Evolution and interaction of defence mechanisms in Pseudomonas aeruginosa

Abstract

Bacteria and their parasites are locked in a continuous arms race that results in an astounding diversity of defence systems and anti-defence systems. In the last few years we have witnessed a revolution in the field of defence biology, with the discovery of dozens of unknown systems. This extraordinary discovery rate stems from the observation that often bacterial defences cluster together in so called “defence-islands”. There is now evidence that bacterial strains can carry multiple systems simultaneously, and in some cases their synergistic interaction has been demonstrated experimentally. However, how these interactions shape phage-bacteria evolutionary dynamics is still largely unexplored. Additionally, the knowledge about the effect of abiotic factors on the activity of defence systems is scarce. Specifically, studies considering the role of temperature, a fundamental abiotic factor regulating microorganisms’ lifestyle and virtually any molecular pathway are only limited to the functionality of Restriction-Modification (RM) and Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR associated genes (CRISPR-Cas). Crucially, how temperature shapes bacteria-phage dynamics when the host carries multiple defences acting in concert to fend off phage attack has been never considered. This thesis contributes to the plethora of bacterial immunity with the description of a novel defence system, named Methylation Associated Defence System (MADS), found in a clinically relevant strain of Pseudomonas aeruginosa. We demonstrate that MADS uses methylation to distinguish between self and non-self, and that phages can escape MADS interference via methylation of their genome. We also identify the mad genes responsible for the methylation and restriction activities. Bioinformatics analysis reveals that MADS is located in a conserved genomic hotspot for defence systems and that it is widespread across Gram-negative and Gram-positive bacteria. Furthermore, we describe the synergistic interaction between MADS and CRISPR-Cas, with phages rapidly falling below detection limit when both systems are present, and phages amplifying when only one of the two defences is carried by the bacteria. We use mathematical modelling and experimental data to show that the synergistic interaction between MADS and a Type IE CRISPR-Cas is due to a disruption of the Anti-CRISPR (Acr) phage cooperation needed to overcome CRISPR-Cas. This is because Acr phages need to acquire the epigenetic modification that allow them to evade MADS. However, the presence of CRISPR-Cas limits the emergence of MADS-escape phages, as it reduces the phage population size. Finally, I demonstrate that temperature is a key determinant of the outcome of phage infection. I compare phage infectivity at 28°C and 37°C and find that phage population quickly decline at 37°C, but it persists at 28°C. This is because it appears that MADS escape phages arise more readily at lower temperature. Altogether, this PhD thesis increases the knowledge on bacterial immunity by describing a new bacterial defence system. Additionally, it elucidates important aspects of phage-bacteria dynamics using an integrative approach that takes into account the interaction between genetics and environmental parameters in shaping bacterial resistance and phage infectivity.