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Background: Alterations in upper respiratory microbiomes have been implicated in shaping host health trajectories, including by limiting mucosal pathogen colonization. However, limited comparative studies of respiratory microbiome development and functioning across age groups have been performed. Herein, we perform shotgun metagenomic sequencing paired with pathogen inhibition assays to elucidate differences in nasal and oral microbiome composition and functioning across healthy 24-month-old infant (n=229) and adult (n=100) populations. Results: We find that beta diversity of nasal and oral microbiomes varies with age, with nasal microbiomes showing greater population-level variation compared to oral microbiomes. Infant microbiome alpha diversity was significantly lower across nasal samples and higher in oral samples, relative to adults. Accordingly, we demonstrate significant differences in genus- and species-level composition of microbiomes between sites and age groups. Antimicrobial resistome patterns likewise varied across body sites, with oral microbiomes showing higher resistance gene abundance compared to nasal microbiomes. Biosynthetic gene clusters encoding specialized metabolite production were found in higher abundance across infant oral microbiomes, relative to adults. Investigation of pathogen inhibition revealed greater inhibition of gram-negative and gram-positive bacteria by oral commensals, while nasal isolates had higher antifungal activity. Conclusions: In summary, we identify significant differences in the microbial communities inhabiting nasal and oral cavities of healthy infants relative to adults. These findings inform our understanding of the interactions impacting respiratory microbiome composition and functioning, with important implications for host health across the lifespan.
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Moraxella catarrhalis is found almost exclusively within the human respiratory tract. This pathobiont is associated with ear infections and the development of respiratory illnesses, including allergies and asthma. Given the limited ecological distribution of M. catarrhalis, we hypothesized that we could leverage the nasal microbiomes of healthy children without M. catarrhalis to identify bacteria that may represent potential sources of therapeutics. Rothia was more abundant in the noses of healthy children compared to children with cold symptoms and M. catarrhalis. We cultured Rothia from nasal samples and determined that most isolates of Rothia dentocariosa and "Rothia similmucilaginosa" were able to fully inhibit the growth of M. catarrhalis in vitro, whereas isolates of Rothia aeria varied in their ability to inhibit M. catarrhalis. Using comparative genomics and proteomics, we identified a putative peptidoglycan hydrolase called secreted antigen A (SagA). This protein was present at higher relative abundance in the secreted proteomes of R. dentocariosa and R. similmucilaginosa than in those from non-inhibitory R. aeria, suggesting that it may be involved in M. catarrhalis inhibition. We produced SagA from R. similmucilaginosa in Escherichia coli and confirmed its ability to degrade M. catarrhalis peptidoglycan and inhibit its growth. We then demonstrated that R. aeria and R. similmucilaginosa reduced M. catarrhalis levels in an air-liquid interface culture model of the respiratory epithelium. Together, our results suggest that Rothia restricts M. catarrhalis colonization of the human respiratory tract in vivo. IMPORTANCE Moraxella catarrhalis is a pathobiont of the respiratory tract, responsible for ear infections in children and wheezing illnesses in children and adults with chronic respiratory diseases. Detection of M. catarrhalis during wheezing episodes in early life is associated with the development of persistent asthma. There are currently no effective vaccines for M. catarrhalis, and most clinical isolates are resistant to the commonly prescribed antibiotics amoxicillin and penicillin. Given the limited niche of M. catarrhalis, we hypothesized that other nasal bacteria have evolved mechanisms to compete against M. catarrhalis. We found that Rothia are associated with the nasal microbiomes of healthy children without Moraxella. Next, we demonstrated that Rothia inhibit M. catarrhalis in vitro and on airway cells. We identified an enzyme produced by Rothia called SagA that degrades M. catarrhalis peptidoglycan and inhibits its growth. We suggest that Rothia or SagA could be developed as highly specific therapeutics against M. catarrhalis.
