications on the WTA-chains.
Assuntos
Infecções Estafilocócicas , Ácidos Teicoicos , Parede Celular/metabolismo , Glicosilação , Humanos , Staphylococcus aureus/metabolismoRESUMO
Staphylococcus aureus is a common skin commensal but is also associated with various skin and soft tissue pathologies. Upon invasion, S. aureus is detected by resident innate immune cells through pattern-recognition receptors (PRRs), although a comprehensive understanding of the specific molecular interactions is lacking. Recently, we demonstrated that the PRR langerin (CD207) on epidermal Langerhans cells senses the conserved ß-1,4-linked N-acetylglucosamine (GlcNAc) modification on S. aureus wall teichoic acid (WTA), thereby increasing skin inflammation. Interestingly, the S. aureus ST395 lineage as well as certain species of coagulase-negative staphylococci (CoNS) produce a structurally different WTA molecule, consisting of poly-glycerolphosphate with α-O-N-acetylgalactosamine (GalNAc) residues, which are attached by the glycosyltransferase TagN. Here, we demonstrate that S. aureus ST395 strains interact with the human Macrophage galactose-type lectin (MGL; CD301) receptor, which is expressed by dendritic cells and macrophages in the dermis. MGL bound S. aureus ST395 in a tagN- and GalNAc-dependent manner but did not interact with different tagN-positive CoNS species. However, heterologous expression of Staphylococcus lugdunensis tagN in S. aureus conferred phage infection and MGL binding, confirming the role of this CoNS enzyme as GalNAc-transferase. Functionally, the detection of GalNAc on S. aureus ST395 WTA by human monocyte-derived dendritic cells significantly enhanced cytokine production. Together, our findings highlight differential recognition of S. aureus glycoprofiles by specific human innate receptors, which may affect downstream adaptive immune responses and pathogen clearance.
Assuntos
Parede Celular/metabolismo , Células Dendríticas/imunologia , Glicosiltransferases/metabolismo , Lectinas Tipo C/imunologia , Staphylococcus aureus/enzimologia , Ácidos Teicoicos/química , Acetilgalactosamina/análogos & derivados , Acetilgalactosamina/química , Citocinas/metabolismo , Derme/imunologia , Derme/microbiologia , Glicerofosfatos/química , Glicosiltransferases/genética , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/imunologia , Mutação , Staphylococcus aureus/química , Staphylococcus aureus/imunologia , Staphylococcus aureus/patogenicidade , Staphylococcus lugdunensis/química , Staphylococcus lugdunensis/enzimologiaRESUMO
The gut microbiome contributes to inflammatory bowel disease (IBD), in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD, and dysfunctional epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. We therefore hypothesized that the combination of ER and mitochondrial stresses significantly disrupt epithelial barrier function. Here, we treated human colonic biopsies, epithelial colonoids, and epithelial cells with an uncoupler of oxidative phosphorylation, dinitrophenol (DNP), with or without the ER stressor tunicamycin and assessed epithelial barrier function by monitoring internalization and translocation of commensal bacteria. We also examined barrier function and colitis in mice exposed to dextran sodium sulfate (DSS) or DNP and co-treated with DAPK6, an inhibitor of death-associated protein kinase 1 (DAPK1). Contrary to our hypothesis, induction of ER stress (i.e. the unfolded protein response) protected against decreased barrier function caused by the disruption of mitochondrial function. ER stress did not prevent DNP-driven uptake of bacteria; rather, specific mobilization of the ATF6 arm of ER stress and recruitment of DAPK1 resulted in enhanced autophagic killing (xenophagy) of bacteria. Of note, epithelia with a Crohn's disease-susceptibility mutation in the autophagy gene ATG16L1 exhibited less xenophagy. Systemic delivery of the DAPK1 inhibitor DAPK6 increased bacterial translocation in DSS- or DNP-treated mice. We conclude that promoting ER stress-ATF6-DAPK1 signaling in transporting enterocytes counters the transcellular passage of bacteria evoked by dysfunctional mitochondria, thereby reducing the potential for metabolic stress to reactivate or perpetuate inflammation.
