Project Title

Cyclic Di-GMP Regulates Biofilm Formation, Desiccation Tolerance, and Motility in Acinetobacter Baumannii

Authors' Affiliations

Garrett Reynolds, Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN Gabrielle Shipstone, Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN Gabriel Smith, Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN Erik Petersen, Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN

Location

Culp Room 304

Start Date

4-6-2022 1:00 PM

End Date

4-6-2022 2:00 PM

Faculty Sponsor’s Department

Health Sciences

Name of Project's Faculty Sponsor

Erik Petersen

Additional Sponsors

Erik Petersen, Bert Lampson, Christopher Pritchett

Classification of First Author

Graduate Student-Master’s

Competition Type

Competitive

Type

Oral Presentation

Project's Category

Microbiology

Abstract or Artist's Statement

Acinetobacter baumannii is an increasingly multidrug-resistant Gram-negative bacterial pathogen and contributes to many hospital-acquired infections. Discovering new treatments against Acinetobacter baumannii infections is necessary as the pathogen adapts to the antimicrobials prescribed by physicians. Cyclic di-GMP (c-di-GMP), a bacterial second messenger, can regulate various phenotypes including biofilm formation, desiccation tolerance, motility, etc.; many of these phenotypes may help A. baumannii better survive a hospital environment, such as dryness on hospital surfaces. Up to twelve c-di-GMP modulating enzymes (CMEs) and two c-di-GMP binding proteins are predicted to be encoded by this pathogen. Diguanylate cyclases (DGCs) produce c-di-GMP, whereas phosphodiesterases (PDEs) degrade c-di-GMP. More c-di-GMP that can bind to its binding proteins means more biofilm formation and less motility. Of the eleven CMEs, 7 are DGCs, 2 are PDEs, and 3 encode both domains (DGCs/PDEs). I hypothesized that biofilm formation, desiccation tolerance, and motility were controlled by c-di-GMP and that we could target these parts of the c-di-GMP signaling network for new treatments. If we disrupt these genes, then we should see a reduction in the regulatory effects of these phenotypes. In this investigation, we generated mutants with a single gene knockout or transposon mutagenesis in two different A. baumannii strains: 17978, a historical laboratory strain that exhibits swarming motility and AB5075, a recent clinical isolate that exhibits twitching motility. To test biofilm formation, we let the mutants grow to their maximum concentration in 96-well plates, stained the plates with crystal violet, and quantified the crystal violet that stained the biofilm. To test for motility, a LB agar plate was stabbed to the plastic surface or dropped on the agar surface with diluted culture to determine the presence of twitching or swarming motility, respectively. To test for desiccation tolerance, we washed the cultures in distilled water to rid the sample of any salt, serially diluted the samples in solution, and plated them out onto LB agar plates. Bacterial counts were quantified before and after desiccation to determine survival of each mutant. From these experiments, 6 DGCs, 1 PDE, and 2 DGCs/PDEs were shown to regulate biofilm formation in AB5075. Furthermore, a PDE and a DGC/PDE were shown to regulate twitching motility in AB5075, while a single DGC was required for tolerating dryness. In strain 17978, we have found a PDE and 4 DGCs that are necessary for swarming motility and are currently conducting biofilm and desiccation tolerance assays. So far, we’ve identified a role for c-di-GMP in A. baumannii biofilm formation, motility, and desiccation survival. Inhibiting the regulation of these pathways could produce novel mechanisms to combat this pathogen in the hospital environment.

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Apr 6th, 1:00 PM Apr 6th, 2:00 PM

Cyclic Di-GMP Regulates Biofilm Formation, Desiccation Tolerance, and Motility in Acinetobacter Baumannii

Culp Room 304

Acinetobacter baumannii is an increasingly multidrug-resistant Gram-negative bacterial pathogen and contributes to many hospital-acquired infections. Discovering new treatments against Acinetobacter baumannii infections is necessary as the pathogen adapts to the antimicrobials prescribed by physicians. Cyclic di-GMP (c-di-GMP), a bacterial second messenger, can regulate various phenotypes including biofilm formation, desiccation tolerance, motility, etc.; many of these phenotypes may help A. baumannii better survive a hospital environment, such as dryness on hospital surfaces. Up to twelve c-di-GMP modulating enzymes (CMEs) and two c-di-GMP binding proteins are predicted to be encoded by this pathogen. Diguanylate cyclases (DGCs) produce c-di-GMP, whereas phosphodiesterases (PDEs) degrade c-di-GMP. More c-di-GMP that can bind to its binding proteins means more biofilm formation and less motility. Of the eleven CMEs, 7 are DGCs, 2 are PDEs, and 3 encode both domains (DGCs/PDEs). I hypothesized that biofilm formation, desiccation tolerance, and motility were controlled by c-di-GMP and that we could target these parts of the c-di-GMP signaling network for new treatments. If we disrupt these genes, then we should see a reduction in the regulatory effects of these phenotypes. In this investigation, we generated mutants with a single gene knockout or transposon mutagenesis in two different A. baumannii strains: 17978, a historical laboratory strain that exhibits swarming motility and AB5075, a recent clinical isolate that exhibits twitching motility. To test biofilm formation, we let the mutants grow to their maximum concentration in 96-well plates, stained the plates with crystal violet, and quantified the crystal violet that stained the biofilm. To test for motility, a LB agar plate was stabbed to the plastic surface or dropped on the agar surface with diluted culture to determine the presence of twitching or swarming motility, respectively. To test for desiccation tolerance, we washed the cultures in distilled water to rid the sample of any salt, serially diluted the samples in solution, and plated them out onto LB agar plates. Bacterial counts were quantified before and after desiccation to determine survival of each mutant. From these experiments, 6 DGCs, 1 PDE, and 2 DGCs/PDEs were shown to regulate biofilm formation in AB5075. Furthermore, a PDE and a DGC/PDE were shown to regulate twitching motility in AB5075, while a single DGC was required for tolerating dryness. In strain 17978, we have found a PDE and 4 DGCs that are necessary for swarming motility and are currently conducting biofilm and desiccation tolerance assays. So far, we’ve identified a role for c-di-GMP in A. baumannii biofilm formation, motility, and desiccation survival. Inhibiting the regulation of these pathways could produce novel mechanisms to combat this pathogen in the hospital environment.