RESUMEN
Malassezia, the most abundant fungal commensal on the mammalian skin, has been linked to several inflammatory skin diseases such as atopic dermatitis, seborrheic dermatitis and psoriasis. This study reveals that epicutaneous application with Malassezia globosa (M. globosa) triggers skin inflammation in mice. RNA-sequencing of the resulting mouse lesions indicates activation of Interleukin-17 (IL-17) signaling and T helper 17 (Th17) cells differentiation pathways by M. globosa. Furthermore, our findings demonstrate a significant upregulation of IL-23, IL-23R, IL-17A, and IL-22 expressions, along with an increase in the proportion of Th17 and pathogenic Th17 cells in mouse skin exposed to M. globosa. In vitro experiments illustrate that M. globosa prompts human primary keratinocytes to secrete IL-23 via TLR2/MyD88/NF-κB signaling. This IL-23 secretion by keratinocytes is shown to be adequate for inducing the differentiation of pathogenic Th17 cells in the skin. Overall, these results underscore the significant role of Malassezia in exacerbating skin inflammation by stimulating IL-23 secretion by keratinocytes and promoting the differentiation of pathogenic Th17 cells.
Asunto(s)
Diferenciación Celular , Interleucina-23 , Queratinocitos , Malassezia , Células Th17 , Malassezia/inmunología , Queratinocitos/microbiología , Queratinocitos/inmunología , Queratinocitos/metabolismo , Células Th17/inmunología , Animales , Interleucina-23/metabolismo , Humanos , Ratones , Transducción de Señal , FN-kappa B/metabolismo , Receptor Toll-Like 2/metabolismo , Interleucina-17/metabolismo , Piel/microbiología , Piel/patología , Piel/inmunología , Modelos Animales de Enfermedad , Factor 88 de Diferenciación Mieloide/metabolismo , Factor 88 de Diferenciación Mieloide/genética , Células Cultivadas , Ratones Endogámicos C57BL , Interleucina-22RESUMEN
Malassezia species are commensal and opportunistic fungi found in human skin. All Malassezia species lack fatty acid synthesis genes and survive by utilizing several lipases to degrade and absorb fatty acids from external lipid sources, but little research has been done on their optimal active pH and temperature. Our skin protects itself from external stimuli and maintains homeostasis, involving bacteria and fungi such as Malassezia species that inhabit our skin. Hence, dysbiosis in the skin microbiome can lead to various skin diseases. The skin's pH is slightly acidic compared to neutral, and changes in pH can affect the metabolism of Malassezia species. We used keratinocyte cell lines to determine the effect of lipids bio-converted by Malassezia furfur, Malassezia japonica, and Malassezia yamatoensis under pH conditions similar to those of healthy skin. Lipids bio-converted from Malassezia species were associated with the regulation of transcripts related to inflammation, moisturizing, and promoting elasticity. Therefore, to determine the effect of pH on lipid metabolism in M. furfur, which is associated with seborrheic dermatitis, changes in biomass, lipid content, and fatty acid composition were determined. The results showed that pH 7 resulted in low growth and reduced lipid content, which had a negative impact on skin health. Given that bio-converted Malassezia-derived lipids show positive effects at the slightly acidic pH typical of healthy skin, it is important to study their effects on skin cells under various pH conditions. KEY POINTS: ⢠pH 6, Malassezia spp. bio-converted lipid have a positive effect on skin cells ⢠Malassezia spp. have different lipid, fatty acid, and growth depending on pH ⢠Malassezia spp. can play a beneficial role by secreting lipids to the outside.
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Ácidos Grasos , Queratinocitos , Metabolismo de los Lípidos , Malassezia , Piel , Malassezia/metabolismo , Concentración de Iones de Hidrógeno , Humanos , Ácidos Grasos/metabolismo , Queratinocitos/microbiología , Queratinocitos/metabolismo , Piel/microbiología , Línea Celular , Lípidos/análisis , Dermatitis Seborreica/microbiologíaRESUMEN
Caffeine affords several beneficial effects on human health, acting as an antioxidant, anti-inflammatory agent, and analgesic. Caffeine is widely used in cosmetics, but its antimicrobial activity has been scarcely explored, namely against skin infection agents. Dermatophytes are the most common fungal agents of human infection, mainly of skin infections. This work describes the in vitro effect of caffeine during keratinocyte infection by Trichophyton mentagrophytes, one of the most common dermatophytes. The results show that caffeine was endowed with antidermatophytic activity with a MIC, determined following the EUCAST standards, of 8 mM. Caffeine triggered a modification of the levels of two major components of the fungal cell wall, ß-(1,3)-glucan and chitin. Caffeine also disturbed the ultrastructure of the fungal cells, particularly the cell wall surface and mitochondria, and autophagic-like structures were observed. During dermatophyte-human keratinocyte interactions, caffeine prevented the loss of viability of keratinocytes and delayed spore germination. Overall, this indicates that caffeine can act as a therapeutic and prophylactic agent for dermatophytosis.