Assuntos
Proteínas Quinases Associadas com Morte Celular/metabolismo , Estresse do Retículo Endoplasmático , Mitocôndrias/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Idoso , Animais , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Feminino , Humanos , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Permeabilidade , Tunicamicina/farmacologiaRESUMO
BACKGROUND: Respiratory mucosal host defense relies on the production of secretory IgA (sIgA) antibodies, but we currently lack a fundamental understanding of how sIgA is induced by contact with microbes and how such immune responses may vary between humans. Defense of the nasal mucosal barrier through sIgA is critical to protect from infection and to maintain homeostasis of the microbiome, which influences respiratory disorders and hosts opportunistic pathogens. METHODS: We applied IgA-seq analysis to nasal microbiota samples from male and female healthy volunteers, to identify which bacterial genera and species are targeted by sIgA on the level of the individual host. Furthermore, we used nasal sIgA from the same individuals in sIgA deposition experiments to validate the IgA-seq outcomes. CONCLUSIONS: We observed that the amount of sIgA secreted into the nasal mucosa by the host varied substantially and was negatively correlated with the bacterial density, suggesting that nasal sIgA limits the overall bacterial capacity to colonize. The interaction between mucosal sIgA antibodies and the nasal microbiota was highly individual with no obvious differences between potentially invasive and non-invasive bacterial species. Importantly, we could show that for the clinically relevant opportunistic pathogen and frequent nasal resident Staphylococcus aureus, sIgA reactivity was in part the result of epitope-independent interaction of sIgA with the antibody-binding protein SpA through binding of sIgA Fab regions. This study thereby offers a first comprehensive insight into the targeting of the nasal microbiota by sIgA antibodies. It thereby helps to better understand the shaping and homeostasis of the nasal microbiome by the host and may guide the development of effective mucosal vaccines against bacterial pathogens. Video Abstract.
Assuntos
Imunoglobulina A Secretora , Microbiota , Humanos , Feminino , Masculino , Imunoglobulina A Secretora/metabolismo , Mucosa Nasal , Microbiota/fisiologiaRESUMO
The success of Staphylococcus aureus as a major cause for endovascular infections depends on effective interactions with blood-vessel walls. We have previously shown that S. aureus uses its wall teichoic acid (WTA), a surface glycopolymer, to attach to endothelial cells. However, the endothelial WTA receptor remained unknown. We show here that the endothelial oxidized low-density lipoprotein receptor 1 (LOX-1) interacts with S. aureus WTA and permits effective binding of S. aureus to human endothelial cells. Purified LOX-1 bound to isolated S. aureus WTA. Ectopic LOX-1 expression led to increased binding of S. aureus wild type but not of a WTA-deficient mutant to a cell line, and LOX-1 blockage prevented S. aureus binding to endothelial cells. Moreover, WTA and LOX-1 expression levels correlated with the efficacy of the S. aureus-endothelial interaction. Thus, LOX-1 is an endothelial ligand for S. aureus, whose blockage may help to prevent or treat severe endovascular infections.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Humanos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Células Endoteliais , Ácidos Teicoicos/metabolismo , Receptores Depuradores/metabolismo , Receptores Depuradores Classe E/genética , Receptores Depuradores Classe E/metabolismoRESUMO
Staphylococcus aureus is the leading cause of skin and soft tissue infections. It remains incompletely understood how skin-resident immune cells respond to invading S. aureus and contribute to an effective immune response. Langerhans cells (LCs), the only professional antigen-presenting cell type in the epidermis, sense S. aureus through their pattern-recognition receptor langerin, triggering a proinflammatory response. Langerin recognizes the ß-1,4-linked N-acetylglucosamine (ß1,4-GlcNAc) but not α-1,4-linked GlcNAc (α1,4-GlcNAc) modifications, which are added by dedicated glycosyltransferases TarS and TarM, respectively, on the cell wall glycopolymer wall teichoic acid (WTA). Recently, an alternative WTA glycosyltransferase, TarP, was identified, which also modifies WTA with ß-GlcNAc but at the C-3 position (ß1,3-GlcNAc) of the WTA ribitol phosphate (RboP) subunit. Here, we aimed to unravel the impact of ß-GlcNAc linkage position for langerin