Joshua D. Hall

Infected-Host-Cell Repertoire and Cellular Response in the Lung following Inhalation of Francisella tularensis Schu S4, LVS, or U112

ABSTRACT Francisella tularensis causes systemic disease in humans and other mammals, with high morbidity and mortality associated with inhalation-acquired infection. F. tularensis is a facultative intracellular pathogen, but the scope and significance of cell types infected during disease is unknown. Using flow cytometry, we identified and quantified infected-cell types and assessed the impact of infection on cell populations following inhalation of F. tularensis strains U112, LVS, and Schu S4. Initially, alveolar macrophages comprised over 70% of Schu S4- and LVS-infected cells, whereas approximately 51% and 27% of U112-infected cells were alveolar macrophages and neutrophils, respectively. After 3 days, roughly half of Schu S4- and LVS- and nearly 80% of U112-infected cells were neutrophils. All strains infected CD11bhigh macrophages, dendritic cells, monocytes, and alveolar type II cells throughout infection. Macrophage, monocyte, and dendritic-cell populations were reduced during U112 infection but not Schu S4 or LVS infection. These results demonstrate directly that F. tularensis is a promiscuous intracellular pathogen in the lung that invades and replicates within cell types ranging from migratory immune cells to structural tissue cells. However, the proportions of cell types infected and the cellular immune response evoked by the human pathogenic strain Schu S4 differ from those of the human avirulent U112.

Francisella tularensis replicates within alveolar type II epithelial cells in vitro and in vivo following inhalation.

ABSTRACT Francisella tularensis replicates in macrophages and dendritic cells, but interactions with other cell types have not been well described. F. tularensis LVS invaded and replicated within alveolar epithelial cell lines. Following intranasal inoculation of C57BL/6 mice, Francisella localized to the alveolus and replicated within alveolar type II epithelial cells.

Use of Transposon-Transposase Complexes To Create Stable Insertion Mutant Strains of Francisella tularensis LVS

ABSTRACT Francisella tularensis is a highly virulent zoonotic bacterial pathogen capable of infecting numerous different mammalian species, including humans. Elucidation of the pathogenic mechanisms of F. tularensis has been hampered by a lack of tools to genetically manipulate this organism. Herein we describe the use of transposome complexes to create insertion mutations in the chromosome of the F. tularensis live vaccine strain (LVS). A Tn5-derived transposon encoding kanamycin resistance and lacking a transposase gene was complexed with transposase enzyme and transformed directly into F. tularensis LVS by electroporation. An insertion frequency of 2.6 × 10−8 ± 0.87 × 10−8 per cell was consistently achieved using this method. There are 178 described Tn5 consensus target sites distributed throughout the F. tularensis genome. Twenty-two of 26 transposon insertions analyzed were within known or predicted open reading frames, but none of these insertions was associated with the Tn5 target site. Analysis of the insertions of sequentially passed strains indicated that the transposons were maintained stably at the initial insertion site after more than 270 generations. Therefore, transformation by electroporation of Tn5-based transposon-transposase complexes provided an efficient mechanism for generating random, stable chromosomal insertion mutations in F. tularensis.

Francisella tularensis Invasion of Lung Epithelial Cells

ABSTRACT Francisella tularensis, a gram-negative facultative intracellular bacterial pathogen, causes disseminating infections in humans and other mammalian hosts. Macrophages and other monocytes have long been considered the primary site of F. tularensis replication in infected animals. However, recently it was reported that F. tularensis also invades and replicates within alveolar epithelial cells following inhalation in a mouse model of tularemia. TC-1 cells, a mouse lung epithelial cell line, were used to study the process of F. tularensis invasion and intracellular trafficking within nonphagocytic cells. Live and paraformaldehyde-fixed F. tularensis live vaccine strain organisms associated with, and were internalized by, TC-1 cells at similar frequencies and with indistinguishable differences in kinetics. Inhibitors of microfilament and microtubule activity resulted in significantly decreased F. tularensis invasion, as did inhibitors of phosphatidylinositol 3-kinase and tyrosine kinase activity. Collectively, these results suggest that F. tularensis epithelial cell invasion is mediated by a preformed ligand on the bacterial surface and driven entirely by host cell processes. Once internalized, F. tularensis-containing endosomes associated with early endosome antigen 1 (EEA1) followed by lysosome-associated membrane protein 1 (LAMP-1), with peak coassociation frequencies occurring at 30 and 120 min postinoculation, respectively. By 2 h postinoculation, 70.0% (± 5.5%) of intracellular bacteria were accessible to antibody delivered to the cytoplasm, indicating vacuolar breakdown and escape into the cytoplasm.

RipA, a Cytoplasmic Membrane Protein Conserved among Francisella Species, Is Required for Intracellular Survival

ABSTRACT Francisella tularensis is a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of the F. tularensis live vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequenced Francisella species yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele in trans. Based on the deletion mutant phenotype, FTL_1914 was termed ripA (required for intracellular proliferation, factor A). Following uptake by J774.A1 cells, F. tularensis LVS ΔripA colocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of the F. tularensis LVS ΔripA mutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. The F. tularensis LVS ΔripA mutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved among Francisella species that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence.

