Identification,Regulatory Mechanism,and Functional Analysis Of A Novel Two-component Signaling System In Uropathogenic Escherichia Coli

Urinary tract infection(UTI)is one of the most common bacterial infections in humans worldwide.Among all the causative agents,uropathogenic E.coli is responsible for the majority of all cases.Many virulence factors important for UPEC infections were identified and well characterized,such as adhesins,toxins,and iron uptake systems.To establish successful infections,UPEC must be able to survive and replicate within hosts.Therefore,the adaptation to local nutrient availability is very important.However,metabolic factors facilitating UPEC fitness in vivo remain largely unknown,especially those help UPEC adapt to renal physiological conditions.Two-component signaling systems(TCSs)are major mechanisms by which bacteria adapt to nutrient availability in a wide range of environments.However,no pathogen-associated TCS has been identified in uropathogenic Escherichia coli(UPEC).Here,we identified a novel TCS,which we termed KguS/KguR(KguS:a-ketoglutarate utilization sensor;KguR:a-ketoglutarate utilization regulator)in UPEC CFT073,a strain isolated from human pyelonephritis.kguS/kguR was strongly associated with UPEC but was found only rarely among other E.coli including commensal and intestinal pathogenic strains.An in vivo competition assay in a mouse UTI model showed that deletion of kguS/kguR in UPEC CFT073 resulted in a significant reduction in its colonization of the bladders and kidneys of mice,suggesting that KguS/KguR contributed to UPEC fitness in vivo.Comparative proteomics identified the target gene products of KguS/KguR,and sequence analysis showed that TCS KguS/KguR and its targeted-genes,c5032 to c5039,are encoded on a genomic island,which is not present in intestinal pathogenic E.coli.Expression of the target genes was induced by a-ketoglutarate(a-KG).These genes were further shown to be involved in utilization of a-KG as a sole carbon source under anaerobic conditions.KguS/KguR contributed to the regulation of the target genes with the direct regulation by KguR verified using an electrophoretic mobility shift assay.In addition,oxygen deficiency positively modulated expression of kguS/kguR and its target genes.We therefore describe the first UPEC-associated TCS that functions in controlling the utilization of a-ketoglutarate in vivo thereby facilitating UPEC adaptation to life inside the urinary tract.a-ketoglutarate(KG)are abundant host-derived metabolites that can be readily utilized by uropathogenic Escherichia coli.Transport of this molecule likely constitutes the very first and most important step in its assimilation.Here we studied the roles of two KG transporters in KG utilization,their transcriptional regulation,and their contributions to UPEC fitness in vivo.UPEC-associated c5038 was acquired through horizontal gene transfer,and C5038 belongs to the DASS family.C5038 shares 49%similarity with CitT.TMHMM method predicted that it has 13 transmembrane segments.It also possesses a SAT signature sequence that is presumably directly involved in the transport of substrate.kgtP is a widely conserved locus in commensal and pathogenic E.coli,and KgtP belongs to the MFS family.Anaerobiosis induced c5038 expression but repressed kgtP expression.c5038 was positively regulated by FNR and ArcA anaerobically and played a major role in anaerobic growth on KG;while KgtP was required for aerobic growth on KG,but its expression was severely repressed by FNR and ArcA anaerobically.c5038 was regulated via KguSR,and kguSR expression was higher under anaerobic conditions.c5038 is induced by KG but kgtP expression does not respond to KG.Notably,kgtP expression is highest in M9 containing glycerol as sole carbon source,but lower in M9 with glucose,and lowest in LB media.The CRP regulator known to regulate carbon utilization genes positively regulates kgtP expression.In addition,c5038 and kgtP expression was further shown to be controlled by different alternative sigma factors RpoN and RpoS,respectively.When driven by the same constitutively expressed promoter,C5038 functions more efficiently anaerobically whereas KgtP more efficiently aerobically.Competition assays in a murine model suggest that c5038 but not kgtP is important for UPEC fitness in vivo.In conclusion,transcriptional regulation distinguished C5038 from KgtP in the utilization of KG,further leading to their differential contributions in vivo.This study therefore greatly expanded our understanding of UPEC pathobiology.Previous studies have shown that KguR directly binds to the promoter regions of its target genes,and positively regulates the expression of the targets.However,the molecular mechanism underlying the regulation is still unclear.Furthermore,KguR affects the production of multiple protein targets,this chapter attempts to identify more targets regulated at the transcriptional level,aiming to gain a more thorough understanding of KguR's role.First of all,we analyzed the DNA sequence of c5038 promoter region,and cloned various length of promoter regions,followed by determining the ?-galactosidase activities.We then found the shortest promoter possible,Pc5038F3R,which is actively responsive to KG.Then,we found a inverted repeat sequence within Pc5038F3R region,TGTGCG-N8-CGCACA.EMSA,promoter-reporter gene fusion and ?-galactosidase activity assay demonstrated that the IR region was critical for binding by KguR as well as promoter activity in vivo.It is noteworthy that deletion of the IR region did not abolish KguR induction completely,in other words KguR can still activate target's expression.In addition,we also found highly similar sequence in c5032 and kguS promoter regions,TGTGTG-N13-CGCGCA and TGTGCG-N6-CACACA.Deletion of IR region both greatly affected the binding ability of KguR to the DNA probe.KguR self-regulates its own expression,which very likely depends on the IR region.Besides,these binding sites are conserved among E.coli strains.Therefore,we generated a binding consensus out of all the binding sites.KguR is 37%similar to nitrogen utilization regulator NtrC.NtrC proteins all possess AAA+ ATPase domain,which is considered to bind sigma54 factor.Hence,this part investigated the roles of different domains in regulation.The results showed that DNA binding HTH domain is not essential for induction,but AAA+ ATPase is required for activation of targets.