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Antifúngicos , Arthrodermataceae , Cafeína , Queratinocitos , Cafeína/farmacología , Queratinocitos/efectos de los fármacos , Queratinocitos/microbiología , Humanos , Antifúngicos/farmacología , Arthrodermataceae/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Pared Celular/efectos de los fármacos , Tiña/tratamiento farmacológico , Tiña/microbiología , Quitina/farmacología , Quitina/químicaRESUMEN
Acne is a chronic inflammatory skin condition that involves Cutibacterium acnes (C. acnes), which is classified into six main phylotypes (IA1, IA2, IB, IC, II and III). Acne development is associated with loss of C. acnes phylotype diversity, characterised by overgrowth of phylotype IA1 relative to other phylotypes. It was also shown that purified extracellular vesicles (EVs) secreted by C. acnes can induce an acne-like inflammatory response in skin models. We aimed to determine if the inflammatory profile of EVs secreted by C. acnes phylotype IA1 from an inflammatory acne lesion was different from C. acnes phylotype IA1 from normal skin, thus playing a direct role in the severity of inflammation. EVs were produced in vitro after culture of two clinical strains of C. acnes phylotype IA1, T5 from normal human skin and A47 from an inflammatory acne lesion, and then incubated with either human immortalised keratinocytes, HaCaT cells, or skin explants obtained from abdominoplasty. Subsequently, quantitative PCR (qPCR) was performed for human ß-defensin 2 (hBD2), cathelicidin (LL-37), interleukin (IL)-1ß, IL-6, IL-8, IL-17α and IL-36γ, and ELISA for IL-6, IL-8 and IL-17α. We found that EVs produced in vitro by C. acnes derived from inflammatory acne lesions significantly increased the pro-inflammatory cytokines and anti-microbial peptides at both transcriptional and protein levels compared with EVs derived from normal human skin. We show for the first time that C. acnes EVs from inflammatory acne play a crucial role in acne-associated inflammation in vitro and that C. acnes phylotype IA1 collected from inflammatory acne lesion and normal skin produce different EVs and inflammatory profiles in vitro.
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Acné Vulgar , Vesículas Extracelulares , Queratinocitos , Propionibacterium acnes , Humanos , Vesículas Extracelulares/metabolismo , Acné Vulgar/microbiología , Queratinocitos/microbiología , Piel/microbiología , Inflamación/microbiología , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Células HaCaT , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Citocinas/metabolismo , Interleucina-17/metabolismo , PropionibacteriaceaeRESUMEN
The Staphylococcus intermedius group (SIG) includes coagulase-positive staphylococci commonly found in animals. The taxonomic classification within the SIG has evolved with molecular techniques distinguishing five species. Despite their similarities, these species exhibit varied host affinities, with unclear implications for virulence and host interaction. This study aimed to investigate the presence of coagulase-positive staphylococci in pigeons and to detect genes encoding for selected virulence factors in isolated strains. Another goal was to determine the adhesion capabilities of randomly selected pigeon S. intermedius, S. delphini, and canine S. pseudintermedius strains to canine and pigeon corneocytes and their adhesion and invasion abilities to canine keratinocytes in vitro. In total, 121 coagulase-positive strains were isolated from domestic and feral pigeons. The most prevalent species were S. delphini B and S. intermedius in domestic and feral pigeons, respectively. We proved that pigeon strains carried genes encoding for exfoliative toxin SIET and leukotoxin Luk-I. Moreover, we found that S. intermedius showed higher adherence to pigeon than to canine corneocytes, aligning with its presumed natural host. No difference in adherence abilities of S. pseudintermedius to canine and pigeon corneocytes was observed. In this study, we also observed that S. pseudintermedius could successfully invade the canine keratinocytes, in contrary to S. delphini and S. intermedius. Moreover, only S. intermedius was not able to invade canine keratinocytes at all. These findings highlight the complex interplay between SIG bacteria, and their hosts, underscoring the need for further research to understand the mechanisms of host adaptation and pathogenicity within this group.
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Adhesión Bacteriana , Columbidae , Especificidad del Huésped , Queratinocitos , Infecciones Estafilocócicas , Staphylococcus intermedius , Staphylococcus , Factores de Virulencia , Animales , Columbidae/microbiología , Perros , Factores de Virulencia/genética , Staphylococcus/genética , Staphylococcus/patogenicidad , Staphylococcus/clasificación , Staphylococcus/aislamiento & purificación , Queratinocitos/microbiología , Virulencia/genética , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/veterinaria , Staphylococcus intermedius/genética , Staphylococcus intermedius/patogenicidad , Coagulasa/metabolismo , Coagulasa/genética , Exfoliatinas/genética , Exfoliatinas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Acne is one of the most common skin conditions worldwide, with multifactorial origins it affects areas of the skin with hair follicles and sebaceous glands that become clogged. Bacterial incidence aggravates treatment due to resistance to antimicrobial agents and production of virulence factors such as biofilm formation. Based on these information, this study aims to conduct in vitro evaluations of the antibacterial activity of essential oils (EOs), alone and in combination, against Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis in planktonic and biofilm forms. This study also assessed the anti-inflammatory potential (TNF-α) and the effects of EOs on the viability of human keratinocytes (HaCaT), murine fibroblasts (3T3-L1), and bone marrow-derived macrophages (BMDMs). Of all EOs tested, 13 had active action against P. acnes, 9 against S. aureus, and 9 against S. epidermidis at concentrations of 0.125-2.0 mg/mL. Among the most active plant species, a blend of essential oil (BEOs) was selected, with Cymbopogon martini (Roxb.) Will. Watson, Eugenia uniflora L., and Varronia curassavica Jacq., the latter due to its anti-inflammatory action. This BEOs showed higher inhibition rates when compared to chloramphenicol against S. aureus and S. epidermidis, and higher eradication rates when compared to chloramphenicol for the three target species. The BEOs did not affect the cell viability of cell lines evaluated, and the levels of TNF-α decreased. According to these results, the BEOs evaluated showed potential for the development of an alternative natural formulation for the treatment of acne.
