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Bacteria in nature can exist in multicellular communities called biofilms. Biofilms also form in the course of many infections. Pseudomonas aeruginosa infections frequently involve biofilms, which contribute materially to the difficulty to treat these infections with antibiotic therapy. Many biofilm-related characteristics are controlled by the second messenger, cyclic-di-GMP, which is upregulated on surface contact. Among these factors is the exopolysaccharide Psl, which is a critically important component of the biofilm matrix. Here we describe the discovery of a P. aeruginosa bacteriophage, which we have called Clew-1, that directly binds to and uses Psl as a receptor. While this phage does not efficiently infect planktonically growing bacteria, it can disrupt P. aeruginosa biofilms and replicate in biofilm bacteria. We further demonstrate that the Clew-1 can reduce the bacterial burden in a mouse model of P. aeruginosa keratitis, which is characterized by the formation of a biofilm on the cornea. Due to its reliance on Psl for infection, Clew-1 does not actually form plaques on wild-type bacteria under standard in vitro conditions. This argues that our standard isolation procedures likely exclude bacteriophage that are adapted to using biofilm markers for infection. Importantly, the manner in which we isolated Clew-1 can be easily extended to other strains of P. aeruginosa and indeed other bacterial species, which will fuel the discovery of other biofilm-tropic bacteriophage and expand their therapeutic use.
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Assembly of NADPH oxidase 2 (NOX2) proteins in neutrophils plays an essential role in controlling microbial infections by producing high levels of reactive oxygen species (ROS). In contrast, the role of the Hv1 voltage-gated proton channel that is required for sustained NOX2 activity is less well characterized. We examined the role of Hv1 in a murine model of blinding Pseudomonas aeruginosa corneal infection and found that in contrast to C57BL/6 mice, Hvcn1 -/- mice exhibit an impaired ability to kill bacteria and regulate disease severity. In vitro, we used a novel Hv1 Inhibitor Flexible (HIF) to block ROS production by human and murine neutrophils and found that HIF inhibits ROS production in a dose-dependent manner following stimulation with PMA or infection with P. aeruginosa. Collectively, these findings demonstrate an important role for Hv1 on controlling bacterial growth in a clinically relevant bacterial infection model.
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Fusarium oxysporum is a cross-kingdom pathogen. While some strains cause disseminated fusariosis and blinding corneal infections in humans, others are responsible for devastating vascular wilt diseases in plants. To better understand the distinct adaptations of F. oxysporum to animal or plant hosts, we conducted a comparative phenotypic and genetic analysis of two strains: MRL8996 (isolated from a keratitis patient) and Fol4287 (isolated from a wilted tomato [Solanum lycopersicum]). In vivo infection of mouse corneas and tomato plants revealed that, while both strains cause symptoms in both hosts, MRL8996 caused more severe corneal ulceration and perforation in mice, whereas Fol4287 induced more pronounced wilting symptoms in tomato. In vitro assays using abiotic stress treatments revealed that the human pathogen MRL8996 was better adapted to elevated temperatures, whereas the plant pathogen Fol4287 was more tolerant of osmotic and cell wall stresses. Both strains displayed broad resistance to antifungal treatment, with MRL8996 exhibiting the paradoxical effect of increased tolerance to higher concentrations of the antifungal caspofungin. We identified a set of accessory chromosomes (ACs) and protein-encoding genes with distinct transposon profiles and functions, respectively, between MRL8996 and Fol4287. Interestingly, ACs from both genomes also encode proteins with shared functions, such as chromatin remodeling and post-translational protein modifications. Our phenotypic assays and comparative genomics analyses lay the foundation for future studies correlating genotype with phenotype and for developing targeted antifungals for agricultural and clinical uses.
