Between-Person Comparison of Metabolite Fitting for NMR-Based Quantitative Metabolomics

Nuclear magnetic resonance (NMR) spectroscopy is widely used as an analytical platform for metabolomics. Many studies make use of 1D spectra, which have the advantages of relative simplicity and rapid acquisition times. The spectral data can then be analyzed either with a chemometric workflow or by an initial deconvolution or fitting step to generate a list of identified metabolites and associated sample concentrations. Various software tools exist to simplify the fitting process, but at least for 1D spectra, this still requires a degree of skilled operator input. It is of critical importance that we know how much person-to-person variability affects the results, in order to be able to judge between different studies. Here we tested a commercially available software package (Chenomx’ NMR Suite) for fitting metabolites to a set of NMR spectra of yeast extracts and compared the output of five different people for both metabolite identification and quantitation. An initial comparison showed good agreement for a restricted set of common metabolites with characteristic well-resolved resonances but wide divergence in the overall identities and number of compounds fitted; refitting according to an agreed set of metabolites and spectral processing approach increased the total number of metabolites fitted but did not dramatically increase the quality of the metabolites that could be fitted without prior knowledge about peak identity. Hence, robust peak assignments are required in advance of manual deconvolution, when the widest range of metabolites is desired. However, very low concentration metabolites still had high coefficients of variation even with shared information on peak assignment. Overall, the effect of the person was less than the experimental group (in this case, sampling method) for almost all of the metabolites

Quantitative RT-PCRs of phagocytosed bacteria.

<p>Transcript analyses of <i>rpsB</i>, <i>infB</i> (category I), <i>ilvC</i> (category II), <i>psmα1-4</i>, <i>psmβ1,2</i> (category III) and <i>RNAIII</i> genes after phagocytosis of the <i>S. aureus</i> strain HG001 and the corresponding <i>rsh_Syn</i> mutant, <i>rsh_Syn</i>, <i>codY</i> double mutant and the complemented <i>rsh_Syn</i> mutant (compl). The transcripts were relatively quantified in reference of <i>gyrB</i> and are shown in a log₁₀ scale relative to the wild-type strain (HG001). RNA was isolated after a 60 and 90-minute interaction of <i>S. aureus</i> with PMNs. Values from four separate experiments were used to calculate the mean expression. The levels of significance compared to the wild-type strain were determined by the two-tailed Student t test (p<0.05).</p

Quantitative analysis of the intracellular ATP concentrations.

<p>The intracellular ATP pools of strain HG001, the corresponding <i>rsh_Syn</i> mutant, the <i>rsh_Syn</i>, <i>codY</i> double mutant and the complemented <i>rsh_Syn</i> mutant (compl) were determined by luciferase activity. The bacteria were grown in CDM to exponential growth phase (OD₆₀₀ = 0.5) followed by further incubation in medium with (control) or without (−) leu/val for 30 minutes. The levels of significance to the corresponding control (with leu/val) were determined by the two-tailed Student t test (p<0.05).</p

Genes positively regulated under stringent response independent of CodY.

1<p>: Fold changes are indicated for each comparison and displayed for genes showing statistically significant differential expression. Values corresponds to expression ratios, i.e. averaged expression levels from three independent replicate experiments (p<0.05).</p

Transcriptome analysis of (p)ppGpp dependent genes during stringent response.

<p>Transcriptional changes of <i>S. aureus</i> in response to leucine/valine starvation (−leu/val) and the overlap of the two regulons CodY and stringent response. (A) Venn diagrams showing genes which are down- (blue) and up-regulated (red) during RSH mediated stringent response. A contribution of CodY to the regulation was determined by analyzing the transcriptional changes in a <i>codY</i>-positive background (WT strains vs. <i>rsh_Syn</i> mutant) indicated as codY+ in the figure and in a <i>codY</i>-negative background (<i>codY</i> mutant vs. <i>rsh_Syn</i>, <i>codY</i> double mutant) indicated as codY−. (B) Functional classes of genes which are down- (blue) and up-regulated (red) during RSH mediated stringent response. (C) Heat maps of gene expression ratios for indicated functional classes prominently altered in their expression. The ratios were analyzed in a <i>codY-</i>positive (codY+) and a <i>codY-</i> negative (codY−) background as described for the Venn diagrams. ** saturated signal *** genes with annotated CodY binding motifs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003016#ppat.1003016-Majerczyk1" target="_blank">[16]</a>.</p

Phagocytosis killing assays and a PMN lysis assay with different <i>S. aureus</i> strains.

