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Temporal Proteomic and Lipidomic Analysis of Lytic and Lysogenic Mycobacteriophage

thesis
posted on 14.04.2022, 20:27 authored by Lauren E NovakLauren E Novak

  

Phages are viruses that infect bacteria and use its host machinery to replicate. They are one of the most ubiquitous and diverse biological entities in the biosphere with a long evolutionary history. There is renewed interest in phage therapeutics due to increased levels of antimicrobial resistance (AMR) and decreased numbers of newly developed antibiotics. Additionally, phages have applications in food safety, water quality, biocontrol, and vaccines. While many phage genomes have been sequenced and annotated, proteomic and lipidomic profiles of phage have barely been explored. The aim of this research is to measure the protein and lipid dynamics of phages with different infection cycles at different timepoints in the host’s growth cycle using mass spectrometry (MS). The data produced from this experiment can verify genomic annotation, discover novel proteins and lipids, and inspire future mechanistic and functional studies. For this study, Krili and PotatoSplit, lytic cluster O and lysogenic cluster A3 mycobacteriophages, were used to infect Mycobacterium smegmatis during the mid-exponential growth stage. Samples were taken in triplicate at hours 0.5 (immediately after infection), 3 (early exponential), 7 (late exponential), and 10 (early stationary). The samples were washed, mechanically lysed, quantified on BCA assay, lipid extracted, protein digested and desalted. For MS analysis, the samples were analyzed using a HPLC-ESI-MS/MS. Protein data was processed using MaxQuant and lipid data using MS-DIAL and MS-FINDER. Normalized data was statistically analyzed on MetaboAnalyst, correlated on JMP, and functionally analyzed using DAVID. The results showed that the protein and lipid expression of both phages differed with time in comparison to the control (uninfected Mycobacterium smegmatis) and to each other. Additionally, phage protein-lipid correlations suggest simultaneous phage infection and bacterial defense mechanisms particularly during late infection. Temporal and correlation data produced from this experiment can be used for future targeted -omics research. As one of the first experiments of its kind, the MS workflow used in this study can provide a scaffold for future phage temporal proteomic and lipidomic analysis. 

History

Degree Type

Master of Science in Agricultural and Biological Engineering

Department

Agricultural and Biological Engineering

Campus location

West Lafayette

Advisor/Supervisor/Committee Chair

Kari Clase

Additional Committee Member 2

Jackeline Franco Marmolejo

Additional Committee Member 3

Stephen Byrn