RESUMEN
UNLABELLED: Engaging large numbers of undergraduates in authentic scientific discovery is desirable but difficult to achieve. We have developed a general model in which faculty and teaching assistants from diverse academic institutions are trained to teach a research course for first-year undergraduate students focused on bacteriophage discovery and genomics. The course is situated within a broader scientific context aimed at understanding viral diversity, such that faculty and students are collaborators with established researchers in the field. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) course has been widely implemented and has been taken by over 4,800 students at 73 institutions. We show here that this alliance-sourced model not only substantially advances the field of phage genomics but also stimulates students' interest in science, positively influences academic achievement, and enhances persistence in science, technology, engineering, and mathematics (STEM) disciplines. Broad application of this model by integrating other research areas with large numbers of early-career undergraduate students has the potential to be transformative in science education and research training. IMPORTANCE: Engagement of undergraduate students in scientific research at early stages in their careers presents an opportunity to excite students about science, technology, engineering, and mathematics (STEM) disciplines and promote continued interests in these areas. Many excellent course-based undergraduate research experiences have been developed, but scaling these to a broader impact with larger numbers of students is challenging. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunting Advancing Genomics and Evolutionary Science (SEA-PHAGES) program takes advantage of the huge size and diversity of the bacteriophage population to engage students in discovery of new viruses, genome annotation, and comparative genomics, with strong impacts on bacteriophage research, increased persistence in STEM fields, and student self-identification with learning gains, motivation, attitude, and career aspirations.
Asunto(s)
Bacterias/virología , Bacteriófagos/genética , Genómica/educación , Microbiología/educación , Adulto , Femenino , Humanos , Masculino , Estudiantes , Adulto JovenRESUMEN
Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.
Asunto(s)
Evolución Biológica , Variación Genética , Genoma Viral/genética , Micobacteriófagos/genética , Secuencia de Bases , ADN Viral/genética , Geografía , Micobacteriófagos/inmunología , Micobacteriófagos/aislamiento & purificación , Análisis de Secuencia de ADN , Estados UnidosRESUMEN
A novel screening method for antimycobacterial agents using Mycobacterium marinum was developed. M. marinum was selected as a model organism because it has a close phylogenetic relationship to M. tuberculosis, a relatively rapid doubling time, similar drug susceptibilities to M. tuberculosis, and less stringent safety requirements. More than 1000 crude marine and plant extracts were screened against M. marinum in a Zone of Inhibition (ZOI) assay, and twenty-one target extracts were identified. The crude organic extract of the marine sponge, Haliclona sp.10, was chosen for further investigation as it yielded a ZOI of 20 mm at a concentration of 80 microg/disk. Following bioassay-guided fractionation, (-)-papuamine was isolated, and yielded a 15 mm ZOI at a concentration of 25 microg/disk. In standard assays using M. marinum, (-)-papuamine exhibited both an MIC and an MBC95 of 6.25 microg/mL. This is the first report of antimycobacterial activity for (-)-papuamine. In addition, when (-)-papuamine and other natural product extracts were tested for activity against both M. marinum and M. tuberculosis, activity was comparable between the two species. These data indicate that (-)-papuamine is a promising lead for the development of new antimycobacterial agents and that M. marinum is a useful surrogate for the screening of antimycobacterial compounds.
Asunto(s)
Antibacterianos/farmacología , Pruebas de Sensibilidad Microbiana , Mycobacterium marinum/efectos de los fármacos , Fitoterapia , Extractos Vegetales/farmacología , Plantas Medicinales , Antibacterianos/administración & dosificación , Antibacterianos/uso terapéutico , Humanos , Extractos Vegetales/administración & dosificación , Extractos Vegetales/uso terapéutico , Reproducibilidad de los Resultados , Agua de MarRESUMEN
The significance of Hansen disease, or leprosy, is underscored by fact that detection of this disease has remained stable over the past 10 yr, even though disease prevalence is reduced. Due to the long incubation time of the organism, health experts predict that leprosy will be with us for decades to come. Despite the fact that Mycobacterium leprae, the causative agent of leprosy, cannot be cultured in the laboratory, researchers are using innovative and imaginative techniques to discern the interactions of M. leprae with host cells both in vitro and in vivo to identify the host and bacterial factors integral to establishment of disease. The studies described in this review present a new and evolving picture of the many interactions between M. leprae and the host. Specific attention will be given to interactions of M. leprae bacilli with host Schwann cells, macrophages, dendritic cells and endothelial cells. The findings described also have implications for the prevention and treatment of leprosy.
