Graduation Year

2021

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Lindsey N. Shaw, Ph.D.

Committee Member

Yu Chen, Ph.D.

Committee Member

Sophie Darch, Ph.D.

Committee Member

James Riordan, Ph.D.

Committee Member

Prahathees Eswara, Ph.D.

Keywords

universal stress protein, antimicrobial peptides, high-throughput, ESKAPE

Abstract

Acinetobacter baumannii is a multi-drug resistant nosocomial pathogen known for causing wound related- and respiratory-infections. It is currently on the WHO’s list of critical pathogens due to its broadly drug resistant nature and the constant appearance of pan-resistant isolates. A majority of the infections caused by this organism are biofilm associated, however there is limited existing knowledge regarding the mechanisms used to engage in this multicellular lifestyle. As such, we set out to explore the factors influencing this behavior using an 10,000+ isolate transposon mutant library of A. baumannii strain AB5075. Of the strains tested, 6.45% demonstrated some level of change to their biofilm forming capacity (either increased or decreased). The screen, coupled with more in-depth (extracellular matrix) ECM analyses and real-time biofilm tracking, allowed us to further characterize 16 of the most influential strains. During this investigation, the most significant biofilm phenotype was observed for a tn mutant of a universal stress protein, which demonstrated an 8-fold increase in biofilm formation compared to the wildtype strain. This led us to investigate the function of this protein. Through this, we have named the protein UspG based on structure prediction tools and demonstrate its essentiality in the survival of AB5075 in whole human blood; likely mediated at least in part by aiding in protection against oxidative stress. In addition, we reveal its importance during exponential growth through expression monitoring and RNA sequencing analysis. These studies reveal that UspG broadly influences cellular behavior, and specifically the processes of virulence, metabolism, and cell envelope homeostasis. Collectively, these studies provide a deeper understanding of pathways important in the formation and maintenance of biofilms in A. baumannii. Further examination of the factors highlighted herein will provide promising insight into potential targets for therapeutic intervention in the clinical setting.

Towards the latter point, the rising rates of multi-drug resistant bacterial infections demonstrate a pressing need for the development of new antibacterial agents with novel mechanisms of action. Medicinal plants are a viable source for antimicrobial peptides and therefore we have worked with collaborators on development of the PepSAVI-MS pipeline for bioactive peptide discovery. This platform uses mass spectrometry coupled with statistics to create a highly versatile approach to isolating bioactive peptides from complex multi-cellular systems. Our primary focus in this regard is ribosomally synthesized post-translationally modified peptides (RiPPs), which have been largely overlooked in standard AMP fractionation techniques. We have validated this approach through the screening of Viola odorata fractions and thereafter assessing the bioactivity of purified AMPs of interest, including cycloviolacin O2, against the ESKAPE pathogens. Herein we report the bioactivity of several additional ethnobotanical species, many of which possess profound and broad-spectrum activity against an array of multi-drug resistant bacterial pathogens. With the evident promise of our preliminary analyses based on bioactivity alone, we are confident that this pipeline will reveal novel antimicrobial peptides with potential as future therapeutics.

Download

Included in

Microbiology Commons

Share

COinS