binding and LC activation. Using genetically modified S. aureus strains, we observed that langerin similarly recognized bacteria that produce either TarS- or TarP-modified WTA, yet tarP-expressing S. aureus induced increased cytokine production and maturation of in vitro-generated LCs compared to tarS-expressing S. aureus. Chemically synthesized WTA molecules, representative of the different S. aureus WTA glycosylation patterns, were used to identify langerin-WTA binding requirements. We established that ß-GlcNAc is sufficient to confer langerin binding, thereby presenting synthetic WTA molecules as a novel glycobiology tool for structure-binding studies and for elucidating S. aureus molecular pathogenesis. Overall, our data suggest that LCs are able to sense all ß-GlcNAc-WTA producing S. aureus strains, likely performing an important role as first responders upon S. aureus skin invasion.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Humanos , Células de Langerhans , Polissacarídeos , Staphylococcus aureus/genética , Ácidos TeicoicosRESUMO
Staphylococcus aureus is a major opportunistic human pathogen that frequently causes disease in community and hospital settings. Nasal colonization is an important risk factor for developing invasive disease. Cell wall-associated glycopolymers called wall teichoic acids (WTAs) contribute to efficient nasal colonization by S. aureus. In addition, WTAs are key targets of the host immune system due to their accessibility and high abundance on the S. aureus cell surface. In this review we discuss the new insights into interactions between the host and S. aureus WTA and the implications of these interactions for preventative and therapeutic approaches against S. aureus-mediated disease.
Assuntos
Interações entre Hospedeiro e Microrganismos/fisiologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/metabolismo , Anticorpos Antibacterianos , Parede Celular/metabolismo , Humanos , Imunidade Inata , Terapia por Fagos , Infecções Estafilocócicas/prevenção & controle , Infecções Estafilocócicas/terapia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/imunologia , Ácidos Teicoicos/química , Ácidos Teicoicos/imunologia , VacinaçãoRESUMO
Antigen-presenting cells (APCs) are present throughout the human body-in tissues, at barrier sites and in the circulation. They are critical for processing external signals to instruct both local and systemic responses toward immune tolerance or immune defense. APCs express an extensive repertoire of pattern-recognition receptors (PRRs) to detect and transduce these signals. C-type lectin receptors (CLRs) comprise a subfamily of PRRs dedicated to sensing glycans, including those expressed by commensal and pathogenic bacteria. This review summarizes recent findings on the recognition of and responses to bacteria by membrane-expressed CLRs on different APC subsets, which are discussed according to the primary site of infection. Many CLR-bacterial interactions promote bacterial clearance, whereas other interactions are exploited by bacteria to enhance their pathogenic potential. The discrimination between protective and virulence-enhancing interactions is essential to understand which interactions to target with new prophylactic or treatment strategies. CLRs are also densely concentrated at APC dendrites that sample the environment across intact barrier sites. This suggests an-as yet-underappreciated role for CLR-mediated recognition of microbiota-produced glycans in maintaining tolerance at barrier sites. In addition to providing a concise overview of identified CLR-bacteria interactions, we discuss the main challenges and potential solutions for the identification of new CLR-bacterial interactions, including those with commensal bacteria, and for in-depth structure-function studies on CLR-bacterial glycan interactions. Finally, we highlight the necessity for more relevant tissue-specific in vitro, in vivo and ex vivo models to develop therapeutic applications in this area.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Infecções Bacterianas/imunologia , Lectinas Tipo C , Polissacarídeos , Bactérias , HumanosRESUMO
