Phage-bacteria interactions

: Discovery of defense systems

Abstract

Phages can follow two types of cycle, the lytic cycle, characterized by the immediate viral production, and the lysogenic cycle, where the phage DNA will be inserted in the phage genome as a prophage. Virulent phages reproduce using only the lytic cycle, while temperate phages can reproduce using both. Prophages can confer multiple advantages to their host, such as toxin acquirement or antibiotic resistance. Interestingly, prophages encode defense systems that help their host fight against other phages. Multiple defense systems exist, working in many different ways. They can target the invading nucleic acids, such as the restriction-modification or CRISPR systems. Others, induce the host death upon phage infection, to protect the whole population. The latter are called abortive infection systems and consist among others, toxin/antitoxin (TA) systems. TAs are composed of a toxin, killing the cell, and an antitoxin inhibiting the toxin’s activity. Recently, new defense systems have been discovered using the “guilty by association” method. Indeed defense genes tend to accumulate in what is called defense islands. By looking in these clusters, new systems can be found and characterized. With this idea in mind, Remy Dugauquier has been developing an automatic tool to uncover these genes, ADAM (Automatic Detection of Antiviral Mechanisms). Using this program, we selected 16 two-gene/three-gene modules to analyse experimentally. These potential TA systems can be easily tested by performing a killing rescue assay. We found one candidate (system 11) showing the typical TA phenotype, but we don’t rule out the other pairs as potential TAs or other defense systems. To better characterize these genes, the different protein structures and functions were predicted using bioinformatics tools. Furthermore, the potential complex formation has also been assessed between each gene pair. These analyses returned five promising candidates; two of unknown function, a second one being a homolog of a known TA and the last one, system 11, which gave a positive result in the killing rescue assay (system 11). Because of its promising features, we decided to characterize system 11. It is composed of gene 36, predicted to encode MOR, that showed toxicity, and gene 37, a kinase, which played the role of the antitoxin. MOR, with another protein called CI, is part of a bacteriophage genetic switch that decides between the lytic and lysogenic cycle. MOR, when overexpressed, might cause the induction of an E. coli prophage or the expression of a toxin, while the role of the predicted kinase remains a mystery. To better characterize the system, we first looked at the genome context and found that, also adjacent to gene 36, there is a gene (35) that seems to encode the CI protein. We also assessed the growth kinetics of this system and could observe killing in liquid culture. Furthermore, we looked at the cells shape upon toxin induction and could witness an elongation of the cells compared to the control. To conclude, optimization is needed for the other candidates testing while further experiments are required to uncover system 11’s operating mode.

Date of Award	30 Jan 2023
Original language	English
Awarding Institution	University of Namur
Supervisor	Gipsi Lima Mendez (Supervisor)