Evolutionary and functional dynamics of a leishmanolysin-like immune multigene family during early infection in...

The evolutionary dynamics of multigene families are central to shaping host-parasite interactions and immune outcomes. In parasitic protists, expansion and diversification of surface-associated gene families enable immune evasion and host modulation, yet these mechanisms remain poorly understood in marine ciliates. Philasterides dicentrarchi, the causative agent of scuticociliatosis, provides a relevant model to investigate how gene family evolution contributes to parasite virulence and immune interaction.

We performed an integrated multi-omics analysis combining genome-wide screening using Hidden Markov Models, repeat annotation, phylogenetic inference, RNA-seq-based transcriptomic profiling, and LC-MS/MS analysis of the surface-associated fraction. Structural features of leishmanolysin-like (LSF) proteins were predicted using domain and membrane-targeting analyses, and expression dynamics were evaluated across early infection time points.

Genome-wide analysis identified 16 annotated LSF genes and a broader repertoire of 73 LSF-related sequences, consistent with a dynamic birth-death model of multigene family evolution. This expansion was associated with a repeat-rich genomic landscape dominated by simple and low-complexity elements, suggesting that genome plasticity contributes to diversification. LSF proteins retained conserved M8 metalloprotease domains and membrane-targeting features but displayed substantial sequence and regulatory divergence. Transcriptomic data revealed rapid and coordinated upregulation at 1 h post-infection, followed by a decline at later time points. Proteomic analysis confirmed the presence of most LSF proteins at the parasite surface and demonstrated a positive correlation between transcript abundance and protein detection.

Our integrative multi-omics analysis reveals that the leishmanolysin-like family (LSF) of Philasterides dicentrarchi is a highly expanded and diversified multigene repertoire. The rapid induction of LSF transcripts during early infection and the detection of most family members in the surface-associated proteome indicate their active deployment at the host-parasite interface. These findings provide new insights into the evolutionary and functional dynamics of immune-interacting multigene families in marine parasitic ciliates.