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Acné Vulgar , Antibacterianos , Antiinflamatorios , Biopelículas , Queratinocitos , Macrófagos , Pruebas de Sensibilidad Microbiana , Aceites Volátiles , Propionibacterium acnes , Staphylococcus aureus , Staphylococcus epidermidis , Factor de Necrosis Tumoral alfa , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Aceites Volátiles/farmacología , Humanos , Acné Vulgar/microbiología , Acné Vulgar/tratamiento farmacológico , Ratones , Antiinflamatorios/farmacología , Antibacterianos/farmacología , Propionibacterium acnes/efectos de los fármacos , Staphylococcus epidermidis/efectos de los fármacos , Animales , Staphylococcus aureus/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Queratinocitos/microbiología , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Factor de Necrosis Tumoral alfa/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/microbiología , Supervivencia Celular/efectos de los fármacos , Células HaCaT , Línea Celular , Aceites de Plantas/farmacologíaRESUMEN
Staphylococcus aureus α-hemolysin (Hla) is a pore-forming toxin critical for the pathogenesis of skin and soft tissue infections, which causes the pathognomonic lesion of cutaneous necrosis (dermonecrosis) in mouse models. To determine the mechanism by which dermonecrosis develops during S. aureus skin infection, mice were given control serum, Hla-neutralizing antiserum, or an inhibitor of Hla receptor [A-disintegrin and metalloprotease 10 (ADAM10) inhibitor] followed by subcutaneous infection by S. aureus, and the lesions were evaluated using immunohistochemistry and immunofluorescence. Hla induced apoptosis in the vascular endothelium at 6 hours post-infection (hpi), followed by apoptosis in keratinocytes at 24 hpi. The loss of vascular endothelial (VE)-cadherin expression preceded the loss of epithelial-cadherin expression. Hla also induced hypoxia in the keratinocytes at 24 hpi following vascular injury. Treatment with Hla-neutralizing antibody or ADAM10 inhibitor attenuated early cleavage of VE-cadherin, cutaneous hypoxia, and dermonecrosis. These findings suggest that Hla-mediated vascular injury with cutaneous hypoxia underlies the pathogenesis of S. aureus-induced dermonecrosis.
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Proteína ADAM10 , Toxinas Bacterianas , Cadherinas , Proteínas Hemolisinas , Queratinocitos , Necrosis , Staphylococcus aureus , Animales , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/toxicidad , Ratones , Toxinas Bacterianas/toxicidad , Toxinas Bacterianas/metabolismo , Staphylococcus aureus/patogenicidad , Queratinocitos/microbiología , Queratinocitos/metabolismo , Proteína ADAM10/metabolismo , Cadherinas/metabolismo , Apoptosis , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Antígenos CD/metabolismo , Proteínas de la Membrana/metabolismo , Infecciones Cutáneas Estafilocócicas/microbiología , Infecciones Cutáneas Estafilocócicas/patología , Infecciones Cutáneas Estafilocócicas/inmunología , Piel/patología , Piel/microbiología , Femenino , Endotelio Vascular/patología , Endotelio Vascular/microbiología , Endotelio Vascular/metabolismo , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/patología , Modelos Animales de EnfermedadRESUMEN
Intracellular pathogens including Staphylococcus aureus contribute to the non-healing phenotype of chronic wounds. Lactobacilli, well known as beneficial bacteria, are also reported to modulate the immune system, yet their role in cutaneous immunity remains largely unknown. We explored the therapeutic potential of bacteria-free postbiotics, bioactive lysates of lactobacilli, to reduce intracellular S. aureus colonization and promote healing. Fourteen postbiotics derived from various lactobacilli species were screened, and Latilactobacillus curvatus BGMK2-41 was selected for further analysis based on the most efficient ability to reduce intracellular infection by S. aureus diabetic foot ulcer clinical isolate and S. aureus USA300. Treatment of both infected keratinocytes in vitro and infected human skin ex vivo with BGMK2-41 postbiotic cleared S. aureus. Keratinocytes treated in vitro with BGMK2-41 upregulated expression of antimicrobial response genes, of which DEFB4, ANG, and RNASE7 were also found upregulated in treated ex vivo human skin together with CAMP exclusively upregulated ex vivo. Furthermore, BGMK2-41 postbiotic treatment has a multifaceted impact on the wound healing process. Treatment of keratinocytes stimulated cell migration and the expression of tight junction proteins, while in ex vivo human skin BGMK2-41 increased expression of anti-inflammatory cytokine IL-10, promoted re-epithelialization, and restored the epidermal barrier via upregulation of tight junction proteins. Together, this provides a potential therapeutic approach for persistent intracellular S. aureus infections.
