Analysis of wzz2 expression in different Pseudomonas aeruginosa serotypes and the correlation of resistance to serum and outer membrane stress Pubblico

Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen that can cause infections in immunocompromised people as well as lung infections in those living with cystic fibrosis (CF). This bacterium produces a virulence factor, lipopolysaccharide (LPS), which is a glycolipid molecule embedded in the bacterial outer membrane. The polysaccharide that extends from the distal portion of LPS is referred to as O antigen, the composition of which categorizes P. aeruginosa into 20 different serotypes. O antigen is further characterized as long or very long based on the number of sugar repeat units. The expression of long O antigen is controlled by the chain-length control protein Wzz1, while the expression of the very long is controlled by the chain-length control protein Wzz2. This study focuses on the production of Wzz2 and very long O antigen with the goal of understanding how they contribute to the fitness of different serotype strains in human serum and other outer membrane disrupting environments. Analysis of LPS in all 20 serotype strains revealed that each serotype produced varying amounts and lengths of O antigen. wzz2 clean deletion mutants of serotype O1 and O8 confirmed that the mutation was responsible for the production of very long O antigen in both strains. Next, we determined whether the expression of very long O antigen contributed to serum resistance of individual serotype strains and found that the deletion of wzz2 caused serotype O1 strain to become sensitive to normal human serum, but had little effect on the serum resistance of the serotype O5 control strain PAO1 or the serotype O8 strain. We also evaluated the relative fitness of individual serotype strains and their wzz2 mutants under different osmotic stress and pH. Deletion of wzz2 caused serotype O1 and O8 strains to grow poorly in low pH condition but the mutation in wzz2 caused PAO1 to grow better. Our experimental results suggest that the role of O antigen in the protection against outer membrane disrupting environments varies across different serotypes. Future experiments aim to elucidate the contribution of very long O antigen to serum resistance and antibiotic resistance across all serotypes.

Table of Contents

INTRODUCTION 1

RESULTS AND DISCUSSION 3

CONCLUSIONS AND FUTURE DIRECTIONS 12

METHODS 14

FIGURES AND TABLES 19

REFERENCES 28

About this Honors Thesis

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School	Emory College
Department	Physics
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell Biology, Microbiology
Parola chiave	O antigen Lipopolysaccharide Pseudomonas aeruginosa
Committee Chair / Thesis Advisor	Goldberg, Joanna, Emory University
Committee Members	Kim, Minsu, Emory University Finzi, Laura, Emory University

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