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Asma , Moraxella catarrhalis , Niño , Adulto , Humanos , Peptidoglicano/metabolismo , Ruidos RespiratoriosRESUMEN
The aerodigestive tract (ADT) is the primary portal through which pathogens and other invading microbes enter the body. As the direct interface with the environment, we hypothesize that the ADT microbiota possess biosynthetic gene clusters (BGCs) for antibiotics and other specialized metabolites to compete with both endogenous and exogenous microbes. From 1,214 bacterial genomes, representing 136 genera and 387 species that colonize the ADT, we identified 3,895 BGCs. To determine the distribution of BGCs and bacteria in different ADT sites, we aligned 1,424 metagenomes, from nine different ADT sites, onto the predicted BGCs. We show that alpha diversity varies across the ADT and that each site is associated with distinct bacterial communities and BGCs. We identify specific BGC families enriched in the buccal mucosa, external naris, gingiva, and tongue dorsum despite these sites harboring closely related bacteria. We reveal BGC enrichment patterns indicative of the ecology at each site. For instance, aryl polyene and resorcinol BGCs are enriched in the gingiva and tongue, which are colonized by many anaerobes. In addition, we find that streptococci colonizing the tongue and cheek possess different ribosomally synthesized and posttranslationally modified peptide BGCs. Finally, we highlight bacterial genera with BGCs but are underexplored for specialized metabolism and demonstrate the bioactivity of Actinomyces against other bacteria, including human pathogens. Together, our results demonstrate that specialized metabolism in the ADT is extensive and that by exploring these microbiomes further, we will better understand the ecology and biogeography of this system and identify new bioactive natural products. IMPORTANCE Bacteria produce specialized metabolites to compete with other microbes. Though the biological activities of many specialized metabolites have been determined, our understanding of their ecology is limited, particularly within the human microbiome. As the aerodigestive tract (ADT) faces the external environment, bacteria colonizing this tract must compete both among themselves and with invading microbes, including human pathogens. We analyzed the genomes of ADT bacteria to identify biosynthetic gene clusters (BGCs) for specialized metabolites. We found that the majority of ADT BGCs are uncharacterized and the metabolites they encode are unknown. We mapped the distribution of BGCs across the ADT and determined that each site is associated with its own distinct bacterial community and BGCs. By further characterizing these BGCs, we will inform our understanding of ecology and biogeography across the ADT, and we may uncover new specialized metabolites, including antibiotics.
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Bacterias/aislamiento & purificación , Bacterias/metabolismo , Microbioma Gastrointestinal , Tracto Gastrointestinal/microbiología , Bacterias/clasificación , Bacterias/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vías Biosintéticas , Mejilla/microbiología , Genoma Bacteriano , Humanos , Microbiota , Boca/microbiología , Cavidad Nasal/microbiología , FilogeniaRESUMEN
Cytochrome P450 2C8 (CYP2C8) epoxygenase is responsible for the metabolism of over 60 clinically relevant drugs, notably the anticancer drug Taxol (paclitaxel, PAC). Specifically, there are naturally occurring polymorphisms, CYP2C8*2 and CYP2C8*3, that display altered PAC hydroxylation rates despite these mutations not being located in the active site. Herein, we demonstrate that these polymorphisms result in a greater uncoupling of PAC metabolism by increasing the amount of hydrogen peroxide formed per PAC turnover. Anaerobic stopped-flow measurements determined that these polymorphisms have altered first electron transfer kinetics, compared to CYP2C8*1 (wildtype), that suggest electron transfer from cytochrome P450 reductase (CPR) is disfavored. Therefore, these data demonstrate that these polymorphisms affect the catalytic cycle of CYP2C8 and suggest that redox interactions with CPR are disrupted.
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Citocromo P-450 CYP2C8/genética , Citocromo P-450 CYP2C8/metabolismo , Polimorfismo de Nucleótido Simple , Antineoplásicos Fitogénicos/metabolismo , Transporte de Electrón , Humanos , Hidroxilación , Modelos Moleculares , Oxidación-Reducción , Paclitaxel/metabolismoRESUMEN
Exploitation of the immune system has emerged as an important therapeutic strategy for acute lymphoblastic leukemia (ALL). However, the mechanisms of immune evasion during leukemia progression remain poorly understood. We sought to understand the role of calcineurin in ALL and observed that depletion of calcineurin B (CnB) in leukemia cells dramatically prolongs survival in immune-competent but not immune-deficient recipients. Immune-competent recipients were protected from challenge with leukemia if they were first immunized with CnB-deficient leukemia, suggesting robust adaptive immunity. In the bone marrow (BM), recipients of CnB-deficient leukemia harbored expanded T-cell populations as compared with controls. Gene expression analyses of leukemia cells extracted from the BM identified Cn-dependent significant changes in the expression of immunoregulatory genes. Increased secretion of IL12 from CnB-deficient leukemia cells was sufficient to induce T-cell activation ex vivo, an effect that was abolished when IL12 was neutralized. Strikingly, recombinant IL12 prolonged survival of mice challenged with highly aggressive B-ALL. Moreover, gene expression analyses from children with ALL showed that patients with higher expression of either IL12A or IL12B exhibited prolonged survival. These data suggest that leukemia cells are dependent upon calcineurin for immune evasion by restricting the regulation of proinflammatory genes, particularly IL12. SIGNIFICANCE: This report implicates calcineurin as an intracellular signaling molecule responsible for immune evasion during leukemia progression and raises the prospect of re-examining IL12 as a therapeutic in leukemia.