Asunto(s)
Queratinocitos , Lactobacillus , Staphylococcus aureus , Humanos , Queratinocitos/microbiología , Queratinocitos/metabolismo , Queratinocitos/efectos de los fármacos , Piel/microbiología , Piel/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Probióticos/farmacología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/metabolismo , Ribonucleasas/metabolismoRESUMEN
Millions of hibernating bats across North America have died from white-nose syndrome (WNS), an emerging disease caused by a psychrophilic (cold-loving) fungus, Pseudogymnoascus destructans, that invades their skin. Mechanisms of P. destructans invasion of bat epidermis remain obscure. Guided by our in vivo observations, we modeled hibernation with a newly generated little brown bat (Myotis lucifugus) keratinocyte cell line. We uncovered the stealth intracellular lifestyle of P. destructans, which inhibits apoptosis of keratinocytes and spreads through the cells by two epidermal growth factor receptor (EGFR)-dependent mechanisms: active penetration during torpor and induced endocytosis during arousal. Melanin of endocytosed P. destructans blocks endolysosomal maturation, facilitating P. destructans survival and germination after return to torpor. Blockade of EGFR aborts P. destructans entry into keratinocytes.
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Nivel de Alerta , Ascomicetos , Quirópteros , Receptores ErbB , Hibernación , Queratinocitos , Animales , Apoptosis , Ascomicetos/fisiología , Ascomicetos/patogenicidad , Línea Celular , Quirópteros/microbiología , Quirópteros/fisiología , Endocitosis , Receptores ErbB/metabolismo , Queratinocitos/microbiología , Melaninas/metabolismoRESUMEN
Cutibacterium acnes is the most abundant bacterium of the human skin microbiome since adolescence, participating in both skin homeostasis and diseases. Here, we demonstrate individual and niche heterogeneity of C. acnes from 1,234 isolate genomes. Skin disease (atopic dermatitis and acne) and body site shape genomic differences of C. acnes, stemming from horizontal gene transfer and selection pressure. C. acnes harbors characteristic metabolic functions, fewer antibiotic resistance genes and virulence factors, and a more stable genome compared with Staphylococcus epidermidis. Integrated genome, transcriptome, and metabolome analysis at the strain level unveils the functional characteristics of C. acnes. Consistent with the transcriptome signature, C. acnes in a sebum-rich environment induces toxic and pro-inflammatory effects on keratinocytes. L-carnosine, an anti-oxidative stress metabolite, is up-regulated in the C. acnes metabolome from atopic dermatitis and attenuates skin inflammation. Collectively, our study reveals the joint impact of genes and the microenvironment on C. acnes function.
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Acné Vulgar , Dermatitis Atópica , Queratinocitos , Propionibacterium acnes , Piel , Humanos , Piel/microbiología , Dermatitis Atópica/microbiología , Dermatitis Atópica/genética , Queratinocitos/microbiología , Acné Vulgar/microbiología , Propionibacterium acnes/genética , Genómica , Genoma Bacteriano , Staphylococcus epidermidis/genética , Transcriptoma , Factores de Virulencia/genética , Propionibacteriaceae/genética , Metaboloma , Metabolómica , Microbiota/genética , MultiómicaRESUMEN
Chronic wounds are a common and costly complication of diabetes, where multifactorial defects contribute to dysregulated skin repair, inflammation, tissue damage, and infection. We previously showed that aspects of the diabetic foot ulcer microbiota were correlated with poor healing outcomes, but many microbial species recovered remain uninvestigated with respect to wound healing. Here, we focused on Alcaligenes faecalis, a Gram-negative bacterium that is frequently recovered from chronic wounds but rarely causes infection. Treatment of diabetic wounds with A. faecalis accelerated healing during early stages. We investigated the underlying mechanisms and found that A. faecalis treatment promotes reepithelialization of diabetic keratinocytes, a process that is necessary for healing but deficient in chronic wounds. Overexpression of matrix metalloproteinases in diabetes contributes to failed epithelialization, and we found that A. faecalis treatment balances this overexpression to allow proper healing. This work uncovers a mechanism of bacterial-driven wound repair and provides a foundation for the development of microbiota-based wound interventions.
