The Role Of Polyphosphate Kinase In The Pathogenicity And Persistence Of Acinetobacter Baumannii And Discovery Of Its Inhibitors

Acinetobacter baumannii（A.baumannii）is an important gram-negative drug-resistant pathogen in clinic,seriously threatening the efficacy of antibiotics.Carbapenem-resistant A.baumannii tops the list of bacteria that need to be addressed urgently by WHO in 2017.However,the research on A.baumannii in veterinary field is relatively lagging behind.Evidence is accumulating that the isolation rate and infection rate of A.baumannii have gradually increased with hypervirulence and hyperresistance in various animals such as livestock,pet and aquatic animals.In addition,the increase in the number of companion animals and the role change of companion animals led to the gradual emergence of A.baumannii as an important drug-resistant pathogen of nosocomial infection in animal hospitals,which increased the transmission of A.baumannii between animals and humans.Additioanlly,A.baumannii is also known for its ability to acquire antimicrobial resistance traits,which further promotes the spread of bacterial resistance,threatens human health and increases the cost of animal treatment.Therefore,it is urgent to develop strategies and lead compounds against A.baumannii infections.Polyphosphate kinase（PPK）is a key enzyme that catalyzes the formation of high-energy phosphate compound polyphosphate（poly P）from ATP or GTP in bacteria.PPK is involved in bacteria mobility,biofilm formation,adhesion and invasion,virulence factors expression and bacteria persistence.Furthermore,PPK is also critical for bacterial resistance to stress such as external oxidative,hypertonic,hypotonic,acid or alkali,and UV radiation.Therefore,PPK is an important target for fighting bacteria pathogenicity and persistence.Thus,characterization of the role of PPK in the pathogenicity and persistence of A.baumannii by gene knockout is of great significance for fighting A.baumannii infections,which has not been reported previously.A.baumannii ATCC 17978 PPK1 deletion strain（Δppk1::Apr）and its complement strains（Δppk1::Apr/PJL02-ppk1）were successfully constructed in this study.Surface associated motility assay,biofilm formation assay,bacterial persistence test and metabolomic analysis were performed to determine the effect of PPK1 on the pathogenicity and persistence of A.baumannii.Furthermore,A.baumannii infected mouse model was established to explore the effect of PPK1 on the pathogenicity and persistence in vivo.Finally,inhibitors targeted to PPK1 were further screened and its effects on the pathogenicity and persistence in vitro/vivo were also explored.Our results indicated that PPK1 plays an indispensable role in the formation of poly P in bacteria and the maintenance of pathogenicity and persistence.Compared with the wild-type strain,the poly P content in the PPK1-deficient strain was reduced about 33%.Transmission electron microscopy analysis revealed a 75%reduction on diameter in mobility assay due to the lack of pili-like structures.In the biofilm formation assay,the biofilm count in the PPK1-deficient strain was reduced by 4.5×log₁₀ units compared to the wild type.Under adverse stress stimulation,Δppk1::Apr was sensitive to 40×MIC ampicillin,H₂O₂ stimulation and heat stimulation.The bacterial survival under these stimulation decreased by 1.1×log₁₀ units,7.0×log₁₀units and 1.0×log₁₀ units,respectively,compared with the wild type.And in starvation stress assay,theΔppk1::Apr exhibited growth inhibition.Bacterial metabolomic analysis showed that PPK1 was involved in bacteria glycerophospholipid metabolism and fatty acid anabolism.The bacteria load in the lungs of theΔppk1::Apr infected group was reduced by 0.97×log₁₀ units compared with the model group,and the level of inflammatory factors was significantly ameliorated in vivo.Phloretin,a natural compound as PPK1 inhibitor,could reduce poly P production in bacteria,impair the ability of mobility and biofilm formation of A.baumannii and inhibit bacteria persistence under the treatment with 40×MIC ampicillin,H₂O₂ stimulation and heat stimulation.Moreover,phloretin could reduce the survival and virulence of A.baumannii in vivo.Taken together,our results established that PPK1 is inaccessible for A.baumannii pathogenicity and persistence,and is a promising target against A.baumannii infections.Phloretin,as an PPK1 inhibitor,showed a favourable inhibition on the pathogenicity and persistence of A.baumannii.Thus,our work provided a new target and potential lead compound for the treatment of A.baumannii infections.