In-silico prediction and modeling of ApxA exotoxins of Actinobacillus pleuropneumoniae: ApxIA, -IIA, -IIIA and –IVA

Abstract

Actinobacillus pleuropneumonia (APP) is a Gram-negative bacterium that serves as the major etiological agent for porcine pleuropneumonia, one of the critical diseases causing substantial socio-economic losses in swine rearing industry world-wide. Apx exotoxins are the members of Repeats-in-Toxin (RTX) family secreted by the Type-I Secretion System (T1SS) of Gram-negative bacteria, reported as the major virulence factor in the pathogenesis of A. pleuropneumoniae. This study demonstrates the in-silico analysis of ApxA exotoxins of A. pleuropneumoniae as the pre-experimental approach, in which the structural and functional characteristics of each exotoxin was annotated. In this study, several computational tools were used to predict the structural and functional properties of ApxA structural exotoxins. The gene sequences of ApxIA(AF363361.1), ApxIIA(AF363362.1), ApxIIIA(AF363363.1), ApxIVA (HM021153.1) and the corresponding amino acid sequences were retrieved from NCBI database. Physiochemical properties of each ApxA protein were computed via ProtParam tool. Hydropathy plots were constructed by the ProtScale server to computationally define the secondary conformations and the hydrophobic natures of ApxA exotoxins, providing structural insights to the protein modeling. Protein motif and domain analysis by MEME, MAST, Genomenet and ProDom provided motif/domain building blocks in each of the four ApxA exotoxin (ApxIA, -IIA, -IIIA and –IVA). Each of the ApxA protein structure was predicted by I-TASSER, a powerful state-of-art three-dimensional modeling tool. Finally, the qualities of the predicted three-dimensional structures of ApxA proteins were validated by the SWISS-MODEL protein structure & model assessment tool and SAVES server. The three-dimensional structures of ApxA proteins were modeled computationally, providing the novel insights to the proteomic natures of ApxA exotoxins that may essentially assist in building successful diagnostic method of APP infection as well as in vaccinology. The in-silico approach constructed in this study to characterize and model the tertiary structures of immunogenic proteins of unknown structures such as ApxA exotoxins will serve as the efficient pre-experimental step to development of vaccines and diagnostic tools. 3-D modeling of ApxA exotoxins were executed to best interpret the proteins of their structures and functions involved in the pathogenesis of A. pleuropneumoniae. The domain-wise interpretations of the proteins were performed to analyze the likely roles of each toxin in the pathogenesis, and the antigenic epitopes were predicted. The structural and functional annotations suggested in this study may aid in the prevention and control of A. pleuropneumoniae infection by utilizing the findings to the development of the diagnostic methods and vaccine candidates.