Characterization of Regulatory Pathways that Control a Virulence-Opacity Switch in Acinetobacter baumannii Pubblico
Tierney, Aimee (Fall 2021)
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 Δ1645/Δ1959/2818::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
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