Asunto(s)
Alcaligenes faecalis , Queratinocitos , Metaloproteinasas de la Matriz , Cicatrización de Heridas , Alcaligenes faecalis/metabolismo , Animales , Queratinocitos/metabolismo , Queratinocitos/microbiología , Humanos , Metaloproteinasas de la Matriz/metabolismo , Metaloproteinasas de la Matriz/genética , Pie Diabético/microbiología , Pie Diabético/patología , Pie Diabético/metabolismo , Ratones , Repitelización , MasculinoRESUMEN
Wound infections are highly prevalent and can lead to delayed or failed healing, causing significant morbidity and adverse economic impacts. These infections occur in various contexts, including diabetic foot ulcers, burns, and surgical sites. Enterococcus faecalis is often found in persistent non-healing wounds, but its contribution to chronic wounds remains understudied. To address this, we employed single-cell RNA sequencing (scRNA-seq) on infected wounds in comparison to uninfected wounds in a mouse model. Examining over 23,000 cells, we created a comprehensive single-cell atlas that captures the cellular and transcriptomic landscape of these wounds. Our analysis revealed unique transcriptional and metabolic alterations in infected wounds, elucidating the distinct molecular changes associated with bacterial infection compared to the normal wound healing process. We identified dysregulated keratinocyte and fibroblast transcriptomes in response to infection, jointly contributing to an anti-inflammatory environment. Notably, E. faecalis infection prompted a premature, incomplete epithelial-mesenchymal transition in keratinocytes. Additionally, E. faecalis infection modulated M2-like macrophage polarization by inhibiting pro-inflammatory resolution in vitro, in vivo, and in our scRNA-seq atlas. Furthermore, we discovered macrophage crosstalk with neutrophils, which regulates chemokine signaling pathways, while promoting anti-inflammatory interactions with endothelial cells. Overall, our findings offer new insights into the immunosuppressive role of E. faecalis in wound infections.
If wounds get infected, they heal much more slowly, sometimes leading to skin damage and other complications, including disseminated infections or even amputation. Infections can happen in many types of wounds, ranging from ulcers in patients with diabetes to severe burns. If infections are not cleared quickly, the wounds can become 'chronic' and are unable to heal without intervention. Enterococcus faecalis is a type of bacteria that normally lives in the gut. Within that environment, in healthy people, it is not harmful. However, if it comes into contact with wounds particularly diabetic ulcers or the site of a surgery it can cause persistent infections and prevent healing. Although researchers are beginning to understand how E. faecalis initially colonises wounds, the biological mechanisms that transform these infections into chronic wounds are still largely unknown. Celik et al. therefore set out to investigate exactly how E. faecalis interferes with wound healing. To do this, Celik et al. looked at E. faecalis-infected wounds in mice and compared them to uninfected ones. Using a genetic technique called single-cell RNA sequencing, Celik et al. were able to determine which genes were switched on in individual skin and immune cells at the site of the wounds. This in turn allowed the researchers to determine how those cells were behaving in both infected and uninfected conditions. The experiments revealed that when E. faecalis was present in wounds, several important cell types in the wounds did not behave normally. For example, although the infected skin cells still underwent a change in behaviour required for healing (called an epithelial-mesenchymal transition), the change was both premature and incomplete. In other words, the skin cells in infected wounds started changing too early and did not finish the healing process properly. E. faecalis also changed the way macrophages and neutrophils worked within the wounds. These are cells in our immune system that normally promote inflammation, a process involved in both uninfected wounds or during infections and is a key part of wound healing when properly controlled. In the E. faecalis-infected wounds, these cells' inflammatory properties were suppressed, making them less helpful for healing. These results shed new light on how E. faecalis interacts with skin cells and the immune system to disrupt wound healing. Celik et al. hope that this knowledge will allow us to find new ways to target E. faecalis infections, and ultimately develop treatments to help chronic wounds heal better and faster.
Asunto(s)
Enterococcus faecalis , Infecciones por Bacterias Grampositivas , Queratinocitos , Cicatrización de Heridas , Enterococcus faecalis/fisiología , Enterococcus faecalis/genética , Animales , Ratones , Infecciones por Bacterias Grampositivas/microbiología , Queratinocitos/microbiología , Queratinocitos/metabolismo , Macrófagos/microbiología , Macrófagos/metabolismo , Macrófagos/inmunología , Modelos Animales de Enfermedad , Infección de Heridas/microbiología , Transcriptoma , Ratones Endogámicos C57BL , Análisis de la Célula Individual , Transición Epitelial-Mesenquimal/genética , Masculino , Fibroblastos/microbiología , Fibroblastos/metabolismoRESUMEN
Trichophyton rubrum is a human fungal pathogen that causes dermatophytosis, an infection that affects keratinized tissues. Integrated molecular signals coordinate mechanisms that control pathogenicity. Transcriptional regulation is a core regulation of relevant fungal processes. Previous RNA sequencing data revealed that the absence of the transcription factor StuA resulted in the differential expression of the MAPK-related high glycerol osmolarity gene (hog1) in T. rubrum. Here we validated the role of StuA in regulating the transcript levels of hog1. We showed through RT-qPCR that transcriptional regulation controls hog1 levels in response to glucose, keratin, and co-culture with human keratinocytes. In addition, we also detected hog1 pre-mRNA transcripts that underwent alternative splicing, presenting intron retention in a StuA-dependent mechanism. Our findings suggest that StuA and alternative splicing simultaneously, but not dependently, coordinate hog1 transcript levels in T. rubrum. As a means of preventing and treating dermatophytosis, our results contribute to the search for new potential drug therapies based on the molecular aspects of signaling pathways in T. rubrum.
