Functions of the Neisseria meningitidis TolC Protein Público

Abstract

Abstract Functions of the Neisseria meningitidis TolC Protein By Nazia Kamal In Escherichia coli, TolC serves as the ubiquitous outer membrane protein (OMP) for many tripartite efflux pumps and participates in the export of bacterial proteins and exogenously added antimicrobials. In the first instance, it has the capacity to remove toxins, such as the repeat-in-toxin (RTX) toxin HlyA, also known as -hemolysin, via the hlyABCD encoded type I secretion system. In the second instance, it participates in the efflux of antimicrobials by the AcrA-AcrB efflux pump amongst others. There are two tolC homologs in Neisseria meningitidis, mtrE and tolC. In both N. meningitidis and closely related pathogen N. gonorrhoeae, MtrE is the OMP for the mtrCDE efflux pump, which exports hydrophobic agents such as drugs (i.e. erythromycin), detergents (i.e. TX- 100), dyes (i.e. crystal violet), and antimicrobial peptides (i.e. LL37). The TolC-like protein is present only in N. meningitidis and is absent from N. gonorrhoeae. In this body of work, I report that unlike the genetic organization in E. coli, the meningococcal tolC gene is co-transcribed with hlyD, whereby hlyD is the first gene in the operon. Expression of this operon is growth phase-dependent, with maximal expression at the late logarithmic phase of growth. I documented that MtrE is the sole OMP required for drug export in N. meningitidis by the MtrC-MtrD-MtrE efflux system. Although TolC does not have a role in drug export in meningococci it is, however, required for secretion of FrpC, an RTX toxin. When the wildtype meningococcal tolC was introduced into RAM1129, an E. coli tolC deletion strain, it could functionally replace the E. coli protein as the OMP channel in drug export. Furthermore, the meningococcal TolC protein could restore extracellular -hemolytic activity in strain RAM1129 mediated by the hlyABCD encoded type I secretion system. This work documented the ability of the meningococcal TolC protein to participate in drug export in E. coli and toxin secretion in both E. coli and N. meningitidis.

Table of Contents

Table of Contents

Abstract

Acknowledgements

Dedication

List of Tables and Figures

Chapter 1: Introduction…………………………………………………………….1

Chapter 2: The TolC-like protein of Neisseria meningitidis is required for extracellular production of the RTX toxin FrpC but not for resistance to antimicrobials recognized by the Mtr efflux pump system………………………...40

Chapter 3: Biologic Activities of the TolC-like Protein of Neisseria meningitidis as Assessed by Functional Complementation in Escherichia coli…………………61

Chapter 4: Unpublished Observations……………………………………………..76

Chapter 5: Summary and Discussion………………………………………………92

List of Figures and Tables

Chapter 1

Figure 1: Efflux pumps possessed by Neisseria

Figure 2: Schematic of type I secretion

Chapter 2

Table 1: MIC values of HA against meningococci

Figure 1: Organization and transcription of the hlyDtolC operon in N. meningitidis strain M7

Figure 2: Complementation of the tolC mutation restores extracellular production of FrpC

Chapter 3

Table 1: Susceptibility of Nm strains to antimicrobials recognized by efflux pumps

Table 2: Expression of Nm tolC in RAM1129 increases resistance to antimicrobials

Figure 1: Extracellular α-hemolysin production in RAM1129 expressing Nm tolC

Chapter 4

Figure 1: Growth curve of M7, M7farR::kan, and M7marR::kan

Figure 2: Transcription of tolC, mtrE, hlyD, and rnpB.

Figure 3: Crystal violet staining of biofilms.

Table 1: Cell viability measured by Trypan Blue Exclusion

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Biology, Microbiology
Palavra-chave	multidrug efflux pump RTX toxins antimicrobial resistance Neisseria meningitidis TolC
Committee Chair / Thesis Advisor	Shafer, William M, Emory University
Committee Members	Rather, Phil N, Emory University Kalman, Daniel, Emory University Moran Jr., Charles, Emory University Tzeng, Yih-Ling, Emory University Churchward, Gordon, Emory University

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