Impact of hcp and vgrG on Acinetobacter baumannii biofilm formation during infection of human pulmonary alveolar epithelial cells

The purpose of this research is to examine the impact of key type VI secretion system (T6SS) proteins hemolysin coregulated protein (Hcp) and valine-glycine repeat protein G (VgrG) on the metabolism of Acinetobacter baumannii (A. baumannii). Homologous recombination technology was used to construct hcp knockout strain (ATCC17978Δhcp), vgrG knockout strain (ATCC17978ΔvgrG), and a combined hcp and vgrG knockout strain (ATCC17978ΔhcpΔvgrG), with the wild-type A. baumannii strain (ATCC17978) used as a control. These strains were co-cultured with human pulmonary alveolar epithelial cells (HPAEpiC), respectively. Subsequently, a non-targeted metabolomic analysis of the co-culture supernatant, bacteria, and cells was conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the bacterial three-pair comparison groups, the major differential metabolites were organic acids and derivatives, as well as organic oxygen compounds (p < 0.05). Further analysis of the major differential metabolites in bacteria revealed five common metabolites with statistically significant differences (p < 0.05), which were N-acetyl-d-glucosamine 6-phosphate, 6-hydroxypseudooxynicotine, 3-deoxy-D-manno-octulosonate, N-Acetylneuraminic acid, and N-acetylmuramoyl-Ala. The annotation of the above five differential metabolites identified five common metabolic pathways with statistically significant differences (p < 0.05). Among these, phosphotransferase system (PTS) showed significant statistical differences (p = 0.01, p = 0.04, p = 0.03) in ATCC17978Δhcp, ATCC17978ΔvgrG, and ATCC17978ΔhcpΔvgrG. The deletion of hcp and the combined deletion of hcp and vgrG led to a downregulation of PTS overall expression, while the deletion of vgrG did not show a significant change in the overall expression level of PTS. The PTS shows a correlation with biofilm formation. The validation experiments demonstrated that ATCC17978Δhcp exhibited significant phenotypic defects, including reduced biofilm formation capacity and visible surface damage under scanning electron microscopy (SEM). In contrast, ATCC17978ΔvgrG maintained wild-type levels of biofilm formation and intact bacterial morphology. Notably, ATCC17978ΔhcpΔvgrG displayed a unique phenotypic reversal, characterized by enhanced biofilm formation, intact bacterial structure, and increased extracellular polymeric substance (EPS) secretion. However, all mutant strains exhibited decreased adhesion ability. The expression levels of biofilm-related genes in each strain showed a positive correlation with their biofilm formation capacity. These results demonstrate that while the PTS influences biofilm formation, it does not serve as the sole regulatory mechanism. The hcp gene plays a crucial role in biofilm formation, whereas the vgrG gene exhibits minimal impact on biofilm formation. Their co-deletion triggers compensatory pathways enhancing biofilm production.