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Empalme Alternativo , Arthrodermataceae , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos , Tiña , Factores de Transcripción , Humanos , Arthrodermataceae/genética , Arthrodermataceae/metabolismo , Glucosa/metabolismo , Queratinocitos/microbiología , Queratinas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Tiña/metabolismo , Tiña/microbiologíaRESUMEN
We have evaluated the inhibitory effects of supernatants and lysates derived from several candidate probiotics, on the growth and biofilm formation of wound pathogens, and their ability to protect human primary epidermal keratinocytes from the toxic effects of pathogens. Supernatants (neutralized and non-neutralized) and lysates (via sonication) from Lactiplantibacillus plantarum, Limosilactobacillus reuteri, Bifidobacterium longum, Lacticaseibacillus rhamnosus GG, and Escherichia coli Nissle 1917 were tested for their inhibitory effects against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumanni. The supernatants of L. plantarum, L. rhamnosus, B. longum, and L. rhamnosus GG reduced the growth of S. aureus, E. coli, and A. baumanni. B. longum additionally inhibited P. aeruginosa growth. However, neutralized Lactobacillus supernatants did not inhibit growth and in some cases were stimulatory. Lysates of L. plantarum and L. reuteri inhibited S. pyogenes while B. longum lysates inhibited E. coli and S. aureus growth. E. coli Nissle 1917 lysates enhanced the growth of S. pyogenes and P. aeruginosa. Biofilm formation by E. coli was reduced by lysates of L. reuteri and neutralized supernatants of all candidate probiotics. P. aeruginosa biofilm formation was reduced by E. coli Nissle supernatant but increased by L. plantarum, L. reuteri, and Bifidobacterium longum lysates. L. reuteri decreased the toxic effects of S. aureus on keratinocytes while E. coli Nissle 1917 lysates protected keratinocytes from S. pyogenes toxicity. In conclusion, lactobacilli and E. coli Nissle lysates confer inhibitory effects on pathogenic growth independently of acidification and may beneficially alter the outcome of interactions between host cell-pathogen in a species-specific manner.IMPORTANCEOne of the attributes of probiotics is their ability to inhibit pathogens. For this reason, many lactobacilli have been investigated for their effects as potential topical therapeutics against skin pathogens. However, this field is in its infancy. Even though probiotics are known to be safe when taken orally, the potential safety concerns when applied to potentially compromised skin are unknown. For this reason, we believe that extracts of probiotics will offer advantages over the use of live bacteria. In this study, we have surveyed five candidate probiotics, when used as extracts, in terms of their effects against common wound pathogens. Our data demonstrate that some probiotic extracts promote the growth of pathogens and highlight the need for careful selection of species and strains when probiotics are to be used topically.
Asunto(s)
Biopelículas , Escherichia coli , Queratinocitos , Probióticos , Pseudomonas aeruginosa , Staphylococcus aureus , Humanos , Queratinocitos/microbiología , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/fisiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/fisiología , Streptococcus pyogenes/efectos de los fármacos , Streptococcus pyogenes/crecimiento & desarrollo , Streptococcus pyogenes/fisiología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/fisiología , Acinetobacter baumannii/crecimiento & desarrollo , Infección de Heridas/microbiologíaRESUMEN
Streptococcus pyogenes [group A streptococcus (GAS)] is a human pathogen capable of infecting diverse tissues. To successfully infect these sites, GAS must detect available nutrients and adapt accordingly. The phosphoenolpyruvate transferase system (PTS) mediates carbohydrate uptake and metabolic gene regulation to adapt to the nutritional environment. Regulation by the PTS can occur through phosphorylation of transcriptional regulators at conserved PTS-regulatory domains (PRDs). GAS has several PRD-containing stand-alone regulators with regulons encoding both metabolic genes and virulence factors [PRD-containing virulence regulators (PCVRs)]. One is RofA, which regulates the expression of virulence genes in multiple GAS serotypes. It was hypothesized that RofA is phosphorylated by the PTS in response to carbohydrate levels to coordinate virulence gene expression. In this study, the RofA regulon of M1T1 strain 5448 was determined using RNA sequencing. Two operons were consistently differentially expressed across growth in the absence of RofA; the pilus operon was downregulated, and the capsule operon was upregulated. This correlated with increased capsule production and decreased adherence to keratinocytes. Purified RofA-His was phosphorylated in vitro by PTS proteins EI and HPr, and phosphorylated RofA-FLAG was detected in vivo when GAS was grown in low-glucose C medium. Phosphorylated RofA was not observed when C medium was supplemented 10-fold with glucose. Mutations of select histidine residues within the putative PRDs contributed to the in vivo phosphorylation of RofA, although phosphorylation of RofA was still observed, suggesting other phosphorylation sites exist in the protein. Together, these findings support the hypothesis that RofA is a PCVR that may couple sugar metabolism with virulence regulation.
