Metabolomics Assay Identified a Novel Virulence-Associated

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Metabolomics Assay identified a novel virulence-associated siderophore encoded by the High-Pathogenicity Island in uropathogenic Escherichia coli Guang Xu, Hao Guo, and Haitao Lv J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.9b00190 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 18, 2019

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Journal of Proteome Research

Metabolomics assay identified a novel virulence-associated siderophore encoded by the high-pathogenicity island in uropathogenic escherichia coli Guang Xu1, 2, Hao Guo2, and Haitao Lv1*

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Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center

for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China 2

Chongqing Key Laboratory of Natural Product Synthesis and Drug Research, School

of Pharmaceutical Sciences, Chongqing University, Chongqing 401331, China

Corresponding Author. Haitao Lv., Ph.D., Professor, Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China Email.: [email protected] Tel.: 86-021-34208623

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ACS Paragon Plus Environment

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ABSTRACT To date, yersiniabactin remains the only identified siderophore encoded by the high pathogenicity island (HPI) in uropathogenic Escherichia coli (UPEC). In the present study, we aim to discover and identify new siderophores in the HPI-dependent biosynthetic pathway using a combinational strategy of metabolomics and genetics. A global metabolome assay of wild-type UTI89, UTI89ΔybtS and UTI89ΔybtS with the substrate addition of salicylic acid found numerous unknown metabolite features that were encoded by the HPI with an obvious substrate dependency on salicylic acid. One metabolite feature with m/z 307.0206 was shown to have a similar phenotype as yersiniabactin. Furthermore, isotope mass spectrum calculations and MS/MS annotations were combined to identify this metabolite as HPTzTn-COOH. HPTzTnCOOH was verified as a new siderophore in this study, and it was observed to have a robust capacity to chelate different metals, including Al3+, Ni2+ and Ca2+, in addition to binding Fe3+. Our data revealed that HPTzTn-COOH has a stronger diagnostic ability over the more conventionally used yersiniabactin, as characterized by its high production throughout UPEC strains harboring HPI. Altogether, our discoveries revise the siderophore family, and HPTzTn-COOH can be classified as an additional key siderophore along with yersiniabactin. Key Words: Metabolomics, Siderophore, Pathogenic E. coli, Drug Target

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INTRODUCTION Urinary tract infection (UTI) is one of most prevalent infectious diseases and has a high frequency of recurrence1. Uropathogenic Escherichia coli (UPEC) accounts for approximately 80% of community-acquired cases, the high probability of its antibiotic-resistance, the lack of diagnostic biomarker, and unclear pathogenesis significantly limits the efficient treatment of UTIs2. Many virulence factors contributed by UPEC, involving adhesins, capsules, lipopolysaccharides, outer membrane proteins, a-hemolysin, cytolysin A, and siderophores, have the capacity to cause urinary tract infection3. Siderophores are chemically diverse secondary metabolites with the fundamental role of chelating iron 4, 5, which have been experimentally verified to contribute to the infectious ability of UPEC 6, also confer the diagnostic ability and therapeutic potential during various infectious conditions. Yersiniabactin is a well-known siderophore whose biosynthesis is encoded by the high-pathogenicity island (HPI) and is capable of governing the onsite development of urinary tract infections; Yersiniabactin has been observed to modulate the infectious ability of UPEC 4, 7, and it has a diagnostic potential for UTIs. HPI carries 11 genes that primarily coordinate the biosynthesis, transportation and uptake of yersiniabactin8-10. The gene ybtS plays a key role in the transformation of chelate into salicylic acid (SA), a fundamental substrate for the biosynthesis of yersiniabactin11. Mutations in ybtS can lead to the complete blockage of yersiniabactin synthesis due to the interrupted production of SA. Due to the limitations of earlier biochemical knowledge and analytical technologies, yersiniabactin remains as the only identified

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siderophore whose biosynthesis is encoded by the HPI (Figure S1). Given siderophores are secondary metabolites produced by primary metabolism, metabolomics method is currently the most powerful tool for characterizing primary and secondary metabolites in different biological matrix 12-14. In the present study, we aim to discover and identify new siderophores from the HPI-dependent biosynthetic pathway using a novel combinational strategy of metabolomics and genetics15 (Figure 1).