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Calcineurina/inmunología , Interleucina-12/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Calcineurina/deficiencia , Calcineurina/genética , Línea Celular Tumoral , Citocinas/biosíntesis , Citocinas/inmunología , Progresión de la Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Interleucina-12/biosíntesis , Interleucina-12/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Escape del TumorRESUMEN
INTRODUCTION: Multidrug-resistant bacterial infections of the gastrointestinal tract pose a serious public health concern. High levels of antibiotic drug resistance, along with the potential for antibiotics to precipitate disease or alter the gut microbiome has prompted research into alternative treatment methods. Evidence suggests that bacteriophage therapy delivered per os may be well-suited to target such infections. Areas covered: Herein, we discuss the specific advantages and challenges of using orally administered phage therapy. Our literature review encompasses recent works using phages to target various clinically-relevant bacteria in vivo. We also provide insights into methods that aim to overcome the barriers to effective phage transit through the harsh gastrointestinal environment. Expert commentary: Evidence from a number of in vivo animal studies suggests that targeting bacterial infections using phages delivered orally holds potential. Efficacious oral phage therapy depends on the delivery of sufficient phage titers to the infection site, which may be hindered by the host's gastrointestinal tract and immune response.
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Bacteriófagos , Tracto Gastrointestinal/microbiología , Infecciones por Bacterias Gramnegativas/terapia , Infecciones por Bacterias Grampositivas/terapia , Terapia de Fagos/métodos , Inmunidad Adaptativa , Administración Oral , Animales , Farmacorresistencia Bacteriana Múltiple , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Grampositivas/inmunología , Infecciones por Bacterias Grampositivas/microbiología , HumanosRESUMEN
Thromboxane synthase (CYP5A1) is a non-classical cytochrome P450 (CYP) expressed in human platelets that mediates vascular homeostasis by producing thromboxane A2 (TXA2) through the isomerization of prostaglandin H2 (PGH2). A homology alignment of CYP5A1 with human CYPs indicates that a highly conserved I-helix threonine residue is occupied by an isoleucine at position 346 in CYP5A1. We find that reverse-engineering CYP5A1 to contain either threonine or serine in this position dramatically increases TXA2 formation. Interestingly, the levels of malondialdehyde (MDA), a homolytic fragmentation product of PGH2 formed via a pathway independent of TXA2 formation, remain constant. Furthermore, spectral analysis using two PGH2 substrate analogs supports the observed activity changes in the hydroxyl-containing mutants. The more constrained active site of the I346T mutant displays altered PGH2 substrate analog binding properties. Together these studies provide new mechanistic insights into CYP5A1 mediated isomerization of PGH2 with respect to a critical active site residue.
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Isoleucina/química , Nanoestructuras/química , Tromboxano-A Sintasa/química , Tromboxano-A Sintasa/metabolismo , Secuencia Conservada/genética , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Ingeniería de Proteínas , Tromboxano-A Sintasa/genéticaRESUMEN
The endogenous cannabinoid system was first uncovered following studies of the recreational drug Cannabis sativa. It is now recognized as a vital network of signaling pathways that regulate several physiological processes. Following the initial discovery of the cannabinoid receptors 1 (CB1) and 2 (CB2), activated by Cannabis-derived analogs, many endogenous fatty acids termed "endocannabinoids" are now known to be partial agonists of the CB receptors. At present, the most thoroughly studied endocannabinoid signaling molecules are anandamide (AEA) and 2-arachidonylglycerol (2-AG), which are both derived from arachidonic acid. Both AEA and 2-AG are also substrates for the eicosanoid-synthesizing pathways, namely, certain cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) enzymes. In the past, research in the endocannabinoid field focused on the interaction of AEA and 2-AG with the COX and LOX enzymes, but accumulating evidence also points to the involvement of CYPs in modulating endocannabinoid signaling. The focus of this review is to explore the current understanding of CYP-mediated metabolism of endocannabinoids.
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Sistema Enzimático del Citocromo P-450/metabolismo , Endocannabinoides/metabolismo , Animales , Sistema Enzimático del Citocromo P-450/genética , Endocannabinoides/genética , Humanos , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/genética , Receptor Cannabinoide CB2/metabolismoRESUMEN
Recombinant expression of membrane-bound cytochrome P450s in bacterial expression systems provide a well-established system capable of producing large yields of catalytically active protein. As the biochemical knowledge regarding cytochrome P450s increases, so does the efficiency of protein expression through various modifications that do not disrupt the functional properties of the protein of interest. Changes such as N-terminal modifications, reduction of secondary mRNA structure, bacterial codon usage, selection of vector and host strain, as well as varying external growth conditions all appear to influence protein expression. Several optimizations are often required for sufficient expression of cytochrome P450s at the desired cellular localization. This review aims to comprehensively summarize and update the significant advances made in membrane protein P450 expression in bacterial expression systems.