Study On The Zinc Uptake System, PA2403-2410 And ZnuABC, In Pseudomonas Aeruginosa

Zinc ?Zn? essential for all organisms can function as a catalytic cofactors or structure components of enzymes and proteins. The equilibrium of Zn²⁺plays an important role on survival and infection in pathogenic bacteria because deficient level of Zn²⁺ does not support cellular growth while excess Zn²⁺ is toxic to cells. Intracellular homeostasis for Zn²⁺ is established by Zn²⁺ transport and regulation system, such as ZnuABC. ZnuABC belongs to the family of ABC ?ATP-binding cassette? transporters and has been characted in many species of bacterial.Pseudomonas aeruginosa ?PA? is an opportunist human pathogen and the third leading cause of nosocomial infections. It is the major cause of morbidity and mortality among burn victims, AIDS patients, malignant tumors and other immunocompromised individuals. The clinical treatment of PA is difficult due to its highly pathogenic and resistance to a variety of antibiotics.In this report, we identified and characted two potential Zn²⁺ transport systems PA2403-2410 and ZnuABC in PAO1. The typical conserved domain of the three components ?periplasmic binding protein, ATPase and membrane permease? of Zn²⁺ transport systems, have been identified via homology alignment and structural analyses. That means the two Zn²⁺ transport systems PA2403-2410 and ZnuABC in PAO1 have the fundamental structures of Zn²⁺ transport.PA2403-2409 has been proved located in one operon by RT-PCR while PA2410 is an individual gene, which differ from the annotation provided at www.Pseudomonas.com.To further explore the function of these two Zn²⁺ transport systems, seven single mutants, ?PA2403, ?PA2407, ?PA2408, ?PA2409, ?PA2410, ?PA2403-2409, ?PA2403-2410 ??znuA was constructed in the previous research? and a double mutant ?znuA?PA2403-2410, were constructed respectively. The growth of the nine mutants in zinc-deficient and zinc-sufficient conditions was determined. The result shown that the growth of all the mutants was decreased significantly compared with that of PAO1 in zinc-restricted condition except for ?znuA. The results suggested that PA2403-2410 plays a more important role in Zn²⁺ transporting compared with ZnuABC in PAO1. The expression of znuA, PA2403 and PA2410 in PAO1 was determined by pKD-znuA, pKD-2403 and pKD-2410. The results indicated that the expression of znuA and PA2403 has been increased correlatedly with zinc concentrations on low Zn²⁺ medium, but not for PA2410. Furthermore, the expression of pKD-2403 in the seven mutants, ?PA2403, ?PA2407, ?PA2408, ?PA2409, ?PA2410, ?PA2403-2409, was detected. The results suggested that PA2403 may act as a repressor of PA2403-2410. The transport system could probably autoregulate its own transcription.The sensitivity of the mutants ?znuA, ?PA2403 and ?znuA ?PA2403-2410 to H₂O₂, was measured. The results indicated that the sensitivity of ?PA2403 and ?znuA?PA2403-2410 to H₂O₂ increased comparied with PAO1. The increased sensitivity is due to Zn²⁺ deficiency which influence the activity of superoxyde dismutase because the enzyme requires Zn²⁺as cofactor. The stability of the outer membrane was subsequently determined by SDS-EDTA sensitivity tests and transmission electron microscopy. The results suggested that ?PA2403-2410 is involved in outer membrane stability because the outer membrane was impaired in the mutants. The mutant of ?znuA showed similar phenotypes to PAO1. That could be due to higher-affinity Zn²⁺ transport system PA2403-2410 existed in ?znuA.The virulence of the mutants as well as the wild type PAO1 has also been determined by a Drosophila melanogaster infection model. The results showed that the virulence of AznuA, ?PA2403-2410 and ?znuA?PA2403-2410 was decreased sifnificantly compared with PAO1. The results indicated that ZnuABC and PA2403-2410 play an important role in virulence in PAO1.The system for Zn²⁺ homeostasis in P. aeruginosa could be a new target for antibactial therapies and drug development.