• epigenetic regulation
• methylation
• phasevariation
• Siphoviridae
• SpnD39III
• Streptococcus pneumoniae

Streptococcus pneumoniae is one of the most dangerous threats to global health, being the leading cause of acute invasive infection including community acquired pneumonia, meningitis and acute otitis media. The switching between two phenotypic phases, the transparent and opaque colony morphology phenotypes is associated to asymptomatic carriage or invasive disease. Despite being described long time ago, no molecular mechanisms underlying such phase variation were identified. Genetic rearrangements in a pneumococcal Type I restriction methylation (R-M) system locus were previously noticed at the time of the whole genome sequencing of the S. pneumoniae invasive isolate TIGR4 and similarly described in Mycoplasma pulmonis and Bacteroides fragilis. In the pneumococcus this R-M system has been named SpnD39III. In particular, all the six possible rearrangements between multiple hsdS genes were described and the different target specificities identified. Single-molecule, real-time (SMRT) methylomics allowed for the association of the distinct methylation patterns to each SpnD39III conformation. SpnD39III locked mutants allowed the identification of distinct gene expression profiles for each enzyme variant. Consistently with this finding, the SpnD39III system was shown to be involved in the transparent and opaque phenotypes. More importantly, experimental animal models of infection showed variable virulence levels related to each hsdS conformation and in vivo selection for switching between SpnD39III variants. In summary, given the abovementioned results, it has been proposed that the phase-variable SpnD39III R-M system epigenetically regulates gene expression and virulence. This part of the thesis, which is reported in chapter II, has been recently published on Nature Communications.

In order to identify the relative amount of each SpnD39III conformation in a pneumococcal population, a quantitative method has been developed and optimised as shown in chapter III. The protocol consists of a first common PCR using a fluorescent-labelled primer, a differentiating restriction digestion, and a final quantitative GeneScan analysis. This method was shown to be sensitive and reproducible.

In chapter IV, interactions between the SpSL1 temperate bacteriophage and its pneumococcal host were investigated in order to better understand their co-evolutionary dynamics. The infection kinetics of the phage as well as the integration in the pneumococcal genome were described. The lytic phage infection showed an impact on global host transcriptome, with the majority of changes, occurring transiently in the early stage of infection. The observed variations were typical of a metabolic stress related response. In addition, the SpnIII R-M system was shown to be effective in restricting invading phage proportionally to the amount of target sites in the phage genome. Phase-variation of the SpnIII R-M system was also shown to limit intra-strain propagation of phage. Interestingly, despite restriction, SpSL1 infected cells showed the induction of cell death. Deletion of the pneumococcal McrBC Type IV R-M system, specific for methylated cytosines, decreased significantly the abortive infection mechanism, thereby indicating that chromosomal methylation by the phage-encoded C5-cytosine methyltransferase might be the cause of induced cell death. These data demonstrate the function of McrBC system as a novel phage abortive infection mechanism that is triggered by phage methylation of the bacterial chromosome.

The appendix section, at the end of the thesis, contains the result of side projects carried out during the PhD that are beyond the aims of this thesis.

In summary the work presented in this thesis describes the discovery of an intricate interplay of methylation systems between the bacterial host and a virus. The phase variable Type I R-M system SpnIII of pneumococci shows both epigenetic control over host phenotypes and inter and intra-strain restriction of phage infection. On the other hand a phage-encoded methylase is involved in a novel abortive infection mechanism by methylating host DNA and determining cell death via the host-encoded SpnMcrBC type IV R-M system. Such interplay between methylases and restriction modules sheds new light on the dynamics of host-phage interaction.