Investigation of Novel Type III Secretion System Protein Effectors of Aeromonas veronii

Abstract

The type III secretion system (T3SS) plays a crucial role in the pathogenesis of various Gram-negative bacteria, enabling them to directly inject effector proteins into host cells. This study focuses on the identification, characterization, and functional analysis of putative T3SS effector proteins (AopX, PteB, and PteA) in Aeromonas veronii. Insertional gene mutants were generated and evaluated using the Galleria mellonella larvae infection model, showing significant alterations in virulence phenotypes. Transcriptomic analysis of an exsD mutant strain provided insights into the regulation of these novel effectors and demonstrated that they were part of theT3SS regulon. The study further characterized these proteins through expressing them in both Saccharomyces cerevisiae and A549 mammalian cells. Fluorescence imaging analysis showed that these proteins had significant impacts on host cell morphology and cytoskeletal organization. Additionally, BioID proximity labelling combined with mass spectrometry proteomics analysis identified host protein interactions and key cellular pathways that influenced by these bacterial effectors. Furthermore, protein purification was optimized using various chromatography approaches and computational modelling via AlphaFold and Phyre2 was used to predict and align protein structures. Overall we found that AopX, PteB, and PteA are regulated by the T3SS regulon. AopX primarily disrupts the actin cytoskeleton in both yeast and A549 cells, most probably through interacting with Filamin A, while PteB prevents bud formation and affects cell cycle progression by interacting with Serine/Threonine phosphatases. This comprehensive investigation enhances our understanding of T3SS effector proteins in A. veronii pathogenesis and provides insights into their regulation, structural features, and specific mechanisms of host cell manipulation.