<p>(A) and (B) Survival of wild-type <i>S. aureus</i> strain HG001 and strain Newman (WT), and their isogenic <i>rsh_Syn</i> mutants, complementation strains (compl), <i>psmα</i>, <i>psmβ</i> double mutants (HG001), <i>rsh_Syn</i>, <i>psmα</i>, <i>psmβ</i> triple mutants (HG001), <i>agr</i> mutant (HG001) and <i>rsh_Syn</i>, <i>agr</i> double mutant (HG001) were determined after phagosomal uptake by counting colony-forming units after 60 min incubation. Bacterial cells used for the experiment were harvested at similar points of growth at an OD₆₀₀ of 1.0. The levels of significance were determined by the two-tailed Student t test (p<0.05). (C) Complementation of survival of the <i>rsh_Syn</i> mutant with tetracycline inducible <i>psmα</i> and <i>psmβ</i>. Therefore membrane permeable anhydrotetracycline (ATc) 0.1 µg/ml was used to induce <i>psm</i> expression after phagosomal uptake of the bacteria. The levels of significance were determined by the two-tailed Student t test (p<0.05). (D) Lysis of PMNs, as measured by release of lactate dehydrogenase (LDH) activity. PMN lysis was determined after a phagocytotic interaction of 60 min with <i>S. aureus</i> strain HG001 wild-type, corresponding <i>rsh_Syn</i> mutant, <i>agr</i> mutant and <i>rsh_Syn</i>, <i>agr</i> double mutant. As a positive control 2% TritonX were used. The levels of significance were determined by the two-tailed Student t test (p<0.05).</p

(p)ppGpp dependent <i>psm</i> expression during stringent response.

<p>(A) (p)ppGpp dependent activation of target genes in two different <i>S. aureus</i> strains. Strain HG001 (WT) and strain Newman (WT), the corresponding <i>rsh_Syn</i> mutants, <i>codY</i> mutants, <i>rsh_sy</i>_n, <i>codY</i> double mutants, the complemented <i>rsh_Syn</i> mutants (compl) and the <i>agr</i> mutants were grown in CDM to the exponential growth phase (OD₆₀₀ = 0.5) followed by further incubation in medium without (−) leu/val for 30 minutes. (B) Wild-type strain HG001 and its isogenic <i>rsh_Syn</i> mutant were incubated with (+) or without (−) leu/val for 30 minutes. RNA was hybridized with digoxigenin-labelled PCR fragments. The 16S rRNA detected in the ethidium bromide-stained gels is indicated as loading control in the lower lane.</p

Overview of the presumed impact of the stringent response on survival after phagocytosis.

<p>The induction of the stringent response (pppGpp) in phagocytosed bacteria leads to increased <i>psms</i> expression. The PSMs in turn mediate a pronounced survival after phagocytosis, most likely through phagosomal escape or intracellular lysis of the phagocytes.</p

Differences in the transcriptome of <i>S. aureus</i> USA300 grown in SNM3 and complex medium (BM).

<p>(A) PCA mapping of transcriptome profiles from BM compared to SNM3 cultures from microarray experiments. Each sphere represents an individual GeneChip result with the plotted location based upon the correlation of each sample relative to the others. Results from three independent BM and SNM cultures are compared. (B) Functional classes of genes (COG, Clusters of Orthologous Groups of proteins) detected in the microarray analysis, which were more than two-fold up- (red) or down-regulated (blue) and p<0.05 in SNM3 compared to complex medium (BM) are shown. The x-axis indicates the number of differentially regulated genes in each COG subgroup. The genes of subgroups E, M, and P are listed in detail in the supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003862#ppat.1003862.s002" target="_blank">Figure S2</a>.</p

MIC values of DL-propargylglycine for <i>S. aureus</i> strains in complex medium (BM) and SNM3.

<p>, Nasal isolates from this study, for which <i>in vivo</i> gene expression was also determined.</p