Demonstrating Correlations Between Structural And Functional Changes In The Three Vibrio Cholerae Pili Open Access

Storms, Rachel Elizabeth (2016)

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Vibrio cholerae, the pathogenic bacteria responsible for the deadly disease cholera, produces three functional type IV pili: the MSHA-type pilus; the Chitin regulated pilus; and the Toxin co-regulated pilus. It was hypothesized that: i) the three pili would be morphologically distinguishable from each other; ii) mutations in the MSHA pilin subunit and hydrolyzing ATPase would be structurally and functionally similar, iii) that strains mutated in ChiRP and MSHA will not form a biofilm in non-Tcp inducing conditions. Negative staining and immunogold labeling was utilized to visualize the pili with transmission electron microscopy. It was determined that the MSHA pilus is a moderately bundled, wispy filament; the chitin-regulated pilus is a relatively unbundled, wispy, web-like filament; and the toxin co-regulated pilus is a highly bundled, fibrous, rigid filament. In order to correlate the structure of each pilus with its function during biofilm formation, strains mutated in each pilus were assessed for their ability to form biofilms on abiotic surfaces. It was found that the MSHA-pilus is sufficient for biofilm formation - though deletions of this pilus do not completely inhibit formation. The chitin-regulated pilus aids in the fitness of the biofilm, but is not crucial for the formation of the biofilm. The toxin co-regulated pilus may inhibit the formation of a biofilm, for deletions in this pilus produced a more robust biofilm.

Table of Contents

Introduction: 1

Cholera: 1

Vibrio cholerae: 2

Biofilms: 3

Type IV Pili: 4

Vibrio cholerae Pili: 5

Hypotheses: 7

Methods: 9

Bacterial Strains: 9

Biofilm Assays: 9

Negative Stain Transmission Electron Microscopy (TEM) of Strains: 10

Immunogold Labeling and Imaging: 11

Results: 12

Biofilm Assays: 12

Negative Staining: 15

Immunogold Labeling: 19

Discussion: 20

Limitations and Future Directions: 21

References: 23

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