Langerhans cells are key sentinel cells of the skin and mucosal lining. They sense microorganisms through their repertoire of pattern-recognition receptors to mount and direct appropriate immune responses. We recently demonstrated that human Langerhans cells interact with the Gram-positive pathogen Staphylococcus aureus through the Langerhans cell-specific receptor langerin (CD207). It was previously hypothesized that two linked single nucleotide polymorphisms (SNPs; N288D and K313I) in the carbohydrate recognition domain of langerin would affect interaction with microorganisms. We show that recognition of S. aureus by recombinant langerin molecules is abrogated in the co-inheriting SNP variant, which is mainly explained by the N288D SNP and further enhanced by K313I. Moreover, introduction of SNP N288D in ectopically-expressed langerin affected cellular distribution of the receptor such that langerin displayed enhanced plasma membraneexpression. Despite this increased binding of S. aureus by the langerin double SNP variant, uptake of bacteria by this langerin variant was compromised. Our findings indicate that in a proportion of the human population, the recognition and uptake of S. aureus by Langerhans cells may be affected, which could have important consequences for proper immune activation and S. aureus-associated disease.
Assuntos
Antígenos CD , Lectinas Tipo C , Lectinas de Ligação a Manose , Polimorfismo de Nucleotídeo Único , Infecções Estafilocócicas , Staphylococcus aureus/imunologia , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Células CHO , Cricetulus , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/imunologia , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/imunologia , Células THP-1RESUMO
Langerhans cells are a subset of dendritic cells residing in the epidermis of the human skin. As such, they are key mediators of immune regulation and have emerged as prime targets for novel transcutaneous cancer vaccines. Importantly, the induction of protective T cell immunity by these vaccines requires the efficient and specific delivery of both tumor-associated antigens and adjuvants. Langerhans cells uniquely express Langerin (CD207), an endocytic C-type lectin receptor. Here, we report the discovery of a specific, glycomimetic Langerin ligand employing a heparin-inspired design strategy and structural characterization by NMR spectroscopy and molecular docking. The conjugation of this glycomimetic to liposomes enabled the specific and efficient targeting of Langerhans cells in the human skin. We further demonstrate the doxorubicin-mediated killing of a Langerin+ monocyte cell line, highlighting its therapeutic and diagnostic potential in Langerhans cell histiocytosis, caused by the abnormal proliferation of Langerin+ myeloid progenitor cells. Overall, our delivery platform provides superior versatility over antibody-based approaches and novel modalities to overcome current limitations of dendritic cell-targeted immuno- and chemotherapy.
RESUMO
Staphylococcus aureus is a major cause of skin and soft tissue infections and aggravator of the inflammatory skin disease atopic dermatitis (AD [eczema]). Epicutaneous exposure to S. aureus induces Th17 responses through skin Langerhans cells (LCs), which paradoxically contribute to host defense but also to AD pathogenesis. The molecular mechanisms underlying the interaction between S. aureus and LCs are poorly understood. Here we demonstrate that human LCs directly interact with S. aureus through the pattern recognition receptor langerin (CD207). Human, but not mouse, langerin interacts with S. aureus through the conserved ß-N-acetylglucosamine (GlcNAc) modifications on wall teichoic acid (WTA), thereby discriminating S. aureus from other staphylococcal species. Importantly, the specific S. aureus WTA glycoprofile strongly influences the level of proinflammatory cytokines that are produced by in vitro-generated LCs. Finally, in a murine epicutaneous infection model, S. aureus strongly upregulated transcripts of Cxcl1, Il6, and Il17, which required the presence of both human langerin and WTA ß-GlcNAc. Our findings provide molecular insight into the unique proinflammatory capacities of S. aureus in relation to skin inflammation.IMPORTANCE The bacterium Staphylococcus aureus is an important cause of skin infections and is also associated with the occurrence and severity of eczema. Langerhans cells (LCs), a specific subset of skin immune cells, participate in the immune response to S. aureus, but it is yet unclear how LCs recognize S. aureus Therefore, we investigated the molecular mechanism underlying the interaction between LCs and S. aureus We identified that wall teichoic acid, an abundant polymer on the S. aureus surface, is recognized by langerin, a receptor unique to LCs. This interaction allows LCs to discriminate S. aureus from other related staphylococcal species and initiates a proinflammatory response similar to that observed in patients with eczema. Our data therefore provide important new insights into the relationship between S. aureus, LCs, and eczema.
