Investigating Impact of Mycobacterial Physiology on Mycobacteriophage Life Cycles by Mass Spectrometry
Creators: name: Li, Yi
Publisher: name: Purdue University Graduate School
Year: 2019
Subjects: Agricultural biotechnology not elsewhere classified

description: Mycobacteriophages are the viruses that infect mycobacteria. Due to the high death rate and antibiotic-resistant strains, phage therapy is considered to be a promising treatment of tuberculosis. Current understanding of phage-bacteria interaction is abstracted as phage lytic and lysogenic life cycles. However, bacterial physiology may impact phage life cycles and bacterial cells with different physiology may have different responses to phage infection. In order to improve the understanding of phage-bacteria interaction and update phage therapy strategy, the impact of mycobacterial physiology on mycobacteriophage life cycles was studied in this research. In this research, a mass spectrometry-based method was first developed to study phage proteins in phage-bacteria mixture. Then five mycobacteriophages isolated at Purdue University were selected to infect exponential and stationary <i>Mycobacterium smegmatis</i> (<i>M. smegmatis</i>) cell cultures. Growth curves of the <i>M. smegmatis</i> cell cultures infected by the five phages were determined. Proteomics and lipidomics of the <i>M. smegmatis</i> cells cultures infected by phages FrenchFry and MrGordo were analyzed by mass spectrometry. The correlations between individual proteins/lipids and the experimental factors (bacterial growth phases, phages and phage infection time) were studied by developing linear regression models using SAS. The mass spectrometry-based method was proved to be able to detect phage proteins other than the structural proteins. It also verified the phage protein annotation that had been accomplished <i>in silico</i>. X! Tandem and a database consisting of six frame translation of the phage genome and the annotated proteins of <i>M. smegmatis</i> were the optimal option for analyzing mass spectra data of phage-bacteria mixture. The growth curves of the <i>M. smegmatis</i> infected by the phages displayed that growth of exponential <i>M. smegmatis</i> cell cultures were depressed by phages (except FrenchFry) and stationary <i>M. smegmatis</i> cell cultures were not actively lysed by any of the phages. The proteomics results showed that MrGrodo infection impacted more proteins than other factors did. Exponential phase up-regulated proteins involved in cell division. Stationary phase up-regulated proteins that may change cell surface properties. FrenchFry up-regulated LuxR protein. Infection time up-regulated the proteins associated with mycobacterial virulence. The lipidomics results indicated that growth phases impacted the most lipids. Phage infection time increased the amount of the lipids related to mycobacterial virulence. In summary, the mass spectrometry-based method developed in this research can be employed to study phage proteins in phage-bacteria mixture and verify phage genome annotation. Mycobacterial physiology alters mycobacteriophage life cycles. Phage-bacteria interaction is the interaction between the two populations instead of between an individual phage particle and an individual bacterial cell. Virulence of <i>M. smegmatis</i> improves as a response to phage infection.