Integration of Microbiology, Pharmacology, Immunology, and Infectious Disease Using Active Teaching and Self-Directed Learning.

In an era of decreasing basic science curriculum at medical schools, we sought to re-imagine how to optimally deliver three core basic science disciplines (microbiology, pharmacology, and immunology) together with infectious disease in a 5-week course. This course, developed as part of a new 1-year pre-clinical basic science curriculum at the recently established Dell Medical School (DMS) at the University of Texas at Austin, featured a fully integrated curriculum in which the majority of the sessions were team-taught. This course, in line with the goals and missions of DMS, presented material using primarily self-directed and active learning approaches. Here, we describe the format and content of the course. We present our strategy and rationale for selecting these particular learning modalities and topics for pre-class and in-class coverage, using educational and cognitive psychology literature as a guide. We also discuss how, based on feedback from both student evaluations and performance data, the course evolved over the first two iterations.

Effects of Discovery, Iteration, and Collaboration in Laboratory Courses on Undergraduates' Research Career Intentions Fully Mediated by Student Ownership.

Course-based undergraduate research experiences (CUREs) provide a promising avenue to attract a larger and more diverse group of students into research careers. CUREs are thought to be distinctive in offering students opportunities to make discoveries, collaborate, engage in iterative work, and develop a sense of ownership of their lab course work. Yet how these elements affect students' intentions to pursue research-related careers remain unexplored. To address this knowledge gap, we collected data on three design features thought to be distinctive of CUREs (discovery, iteration, collaboration) and on students' levels of ownership and career intentions from ∼800 undergraduates who had completed CURE or inquiry courses, including courses from the Freshman Research Initiative (FRI), which has a demonstrated positive effect on student retention in college and in science, technology, engineering, and mathematics. We used structural equation modeling to test relationships among the design features and student ownership and career intentions. We found that discovery, iteration, and collaboration had small but significant effects on students' intentions; these effects were fully mediated by student ownership. Students in FRI courses reported significantly higher levels of discovery, iteration, and ownership than students in other CUREs. FRI research courses alone had a significant effect on students' career intentions.

Metabolism and Pathogenicity of Pseudomonas aeruginosa Infections in the Lungs of Individuals with Cystic Fibrosis.

Individuals with the genetic disease cystic fibrosis (CF) accumulate mucus or sputum in their lungs. This sputum is a potent growth substrate for a range of potential pathogens, and the opportunistic bacterium Pseudomonas aeruginosa is generally most difficult of these to eradicate. As a result, P. aeruginosa infections are frequently maintained in the CF lung throughout life, and are the leading cause of death for these individuals. While great effort has been expended to better understand and treat these devastating infections, only recently have researchers begun to rigorously examine the roles played by specific nutrients in CF sputum to cue P. aeruginosa pathogenicity. This chapter summarizes the current state of knowledge regarding how P. aeruginosa metabolism in CF sputum affects initiation and maintenance of these infections. It contains an overview of CF lung disease and the mechanisms of P. aeruginosa pathogenicity. Several model systems used to study these infections are described with emphasis on the challenge of replicating the chronic infections observed in humans with CF. Nutrients present in CF sputum are surveyed, and the impacts of these nutrients on the infection are discussed. The chapter concludes by addressing the future of this line of research including the use of next-generation technologies and the potential for metabolism-based therapeutics.

Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum.

Defining the essential genome of bacterial pathogens is central to developing an understanding of the biological processes controlling disease. This has proven elusive for Pseudomonas aeruginosa during chronic infection of the cystic fibrosis (CF) lung. In this paper, using a Monte Carlo simulation-based method to analyze high-throughput transposon sequencing data, we establish the P. aeruginosa essential genome with statistical precision in laboratory media and CF sputum. Reconstruction of the global requirements for growth in CF sputum compared with defined growth conditions shows that the latter requires several cofactors including biotin, riboflavin, and pantothenate. Comparison of P. aeruginosa strains PAO1 and PA14 demonstrates that essential genes are primarily restricted to the core genome; however, some orthologous genes in these strains exhibit differential essentiality. These results indicate that genes with similar molecular functions may have distinct genetic roles in different P. aeruginosa strains during growth in CF sputum. We also show that growth in a defined growth medium developed to mimic CF sputum yielded virtually identical fitness requirements to CF sputum, providing support for this medium as a relevant in vitro model for CF microbiology studies.

The role of two Pseudomonas aeruginosa anthranilate synthases in tryptophan and quorum signal production.

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes infections in the lungs of individuals with the genetic disease cystic fibrosis. Density-dependent production of toxic factors regulated by the Pseudomonas quinolone signal (2-heptyl-3-hydroxy-4-quinolone; PQS) have been proposed to be involved in P. aeruginosa virulence. PQS biosynthesis requires conversion of the central metabolite chorismate to anthranilate by anthranilate synthase. This reaction is also the first step in tryptophan biosynthesis. P. aeruginosa possesses two functional anthranilate synthases, TrpEG and PhnAB, and these enzymes are not functionally redundant, as trpEG mutants are tryptophan auxotrophs but produce PQS while mutants in phnAB are tryptophan prototrophs but do not produce PQS in minimal media. The goal of the work described in this paper was to determine the mechanism for this lack of functional complementation of TrpEG and PhnAB. Our results reveal that overexpression of either enzyme compensates for tryptophan auxotrophy and PQS production in the trpEG and phnAB mutants respectively, leading to the hypothesis that differential regulation of these genes is responsible for the lack of functional complementation. In support of this hypothesis, trpEG was shown to be expressed primarily during low-density growth while phnAB was expressed primarily at high density. Furthermore, dysregulation of phnAB expression eliminated tryptophan auxotrophy in the P. aeruginosa trpEG mutant. Based on these data, we propose a model for anthranilate sequestration by differential transcriptional regulation of the two P. aeruginosa anthranilate synthase enzymes.

