Characterization of Regulatory Pathways that Control a Virulence-Opacity Switch in Acinetobacter baumannii Pubblico

Tierney, Aimee (Fall 2021)

Permanent URL: https://etd.library.emory.edu/concern/etds/7p88ch76w?locale=it
Published

Abstract

Acinetobacter baumannii is a Gram-negative nosocomial pathogen that is highly resistant to a wide variety of antibiotics. A. baumannii cells exhibit phenotypic heterogeneity and switch rapidly between variants that form opaque or translucent colonies. These variants have unique transcriptional profiles and display differences in quorum sensing signal secretion, surface-associated motility, levels of polysaccharide capsule, biofilm formation, and carbon-source utilization. Most interestingly, the opaque variant is virulent while the translucent variant is avirulent, so they are designated VIR-O and AV-T, respectively. These observations suggest that manipulation of the genetic controls of the switch may attenuate A. baumannii virulence, thereby providing new methods of treatment that are not dependent on antibiotic susceptibility. The research described herein details the characterization of several regulatory genes that contribute to the control of the virulence-opacity switch in the clinical isolate AB5075. The stringent response regulator relA is one such gene, and upon its deletion cells become locked in the VIR-O state and display a strong increase in quorum sensing signal secretion and motility. The latter two phenotypes are enacted through the LysR-type transcriptional regulator ABUW_1132, which becomes overexpressed in the absence of relA. Investigation of ABUW_1132 revealed that its deletion decreases VIR-O to AV-T switching 16-fold, halts quorum sensing signal secretion, and reduces motility. Further, ABUW_1132 deletion increases levels of polysaccharide capsule and virulence in the AV-T variant. Finally, this work additionally characterizes a family of at least twelve TetR-type transcriptional regulators, some of which constitute the primary pathway through which switching is controlled. As VIR-O cells grow to high density, a stochastic increase in the transcription of one or more of these TetRs occurs, activating the switch to AV-T. The TetRs display high levels of homology to one another and act in a redundant manner such that if one TetR is deleted, others can serve to activate the switch. Four TetRs—ABUW_1645, ABUW_1959, ABUW_2818, and ABUW_3353—appear to be the most utilized, and deletion of these four genes results in a 1,245-fold decrease in switching. This research adds significantly to the body of knowledge surrounding switching, quorum sensing, motility, and virulence in AB5075.

Table of Contents

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

 

Chapter 2: Characterization of RelA in Acinetobacter baumannii………………………………45

           Table 1: Analysis of switching frequency between VIR-O and AV-T variants………....78

Table 2: Real-time qRT-PCR analysis of gene expression………………………………79

Figure 1: Colony morphology of wild-type and Δ3302 mutants………………………...82

           Figure 2: TLC autoradiogram of 32P-labeled nucleotides………………………………..83

           Figure 3: Cell morphology at low and high cell densities…………………………….…84

Figure 4: Surface motility of A. baumannii strains………………………………………85

Figure 5: Virulence assays using Galleria mellonella…………………………………...86

Figure 6: A model for the RelA-dependent control of downstream pathways………..…87

 

Chapter 3: A LysR-Type Transcriptional Regulator Controls Multiple Phenotypes in Acinetobacter baumannii………………………………………………………………………...88

Figure 1: AB5075 colony opacity morphotypes, effects of 1132 deletion on AHL secretion and motility………………………………………………………………...…123

Figure 2: The Reduced Switching Phenotypes in VIR-O Δ1132……………………....125

Figure 3: Deletion of 1132 in the AV-T background alters the opacity phenotype and capsule expression……………………………………………………………………...126

Figure 4: Deletion of 1132 in AV-T background increases virulence……………….....128

Supplemental Figure 1: Deletion of 1132 does not affect abaI expression…………….129

Supplemental Figure 2: VIR-O Δ1132 cells contain quorum sensing signal (AHL), but do not secrete it…………………………………………………………………………….130

Supplemental Figure 3: AV-T Δ1132 switches to the VIR-O variant at the same frequency as wild-type AV-T………………………………………………………..…131

Supplementary Figure 4: K locus genes for capsule polysaccharide synthesis and export are not transcriptionally regulated by 1132…………………………………………….132

Supplementary Figure 5: Growth curves for 1132 mutants and VIR-O Δ1132 virulence………………………………………………………………………………...133

Supplementary Table 1: RNA Sequencing Data for VIR-O Δ1132……………………134

Supplementary Table 2: Primers used in this study…………………………………….139

 

Chapter 4: A Family of TetR-Type Transcriptional Regulators Controls a Phenotypic Switch in Acinetobacter baumannii………………………………………………………………….……140

Table 1: Phenotypes of VIR-O cells overexpressing TTTRs………………………….……163

Figure 1: Phenotypes associated with overexpression of 1645, 1959, and 2818……….169

Figure 2: TTTR activation profiles during VIR-O to AV-T switching.…...…………...170

Figure 3: Analysis of VIR-O to AV-T switching frequencies in TTTR single, triple, and quadruple mutants………………………………………………………………………172

Supplementary Figure 1: ABUW_1645 overexpression converts other A. baumannii strains from VIR-O to AV-T………………………...………………………………….173

Supplementary Figure 2: The DNA-binding HTH region of 1645 is highly homologous to eleven other TetR-type transcriptional regulators in AB5075………………….………174

Supplementary Figure 3: Phenotypes of inactivating a TTTR in the ON state in AV-T cells……………………………………….…………………………………………….175

Supplementary Figure 4: Passage through the VIR-O state allows TetR-type transcriptional regulators (TTTRs) to reset……………………………………………..176

Supplementary Figure 5: Introduction of a 3353::T26 insertion in the triple mutant AV-T Δ16451959/2818::T26scar forces a switch back to the VIR-O state, evidenced by the change in colony opacity and the restoration of 3-OH C12-HSL secretion……………..177

Supplementary Figure 6: Analysis of inter-regulation between TTTRs by overexpression of TTTRs in the AV-T background…………………………………………………….178

Supplementary Figure 7: Analysis of inter-regulation between TTTRs in single mutants of 1645, 1959, 2818, and 3353 in the AV-T background……………………………....179

Supplementary Figure 8: Analysis of autoregulation of 1645, 1959, and 2818………..180

Supplementary Table 1: Oligonucleotides used in this study…………………………..181

 

Chapter 5: Methods for Detecting N-Acyl Homoserine Lactone Production in Acinetobacter baumannii………………………………………………………………………………………184

Figure 1: Cross-streak of Agrobacterium tumefaciens indicator strain and Acinetobacter baumannii………………………………………………………………………………194

Figure 2: Detection of AHL secretion on a soft agar lawn containing Agrobacterium tumefaciens biosensor strain…………………………………………………………....195

Figure 3: Methods for incubating TLC plates with a soft agar overlay………………...196

 

Chapter 6: Discussion…………………………………………………………………………..197

Figure 1: A model for VIR-O to AV-T to VIR-O switching as directed by the stochastic activation and subsequent deactivation of TTTRs………………………………………199

 

Appendix:

Roles of two-component regulatory systems in antibiotic resistance…………………………..207

Figure 1: The basic process of two-component signal transduction……………………259

Table 1: Summary of two-component regulatory systems that increase antibiotic resistance………………………………………………………………………………..260

About this Dissertation

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Subfield / Discipline
Degree
Submission
Language
  • English
Research Field
Parola chiave
Committee Chair / Thesis Advisor
Committee Members
Ultima modifica

Primary PDF

Supplemental Files