Asunto(s)
Células Dendríticas/fisiología , Células Endoteliales/fisiología , Lepra/microbiología , Macrófagos/fisiología , Mycobacterium leprae/fisiología , Células de Schwann/fisiología , Adhesinas Bacterianas/metabolismo , HumanosRESUMEN
Abstract The emergence of the nontuberculosis mycobacteria (NTM) as clinically relevant pathogens has warranted the study of these ubiquitous organisms in the context of their likely environmental niche, the biofilm. We assayed the NTM bacterium Mycobacterium marinum strain 1218R, a fish outbreak isolate, for biofilm formation on different surfaces over time using three different methods. Using the MBEC system, biofilm development occurred continually over the 14-day culture period reaching a mature or stable biofilm state after 7 days postinoculation. Quantification of M. marinum biofilm formation on high-density polyethylene (HDPE), polycarbonate (PC) and silicon (Si) coupons over a 14-day period was evaluated using a continuous flow reactor system. M. marinum developed biofilms on all of the surfaces tested. However, substantially more biofilm accumulated on the silicon than on the other substrates (Si>HDPE>PC) under the same growth conditions indicating that silicon was the most effective substratum studied for the generation of M. marinum biofilms and suggesting a correlation between surface hydrophobicity and attachment. Finally, confocal laser scanning microscopy was used to visualize M. marinum biofilm development in situ over time and revealed an unusual biofilm ultrastructure. Large cell clusters attached to the surface grew in parallel sinuous arrays of cells that formed large cords.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Mycobacterium marinum/crecimiento & desarrollo , Animales , Adhesión Bacteriana , Reactores Biológicos , Recuento de Colonia Microbiana , Medios de Cultivo , Microscopía Confocal , Mycobacterium marinum/citología , Cemento de Policarboxilato , Polietileno , SilicioRESUMEN
The Mycobacterium marinum G13 promoter is a sigma 70-like promoter that is more active by green fluorescent protein (gfp) differential fluorescence induction (DFI) assays when M. marium resides in an intracellular compartment as compared with growth in media. In assays using DFI, we found that the mycobacterial G13 promoter was also more active in a background of lower nutrient availability during logarithmic growth. This promoter, contained in an insert cloned upstream of a gfp reporter gene, is also active in Escherichia coli. When gfp expression assays of different plasmid constructs were performed in parallel with E. coli and M. marinum, expression in E. coli was maintained after deletion of both upstream and/or downstream regions proximal to the core promoter sequence. In M. marinum, however, although upstream deletions had no appreciable effect on gfp expression, promoter constructs with deleted downstream regions expressed 20- to 40-fold less gfp over all growth phases. The high-level expression of gfp was restored, however, in a clone containing approximately 100 bp downstream of the transcriptional start point. We have therefore utilized this gfp reporter assay of promoter activity to distinguish possible differences in requirements for gfp expression between different genera that utilize sigma 70-like promoter elements. We found that high levels of expression of gfp from the G13 promoter in M. marinum require downstream regions not necessary for gfp expression in E. coli.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Mycobacterium/genética , Regiones Promotoras Genéticas , Clonación Molecular , Medios de Cultivo , ARN Polimerasas Dirigidas por ADN , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Mycobacterium/crecimiento & desarrollo , Mycobacterium/metabolismo , Regiones Promotoras Genéticas/genética , Factor sigma , Transformación BacterianaRESUMEN
Mycobacterium leprae, an obligate intracellular pathogen, shows a unique tropism for Schwann cells (SC). This leads to the peripheral neuropathy disorder observed in leprosy. In this study, we investigated signal transduction events and the intracellular fate of M. leprae during the interaction of the microorganism with SC. First, we demonstrated that the human schwannoma cell line ST88-14 readily phagocytized the bacteria as observed by time-lapse microscopy, actin staining and electron microscopy. The effect of specific kinase inhibitors on M. leprae internalization was then investigated showing that functional protein tyrosine kinase, calcium-dependent protein kinase and phosphatidylinositol 3-kinase, but not cAMP-dependent protein kinase are essential for phagocytosis of the bacteria. Similar results were obtained when irradiated and live bacteria were compared and when M. leprae was pre-coated with recombinant histone-like-protein/laminin binding protein, a bacterial adhesin. In addition, experiments were performed to analyze the bacterial trafficking within the endosomal network by labeling the acidified intracellular compartments of M. leprae-infected SC with the Lysotracker acidotrophic probe. Acidification of vesicles containing live M. leprae was minimal in both RAW murine macrophages and SC, although phagosomes containing heat-killed bacteria seem to follow normal endocytic maturation. These data indicate that the invading bacteria interfere with normal endocytic pathway maturation of bacteria-containing phagosomes within SC.
Asunto(s)
Mycobacterium leprae/fisiología , Proteínas Quinasas/fisiología , Células de Schwann/microbiología , Transducción de Señal/fisiología , Animales , Adhesión Bacteriana , Humanos , Macrófagos/microbiología , Macrófagos/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Células de Schwann/fisiologíaRESUMEN
We report here the development of a pathogenesis model utilizing Mycobacterium marinum infection of zebrafish (Danio rerio) for the study of mycobacterial disease. The zebrafish model mimics certain aspects of human tuberculosis, such as the formation of granuloma-like lesions and the ability to establish either an acute or a chronic infection based upon inoculum. This model allows the genetics of mycobacterial disease to be studied in both pathogen and host.