Asunto(s)
Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Streptococcus pyogenes , Factores de Virulencia , Streptococcus pyogenes/patogenicidad , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Virulencia , Fosforilación , Humanos , Regulón , Operón , Infecciones Estreptocócicas/microbiología , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Queratinocitos/microbiologíaRESUMEN
BACKGROUND: Oral lichen planus (OLP) is one of the most common oral mucosal diseases, exhibiting a higher prevalence in women than men, but its pathogenesis is still unclear. Current research suggests that microbial dysbiosis may play an important role in the pathogenesis of OLP. Our previous research has found that the increase of Prevotella melaninogenica and decrease of Streptococcus salivarius have been identified as a potential pathogenic factor in OLP. Consequently, the objective of this study is to examine whether S. salivarius can counteract the detrimental effects of P. melaninogenica on the integrity of the epithelial barrier function. MATERIALS AND METHODS: Epithelial barrier disruption was induced by P. melaninogenica in human keratinocytes (HaCaT cells). HaCaT cells were pretreated with S. salivarius(MOI = 20) or cell-free supernatant for 3 h, followed by treatment with P. melaninogenica (MOI = 5) for 3 h. The epithelial barrier integrity of HaCaT cells was detected by FD4 permeability. The mRNA level of tight junction protein was detected by quantitative real-time polymerase chain reaction (PCR). Immunofluorescence and Western Blot were used to detect the protein expression of zonula occludin-1 (ZO-1). The serial dilution-spotting assay was applied to monitor the viability of P. melaninogenica at the end of 8 and 24 h incubation. RESULTS: Challenge by P. melaninogenica decreased the levels of tight junction proteins, including occludin, ZO-1, and claudin in HaCaT cells. S. salivarius or its cell-free supernatant inhibited the down-regulation of ZO-1 mRNA and protein expression levels induced by P. melaninogenica and thus improved the epithelial barrier function. The inhibitory effect of the cell-free supernatant of S. salivarius on the growth of P. melaninogenica is associated with metabolic acid production rather than with bacteriocins and hydrogen peroxide. CONCLUSIONS: These results suggest that live S. salivarius or its cell-free supernatant significantly ameliorated the disruption of epithelial tight junctions induced by P. melaninogenica, likely through the inhibition of P. melaninogenica growth mediated by metabolic acid production.
Asunto(s)
Queratinocitos , Prevotella melaninogenica , Streptococcus salivarius , Humanos , Prevotella melaninogenica/metabolismo , Queratinocitos/microbiología , Queratinocitos/metabolismo , Streptococcus salivarius/metabolismo , Uniones Estrechas/metabolismo , Ocludina/metabolismo , Células HaCaT , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
The utilization of ATP within cells plays a fundamental role in cellular processes that are essential for the regulation of host-pathogen dynamics and the subsequent immune response. This study focuses on ATP-binding proteins to dissect the complex interplay between Staphylococcus aureus and human cells, particularly macrophages (THP-1) and keratinocytes (HaCaT), during an intracellular infection. A snapshot of the various protein activity and function is provided using a desthiobiotin-ATP probe, which targets ATP-interacting proteins. In S. aureus, we observe enrichment in pathways required for nutrient acquisition, biosynthesis and metabolism of amino acids, and energy metabolism when located inside human cells. Additionally, the direct profiling of the protein activity revealed specific adaptations of S. aureus to the keratinocytes and macrophages. Mapping the differentially activated proteins to biochemical pathways in the human cells with intracellular bacteria revealed cell-type-specific adaptations to bacterial challenges where THP-1 cells prioritized immune defenses, autophagic cell death, and inflammation. In contrast, HaCaT cells emphasized barrier integrity and immune activation. We also observe bacterial modulation of host processes and metabolic shifts. These findings offer valuable insights into the dynamics of S. aureus-host cell interactions, shedding light on modulating host immune responses to S. aureus, which could involve developing immunomodulatory therapies. IMPORTANCE: This study uses a chemoproteomic approach to target active ATP-interacting proteins and examines the dynamic proteomic interactions between Staphylococcus aureus and human cell lines THP-1 and HaCaT. It uncovers the distinct responses of macrophages and keratinocytes during bacterial infection. S. aureus demonstrated a tailored response to the intracellular environment of each cell type and adaptation during exposure to professional and non-professional phagocytes. It also highlights strategies employed by S. aureus to persist within host cells. This study offers significant insights into the human cell response to S. aureus infection, illuminating the complex proteomic shifts that underlie the defense mechanisms of macrophages and keratinocytes. Notably, the study underscores the nuanced interplay between the host's metabolic reprogramming and immune strategy, suggesting potential therapeutic targets for enhancing host defense and inhibiting bacterial survival. The findings enhance our understanding of host-pathogen interactions and can inform the development of targeted therapies against S. aureus infections.
Asunto(s)
Adenosina Trifosfato , Interacciones Huésped-Patógeno , Queratinocitos , Macrófagos , Staphylococcus aureus , Humanos , Staphylococcus aureus/metabolismo , Adenosina Trifosfato/metabolismo , Interacciones Huésped-Patógeno/inmunología , Macrófagos/microbiología , Macrófagos/metabolismo , Macrófagos/inmunología , Queratinocitos/microbiología , Queratinocitos/metabolismo , Queratinocitos/inmunología , Células THP-1 , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Proteómica/métodos , Proteínas Bacterianas/metabolismo , Células HaCaTRESUMEN
Aim: To evaluate the behavior of oral keratinocytes in the presence of Vitamin C (Vit C) and its anti-inflammatory potential. Materials & methods: Oral keratinocytes were initially exposed to 0.1-2.5 mM of Vit C and the metabolic activity and cell migration were evaluated using MTS assay and Ibidi culture inserts, respectively. After, the cells were challenged with Candida albicans and inflammatory markers were analyzed by qPCR. Results: The treatment was not cytotoxic, and the highest concentrations increased the metabolic activity at 24 h. Vit C delayed the cell migration at 48 and 72 h. Interestingly, it downregulated the genes IL-8 and IL-1ß. Conclusion: Vit C could be an interesting adjuvant to anti-fungal treatment due to its anti-inflammatory potential.
