Time-course analysis of Streptococcus sanguinis after manganese depletion reveals changes in glycolytic and nucleic acid metabolites

Introduction Manganese is important for the endocarditis pathogen Streptococcus sanguinis. Little is known about why manganese is required for virulence or how it impacts the metabolome of streptococci. Objectives We applied untargeted metabolomics to cells and media to understand temporal changes resulting from manganese depletion. Methods EDTA was added to a S. sanguinis manganese-transporter mutant in aerobic fermentor conditions. Cell and media samples were collected pre- and post-EDTA treatment. Metabolomics data were generated using positive and negative modes of data acquisition on an LC–MS/MS system. Data were subjected to statistical processing using MetaboAnalyst and time-course analysis using Short Time series Expression Miner (STEM). Recombinant enzymes were assayed for metal dependence. Results We observed quantitative changes in 534 and 422 metabolites in cells and media, respectively, after EDTA addition. The 173 cellular metabolites identified as significantly different indicated enrichment of purine and pyrimidine metabolism. Further multivariate analysis revealed that the top 15 cellular metabolites belonged primarily to lipids and redox metabolites. The STEM analysis revealed global changes in cells and media in comparable metabolic pathways. Glycolytic intermediates such as fructose-1,6-bisphosphate increased, suggesting that enzymes that utilize them require manganese for activity or expression. Recombinant enzymes were confirmed to utilize manganese in vitro. Nucleosides accumulated, possibly due to a blockage in conversion to nucleobases resulting from manganese-dependent regulation. Conclusion Differential analysis of metabolites revealed the activation of a number of metabolic pathways in response to manganese depletion, many of which are connected to carbon catabolite repression. Supplementary Information The online version contains supplementary material available at 10.1007/s11306-021-01795-2.


Metal analysis
As described in Puccio et al. (2020), 40-mL cell culture samples were collected from ΔssaACB cells at the same fermentor growth time points (T-20, T25, T50). The cells were immediately centrifuged at 3,740 x g for 10 min at 4°C. The supernatant was decanted and the cell pellet was washed twice with cold cPBS (PBS treated with Chelex-100 resin (Bio-Rad) for 2 h, then filter sterilized and supplemented with EDTA to 1 mM). The pellet was then divided for subsequent acid digestion or protein concentration determination. Trace metal grade (TMG) nitric acid (15%) (Fisher Chemical) was added to one portion of the pellet. The pellet was digested using an Anton Paar microwave digestion system using a modified Organic B protocol: 120°C for 10 min, 180°C for 20 min, with the maximum temperature set to 180°C.
The digested samples were then diluted 3-fold with Chelex-treated dH2O. Metal concentrations were determined using an Agilent 5110 inductively coupled plasma-optical emission spectrometer (ICP-OES).
Concentrations were determined by comparison with a standard curve created with a 10 μg ml −1 multielement standard (CMS-5; Inorganic Ventures) diluted in 5% TMG nitric acid. Pb (Inorganic Ventures) was used as an internal standard (10 μg ml −1 ). The other portion of the pellet was resuspended in PBS and mechanically lysed using a FastPrep-24 instrument with Lysing Matrix B tubes (MP Biomedicals) as described previously (Rhodes et al., 2014). Insoluble material was removed by centrifugation. Protein concentrations were determined using a bicinchoninic acid (BCA) Protein Assay Kit (Pierce) as recommended by the manufacturer, with bovine serum albumin as the standard. Absorbance was measured in a black, flat-bottom 96-well plate (Greiner) using a Synergy H1 plate reader (BioTek).

H2O2 quantitation
Culture supernatants were collected at each time point and stored at -20°C. H2O2 concentration was measured using a Fluorometric Hydrogen Peroxide Assay Kit (Sigma). Standards were prepared from 3% H2O2 provided with the kit as recommended by the manufacturer. Fluorescence was measured in a black, flat-bottom 96-well plate (Greiner) using a Synergy H1 plate reader (BioTek).

Culture enumeration
Sample culture aliquots (1 mL) from each fermentor time point were stored at 4°C. On the same day as the fermentor run, these aliquots were sonicated for 90 s using an ultrasonic homogenizer (Biologics, Inc) to disrupt chains prior to dilution in phosphate buffered saline (PBS). Diluted cultures were plated using an Eddy Jet 2 spiral plater (Neutec Group, Inc.). The plates were incubated for 24 h at 37°C at 0% O2, where atmospheric composition was adjusted using a programmable Anoxomat Mark II jar filling system (AIG, Inc.) and a palladium catalyst was included in the jars.

Sample preparation for metabolomics analysis
Metabolomics samples were stored at -80°C upon shipment. Samples were accessioned into the Metabolon LIMS system and were prepared using the automated MicroLab STAR® system from Hamilton Company.
Several recovery standards were added prior to the first step in the extraction process for QC purposes.
Samples were extracted with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) to precipitate protein and dissociate small molecules bound to protein or trapped in the precipitated protein matrix, followed by centrifugation to recover chemically diverse metabolites. The resulting extract was divided into five fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods using positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS using negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS using negative ion mode ESI, and one reserved for backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen before preparation for analysis.

