Insights into the mechanisms used by phage-inducible chromosomal islands to interfere with helper phage reproduction

Abstract

The phage inducible chromosomal islands (PICIs) are a major class of molecular parasites that significantly contribute to the bacterial evolution. They are clinically relevant because they are responsible for intra- and intergeneric transfer of major virulence genes. PICIs manipulate the lifecycle of certain helper phages for their own spread. At the same time, they use remarkable strategies to interfere with phage reproduction to ensure their sustainability in bacteria. Typically, PICIs interference is achieved by i) forming small PICI-sized heads using phage proteins, which only can accommodate their small genomes, ii) changing the DNApackaging specificity to only recognise the PICI-genome, or iii) inhibiting the transcription of phage late genes. Among the wider PICI family, the Staphylococcus aureus pathogenicity islands (SaPIs) are the most extensively studied elements. SaPIs reside quiescently and replicate together with the chromosomal DNA through the action of Stl master repressor. However, subsequent to the infection by a helper phage or after the SOS-induction of an endogenous helper prophage, the Stl-DNA complex is disrupted by specific phage proteins, activating the SaPI excision-replication-packaging (ERP) cycle. Although SaPIs encode homologues of Stl repressors, these proteins are poorly conserved in term of sequence and folding. Accordingly, SaPIs require divergent phage-encoded proteins for derepression. Unlike most SaPIs that can be induced by multiple structurally unrelated proteins, SaPI1 is exclusively induced by a unique phage-encoded protein named Sri. Primarily, Sri is encoded by the phage to bind to the bacterial helicase loader protein (DnaI), blocking bacterial replication, thus facilitating phage replication. Here in this work, the strategy used by SaPI1 to pirate the phage Sri protein for induction was deciphered using various in vitro and in vivo approaches. Although the results indicate that Stl and DnaI are highly unrelated in their protein sequences and structural folding, our results confirm that Sri uses identical residues to bind to both proteins. Therefore, instead of using molecular mimicry, SaPI1 uses functional mimicry to steal the phage inducer. The findings reveal that any attempt from the phage to avoid SaPI interference will add a transmission cost by limiting the phage ability to block the host replication. It also highlights the constant arms race between phages that try to avoid SaPI-mediated interference, and SaPIs, which must cope with any escape attempt from the phage to ensure their promiscuous spread in nature. Moreover, this work also reports the existence of SaPI1-like transcriptional regulation models in Staphylococcus hominis and Staphylococcus simulans, providing a fascinating example of inter-species transfer of SaPI elements. The work was also extended to investigate the mechanisms of PICI interference with phage reproduction. Generally, pac SaPIs encode two proteins (TerSSP + Ppi) to redirect the specificity and interfere with phage DNA packaging, respectively. Ppi binds to the phage TerSφ and blocks its function, ensuring an advantageous transfer of the SaPIs. The study highlights that, although the phage and SaPI small terminases belong to the same family, the TerSSP includes an extra C-terminus motif, which is essential to avoid Ppi binding to the TerSSP. On the other side, and more fascinatingly, the structural studies in this work also reveal a novel molecular parasitism strategy employed by Gram-negative cos PICIs. In contrast to the aforementioned two-shot strategy (Ppi +TerSSP) used by the SaPIs, the E. coli EcCICFT073 island was found to encode a sole protein to perform both interference and packaging redirection functions. Therefore, this protein was named Rpp protein (redirecting phage packaging). Rpp forms a heterod