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Patients with decreased levels of CD18 (ß2 integrins) suffer from life-threatening bacterial and fungal infections. CD11b, the α subunit of integrin CR3 (CD11b/CD18, αMß2), is essential for mice to fight against systemic Candida albicans infections. Live elongating C. albicans activates CR3 in immune cells. However, the hyphal ligands that activate CR3 are not well defined. Here, we discovered that the C. albicans Als family proteins are recognized by the I domain of CD11b in macrophages. This recognition synergizes with the ß-glucan-bound lectin-like domain to activate CR3, thereby promoting Syk signaling and inflammasome activation. Dectin-2 activation serves as the "outside-in signaling" for CR3 activation at the entry site of incompletely sealed phagosomes, where a thick cuff of F-actin forms to strengthen the local interaction. In vitro, CD18 partially contributes to IL-1ß release from dendritic cells induced by purified hyphal Als3. In vivo, Als3 is vital for C. albicans clearance in mouse kidneys. These findings uncover a novel family of ligands for the CR3 I domain that promotes fungal clearance.
Assuntos
Antígenos CD18 , Candidíase , Proteínas Fúngicas , Lectinas Tipo C , Macrófagos , Animais , Camundongos , beta-Glucanas/metabolismo , beta-Glucanas/imunologia , Candida albicans/imunologia , Candidíase/imunologia , Candidíase/microbiologia , Antígeno CD11b/metabolismo , Antígeno CD11b/imunologia , Antígenos CD18/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/imunologia , Lectinas Tipo C/metabolismo , Lectinas Tipo C/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Transdução de SinaisRESUMO
Macrophages infected with Gram-negative bacteria expressing Type III secretion system (T3SS) activate the NLRC4 inflammasome, resulting in Gasdermin D (GSDMD)-dependent, but GSDME independent IL-1ß secretion and pyroptosis. Here we examine inflammasome signaling in neutrophils infected with Pseudomonas aeruginosa strain PAO1 that expresses the T3SS effectors ExoS and ExoT. IL-1ß secretion by neutrophils requires the T3SS needle and translocon proteins and GSDMD. In macrophages, PAO1 and mutants lacking ExoS and ExoT (ΔexoST) require NLRC4 for IL-1ß secretion. While IL-1ß release from ΔexoST infected neutrophils is also NLRC4-dependent, infection with PAO1 is instead NLRP3-dependent and driven by the ADP ribosyl transferase activity of ExoS. Genetic and pharmacologic approaches using MCC950 reveal that NLRP3 is also essential for bacterial killing and disease severity in a murine model of P. aeruginosa corneal infection (keratitis). Overall, these findings reveal a function for ExoS ADPRT in regulating inflammasome subtype usage in neutrophils versus macrophages and an unexpected role for NLRP3 in P. aeruginosa keratitis.
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Doenças da Córnea , Pseudomonas aeruginosa , Animais , Camundongos , Inflamassomos , Neutrófilos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Gravidade do PacienteRESUMO
Pseudomonas aeruginosa is an important cause of dermal, pulmonary, and ocular disease. Our studies have focused on P. aeruginosa infections of the cornea (keratitis) as a major cause of blinding microbial infections. The infection leads to an influx of innate immune cells, with neutrophils making up to 90% of recruited cells during early stages. We previously reported that the proinflammatory cytokines IL-1α and IL-1ß were elevated during infection. Compared with wild-type (WT), infected Il1b-/- mice developed more severe corneal disease that is associated with impaired bacterial killing as a result of defective neutrophil recruitment. We also reported that neutrophils are an important source of IL-1α and IL-1ß, which peaked at 24 h postinfection. To examine the role of IL-1α compared with IL-1ß in P. aeruginosa keratitis, we inoculated corneas of C57BL/6 (WT), Il1a-/-, Il1b-/-, and Il1a-/-Il1b-/- (double-knockout) mice with 5 × 104 ExoS-expressing P. aeruginosa. Il1b-/- and double-knockout mice have significantly higher bacterial burden that was consistent with delayed neutrophil and monocyte recruitment to the corneas. Surprisingly, Il1a-/- mice had the opposite phenotype with enhanced bacteria clearance compared with WT mice. Although there were no significant differences in neutrophil recruitment, Il1a-/- neutrophils displayed a more proinflammatory transcriptomic profile compared to WT with elevations in C1q expression that likely caused the phenotypic differences observed. To our knowledge, our findings identify a novel, non-redundant role for IL-1α in impairing bacterial clearance.
