Investigation Of The Role Of PA1548 In Cbb3-cox Maturation And Its Effect On Virulence Factors

Pseudomonas aeruginosa,an opportunistic pathogen,is able to survive in different environments,such as soil,air,traumatic wounds and the lungs CF patients.This is closely related to the presence of multiple virulence factors and survival at different oxygen concentrations.Pseudomonas aeruginosa has a variety of respiratory chains that can cope with different oxygen environments,and a variety of terminal enzymes support the possibility that Pseudomonas aeruginosa has a variety of respiratory chain.Therefore,the study of respiratory chain correlation will provide theoretical basis for revealing the adaptability of Pseudomonas aeruginosa to various environments.Based on previous studies,the unknown function gene PA1548 in Pseudomonas aeruginosa PAO1 was studied in depth,and it was confirmed that PA1548 plays an important role in the respiratory chain.Meanwhile,the molecular mechanism of its influence on Cbb3 terminal oxidase(Cbb3-Cox)and virulence factors was explored.In this study,we demonstrated that in PA1548 deletion mutant the respiratory chain is impaired due to the loss of Cbb3-Cox activity.To explore how PA1548 affected the activity of Cbb3-Cox,we measured the subunits Cco O and Cco P in?PA1548.The results showed that Cco O and Cco P amount did not change in the mutant,and expression of Cbb3-1 or Cbb3-2 in?PA1548(resulting strains:?PA1548/p-cbb₃-1 and?PA1548/p-cbb₃-2)did not restore Cbb3-Cox activity.These results suggested that PA1548 has no effect on the amount of Cbb3-Cox,but only its activity.Adding Cu to the growth medium rescued the Cbb3-Cox activity in?PA1548,suggesting that the effect of PA1548 on Cbb₃-Cox was related to Cu.Using purified PA1548 protein and Cu SO₄,we found that the fluorescence intensity of the PA1548-Cu complex was continuously decreased with the increasing amount of Cu SO₄,indicating that PA1548 has Cu-binding ability.The result of inductively coupled plasma mass spectrometry(ICP-MS)showed that the Cu content of?PA1548 did not change,compared to the PAO1.Therefore,PA1548 had no effect on cellular Cu levels but specifically affected Cbb3-Cox's Cu acquisition.To further explore the effect of PA1548 on respiratory chain.We also examined the effect of PA1548 denitrification.The results indicate that the intermediate products(NO)of denitrification process was decreased in?PA1548.Based on it,the expression of denitrifying respiration associated gene was downregulated in?PA1548.These results suggest that PA1548 affected the amount of NO via its effect on the expression of genes encoding denitrifying respiratory enzymes.Further investigation indicates the downregulation of denitrifying respiratory enzyme genes was due to its effect on anr,dnr(dissimilatory nitrate respiration regulator)and nar XL(two-component transcriptional regulator).The redox related virulence factors contain pyocyanin and siderophore.Thus,we firstly explore the mechanism of the effect of PA1548 on pyocyanin.The result showed that?cbb₃ produced same amunt of pyocyanin as?PA1548,suggesting that the reduced pyocyanin synthesis in?PA1548 was attributed to impaired function of Cbb3-Cox.Cbb3-Cox can affect the balance of redox in magnetospirillum gryphiswaldense.And the production of pyocyanin was down-regulated in gsh A mutant.Therefore,we detected the ratio of NAD/NADH in?PA1548.The result showed the ratio of NAD/NADH was increased in?PA1548.Meanwhile,when gsh A was transferred into?PA1548,the amount of pyocyanin was partially restored.Thus,the decrease of pyocyanin in?PA1548 is partly due to the change of intracellular redox state.However,we detected the amount of pyoverdine and pyochelin in?PA1548.The result showed that the amount of pyoverdine was down-regulated and the amount of pyochelin was up-regulated in?PA1548.However,the same amount of pyoverdine in?cbb₃and?PA1548 was found.The content of pyoverdine and pyochelin in?cbb₃ is the same as that in PAO1.Therefore,the results showed that the effect of PA1548 on siderophores was independent of Cbb3-CoxAs PA1548 can affect the activity of all Cbb3-Cox and some virulence factor genes,we screened Chinese herbal medicine extracts for compounds that can inhibit the expression of PA1548.We found that the extracts of pomegranaterind and feather cockscomb seed could inhibit the expression of PA1551-PA1547 and thus inhibit the respiratory chain in P.aeruginosa.Further study identified that tannin,a main component in pomegranaterind,could inhibit the expression of PA1551-PA1547.In conclusion,this paper clarified the relationship between PA1548 and respiratory chain,pyocyanine and pyoverdine,and screened substances that could inhibit the expression of PA1548 gene,providing theoretical basis for future treatment of Pseudomonas aeruginosa.