Structural Requirements for NADase activity of bacterial Toll/interleukin-1 receptor (TIR) domain-containing proteins

Michelle Snyder, Towson University
Greg Snyder, University of Maryland, School of Medicine
Location: Maryland

Context

The course is a 400-level laboratory course designed to engage 20-24 students each semester. The course is a three credit stand-alone laboratory course and meets twice weekly—with one two hour class period and one four hour class period. Depending on the major concentration, the course fulfills an upper-level lab requirement or elective.

The typical student in the course is a junior or senior who has completed one or more upper level cell or molecular biology courses. Departmental efforts have been made, however, to encourage enrollment earlier in students' course sequences, so the only prerequisite requirements are that students enrolled in the course have completed the introductory biology majors sequence (Intro to Cell/Molecular Biology, Intro to Ecology/Evolution, and Genetics).

Target Audience: Major, Upper Division
CURE Duration:A full term

CURE Design

The course is designed to examine the structure-function relationships of the class of bacterial Toll-interleukin-1 receptor (TIR) domain-containing proteins, which play a role in bacterial evasion of innate immune defenses. Previous studies have shown that bacterial TIRs both directly block innate immune signaling responses as well exhibit enzymatic NADase activity in both bacteria and in host cells. Working collaboratively with researchers from the University of Maryland, School of Medicine, undergraduate students in the Cell Biology Laboratory course at Towson University identify structural motifs that they predict might have a role in TIR NADase activity and create amino acid mutations in recombinantly-expressed bacterial TIR proteins to test their hypotheses.

Students spend the first few weeks of the course learning basic laboratory techniques, reading background papers related to the overall project, and examining structural data to identify amino acids they predict would be important for TIR function. Working in groups of four, students from each group then choose one of their selected amino acids to further investigate for the remainder of the semester. Each group creates a unique amino acid mutation and tests their mutant for NADase enzymatic activity. At the completion of the course, each group presents a summary of their findings at the annual Towson University Biology Department Research Symposium. Students from the CURE course are invited to continue independent research projects in the instructor's laboratory following completion of the course.

The CURE course has created opportunities for a large and diverse group of students to engage in authentic research experiences. In addition, the data generated from the course appears to support data derived from recent structural studies of bacterial TIRs, highlighting the potential value of CURE courses for making valuable research contributions to the broader scientific field.

Stakeholders include the general scientific community, and specifically immunology and microbiology researchers. The course is designed in collaboration with my research laboratory at Towson University and Dr. Greg Snyder's research laboratory at the University of Maryland, School of Medicine. At the end of each semester, students present the data as poster presentations at Towson's Biology Department Research Symposium. The data and mutants generated are used in ongoing research in the laboratories at Towson and the University of Maryland. Information about course design and data generated in the CURE course have been presented at the Annual Meeting of the American Association of Immunologists, and a manuscript detailing scientific findings from the CURE course that corroborate recent findings in the field currently is being prepared.

Core Competencies: Analyzing and interpreting data, Asking questions (for science) and defining problems (for engineering), Constructing explanations (for science) and designing solutions (for engineering), Developing and using models, Planning and carrying out investigations
Nature of Research: Basic Research, Wet Lab/Bench Research

Tasks that Align Student and Research Goals

Instructional Materials

Instructional Materials:
-Course syllabus
-Course Schedule/Matrix
-Course Assignments (zipped folder)
-Journal Club Reading Guides
-Bionformatics Assignments
-Experimental Design and Analysis Worksheets

Course Syllabus (Microsoft Word 2007 (.docx) 50kB Jun27 23)
Course Schedule/Matrix (Microsoft Word 2007 (.docx) 30kB Jun27 23)
Course Assignments (Zip Archive 182kB Jun27 23)

Assessment

Miniproposal Rubric (Microsoft Word 2007 (.docx) 16kB Jun27 23)
Poster Rubric (Acrobat (PDF) 106kB Jun27 23)

Instructional Staffing

Instructor-Involved with all aspects of the course, including overall course development and experimental design, oversight of lab preparation, teaching of the class, facilitating students in carrying out the experiment and interpreting the results, and advice and mentoring in regards to preparation of the final poster.

Undergraduate Learning Assistants (ULAs)--One to two ULAs assist with preparation of materials and assistance with student groups during the laboratory periods. ULAs for my class have been students in the instructor's research lab, who often have completed the course previously.

Scientific Collaborator-Dr. Greg Snyder (University of Maryland, School of Medicine) is a scientific collaborator. Dr. G. Snyder collaborates with CURE project design and experimental troubleshooting. Dr. G. Snyder developed the worksheets for bioinformatics analysis and molecular visualization.

Towson University STEM Career Coach (Ms. Tanja Swain and previously Mr. Matthew Smith)-One goal for the course is that students create strategies and tools for career preparation. Three one-hour workshops are designed by the career coach to highlight tools available in the Career Center, including assistance in preparing resumes/CVs and cover letters, in job search strategies including the use of LinkedIn and in practicing interview strategies.

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Provenance: Michelle Snyder, Towson University
Reuse: This item is offered under a Creative Commons Attribution-NonCommercial-ShareAlike license http://creativecommons.org/licenses/by-nc-sa/3.0/ You may reuse this item for non-commercial purposes as long as you provide attribution and offer any derivative works under a similar license.