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Candida albicans is a major opportunistic pathogen of humans. It can grow as morphologically distinct yeast, pseudohyphae and hyphae, and the ability to switch reversibly among different forms is critical for its virulence. The relationship between morphogenesis and innate immune recognition is not quite clear. Dectin-1 is a major C-type lectin receptor that recognizes ß-glucan in the fungal cell wall. C. albicans ß-glucan is usually masked by the outer mannan layer of the cell wall. Whether and how ß-glucan masking is differentially regulated during hyphal morphogenesis is not fully understood. Here we show that the endo-1,3-glucanase Eng1 is differentially expressed in yeast, and together with Yeast Wall Protein 1 (Ywp1), regulates ß-glucan exposure and Dectin-1-dependent immune activation of macrophage by yeast cells. ENG1 deletion results in enhanced Dectin-1 binding at the septa of yeast cells; while eng1 ywp1 yeast cells show strong overall Dectin-1 binding similar to hyphae of wild-type and eng1 mutants. Correlatively, hyphae of wild-type and eng1 induced similar levels of cytokines in macrophage. ENG1 expression and Eng1-mediated ß-glucan trimming are also regulated by antifungal drugs, lactate and N-acetylglucosamine. Deletion of ENG1 modulates virulence in the mouse model of hematogenously disseminated candidiasis in a Dectin-1-dependent manner. The eng1 mutant exhibited attenuated lethality in male mice, but enhanced lethality in female mice, which was associated with a stronger renal immune response and lower fungal burden. Thus, Eng1-regulated ß-glucan exposure in yeast cells modulates the balance between immune protection and immunopathogenesis during disseminated candidiasis.
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Candida albicans/patogenicidade , Candidíase/imunologia , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Virulência/fisiologia , beta-Glucanas/imunologia , Animais , Candida albicans/imunologia , Candida albicans/metabolismo , Candidíase/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , beta-Glucanas/metabolismoRESUMO
Neutrophils are an important source of interleukin (IL)-1ß and other cytokines because they are recruited to sites of infection and inflammation in high numbers. Although secretion of processed, bioactive IL-1ß by neutrophils is dependent on NLRP3 and Gasdermin D (GSDMD), IL-1α secretion by neutrophils has not been reported. In this study, we demonstrate that neutrophils produce IL-1α following injection of Aspergillus fumigatus spores that express cell-surface ß-glucan. Although IL-1α secretion by lipopolysaccharide (LPS)/ATP-activated macrophages and dendritic cells is GSDMD dependent, IL-1α secretion by ß-glucan-stimulated neutrophils occurs independently of GSDMD. Instead, we found that bioactive IL-1α is in exosomes that were isolated from cell-free media of ß-glucan-stimulated neutrophils. Further, the exosome inhibitor GW4869 significantly reduces IL-1α in extracellular vesicles (EVs) and total cell-free supernatant. Together, these findings identify neutrophils as a source of IL-1α and demonstrate a role for EVs, specifically exosomes, in neutrophil secretion of bioactive IL-1α.