MATERIALS AND METHODS Chemicals and reagents Ultrapure water was produced by an Ultrapure Water Polishing System (Huachuang, Chongqing, China). Acetonitrile, methanol, and formic acid (all HPLC grade) were purchased from Fisher Scientific (Fisher Scientific, Shanghai, China). LB broth (Luria–Bertani) and LB agar were purchased from Becton Dickinson (BD, Sparks, MD, USA). Salicylic acid, potassium phosphate, ammonium sulfate, niacin, magnesium sulfate, calcium chloride, glycerol and potassium hydroxide were of USP grade and were purchased from Sigma (Sigma-Aldrich Corp., Saint Louis, MO, USA) and Amresco (Solon Industrial Parkway, Ohio, USA). All other reagents were of ACS grade.

Mutant Strains Construction Mutant UTI89ΔybtS strains were made using the red recombinase method with pKD4

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or pKD13 as a template and were confirmed by PCR with flanking primers, as listed in the references16-18. Antibiotic insertions were removed by transforming the mutant strains with pCP20 expressing FLP recombinase19, 20.

Bacterial Strains and Cultivation After 24 hour LB-agar plate cultivation, the UTI89 and UTI89ΔybtS strains were routinely cultured in LB broth for 4 hours15. Different concentrations of precursor salicylic acid were added into M63 medium supplemented with 0.2% glycerol and 10 mg/L nicotinic acid, to rescue the production of yersiniabactin and unknown siderophores (UTI89ΔybtS with the substrate addition of salicylic acid). The incubated LB cultures were then diluted 1:100 into M63 medium in different groups and incubated for 18 h at 37 °C in a rotary shaker21.

Sample preparation All of the 50 ml culture solutions were centrifuged at 3400 × g at 4 °C for 15 min; the pellets were used for metabolite extraction and the supernatants were used for siderophore detection. Aliquots of 1 ml supernatant were filtered (0.22 µm, 13 mm) and centrifuged at 16000 × g and 4 °C for 15 min, and 200 µl of the final liquid sample was used for UHPLC-MS15, 19.

Ultra-High-Performance Liquid Chromatography−Mass Spectrometry (UHPLC−MS)

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The samples were injected sequentially for the UHPLC-QTOF/MS analysis, which were performed using an UHPLC system (Agilent 1290 Infinity, Agilent Technologies, USA) coupled to an ion mobility quadruple time-of-flight (IM/Q-TOF) mass spectrometer (Agilent 6560 Ion Mobility Q-TOF, Agilent Technologies, USA). The sample separation was carried out on a Waters ACQUITY UPLC HSS T3 column (2.1×100 mm, 1.8 µm). The injection volume was 5 µl, the flow rate was 0.3 ml/min, and the column temperature was kept at 35 °C. The mobile phases A and B were 0.1% formic acid in water and formic acid in acetonitrile, respectively. The gradient program is summarized as follows: 0-1 min: 2% B, 8 min: 35% B, 10 min: 80% B, 11-17 min: 98% B. The parameters of MS acquisition are as follows: sheath gas temperature: 350 °C; sheath gas flow: 12 L/min; dry gas temperature: 250 °C; dry gas flow: 10 L/min; capillary voltage: 3500 V in positive mode and 3000 V in negative mode; nozzle voltage: 500 V; and nebulizer pressure: 40 psi in positive mode or 40 psi in negative mode. The acquisition rate was set at 1.5 spectra/s, and the mass range was set at m/z 100–1200 m/z. The reference masses (m/z) were 121.0508 and 922.0098; the acquisition time was set at 15 min 15, 19. The targeted MS/MS mode was applied for tandem mass spectrometry (MS/MS) data acquisition. The parameters of the ion source corresponded with the MS mode. The scan model was ESI and the acquisition rate was set at 1.5 spectra/s in MS and MS/MS modules. The mass range was set at m/z 100–1200 for the MS module and m/z 50–800 for the MS/MS module. The collision energy was set at 15, 20, 23, 27, and 30 volts, and the targeted precursor ions were m/z: 307.0206, m/z: 482.1236, m/z:

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665.9453, and m/z: 535.0351 (ESI+)22, 23.

