Analysis of virulence proteins in pathogenic Acinetobacter baumannii to provide early warning of zoonotic risk

Acinetobacter baumannii is a ubiquitous opportunistic pathogen usually with low virulence. In recent years, reports of increased pathogenicity of A. baumannii in livestock due to the migratory behaviour of wildlife have attracted public health attention. Our previous study reported that an A. baumannii strain isolated from dead chicks, CCGGD201101, showed enhanced pathogenicity, but the mechanism for increased virulence is not understood. Here, to screen potential virulence factors, the proteomes of the isolated strain CCGGD201101 and the standard strain ATCC19606 of A. baumannii were compared, and the possible virulence-enhancing mechanisms were further analysed. The 50 % lethal dose (LD₅₀) values of CCGGD201101 and standard strain ATCC19606 in ICR mice were determined to verify their bacterial toxicity. 2D fluorescence difference gel electrophoresis (2D-DIGE) combined with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS) and quantitative real-time PCR (RTqPCR) were applied to screen and identify differentially expressed proteins or genes that may be related to virulence enhancement. Bioinformatics analyses based on proteinprotein interaction (PPI) networks were used to explore the function of potential virulence proteins. The pathogenicity of potential virulence factors was assessed by phylogenetic analyses and an animal infection model. The results showed that the LD₅₀ of CCGGD201101 for mice was 1.186 × 10⁶ CFU/mL, and the virulence was increased by 180.5-fold compared to ATCC19606. Forty-seven protein spots were significantly upregulated for the A. baumannii CCGGD201101 strain (fold change ≥1.5, p < 0.05). In total, 14 upregulated proteins were identified using proteomic analysis, and the mRNA expression levels of these proteins were nearly identical, with few exceptions. According to the PPI network and phylogenetic analyses, the I78 family peptidase inhibitor, 3-oxoacyl-ACP reductase FabG, and glycine zipper were screened as being closely related to the pathogenicity of bacteria. Furthermore, the I78 overexpression strains exhibited higher lethality in mouse infection models, which indicated that the I78 family peptidase inhibitor was a potential new virulence factor to enhance the pathogenicity of the A. baumannii CCGGD201101 strain. The present study helped us to better understand the mechanisms of virulence enhancement and provided a scientific basis for establishing an early warning system for enhanced virulence of A. baumannii from animals.