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Vesículas Extracelulares/metabolismo , Interleucina-1alfa/metabolismo , Neutrófilos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , beta-Glucanas/metabolismo , Animais , Feminino , Humanos , Masculino , CamundongosRESUMO
The circadian clock coordinates a variety of immune responses with signals from the external environment to promote survival. We investigated the potential reciprocal relationship between the circadian clock and skin inflammation. We treated mice topically with the Toll-like receptor 7 (TLR7) agonist imiquimod (IMQ) to activate IFN-sensitive gene (ISG) pathways and induce psoriasiform inflammation. IMQ transiently altered core clock gene expression, an effect mirrored in human patient psoriatic lesions. In mouse skin 1 d after IMQ treatment, ISGs, including the key ISG transcription factor IFN regulatory factor 7 (Irf7), were more highly induced after treatment during the day than the night. Nuclear localization of phosphorylated-IRF7 was most prominently time-of-day dependent in epidermal leukocytes, suggesting that these cell types play an important role in the diurnal ISG response to IMQ. Mice lacking Bmal1 systemically had exacerbated and arrhythmic ISG/Irf7 expression after IMQ. Furthermore, daytime-restricted feeding, which affects the phase of the skin circadian clock, reverses the diurnal rhythm of IMQ-induced ISG expression in the skin. These results suggest a role for the circadian clock, driven by BMAL1, as a negative regulator of the ISG response, and highlight the finding that feeding time can modulate the skin immune response. Since the IFN response is essential for the antiviral and antitumor effects of TLR activation, these findings are consistent with the time-of-day-dependent variability in the ability to fight microbial pathogens and tumor initiation and offer support for the use of chronotherapy for their treatment.
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Ritmo Circadiano , Imunidade Inata/genética , Interferons/genética , Glicoproteínas de Membrana/genética , Pele/metabolismo , Receptor 7 Toll-Like/genética , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Imiquimode/farmacologia , Indutores de Interferon/farmacologia , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Interferons/metabolismo , Masculino , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pele/efeitos dos fármacos , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/metabolismoRESUMO
MITF, a gene that is mutated in familial melanoma and Waardenburg syndrome, encodes multiple isoforms expressed from alternative promoters that share common coding exons but have unique amino termini. It is not completely understood how these isoforms influence pigmentation in different tissues and how the expression of these independent isoforms of MITF is regulated. Here, we show that melanocytes express two isoforms of MITF, MITF-A and MITF-M. The expression of MITF-A is partially regulated by a newly identified retinoid enhancer element located upstream of the MITF-A promoter. Mitf-A knockout mice have only subtle changes in melanin accumulation in the hair and reduced Tyr expression in the eye. In contrast, Mitf-M-null mice have enlarged kidneys, lack neural crest-derived melanocytes in the skin, choroid, and iris stroma, yet maintain pigmentation within the retinal pigment epithelium and iris pigment epithelium of the eye. Taken together, these studies identify a critical role for MITF-M in melanocytes, a minor role for MITF-A in regulating pigmentation in the hair and Tyr expression in the eye, and a novel role for MITF-M in size control of the kidney.
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Homeostase , Fator de Transcrição Associado à Microftalmia/metabolismo , Pigmentação , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Olho/patologia , Células HEK293 , Homeostase/efeitos dos fármacos , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanócitos/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator de Transcrição Associado à Microftalmia/genética , Fenótipo , Pigmentação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Receptor alfa de Ácido Retinoico/metabolismo , Retinoides/farmacologiaRESUMO
Despite ongoing efforts and technology development, the contamination of medical device surfaces by disease-causing microbes remains problematic. Two approaches to producing antimicrobial surfaces are using antimicrobial materials and applying physical topography such as nanopatterns. In this work, we describe the use of physical topography on a soft hydrogel to control microbial growth. We demonstrate this approach by using chitosan hydrogel films with nanopillars having periodicities ranging from 300 to 500 nm. The flat hydrophilic chitosan films exhibit antimicrobial activity against the pathogenic bacteria Pseudomonas aeruginosa and filamentous fungi Fusarium oxysporum. The addition of nanopillars to the hydrogel surface further reduces the growth of P. aeruginosa and F. oxysporum up to â¼52 and â¼99%, respectively. Multiple modes of antimicrobial action appear to act synergistically to inhibit microbial growth on the nanopillar hydrogels. We verified that the strongly bactericidal and fungicidal nanopillared material retains biocompatibility to human epithelial cells with the MTT assay. The nanopillared material is a promising candidate for applications that require a biocompatible and antimicrobial film. The study demonstrates that taking advantage of multiple modes of antimicrobial action can effectively inhibit pathogenic microbial growth.