Data Analysis MS raw data (.d) files of MS mode acquisition were converted to the 32-bit mzXML format using the ProteoWizard software and were preprocessed by XCMS (version 3.5.1) (https://xcmsonline.scripps.edu) to generate a three-dimensional data matrix with the retention time (RT), mass to charge ratio (m/z) and peak intensity, which was then transferred to the Metaboanalyst 3.0 online database for pattern-recognition analysis (http://www.metaboanalyst.ca). The partial least squares-discriminant analysis (PLS-DA) was used for group classification of metabolomics data24, 25; metabolite features were recognized according statistical criteria (p value 1) that resulted from the PLS-DA analysis. A heatmap was used for the overview of all metabolite features throughout the differently treated groups26. MS raw data (.d) files of targeted MS/MS mode acquisition were processed by Agilent MassHunter Workstation Qualitative Analysis software (Version B.07.00 SP1), with peak extraction and targeted MS/MS finding19, 22.

Identification of Metabolite Feature Differential metabolite features were selected with VIP values greater than 1 and P values less than 0.05 in positive MS mode. The features were first identified by accurate mass using multiple databases (Metlin databases (https://metlin.scripps.edu/index.php), Human Metabolome database (HMDB)

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(http://www.hmdb.ca), EcoCyc database (www.ecocyc.org), ECMDB database (http://ecmdb.ca/), siderophorebase (http://bertrandsamuel.free.fr/siderophore_base) and KEGG (http://www.kegg.jp/)). MS/MS spectra and isotope-spectra annotation further confirmed the identities.

Statistical Analysis The bar plot graphs and all other statistics were generated using the Microsoft Office program (Excel 2013).

RESULTS AND DISCUSSION Discovery and identification of a novel yersiniabactin like natural product that was encoded by high pathogenicity island Untargeted metabolomics was implemented to investigate the differential metabolome among the wild-type UTI89, UTI89ΔybtS and UTI89ΔybtS that is related to salicylic acid (SA) in a novel effort to discover and identify new metabolite features whose biosynthetic phenotypes are similar to that of yersiniabactin. Our data revealed the biosynthesis of many metabolites encoded by the HPI that are dependent on SA (Figure 2A and B). Excitingly, the metabolite with a mass-to-charge ratio of 307.0206 and a retention time of 10.39 min were found to share a similar biosynthetic phenotype as yersiniabactin (Figures S2-S4), and the biosynthetic capability of this metabolite was much higher than that of yersiniabactin (Figure 2C and D, Figure S5).

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To verify the identity of this metabolite, the signal-peak was extracted from the full-scan metabolic profile with the exact mass of this metabolite feature, and it was then screened for comparison to the proposed compounds derived from multiple databases, illustrated in the discovery and identification of metabolites section, and was preliminarily deduced to be 2’-(2-hydroxyphenyl)-4’-thiazolyl-2, 4-thiazolinyl-4carboxylicacid (HPTzTn-COOH) (Figure 3A and B). Additionally, isotope mass spectrum annotation was carried out to verify this identification, and the results confirmed that this metabolite is, indeed, HPTzTn-COOH (Figure 3C). Finally, an MS2 assay with a reference compound was used to accurately identify this metabolite feature as 2’-(2-hydroxyphenyl)-4’-thiazolyl-2, 4-thiazolinyl-4carboxylicacid (HPTzTn-COOH) (Figure 4). In short, our study was the first to discover the involvement of HPTzTn-COOH in uropathogenic E. coli UTI89. The biosynthetic pathway of HPTzTn-COOH was characterized by hydrolysis and autoxidation, as is yersiniabactin, with a high-level of biosynthetic capability 10, 27, 28. It may have the capacity to coordinate the processes of metal chelating and the transmembrane pathway exerted by siderophores29. In other systems, HPTzTn-COOH has been found to be a key intermediate for facilitating the biosynthesis of the siderophores yersiniabactin and pyochelin in Yersinia pestis and Pseudomonas aeruginos27, 28, 30.