Author Experience

Michelle Snyder, Towson University

The benefits of undergraduate research for promoting student content knowledge, critical thinking and analysis, transferable job skills, and science self-efficacy are well-documented. In the traditional apprenticeship model, however, slots for undergraduate researchers are limited. CUREs expand capacity for research training, and because CUREs make use of class time for research, they often incorporate students with outside obligations, including jobs, commutes or family responsibilities, who might not otherwise seek independent research experiences. While my major motivation for developing and teaching my CURE class focused on this ability to engage a larger, more diverse groups of students in research, I also recognized the potential benefits for my own research program for exploring new research questions and building datasets for future publication. These faculty benefits are especially compelling for faculty at primarily undergraduate institutions balancing relatively heavy teaching loads and research programs supported by relatively limited resources.

Advice for Implementation

-Plan catch-up days through the semester to account for experimental failures, reagents on back-order, or other scenarios for which you can't plan.

-Utilize multiple modes of assessment besides exams. The course gives an opportunity for students to practice the research enterprise and gain science self-efficacy, and there is value in reducing student anxiety around grades for this course.

-Be purposeful about naming the skills that students are demonstrating in the classroom. Students tend to make mistakes, but find ways to respond positively--"I'm impressed with the way your group problem-solved to still generate data from that experiment. Problem-solving is a critical component of being a scientist."

-I taught one iteration of the course on-line during the pandemic when students could not come to campus, so all experimental procedures that semester were carried out by the instructor and ULA. The time that would have been used for experimental procedures was devoted instead to various exercises, many of which I have continued to implement post-pandemic:

a) Among the most valuable new exercises involve extended time for designing experiments and interpreting experimental results. Students discuss these topics and complete worksheets (shared in the instructional materials here). I find that building this time into the course deliberately slows the pace of the course--even if the tradeoff involves fewer experimental procedures completed--so that students take the time to consider these important topics, giving a chance for all group members to digest the overall goals, designs or outcomes of the experiments before rushing to begin the experimental procedures.

b) A second valuable addition to the course during the pandemic was the implementation of career preparation workshops by the STEM Career Coach.

c) I also implemented completion of a short written proposal, with scaffolded short assignments, in which students use the literature to define a novel question related to bacterial TIRs and develop a scientific plan that could be used in the future to address the question. Now that students are back in-person 6 hours a week in the laboratory, I have been working to shorten the assignment and still have students gain the benefits of practicing literature searches, hypothesis development, experimental design, and writing.

Iteration

Students work in groups of four, with pairs of students completing duplicate experimental runs. This allows for back-up experiments, for example, when creation of the mutant or verification of expression is required to move forward with future experiments.

Most experimental procedures are repeated at least twice to allow for problem-solving and trouble-shooting. Even in cases where experimental procedures are not repeated (for example, if only one student's PCR mutagenesis experiment is successful, there tend to be enough subsequent transformants for each student to perform independent plasmid extraction and sequencing), I will incorporate additional PCR practice where long experimental incubation times create down-time in future weeks.

Using CURE Data

Data generated in the course is shared on a course Blackboard site and transferred to the instructor's Onedrive following completion of the course. During the course, all experimental results are performed as technical replicates by pairs of students in the groups of four. Most experiments are repeated at least once as biological replicates during the CURE course. The results of all NADase experiments are repeated as biological replicates by advanced undergraduate research students in Dr. M. Snyder's laboratory or by Dr. M. Snyder.

All students in the CURE course are given the opportunity to be included as co-authors on publications derived from the CURE course data, provided they review the manuscript prior to submission. Students who contribute to the project, but do not respond to requests for manuscript review will be acknowledged for their contributions. ULAs and students from the CURE course are invited to continue independent research in Dr. M. Snyder's laboratory, and those who do and make more significant contributions to the datasets will be listed earlier in the author order.

Resources

Papers used for Journal Clubs:
1. Cirl, C., et al. (2008). Subversion of the Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins. Nat. Med. 14:399-406. 
2. Essuman, K., et al. (2018) TIR domain proteins are an ancient family of NAD+-consuming enzymes. Curr. Biol. 28:421-430. doi: 10.1016/j.cub.2017.12.024.

Additional Key Papers:

1. Essuman, K., et al. (2017) The SARM1 Toll/Interleukin-1 receptor domain possesses intrinsic NAD+ cleavage activity that promotes pathological axonal degeneration. Neuron, 93:1334-43. doi: 10.1016/j.neuron.2017.02.022.

2. Klontz, et al., (2023) The structure of NAD+ consuming protein Acinetobacter baumannii TIR domain shows unique kinetics and conformations. bioRxiV 2023.05.19.541320 doi.org/10.1101/2023.05.19.541320

3. Li, S. (2023) Toll/interleukin-1 receptor domains in bacterial and plant immunity. Curr. Opin. Microbiol. Apr 19;74:102316. doi:10.1016/j.mib.2023.102316

4. Manik, M.K., et. al. (2022) Cyclic ADP ribose isomers: production, chemical structures and immune signaling. Science, 377:eadc8969. doi: 10.1126/science.adc8969.

5. Shi, et. al. (2022) Structural basis of SARM1 activation, substrate recognition and inhibition by small molecules. Mol. Cell., 82:1643-59. https://doi.org/10.1016/j.molcel.2022.03.007

6. Snyder, G.A., et al. (2013) Molecular mechanisms for the subversion of MyD88 signaling by TcpC from virulent uropathogenic Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 110:6985-90. doi: 10.1073/pnas.1215770110.