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During oropharyngeal candidiasis (OPC), Candida albicans proliferates and invades the superficial oral epithelium. Ephrin type-A receptor 2 (EphA2) functions as an oral epithelial cell ß-glucan receptor that triggers the production of proinflammatory mediators in response to fungal infection. Because EphA2 is also expressed by neutrophils, we investigated its role in neutrophil candidacidal activity during OPC. We found that EphA2 on stromal cells is required for the accumulation of phagocytes in the oral mucosa of mice with OPC. EphA2 on neutrophils is also central to host defense against OPC. The interaction of neutrophil EphA2 with serum-opsonized C. albicans yeast activates the MEK-ERK signaling pathway, leading to NADPH subunit p47phox site-specific phospho-priming. This priming increases intracellular reactive oxygen species production and enhances fungal killing. Thus, in neutrophils, EphA2 serves as a receptor for ß-glucans that augments Fcγ receptor-mediated antifungal activity and controls early fungal proliferation during OPC.
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Antifúngicos/metabolismo , Candida albicans/patogenicidade , Mucosa Bucal/patologia , Micoses/genética , Neutrófilos/metabolismo , Receptor EphA2/metabolismo , HumanosRESUMO
Aspergillus fumigatus is an important cause of pulmonary and systemic infections in immune compromised individuals, and of corneal ulcers and blindness in immune competent patients. To examine the role of chitin synthases in Aspergillus corneal infection, we analyzed Aspergillus mutants of chitin synthase family 1 and family 2, and found that compared with the parent strain, the quadruple mutants from both families were more readily killed by neutrophils in vitro, and that both also exhibited impaired hyphal growth in the cornea. Further, inhibition of chitin synthases using Nikkomycin Z enhanced neutrophil killing in vitro and in vivo in a murine model of A. fumigatus corneal infection. Acidic mammalian chitinase (AMCase) is mostly produced by macrophages in asthmatic lungs; however, we now demonstrate that neutrophils are a major source of AMCase, which inhibits hyphal growth. In A. fumigatus corneal infection, neutrophils are the major source of AMCase, and addition of AMCase inhibitors or adoptive transfer of neutrophils from AMCase-/- mice resulted in impaired hyphal killing. Together, these findings identify chitin synthases as important fungal virulence factors and neutrophil-derived AMCase as an essential mediator of host defense.
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Aspergilose/imunologia , Quitina Sintase/imunologia , Quitinases/metabolismo , Ceratite/imunologia , Neutrófilos/imunologia , Animais , Aspergillus fumigatus/imunologia , Aspergillus fumigatus/patogenicidade , Quitina Sintase/biossíntese , Humanos , Ceratite/metabolismo , Ceratite/microbiologia , Camundongos Endogâmicos C57BL , Neutrófilos/enzimologia , VirulênciaRESUMO
Filamentous fungi are invasive and multidrug resistant pathogens that commonly contaminate biomedical devices and implants. Once spherical fungal spores attach to a surface, they exhibit germ tube development, hyphal growth, and robust biofilm formation. Nanotopography found on plants, reptiles, and insect wings possess bactericidal properties during prokaryotic cell adhesion. Here, we demonstrate the application of biomimetic nanopillars that inhibit eukaryotic filamentous fungal growth and possess fungicidal properties. Furthermore, many spores on the nanopillars appeared deflated, while those on the flat surfaces remained spherical and intact. These antifungal phenomena provide promising applications in antifouling biointerfaces for biomedical devices and implants.