A Phenotype at Last: Essential Role for the Yersinia enterocolitica Ysa Type III Secretion System in a Drosophila melanogaster S2 Cell Model

ABSTRACT The highly pathogenic Yersinia enterocolitica strains have a chromosomally encoded type III secretion system (T3SS) that is expressed and functional in vitro only when the bacteria are cultured at 26°C. Mutations that render this system nonfunctional are slightly attenuated in the mouse model of infection only following an oral inoculation and only at early time points postinfection. The discrepancy between the temperature required for the Ysa gene expression and the physiological temperature required for mammalian model systems has made defining the role of this T3SS challenging. Therefore, we explored the use of Drosophila S2 cells as a model system for studying Ysa function. We show here that Y. enterocolitica is capable of infecting S2 cells and replicating intracellularly to high levels, an unusual feature of this pathogen. Importantly, we show that the Ysa T3SS is required for robust intracellular replication. A secretion-deficient mutant lacking the secretin gene, ysaC, is defective in replication within S2 cells, marking the first demonstration of a pronounced Ysa-dependent virulence phenotype. Establishment of S2 cells as a model for Y. enterocolitica infection provides a versatile tool to elucidate the role of the Ysa T3SS in the life cycle of this gastrointestinal pathogen.

Predictors of Student Productivity in Biomedical Graduate School Applications

Many US biomedical PhD programs receive more applications for admissions than they can accept each year, necessitating a selective admissions process. Typical selection criteria include standardized test scores, undergraduate grade point average, letters of recommendation, a resume and/or personal statement highlighting relevant research or professional experience, and feedback from interviews with training faculty. Admissions decisions are often founded on assumptions that these application components correlate with research success in graduate school, but these assumptions have not been rigorously tested. We sought to determine if any application components were predictive of student productivity measured by first-author student publications and time to degree completion. We collected productivity metrics for graduate students who entered the umbrella first-year biomedical PhD program at the University of North Carolina at Chapel Hill from 2008–2010 and analyzed components of their admissions applications. We found no correlations of test scores, grades, amount of previous research experience, or faculty interview ratings with high or low productivity among those applicants who were admitted and chose to matriculate at UNC. In contrast, ratings from recommendation letter writers were significantly stronger for students who published multiple first-author papers in graduate school than for those who published no first-author papers during the same timeframe. We conclude that the most commonly used standardized test (the general GRE) is a particularly ineffective predictive tool, but that qualitative assessments by previous mentors are more likely to identify students who will succeed in biomedical graduate research. Based on these results, we conclude that admissions committees should avoid over-reliance on any single component of the application and de-emphasize metrics that are minimally predictive of student productivity. We recommend continual tracking of desired training outcomes combined with retrospective analysis of admissions practices to guide both application requirements and holistic application review.

Diversity Exiting the Academy: Influential Factors for the Career Choice of Well-Represented and Underrepresented Minority Scientists

The relative importance of reasons for current career choices for science, technology, engineering, and mathematics PhDs was examined. Reasons why underrepresented minority scientists chose faculty careers differed in some respects from those of well-represented scientists, with implications for graduate/postdoctoral training, formal and informal social support networks, and faculty career decisions.

An evidence-based evaluation of transferrable skills and job satisfaction for science PhDs.

PhD recipients acquire discipline-specific knowledge and a range of relevant skills during their training in the life sciences, physical sciences, computational sciences, social sciences, and engineering. Empirically testing the applicability of these skills to various careers held by graduates will help assess the value of current training models. This report details results of an Internet survey of science PhDs (n = 8099) who provided ratings for fifteen transferrable skills. Indeed, analyses indicated that doctoral training develops these transferrable skills, crucial to success in a wide range of careers including research-intensive (RI) and non-research-intensive (NRI) careers. Notably, the vast majority of skills were transferrable across both RI and NRI careers, with the exception of three skills that favored RI careers (creativity/innovative thinking, career planning and awareness skills, and ability to work with people outside the organization) and three skills that favored NRI careers (time management, ability to learn quickly, ability to manage a project). High overall rankings suggested that graduate training imparted transferrable skills broadly. Nonetheless, we identified gaps between career skills needed and skills developed in PhD training that suggest potential areas for improvement in graduate training. Therefore, we suggest that a two-pronged approach is crucial to maximizing existing career opportunities for PhDs and developing a career-conscious training model: 1) encouraging trainees to recognize their existing individual skill sets, and 2) increasing resources and programmatic interventions at the institutional level to address skill gaps. Lastly, comparison of job satisfaction ratings between PhD-trained employees in both career categories indicated that those in NRI career paths were just as satisfied in their work as their RI counterparts. We conclude that PhD training prepares graduates for a broad range of satisfying careers, potentially more than trainees and program leaders currently appreciate.

Preparing Postbaccalaureates for Entry and Success in Biomedical PhD Programs

An intensive, 1-year biomedical training program to support underrepresented scholars during the critical transition from baccalaureate to PhD is described. In 5 years, this program has transitioned 91% of scholars to PhD programs with 95% retention through a combination of skill- and confidence-building interventions.