Expanded screening and analysis of Photorhabdus virulence cassette (PVC) signal peptides reveals an anti-phagocytotic effector

As one of the typical extracellular contractile injection systems (eCISs), Photorhabdus virulence cassette (PVC) can be released from bacterial cells to inject into eukaryotic cells, while the signal peptides (SPs) direct the loading of heterologous proteins into PVC, making PVC an ideal model for in vitro protein delivery. In this study, the N-terminal sequences (NTSs) of 64 genes were tested for the loading effect, among which 45 NTSs were verified as SPs. Sequence analysis revealed that hydrophilic NTSs were more likely to guide cargo loading, possibly due to their interaction with PVC15 ATPase. Additionally, through SPs identification, an unclassified effector named Photorhabdus asymbiotica anti-phagocytic protein (PAAP) was identified. When delivered by PVC into J774A.1 murine macrophage cells, significant cell rounding was observed. Moreover, the phagocytic activity of macrophages against bacteria was markedly reduced following PVC-PAAP treatment. Transcriptomic and western blotting analyses revealed that the upregulation of Klf2, RhoB, Tsc22d3, and Ezr genes, and the reduction in P21-activated kinase (PAK) expression, potentially lead to cytoskeletal changes and decreased phagocytosis. In summary, this study broadened the scope of SPs of PVC, elucidated the differences in physicochemical properties between SPs and nSPs, and identified a new PVC effector protein along with its biological function in cell morphology and phagocytosis.