Nutrient limitation governs Staphylococcus aureus metabolism and niche adaptation in the human nose

Abstract

Colonization of the human nose by Staphylococcus aureus in one-third of the population represents a major risk factor for invasive infections. The basis for adaptation of S. aureus to this specific habitat and reasons for the human predisposition to become colonized have remained largely unknown. Human nasal secretions were analyzed by metabolomics and found to contain potential nutrients in rather low amounts. No significant differences were found between S. aureus carriers and non-carriers, indicating that carriage is not associated with individual differences in nutrient supply. A synthetic nasal medium (SNM3) was composed based on the metabolomics data that permits consistent growth of S. aureus isolates. Key genes were expressed in SNM3 in a similar way as in the human nose, indicating that SNM3 represents a suitable surrogate environment for in vitro simulation studies. While the majority of S. aureus strains grew well in SNM3, most of the tested coagulase-negative staphylococci (CoNS) had major problems to multiply in SNM3 supporting the notion that CoNS are less well adapted to the nose and colonize preferentially the human skin. Global gene expression analysis revealed that, during growth in SNM3, S. aureus depends heavily on de novo synthesis of methionine. Accordingly, the methionine-biosynthesis enzyme cysteine-γ-synthase (MetI) was indispensable for growth in SNM3, and the MetI inhibitor DL-propargylglycine inhibited S. aureus growth in SNM3 but not in the presence of methionine. Of note, metI was strongly up-regulated by S. aureus in human noses, and metI mutants were strongly abrogated in their capacity to colonize the noses of cotton rats. These findings indicate that the methionine biosynthetic pathway may include promising antimicrobial targets that have previously remained unrecognized. Hence, exploring the environmental conditions facultative pathogens are exposed to during colonization can be useful for understanding niche adaptation and identifying targets for new antimicrobial strategies.

Figure 1. Metabolite composition of nasal secretions in S. aureus carriers and non-carriers.

(A, B) Metabolite quantification by GC-MS from eight sample donors, micromolar concentrations are given for compounds with standard dilutions measured (A), for other substances relative abundances are given against internal standards (B). Upper and lower box limits and the horizontal lines within the boxes represent 25 and 75% percentiles and the medians, respectively. The whiskers of the plots indicate minimum and maximum range, additionally showing the data points. (C) Differences in metabolite profiles in nasal secretions from six carriers (crosses) and seven non-carriers (circles) analyzed by principal component analysis of all metabolite levels. Principal component 1 vs. 2 is shown, explaining in total 67.9% of the PCA model. Metabolite values were log-transformed and scaled by unit variance.

Figure 2. Capacities of S. aureus and CoNS strains to grow in synthetic nasal medium (SNM).

(A), growth of S. aureus USA300 LAC and S. aureus Newman in SNM with increasing (x-fold) concentrations of amino acids, organic acids and glucose after 90 h growth. The mean values and SEM for three independent cultures of each concentration is shown. (B) Growth of nasal isolates of S. aureus (filled circles), S. epidermidis (triangles) and other coagulase-negative staphylococci (CoNS) (open circles) in liquid SNM3 after the indicated incubation times. The means at each time point are shown as horizontal lines for each group. Statistically significant differences vs. S. aureus calculated by the ANOVA Kruskal-Wallis test with Post-test Dunns and 95% confidence intervals are indicated: *, p≤0.05; **, p≤0.01. Cultures were vigorously shaken at 37°C in A and B.

Figure 3. Colony formation abilities on SNM agar of S. aureus, S. epidermidis and other CoNS strains isolated from human nares.