HPTzTn-COOH was characterized as a novel siderophore that has a widespread capacity to chelate different metals besides iron

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To verify whether or not HPTzTn-COOH was a novel siderophore, a metal chelating assay was carried out, and the signal peaks of HPTzTn-COOH binding to different metals were extracted and annotated. Our data clearly showed that HPTzTn-COOH had a robust ability to chelate many metals, such as Al3+ Ni2+ and Ca2+, in addition to binding Fe3+, as does yersiniabactin (Figures S6-7). According to the working principle of siderophores binding to metals, the siderophores interact with the donoratom structures and hexadentate complex, which often employs different patterns of chelating metals according to the inherent structures involving catecholates, hydroxamates and carboxylates, in which the ligating atoms of the siderophore is oxygen or nitrogen. Therefore, it was logically deduced that two molecules of HPTzTn-COOH and one metal molecule were integrated into one siderophore-metal complex with an octahedron conformation. We verified the signal peaks of HPTzTnCOOH binding to different metals. The differential HPTzTn-COOH chelates were m/z 665.9453 ([2M+Fe-2H]+) (Figure 5), m/z 636.9919 ([2M+Al-2H]+), m/z 668.9535 ([2M+Ni-H]+) and m/z 650.9808 ([2M+Ca-H]+) (Figure S8). Furthermore, these complexes were experimentally verified by the extracted MS features and a natural isotope MS assay. In short, all of the acquired data confirmed that HPTZTNCOOH possessed the fundamental characteristics of a siderophore, which are characterized by secondary metabolites biosynthesized by the HPI with a dependence on salicylic acid, the capability of chelating metals, especially ion (III), and the cellular function of membrane transportation19,.Our study was the first to discover and verify HPTzTn-COOH as a novel siderophore encoded by the HPI in a UPEC strain.

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Pathogenic virulence association of HPTzTn-COOH is more prevalent than yersiniabactin We also attempted to verify whether the biosynthesis of HPTzTn-COOH was closely associated with the virulence of pathogenic strains harboring the HPI, because virulence is increased by the biosynthesis of yersiniabactin. We analyzed the production of HPTzTn-COOH and yersiniabactin in UPECs containing the HPI and non-UPEC strains without the HPI. Our data illustrated that HPTzTn-COOH was produced by both of the UPEC strains containing the HPI (CFT073 and UTI89) and was not produced in the non-UPEC MG1655, which did not contain the HPI. Yersiniabactin was only detected in UTI89 and was not detected in either UPEC CFT073 or non-UPEC MG1655 (Figure 6). Our aim was to first verify HPTzTnCOOH as a novel siderophore associated with pathogenic virulence, whose biosynthesis had the ability to distinguish UPEC from non-UPEC strains. Herein, UTI-pathogenesis by uropathogenic E. coli perfectly correlates with the HPTzTnCOOH detectability, rather than with the well-known virulence-associated siderophore yersiniabactin.

Proposed biosynthetic pathway of HPTzTn-COOH with HPI dependency To better understand the biosynthesis of HPTzTn-COOH, characterized as a novel siderophore, we proposed the following biosynthetic pathway. Shikimic acid is first transformed into salicylic acid (SA) via the YbtS protein encoded by the ybtS gene,

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and then, SA is linked to the ArCP domain of the HMWP1 protein encoded by the irp1 gene through the YbtE protein encoded by ybtE. The product is incorporated into the cysteine-S-PCP complex through a condensation process. The first thiazolinyl product is formed through cyclization, followed by an additional cyclization that is carried out after the other cysteine-S-PCP complex is combined with the first product, and the second thiazolinyl product is formed from a hydrosulphonyl and the PCP domain of the HMWP1 protein encoded by irp1. Finally, HPTZTN-COOH is released through the hydrolysis of thioesterase and the autoxidation of the first thiazolinyl, being oxidized into thiazolyl by oxidative factors; the second thiazolinyl exists in a steady state10, 27, 30, 31 (Figure S9).