Quantifying Pseudomonas aeruginosa quinolones and examining their interactions with lipids.

Pseudomonas aeruginosa produces a quorum sensing molecule termed the Pseudomonas Quinolone Signal (2-heptyl-3-hydroxy-4-quinolone; PQS) that regulates an array of genes involved in virulence. This chapter addresses four related techniques useful for detecting and quantifying PQS. First, extraction of PQS from complex mixtures (e.g. cell cultures) is described. Separation of PQS from extracts by Thin-Layer Chromatography (TLC) is used in combination with the natural fluorescence of the molecule for quantification. A second separation technique for the PQS precursor HHQ using High-Performance Liquid Chromatography (HPLC) is also described, and this assay exploits the molecule's characteristic absorbance for quantification. A third method for quantification of PQS from simple mixtures (e.g. enzyme assays) using fluorescence is outlined. Finally, a protocol for determining PQS interactions with membrane lipids through Fluorescence Resonance Energy Transfer (FRET) is presented. These techniques allow for quantification and characterization of PQS from diverse environments, a prerequisite to understanding the biological functions of QS molecules.

Characterization of the Pseudomonas aeruginosa transcriptional response to phenylalanine and tyrosine.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen often associated with chronic infections in the lungs of individuals with the heritable disease cystic fibrosis (CF). Previous work from our laboratory demonstrated that aromatic amino acids within CF lung secretions (sputum) not only serve as carbon and energy sources but also enhance synthesis of the cell signaling molecule Pseudomonas quinolone signal (PQS). The present study investigates the role of the aromatic amino acid-responsive regulator PhhR in mediating these phenotypes. Transcriptome analysis revealed that PhhR controls four putative transcriptional units (phhA, hpd, hmgA, and dhcA) involved in aromatic amino acid catabolism; however, genes involved in PQS biosynthesis were unaffected. The phhA, hpd, hmgA, and dhcA promoters were mapped by primer extension, and purified His(6)-PhhR was shown to bind the phhA, hpd, and dhcA promoters in vitro by use of electrophoretic mobility shift assays. Our work characterizes a transcriptional regulator of catabolic genes induced during P. aeruginosa growth in CF sputum.

The different effects of structurally related sulfakinins on Drosophila melanogaster odor preference and locomotion suggest involvement of distinct mechanisms.

Sulfakinins are myoactive peptides and antifeedant factors. Naturally occurring drosulfakinin I (DSK I; FDDYGHMRFNH(2)) and drosulfakinin II (DSK II; GGDDQFDDYGHMRFNH(2)) contain sulfated or nonsulfated tyrosine. We discovered sDSK II and nsDSK II influenced Drosophila melanogaster larval odor preference. However, sDSK I, nsDSK I, MRFNH(2), and saline did not influence odor preference. We discovered sDSK I and nsDSK I influenced larval locomotion. However, sDSK II, nsDSK II, MRFNH(2), and saline did not influence locomotion. Our novel data suggest distinct mechanisms underlie the effects of DSK I and DSK II peptides on odor preference and locomotion, parameters important to many facets of animal survival.

The drosulfakinin 0 (DSK 0) peptide encoded in the conserved Dsk gene affects adult Drosophila melanogaster crop contractions.

We report that the drosulfakinin 0 (DSK 0; NQKTMSFNH2) structure and genomic organization are conserved. The DSK 0 C-terminus, SFNH2, is widely distributed in the animal kingdom suggesting it defines a novel peptide family. We also report the first description of DSK 0 activity. DSK 0, I (DSK I, FDDYGHMRFNH2), and II (DSK II, GGDDQFDDYGHMRFNH2) are encoded in sulfakinin (Dsk). Drosophila erecta, Drosophila sechellia, Drosophila simulans, and Drosophila yakuba shared 62.5-87.5% identity to Drosophila melanogaster DSK 0; Drosophila pseudoobscura shared 37.5% identity; numerous amino acids were one nucleotide different from a corresponding residue in D. melanogaster. DSK I and II were identical among the drosopholids. DSK 0 proteolytic processing sites were RR except D. yakuba contained KR and D. pseudoobscura contained HR, one nucleotide different from RR. DSK I and II processing sites were identical among the drosopholids. We established DSK 0 decreased adult (EC50=237nM and R(2)=0.941), but not larval gut contractions. DSK 0 exists in the central nervous system including the subesophageal ganglion and an abdominal ganglion. Peptide and genomic conservation, activity, and spatial and temporal distribution support the conclusion that DSK 0 plays diverse biological roles in drosopholids including regulating gut muscle contraction.