Assuntos
Antígenos CD/genética , Antígenos de Superfície/genética , Células de Langerhans/imunologia , Lectinas Tipo C/genética , Lectinas de Ligação a Manose/genética , Infecções Estafilocócicas/imunologia , Ácidos Teicoicos/imunologia , Acetilglucosamina , Animais , Antígenos CD/imunologia , Antígenos de Superfície/imunologia , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Humanos , Inflamação , Interleucina-17/genética , Interleucina-17/imunologia , Lectinas Tipo C/imunologia , Lectinas de Ligação a Manose/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Pele/imunologia , Pele/microbiologia , Staphylococcus aureusRESUMO
Multiple cytosolic innate sensors form large signalosomes after activation, but this assembly needs to be tightly regulated to avoid accumulation of misfolded aggregates. We found that the eIF2α kinase heme-regulated inhibitor (HRI) controls NOD1 signalosome folding and activation through a process requiring eukaryotic initiation factor 2α (eIF2α), the transcription factor ATF4, and the heat shock protein HSPB8. The HRI/eIF2α signaling axis was also essential for signaling downstream of the innate immune mediators NOD2, MAVS, and TRIF but dispensable for pathways dependent on MyD88 or STING. Moreover, filament-forming α-synuclein activated HRI-dependent responses, which suggests that the HRI pathway may restrict toxic oligomer formation. We propose that HRI, eIF2α, and HSPB8 define a novel cytosolic unfolded protein response (cUPR) essential for optimal innate immune signaling by large molecular platforms, functionally homologous to the PERK/eIF2α/HSPA5 axis of the endoplasmic reticulum UPR.
Assuntos
Citosol/enzimologia , Citosol/imunologia , Imunidade Inata , Proteínas Serina-Treonina Quinases/fisiologia , Resposta a Proteínas não Dobradas/imunologia , Fator 4 Ativador da Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Linhagem Celular , Chaperona BiP do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/metabolismo , Fibroblastos , Proteínas de Choque Térmico/metabolismo , Humanos , Listeria/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Chaperonas Moleculares/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Proteína Adaptadora de Sinalização NOD1/química , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteínas Serina-Treonina Quinases/genética , Salmonella/imunologia , Infecções por Salmonella , Shigella/imunologia , Transdução de SinaisRESUMO
In physiological settings, the complement protein C3 is deposited on all bacteria, including invasive pathogens. However, because experimental host-bacteria systems typically use decomplemented serum to avoid the lytic action of complement, the impact of C3 coating on epithelial cell responses to invasive bacteria remains unexplored. Here, we demonstrate that following invasion, intracellular C3-positive Listeria monocytogenes is targeted by autophagy through a direct C3/ATG16L1 interaction, resulting in autophagy-dependent bacterial growth restriction. In contrast, Shigella flexneri and Salmonella Typhimurium escape autophagy-mediated growth restriction in part through the action of bacterial outer membrane proteases that cleave bound C3. Upon oral infection with Listeria, C3-deficient mice displayed defective clearance at the intestinal mucosa. Together, these results demonstrate an intracellular role of complement in triggering antibacterial autophagy and immunity against intracellular pathogens. Since C3 indiscriminately associates with foreign surfaces, the C3-ATG16L1 interaction may provide a universal mechanism of xenophagy initiation.