CONCLUSION Altogether, our study identified a novel siderophore with substrate dependence on SA, thereby increasing the number of siderophore members biosynthesized by HPI. HPTzTn-COOH is a novel and key molecule with higher diagnostic capability than than yersiniabactin and will help to better our understanding of the pathogenic contribution of siderophore biosynthesis to E. coli during infections. This will enable us to not only improve the diagnosis of UPECs causing UTIs by employing HPTT as a new biomarker but will also help to delineate the pathogenesis and advance therapeutic discoveries for urinary tract infections from the perspective of siderophore biosynthesis modulating host-pathogen interactions.

However, additional in vivo

functional experiments would be required to further confirm the pathological role of

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this compound.

SUPPORTING INFORMATION: The following supporting information is available free of charge at ACS website http://pubs.acs.org Figure S1. The biosynthetic pathway of yersiniabactin encoded by the HPI in the UTI89 strain. Figure S2. The extract mass signals of molecular features with m/z 307.0206 and 482.1236. Figure S3. Metabolite feature with a mass-to-charge ratio of 482.1236 was confirmed as yersiniabactin. Figure S4. MS 2 assisted in identifying yersiniabactin. Figure S5. The expression levels of a metabolite feature with m/z 307.0206 and yersiniabactin. Figure S6. Identity of yersiniabactin-Iron complex. Figure S7. MS2 assisted in identifying the yersiniabactin-iron complex. Figure S8. Identifies of the complexes of HPTzTn-COOH binding to different metals. Figure S9. The proposed biosynthetic pathway of HPTzTn-COOH.

ACKNOWLEDGEMENTS This work was supported by National Natural Science Foundation of China grants (No. 81274175 and c010201), National Key R&D Program of China (No.

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2017YFC1308600), and the Startup Funding for Specialized Professorship provided by Shanghai Jiao Tong University (No. WF220441502). Authors give thanks to Dr. Tian Tian in the Chongqing University for scientific discussion.

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The legend of Figures

Figure 1. The discovery strategy for identifying new siderophores encoded by the HPI with observable substrate dependency of salicylic acid by combining metabolomics and genetics methods. By exploring the virulence capacity of novel siderophores compared to the known yersiniabactin in pathogens causing infections we can better understand different aspects relating to urinary tract infections caused by UPEC strains, including biomarker potentials, disease pathogenesis and the development of novel antibiotics.

Figure 2. Metabolomics-genetics combinational strategy identified novel metabolite features whose biosynthetic phenotype was very similar to the known yersiniabactin. (A) (B) Untargeted metabolomics score plot and heatmap revealed differentiable metabolites encoded by the HPI that have a remarkable substrate dependence on salicylic acid; (C) VIP plot generated by pattern-recognition analysis of the global metabolome that discovered and identified many metabolite features whose production was governed by the HPI, with noticeable substrate dependency on salicylic acid. The two metabolite features were recognized as yersiniabactin and a molecular feature with m/z 307.0206, and they shared a very similar expressional pattern in the biosynthetic phenotype visualized in (D).

Figure 3. Identity of metabolite feature with mass-to-charge ratio of 307.0210 by the

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standard analysis of signal peak mass extraction (A) database reference (B) and isotope mass annotation (C).

Figure 4. MS 2 assisted in identifying HPTzTn-COOH with the help of daughter ion matching with a structural reference compound.

Figure 5. Identity of HPTzTn-COOH-Iron complex. (A) The peak signal of HPTzTnCOOH-Iron complex with the extract mass of 665.9453. (B) database reference. (C) isotope mass annotation. (D) MS 2 assisted in identifying the HPTzTn-COOH-iron complex.

Figure 6. Virulence selection of HPTzTn-COOH and yersiniabactin in different strains with and without the HPI. (A) The extract mass signals of two siderophores in different selected strains. (B) The relative levels of two siderophores in different selected strains.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Table of Content

Novel siderophore HPTzTn-COOH biosynthesized by High Pathogenicity Island renders higher pathogenic dependency than the known yersiniabactin.

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Metabolomics Assay Identified a Novel Virulence-Associated

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