Siderophore Biosynthesis Coordinately Modulated The Virulence-Associated Metabolome In Uropathogenic Escherichia Coli

Urinary tract infection(UTI),one of the most common infectious diseases,has severely degraded the health of approximate 60% of women worldwide.The complex pathogenesis,high-recurrence and drug-resistance were observed to markedly impede its clinical diagnosis,treatment and antibiotic development.The fact is that uropathogenic Escherichia coli(UPEC)almost accounts for 80% of clinical cases with UTI.In recent years,a line of evidence manifested that siderophores might hold the capacity to interrogate the pathogenesis of UPEC causing infection in terms of their biosynthesis was confirmed to closely associate with the virulence formation of UPEC strains.Our effort employed a metabolomics-genetics combinational strategy to investigate whether and how siderophore biosynthesis modulated the virulence formation of UPEC strain while compared to non-UPEC one.In this study,we were first to systematically optimize both the protocols for metabolite extraction and the analytical parameters of Nuclear Magnetic Resonance Spectrometer(NMR),consequently a high-sensitivity NMR based metabolomics combined with genetic method was explored and exploited for globally characterizing untargeted metabolome present in pre-culture and post-culture human urinary samples,as well as pellets treated with wild type UPEC 83972,associated mutants with the partial and complete deletion of different patterns of four siderophores,and non-UPEC MG1655 strain as well.Furthermore,multidimensional bioinformatics assay was used to discover and identify virulence associated interactive metabolome between pre-culture and post-culture human urinary samples of UPEC in correspongding to non-UPEC.In addition,virulence associated targeted metabolome between UPEC and non-UPEC was also visualized and characterized,which were highly agreeable with the above results acquired by interactive metabolome assay.Surprisingly,siderophore biosynthesis coordinately modulated the virulence associated interactive and targeted metabolome characterized above as UPEC 83972 with complete deletion of four siderophores was observed to almost share the agreeable metabolic-phenotype with non-UPEC MG1655.Then,23 differential metabolites(targeted meabolome)were discovered and identified through screening the holistic metabolome of UPEC and non-UPEC strains,as 12 metabolites were significantly up-regulated in UPEC involving methylhistidine,acetone,phenylacetylglycine,hypoxanthine,guanine,L-cystine,thymidine,indoleacetate,4-hydroxyphenylacetate,creatine,fumarate and phenylalanine,and 11 metabolites including NAD,adenine,alanine,formate,L-cysteine,leucine,guanidinoacetate,5-aminovalerate,threonine,lactate and glycerate were markedly down-regulated in UPEC in relevant to non-UPEC.Similarly,31 interactive metabolites were identified in pre-and post-culture human urinary samples of UPEC and non-UPEC strains,where 14 metabolites were remarkably increased in post-culture urinary samples of UPEC strain that were termed as excreted metabolome,they were L-cysteine,scyllo-inositol,cytosine,creatinine,vanillin,bilirubin,dihydrothymine,benzaldehyde,NADP,NAD,niacinamide,thiamine,epicatechin and dihydrouracil.Moreover,17 metabolites were termed as utilized metabolome,including urocanic acid,fumarate,p-aminobenzoic acid,p-hydroxyphenylacetic acid,uridine 5-monophosphate(UPM),4-hydroxybenzoic acid,3-hydroxycinnamic acid,trans-2-hydroxycinnamate,m-cresol,o-cresol,p-cresol,4-aminophenol,N-methylnicotinamide,phenylpyruvic acid,deoxyadenosine,guanosine triphosphate,pyridoxal and L-palmitoylcarnitine,whose expressional levels were noticeably lowered in post-culture urinary samples of UPEC strain.Our data further revealed that the expressional levels of those metabolites were significantly and coordinately modulated by the biosynthesis of four siderophores that were observably implicated in the virulence formation of UPEC strain.At last,17 metabolic pathways were characterized to cover the targeted metabolome between UPEC and non-UPEC strains,while 16 metabolic pathways hosted the interactive metabolome(Utilized Metabolome and Excreted Metabolome)between pre-culture and post-culture urinary samples of UPEC strain that were significantly different from that with non-UPEC one.Excitingly,these metabolic pathways are mostly involved in energy metabolism,amino acid metabolism,nucleotide metabolism and intestinal microbial metabolism,etc,whose level changes mostly accounted for the coordinate modulation of siderophore biosynthesis upon the virulence associated targeted and interactive metabolome.Take altogether,this study was first to utilize metabolomics-genetics combinational strategy for delineating the coordinate modulation of siderophore biosynthesis upon the virulence associated metabolome whose functional-annotation could provide us novel insights into diagnosis,pathogenesis and therapeutic discovery of UPEC causing urinary tract infection at metabolic perspective.