Discovery of new phage defense systems in Vibrio cholerae

Abstract: Over a century ago, Felix d’ Herelle began using bacterial viruses which he termed “bacteriophages” to treat bacterial infections. Such “phage therapy” has gained new interest due to the emergence of multi-drug resistant bacterial pathogens. An advantage of phage therapy is the ability to lyse specific bacteria without

disrupting the host microbiota. Although phages can infect and lyse bacteria, bacteria have evolved a myriad of molecular defense systems to protect against phage infection. This has led to the search and identification of a plethora of novel phage defense mechanisms; however, there are still a vast number of phage defense

systems that have yet to be discovered and the molecular mechanism by which these systems mediate phage defense is often not well understood. My research centers on the bacterial pathogen Vibrio cholerae as it is intimately linked to phage predation both during environmental persistence and human infection. Moreover, V.

cholerae has been a catalyst in the discovery of multiple novel phage defense systems. Due to its intricate relationship with phage, the central hypothesis of my thesis research is that V. cholerae encodes additional novel phage defense systems. To identify these systems, I screened a V. cholerae cosmid genomic library in

Escherichia coli for segments of V. cholerae’s genome that protected E. coli from infection by T2 phage. I identified one cosmid encoding 25 kB of DNA that provided E. coli protection against T2, T4, T5, and secΦ18 infection. Transposon and deletion mutagenesis on this cosmid revealed two distinct phage defense systems.

The first novel defense system involves two genes VC1767 and VC1766. Both genes are closely related to Type IV Modification Dependent systems which are known to defend against phage infection by targeting special modifications on the phage DNA. To uncover the mechanism of VC1767-66, I propose: 1) evolutionary

passage to determine whether phage DNA is being modified or if escape mutants arise, 2) purification of VC1767 and VC1766 to determine their biochemical activity, and 3) quantification of phage genome replication during infection to determine when protection occurs. The second novel defense involves three genes VC1764,

VC1763 and VC1762, which belong to a newly discovered but uncharacterized Zorya system. These genes are hypothesized to be an abortive infection system that kills phage infected cells to protect the surrounding population. To understand this system, I propose: 1) determine whether these three genes are sufficient to

protect against phage infection, 2) evaluate proton motive force disassociation to determine if this drives defense, and 3) determine if this system exhibits abortive infection. Both systems will be further examined in V. cholerae using mutagenesis and infection with V. cholerae phage. My studies will uncover the molecular

mechanisms by which these systems protect bacteria from phage infection, increasing our understanding of the evolution and ecology of V. cholerae while highlighting important mechanisms by which bacteria can resist phage therapy.