Periplasmic Protein GltB Activates GtrS-gltR Two-component System In Pseudomonas Aeruginosa

Glucose is an essential nutrient for the human body,serving as a primary fuel for energy production.Many studies have also suggested that pathogens can exploit glucose as either a nutrient or a signal molecule that induces virulence functions of pathogenic bacteria including Staphylococcus aureus and Pseudomonas aeruginosa.In2017,the World Health Organization has categorized P.aeruginosa as a critical priority pathogen that is in urgent need of new antibiotics.Establishing a deeper understanding of how P.aeruginosa senses and responds to glucose may allow for the rational design of improved therapies to treat or prevent infections caused by this formidable pathogen.It has been proposed that,in P.aeruginosa,when glucose passes the outer membrane through the Opr B porin and reaches the periplasm,it can be metabolized through the phosphorylative pathway and the oxidative pathway.Genes encoding the P.aeruginosa glucose catabolism are organized in operons and are under the control of different regulators(i.e.,Hex R,Ptx S,Ptx R,Gnt R,and GtrS-GltR).Among them,response regulator(RR)GltR,which forms a two-component system with its cognate sensor kinase(SK)GtrS,is required for glucose transport activity in P.aeruginosa PAO1,presumably by activating the transcription of gltBFGK(gts ABCD)-opr B operon that encodes Opr B porin and the glucose transport system ABC^GltBFGK.However,these results were somehow contrast to the findings of Daddaoua et al.,where GltR is a transcriptional repressor that is released from its target operators of glucose metabolism genes upon phosphorylation.Although discrepancy exists,results from previous studies showing a conserved role of GtrS-GltR on glucose utilization in different Pseudomonas species including P.aeruginosa.Interestingly,2-ketogluconate(2-KG)and 6-phosphogluconate(6PG),two metabolic intermediates of glucose utilization pathways,can bind to ligand-binding domain of GtrS,leading to increase GtrS autophosphorylation and GltR phosphorylation activity.Importantly,GtrS plays a key role during P.aeruginosa-host interactions and is required for optimal colonization and dissemination in a mouse model of infection.Intrigued by these findings,in this study we decided to investigate the mechanism of action of GtrS-GltR TCS in P.aeruginosa PAO1.In this study,we first used RNA-seq to identify glucose responsive genes in P.aeruginosa.Genes involved in glucose and its metabolite transport and metabolism were differentially up-regulated.The qRT-PCR analysis showed similar results to those of RNA-seq analysis.Then,we constructed three mutants(?gtrS-gltR,?gcd,?gcd?gtrS-gltR)and complemented strains(?gcd?gtrS-gltR/p-gtrS-gltR,?gcd?gtrS-gltR/p-gcd).The growth of?gcd?gtrS-gltR triple mutant strain lacking both gcd and gtrS-gltR on glucose used as the sole carbon source was abolished,indicating that GtrS-GltR TCS modulates glucose utilization independently of Gcd,and vice versa.Using promoter-fusion analysis,we found that in the absence of glucose the promoter activity of gltB(i.e.,gltB-lux)was low,and it was not obviously affected by the deletion of gtrS-gltR locus.In the presence of glucose,the expression level of gltB-lux in WT PAO1 was higher than in the absence of glucose.However,the addition of glucose failed to increase the expression of gltB-lux in the mutant?gtrS-gltR,although it did this for the complemented strain(?gtrS-gltR/p-gtrS-gltR).These data suggest that GtrS-GltR TCS positively regulates the gltBFGK-opr B operon in response to glucose.Using reporter gene assays and a screen of P.aeruginosa PAO1 transposon mutants,we found that 2-KG and 6PG play a far lesser role in determining the activation of GtrS-GltR than glucose.However,gltB,which encodes a periplasmic glucose binding protein,is essential for the glucose-induced activation of GtrS-GltR TCS in P.aeruginosa.Then,we found that GltB interacts with GtrS in P.aeruginosa using ELISA,SPR,Co IP-MS,and BACTH assays.Moreover,GltB binds to a number of membrane-spanning proteins,indicating that GltB may have broad function in P.aeruginosa.To examine the role of the substrate binding pocket of P.aeruginosa GltB in the activation of GtrS-GltR,five residues W33,W34,K90,W268,and D301,were respectively mutated to alanine using site-directed mutagenesis.As a result,amino acid substitutions decreased the glucose-induced expression of gltB-lux,indicating GltB requires its glucose-binding pocket to activate GtrS-GltR.Importantly,like the gtrS deletion mutant,gltB deletion mutant showed attenuated virulence in both Drosophila melanogaster and mouse models of infection.In addition,using CHIP-seq experiments,we provide the genome-wide analysis of the GltR targets.We showed that the promoter of gltB is the major in vivo target of GltR.Collectively,these data suggest that periplasmic protein GltB and GtrS-GltR TCS form a complex regulatory circuit that regulates the virulence of P.aeruginosa in response to glucose.