Characterization of the role of Hfq in ABUW_1645 expression and phenotypic switching in Acinetobacter baumannii Öffentlichkeit

Wolin, Sydney (Spring 2019)

Permanent URL: https://etd.library.emory.edu/concern/etds/5x21tg45r?locale=de
Published

Abstract

Acinetobacter baumannii is a Gram-negative nosocomial pathogen that predominately affects immunocompromised individuals. Recently, A. baumannii strains have exhibited a stark increase in antibiotic resistance. Therefore, new treatment methods are crucial for overcoming infection. Prior research has shown that A. baumannii interconverts between two colony variants, opaque (O) and translucent (T). It has been demonstrated that the O variant is more virulent and antibiotic resistant than the T variant. Therapeutics that force A. baumannii into the T state could be an effective means of treating infection. The purpose of this study was to better comprehend the regulation of phase variation in A. baumannii. Several regulatory genes have been identified that regulate the O to T switch. The TetR transcriptional regulator ABUW_1645 exhibits a 60-fold increase in expression in the T variant, and overexpression of ABUW_1645 drives the O to T switch. Hfq is an RNA chaperone protein that simultaneously binds sRNA and mRNA acting as a post-transcriptional regulator. Hfq has also been previously identified to positively regulate O to T switching. In this study, we found that overexpression of Hfq increases ABUW_1645 expression, and confirmed that Hfq positively regulates O to T switching. Site-directed mutagenesis of both the distal and proximal faces of Hfq reversed its effects on ABUW_1645expression and phenotypic switching. These results demonstrate the importance of Hfq in regulating these processes, and show that both mRNA and sRNA binding are essential for Hfq to have an effect on ABUW_1645expression and phenotypic switching. This work establishes Hfq as a potential target to combat A. baumannii infection. This research suggests that interventions manipulating Hfq may effectively reduce A. baumannii virulence as well as antibiotic resistance by forcing cells into the T state. 

Table of Contents

Introduction……….………..………..………..………..……….……..………...………..………..1

Materials and Methods………..………..………………..………..………..………………….....4

Table 1………..………..………………..………..……..………..…..………..………..….............4

Table 2………..…….....…………….……..………..……..………...………..………..……..........4

Results.………..………..………………..…………..……..………..………..……………….........9

Figure 1………..………………..………..……..………..………..………..………..………….......9

Figure 2...………..………………..………..……..………..………..………..………..…….........10

Figure 3..……..………………..………..……..………..………..………..………….………....….11

Figure 4….………..………………..………..……..………..………..………..………..…..…......11

Figure 5a…..………..………………..………..……..…………..………..………..………..........12

Figure 5b..………..………………..…………..……..…………....………..………..……...........12

Figure 6...……..………………..……..……..………..………..…..………..………..……..........13

Figure 7a……………………………………………………………………………………….......…14

Figure 7b……………………………………………………………………………………......……14

Figure 7c…………………………………………………………………………………….......……14

Figure 7d………………………………………………………………………………………......…14

Discussion………..………..………………..………..………....………..………..………..…..…15

References………..………..………………..………..……….…..………..………..…….…....…18

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