(A) Serial dilutions of all nasal isolates grown in BM overnight were spotted on BM or 3× SNM (SNM3) agar. The y-axis shows the percentage of cells forming colonies on SNM3 compared to BM. The influence of enhanced nutrient concentrations in 5×, 10×, and 20× SNM (B) or of 10–100 µM methionine in SNM3 (C) on the colony-forming ability was analyzed with a subset of strains shown in Figure 3A. S. aureus is shown as filled circles, S. epidermidis as triangles and other coagulase-negative staphylococci (CoNS) as open circles. The horizontal bar indicates the median of each group of strains. Statistically significant differences calculated by the ANOVA Kruskal-Wallis test with Post-test Dunns and 95% confidence intervals are indicated: *, p≤0.05; **, p≤0.01; ***, p≤0.001.

Figure 4. Relative gene expression of selected genes of S. aureus USA300 LAC in BM and SNM.

Gene expression was assayed by qRT-PCR for at least 6 independent cultures growing in BM or SNM3 or in stationary phase BM cultures (BM stat) each. Analyzed transcripts represent RNAIII, phenol soluble modulins β1 and β2 (psm), clumping factor B (clfB), iron-regulated surface determinant A (isdA), and a lytic transglycosylase (sceD). The upper and lower box limits and the horizontal lines within the boxes represent 25 and 75% percentiles and the means, respectively. The whiskers of the plots indicate minimum and maximum values. Statistically significant differences vs. BM calculated by the unpaired, two-tailed Student's t test with Welch's correction are indicated: *, p≤0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001.

Figure 5. Differences in the transcriptome of S. aureus USA300 grown in SNM3 and complex medium (BM).

(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 Figure S2.

Figure 6. Schematic representation of the methionine-biosynthetic pathway of S. aureus USA300 (adapted from KEGG pathway database).

All genes shown in red were up-regulated in SNM3 compared to BM as determined by microarray analysis, with SAUSA300_0357 to SAUSA300_0360 exhibiting the strongest effects (13 to 32-fold). The metK gene, shown in green, was expressed at similar levels in BM and SNM3. The cystathionine-γ-synthase (MetI) represents the target for DL-propargylglycin.

Figure 7. Validation of relative gene expression of selected genes up-regulated in the microarrays by qRT-PCR.

Results from at least six independent BM or SNM3 cultures each are depicted as the x-fold change of expression of the respective genes in SNM3 vs. BM. Analyzed transcripts represent cysteine-γ-synthase (metI), a methionine transporter (metN), aspartate kinase, histidine biosynthesis gene (hisC), oligopeptide permease (oppB), and staphyloferrin B biosynthesis gene (sbnC). The upper and lower box limits and the horizontal lines within the boxes represent 25 and 75% percentiles and the means, respectively. The whiskers of the plots indicate minimum and maximum range. Significant differences calculated by the unpaired, two-tailed Student's t test with Welch's correction are indicated: * p≤0.05; **p≤0.01; *** p≤0.001; **** p≤0.0001.

Figure 8. Relative expression of selected genes in nasal swabs from six S. aureus carriers (in vivo) compared to growth of the corresponding strains in BM or SNM3.

The relative gene expression levels in nasal swabs (in vivo) and SNM3 cultures measured by qRT-PCR were compared with mRNA level of the corresponding strain in BM, which was defined as 1. Each symbol per group (SNM3 or in vivo) represents an independent nasal isolate. The means per group are indicated as horizontal lines. Less than six data points per group are presented for some of the genes since some samples yielded no expression signals. Analyzed transcripts represent cysteine-γ-synthase (metI), iron-regulated surface determinant A (isdA), a lytic transglycosylase (sceD), aspartate kinase, oligopeptide permease (oppB), histidine biosynthesis gene (hisC), and staphyloferrin B biosynthesis gene (sbnC).

Figure 9. Cotton rat nasal colonization model.

Bacterial numbers were determined six days after intranasal inoculation of cotton rats with 1×10⁸ colony forming units (CFU) of S. aureus (Newman). After six days the noses were dissected and bacterial CFUs were enumerated on S. aureus-selective HighChrome agar. Statistical analysis was performed by D'Agostino & Pearson omnibus normality test and a subsequent Mann Whitney test. Significant differences between groups are indicated by one (P<0.05) asterisk (*).