Vitamin C, also known as ascorbic acid, is a vitamin commonly found in fruits and vegetables. It is popular for supporting our immune system, so is commonly taken as a supplement. We looked at the action of vitamin C on cells from the mouth and its potential to reduce inflammation in a fungal disease of the mouth oral candidiasis. We showed that vitamin C is not toxic to cells of the mouth and may reduce inflammation in cells infected by the fungus. This suggests that vitamin C could be used as a complementary therapy for oral candidiasis.
Asunto(s)
Antiinflamatorios , Ácido Ascórbico , Candida albicans , Movimiento Celular , Queratinocitos , Candida albicans/efectos de los fármacos , Candida albicans/inmunología , Ácido Ascórbico/farmacología , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/inmunología , Queratinocitos/microbiología , Queratinocitos/metabolismo , Antiinflamatorios/farmacología , Movimiento Celular/efectos de los fármacos , Interleucina-8/metabolismo , Interleucina-8/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , Inflamación , Antifúngicos/farmacologíaRESUMEN
Hidradenitis Suppurativa (HS) is a chronic autoinflammatory skin disease with activated keratinocytes, tunnel formation and a complex immune infiltrate in tissue. The HS microbiome is polymicrobial with an abundance of commensal gram-positive facultative (GPs) Staphylococcus species and gram-negative anaerobic (GNA) bacteria like Prevotella, Fusobacterium and Porphyromonas with increasing predominance of GNAs with disease severity. We sought to define the keratinocyte response to bacteria commonly isolated from HS lesions to probe pathogenic relationships between HS and the microbiome. Type strains of Prevotella nigrescens, Prevotella melaninogenica, Prevotella intermedia, Prevotella asaccharolytica, Fusobacterium nucleatum, as well as Staphylococcus aureus and the normal skin commensal Staphylococcus epidermidis were heat-killed and co-incubated with normal human keratinocytes. RNA was collected and analysed using RNAseq and RT-qPCR. The supernatant was collected from cell culture for protein quantification. Transcriptomic profiles between HS clinical samples and stimulated keratinocytes were compared. Co-staining of patient HS frozen sections was used to localize bacteria in lesions. A mouse intradermal injection model was used to investigate early immune recruitment. TLR4 and JAK inhibitors were used to investigate mechanistic avenues of bacterial response inhibition. GNAs, especially F. nucleatum, stimulated vastly higher CXCL8, IL17C, CCL20, IL6, TNF and IL36γ transcription in normal skin keratinocytes than the GPs S. epidermidis and S. aureus. Using RNAseq, we found that F. nucleatum (and Prevotella) strongly induced the IL-17 pathway in keratinocytes and overlapped with transcriptome profiles of HS patient clinical samples. Bacteria were juxtaposed to activated keratinocytes in vivo, and F. nucleatum strongly recruited murine neutrophil and macrophage migration. Both the TLR4 and pan-JAK inhibitors reduced cytokine production. Detailed transcriptomic profiling of healthy skin keratinocytes exposed to GNAs prevalent in HS revealed a potent, extensive inflammatory response vastly stronger than GPs. GNAs stimulated HS-relevant genes, including many genes in the IL-17 response pathway, and were significantly associated with HS tissue transcriptomes. The close association of activated keratinocytes with bacteria in HS lesions and innate infiltration in murine skin cemented GNA pathogenic potential. These novel mechanistic insights could drive future targeted therapies.
Asunto(s)
Hidradenitis Supurativa , Queratinocitos , Queratinocitos/inmunología , Queratinocitos/microbiología , Queratinocitos/metabolismo , Humanos , Animales , Ratones , Hidradenitis Supurativa/microbiología , Hidradenitis Supurativa/inmunología , Staphylococcus aureus/inmunología , Staphylococcus epidermidis/inmunología , Fusobacterium nucleatum/inmunología , Transcriptoma , Citocinas/metabolismo , Bacterias Anaerobias , Interleucina-17/metabolismo , Microbiota , Prevotella/inmunologíaRESUMEN
Staphylococcus aureus causes the majority of skin and soft tissue infections, but this pathogen only transiently colonizes healthy skin. However, this transient skin exposure enables S. aureus to transition to infection. The initial adhesion of S. aureus to skin corneocytes is mediated by surface protein G (SasG). Here, phylogenetic analyses reveal the presence of two major divergent SasG alleles in S. aureus: SasG-I and SasG-II. Structural analyses of SasG-II identify a nonaromatic arginine in the binding pocket of the lectin subdomain that mediates adhesion to corneocytes. Atomic force microscopy and corneocyte adhesion assays indicate that SasG-II can bind to a broader variety of ligands than SasG-I. Glycosidase treatment results in different binding profiles between SasG-I and SasG-II on skin cells. In addition, SasG-mediated adhesion is recapitulated using differentiated N/TERT keratinocytes. Our findings indicate that SasG-II has evolved to adhere to multiple ligands, conferring a distinct advantage to S. aureus during skin colonization.