How thermostable direct haemolysin (TDH) diverges from TDH-related haemolysin (TRH)? Reassessing haemolysins in Vibrio parahaemolyticus through functional and structural representation learning

Vibrio parahaemolyticus (Vp) is a significant foodborne pathogen, causing harm to public health and the seafood industry. Among its pathogenic traits, thermostable direct haemolysin (TDH) and TDH related haemolysin (TRH) serve as major virulence indicators. However, the differences between TDH and TRH at various levels have not been thoroughly explored. This study aimed to analyze TDH and TRH comprehensively using sequence data from databases, representation learning, and structure prediction.

Analyzing the amino acid sequences of TDH and TRH from 2131 and 99 records, respectively, showed diverse mutations and a distinct separation between the two toxins at the amino acid level. The identity between TDH and TRH varied from 56.1 to 67.4%. Despite this, a protein language model derived embeddings indicated high functional similarity, while structural inference by AlphaFold revealed distinct clustering patterns.

The study found that the transition from TDH to TRH is sparked by accumulated genome-wide residue changes, rather than a small number of key mutations. These point mutations cause significant structural differences between TDH and TRH. TRH forms an alpha-helical tail, which TDH lacks, resulting in different mechanisms for disrupting host membranes, such as pore-forming and ion flux induction.

These findings provide a detailed understanding of the functional and structural properties of Vp haemolysins, highlighting how multi-dimensional sequence variations impact their role in Vp pathogenicity. Understanding these differences can help researchers design experiments using Vp strains with tdh and trh genes to investigate environmental fitness.