Application of the Microbiology Concept Inventory to improve programmatic curriculum.

The Microbiology Concept Inventory is an assessment tool derived from the fundamental statements created by the American Society for Microbiology. This two-tier, multiple-choice question inventory requires students to choose the most correct answer for each question and provide a brief justification of their reasoning. Educators can utilize this tool to identify common misconceptions held by students and adjust curriculum to address and prevent the persistence of student misconceptions. Over the course of 5 years, the Microbiology Concept Inventory was annually administered to undergraduate students enrolled in entry-level, mid-level, and senior capstone microbiology courses at a mid-western rural university. Analysis was completed to compare course, year, majors and minors, gender, ethnicity, and cumulative GPA. Results of this study showed a significant difference in Microbiology Concept Inventory scores between students with high cumulative GPAs (3.5-4.0) and students with comparatively lower cumulative GPAs (2.5-2.99, 3.0-3.49). Results between the other demographic categories revealed statistically different scores in favor of white students, but no differences in scores between genders. The results suggest evidence of ethnic bias, but no gender bias as measured by the Microbiology Concept Inventory. Additionally, significant differences in scores across cohorts are indicative of improvements in the curricula due to prior targeted changes. Analysis of concept inventory results can guide curriculum changes for course instructors. Implementation of curriculum changes can enrich students' academic success.

Introduction to Type III Secretion Systems.

Type III secretion (T3S) systems are found in a large number of gram-negative bacteria where they function to manipulate the biology of infected hosts. Hosts targeted by T3S systems are widely distributed in nature and are represented by animals and plants. T3S systems are found in diverse genera of bacteria and they share a common core structure and function. Effector proteins are delivered by T3S systems into targeted host cells without prior secretion of the effectors into the environment. Instead, an assembled translocon structure functions to translocate effectors across eukaryotic cell membranes. In many cases, T3S systems are essential virulence factors and in some instances they promote symbiotic interactions.

Isolation of Type III Secretion System Needle Complexes by Shearing.

Type III secretion (T3S) needle proteins are essential for the pathogenesis of many gram-negative bacteria. The needle component of the T3S system serves as the conduit for the translocation of effector proteins from the cytoplasm of many gram-negative bacteria into their target eukaryotic cells. Despite substantial advances that have been made in their characterization, a lot is still unknown about their interactions with other T3S system proteins and their roles in modulating host immune responses during infections. Critical to achieving this knowledge is the ability to isolate these needle proteins in their stable, native form. In this chapter, we describe a modified, streamlined isolation strategy for native forms of these T3S system needle proteins. We also present assays to detect the presence and quantification of these needle proteins.

Analysis of Type III Secretion System Secreted Proteins.

Secreted proteins of the T3SS vary from genus to genus. How secretion is induced in vitro also depends on the genus of bacteria. However, once those proteins are isolated the method for analyzing those proteins is largely the same. The following chapter outlines the specific induction of Yersinia secreted proteins and uniform analysis of those secreted proteins.

Expression and Purification of N-Terminally His-Tagged Recombinant Type III Secretion Proteins.

The ability to express and purify recombinant needle proteins from the Type III Secretion System (T3SS) of many gram-negative bacteria has allowed us to develop novel experimental approaches, both in vitro and in vivo, to identify unique roles for T3SS in bacterial pathogenesis. In addition, these purified needle proteins have shown to be promising immunotherapies acting as both protective antigens and adjuvants, presumably due to their immune activating properties. Here, we describe the expression and purification of recombinant T3SS needle proteins.

Identification of the Targets of Type III Secretion System Inhibitors.

A type III secretion system (T3SS) Inhibitor can be utilized for study in the research lab but also progressed into drug development. Since many pathogenic Gram-negative bacteria utilize this highly conserved system as a virulence factor, the prospect of the T3SS as a drug target is promising. To effectively move a T3SS inhibitor into the route of either research or pharmaceuticals an understanding of the target and mechanism of the inhibitor is required. Several methods can be utilized to identify the target. Included here is the use of knockout mutations, tagged inhibitor pull-down assays, and targeted identification methods.