Mass Spectroscopy (UPLC-MS/MS)
All methods utilized a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution.
The sample extract was dried then reconstituted in solvents compatible to each of the four methods. Each reconstitution solvent contained a series of standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic positive ion conditions, chromatographically optimized for more hydrophilic compounds. In this method, the extract is gradienteluted from a C18 column (Waters UPLC BEH C18-2.1x100 mm, 1.7 µm) using water and methanol, containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA). A second aliquot was also analyzed using acidic positive ion conditions, but chromatographically optimized for more hydrophobic compounds. In this method, the extract is gradient eluted from the aforementioned C18 column using methanol, acetonitrile, water, 0.05% PFPA and 0.01% FA, and is operated at an overall higher organic content. A third aliquot was analyzed using basic negative ion optimized conditions using a separate Time-course metabolomics of Mn-deplete S. sanguinis 4 dedicated C18 column. The basic extracts were gradient-eluted from the column using methanol and water, however with 6.5 mM ammonium bicarbonate at pH 8. The fourth aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10 mM Ammonium Formate, pH 10.8. The MS analysis alternated between MS and data-dependent MS n scans using dynamic exclusion. The scan range varies slightly between methods, but covers approximately 70-1000 m/z. Raw data files were archived and extracted as described below.

Data Extraction and Compound Identification
Raw data were extracted, peak-identified, and QC processed using Metabolon's hardware and software.

Curation
A variety of curation procedures were performed to ensure that a high quality data set was made available for statistical analysis and data interpretation. The QC and curation processes were designed to ensure accurate and consistent identification of true chemical entities, and to remove those representing system artifacts, mis-assignments, redundancy, and background noise. Metabolon data analysts used internally-developed visualization and interpretation software to confirm the consistency of peak identification among the various samples. Library matches for each compound were checked for each sample and corrected if necessary. http://cts.fiehnlab.ucdavis.edu/conversion/batch) was used to convert the common chemical names into their KEGG, HMDB, Metlin, PubChem CID, and ChEBI identifiers.

Expression and purification of recombinant enzymes
The genes encoding fructose-1,6-bisphosphate aldolase (fba; SSA_1992) and fructose-1,6-bisphosphatase (fbp; SSA_1056) were codon-optimized for expression in Escherichia coli (Bio Basic). The optimized genes were cloned into a pET24dTEV vector with an in-frame N-terminal His-Tag. These plasmids were transformed into BL21 (λDE3) E. coli cells which were then grown overnight at 37°C in Luria Broth (LB; Difco) with 100 µg/mL ampicillin. This overnight culture was used to inoculate 1 L of LB, which was incubated with shaking at 30°C until the OD600 reached 0.7-1.0. IPTG (100 mg) was added and cells were incubated for an additional 3 h. Bacteria were harvested by centrifugation at 7000 rpm for 10 min and the pellet was resuspended in 25 mM Tris pH 8.0. Nuclease from Serratia marcenscens was added to 1 mg.
Bacteria were lysed using a single passage through an Avestin Emulsiflex operating at 25,000 psi. The lysate was clarified by centrifugation at 20,000 rpm for 15 min.
The clarified lysate was applied to an NTA agarose column (BioRad) with a column volume of 10 mL.
The column was washed with the following buffer until the A280 returned to baseline: 25 mM Tris pH 8.0, 6 mM imidazole. Fractions were collected and analyzed by 10% or 12% SDS gels. Fractions were pooled according to the gel. Figure S1. Densities of S. sanguinis ΔssaACB mutant fermentor cultures subjected to metabolomic analysis Aliquots from fermentor cultures collected at each time point were plated on BHI agar and colony forming units per mL (CFU mL -1 ) were calculated. EDTA was added to the fermentor at T0. The color of each circle corresponds to each of the six independent fermentor runs. Horizontal lines depict the geometric mean. Significance (P < 0.05) was determined by one-way ANOVA using log-transformed values. Figure S2. Pathway enrichment analysis for differential metabolites Pathway enrichment analysis for significantly differential metabolites (ANOVA) in cells (a) and spent media (b). Red represents lower P-values (more significant in the Hypergeometric Test) and yellow/white represents greater P-values (less significant in the Hypergeometric Test). Nominal P-value < 0.05. The larger the size of the circles represents larger impact of the pathway compared to others in the pathway enrichment analysis function in MetaboAnalyst 4.0.   Models #19 and #18 were statistically significant in cells among the 20 models interrogated. Models # 18, #19, and #14 were statistically significant in media.

Figure S6. Changes in nucleic acid metabolites
Purine nucleosides in media (a) and cells (e). Pyrimidine nucleosides in media (b) and cells (f). Purine nucleobases in media (c) and cells (g). Pyrimidine nucleobases in media (d) and cells (h). Whiskers indicate the range; horizontal bars represent the mean. A two-tailed t-test was used to compare the preinoculum (PI) media samples to post-inoculum (T-20). Red asterisks indicate when P-value < 0.05. Spent media and cell metabolite levels were compared using an ANOVA with a Fisher's least significant difference test to compare the post-EDTA samples to pre-EDTA. Black asterisks indicate P-value < 0.05.