Hemoglobin stimulates vigorous growth of Streptococcus pneumoniae and shapes the pathogen's global transcriptome

Abstract

Streptococcus pneumoniae (Spn) must acquire iron from the host to establish infection. We examined the impact of hemoglobin, the largest iron reservoir in the body, on pneumococcal physiology. Supplementation with hemoglobin allowed Spn to resume growth in an iron-deplete medium. Pneumococcal growth with hemoglobin was unusually robust, exhibiting a prolonged logarithmic growth, higher biomass, and extended viability in both iron-deplete and standard medium. We observed the hemoglobin-dependent response in multiple serotypes, but not with other host proteins, free iron, or heme. Remarkably, hemoglobin induced a sizable transcriptome remodeling, effecting virulence and metabolism in particular genes facilitating host glycoconjugates use. Accordingly, Spn was more adapted to grow on the human α - 1 acid glycoprotein as a sugar source with hemoglobin. A mutant in the hemoglobin/heme-binding protein Spbhp-37 was impaired for growth on heme and hemoglobin iron. The mutant exhibited reduced growth and iron content when grown in THYB and hemoglobin. In summary, the data show that hemoglobin is highly beneficial for Spn cultivation in vitro and suggest that hemoglobin might drive the pathogen adaptation in vivo. The hemoglobin receptor, Spbhp-37, plays a role in mediating the positive influence of hemoglobin. These novel findings provide intriguing insights into pneumococcal interactions with its obligate human host.

Figure 1

Hemoglobin-dependent growth of Spn D39 in iron-deplete medium. THYB was inoculated with D39 cells grown on BAPs (18 h, starting O.D.₆₀₀ = 0.05). Shown is growth in (A) THYB, THYB with 3 mM 2, 2′-Dipyridyl (DP), THYB with DP and 0.5–3 mM of FeNO₃ (Fe); (B) THYB with DP supplemented with 0.5 µM -20 µM hemoglobin (Hb); and (C) THYB with DP supplemented with 5–60 µM heme (Hm). The data are representative of three independent experiments performed in triplicates; error bars indicate SD.

Figure 2

Hemoglobin stimulates a robust growth of Spn D39 in standard (iron-complete) medium. THYB was inoculated with D39 cells grown on BAPs (18 h, starting O.D.₆₀₀ = 0.05. Shown is growth in (A) THYB with 0–20 µM hemoglobin (Hb); (B) THYB with 20 µM BSA, denatured hemoglobin, filtered hemoglobin, or the flow-through; (C) THYB with or without catalase. (D) THYB supplemented with 0–80 µM FeNO₃ (Fe); (E) THYB with 0–40 µM heme (Hm). The data are representative of three independent experiments performed in triplicates; error bars indicate SD. (F) Total intracellular iron content measured by ICP-MS in culture samples (normalized to optical density) grown in THYB, THYB with 20 µM hemoglobin (Hb), or THYB with 80 µM FeNO₃ (Fe). The data represents the average of three independent biological replicates; error bars indicate SD. The asterisks denote statistical significance, P ≤ 0.05 (THYB vs. Fe, and Hb vs. Fe, Student's t-test).

Figure 3

Hemoglobin stimulates Spn growth independently of the strain or the growth assay. Shown is Spn growth in fresh THYB (empty symbols) or THYB with 20 µM hemoglobin (full symbols). The culture starting O.D.₆₀₀ is indicated. (A) TIGR4 (red), and the clinical isolates 3,875 (blue), and 8,655 (yellow) grown on BAPs (18 h) was used as the inoculum (O.D.₆₀₀ = 0.05). (B) THYB was inoculated with D39 cells from frozen logarithmic cultures (O.D.₆₀₀ = 0.02, red), THYB cultures (18 h, O.D.₆₀₀ = 0.05, blue), or cell from BAPs (18 h, O.D.₆₀₀ = 0.05, green). (C) D39 growth in CAT medium (empty symbols), CAT medium with 20 µM hemoglobin (red, full symbols) or 20 µM human serum albumin (green, full symbols). The data are representative of three independent experiments performed in triplicates; error bars indicate SD.

Figure 4

The addition of hemoglobin to the culture medium triggers a significant transcriptome remodeling in Spn. Venn diagram (using R) of differentially expressed genes in D39 culture (fold change ≥ 2). (A) Genes up-regulated by hemoglobin. (B) Genes down-regulated by hemoglobin.

Figure 5

Hemoglobin activates Spn genes vital for host colonization. The relative expression of genes involved in metabolism, nutrient uptake, virulence, or regulation, at 1 h and 2 h post hemoglobin addition (Y-axis) is plotted for D39 genes (X-axis). (A) Up-regulated genes. Stripes indicate involvement in nasopharyngeal colonization. (B) Down-regulated genes. (C) Dot plot representation of gene expression levels depicting average normalized RNA-seq read counts (Y-axis) for cells with 1 h post-hemoglobin treatment is plotted for D39 genes (X-axis). Genes encoding heme/hemoglobin binding (red) or virulence factors (blue) are highlighted.

Figure 6

Hemoglobin up-regulates Spn genes involved in host glycoconjugate use. Log₂-fold changes in gene expression levels (Y-axis) is plotted for D39 genes (X-axis). (A) PTSs, enzymes, and hypothetical protein (HP). (B) down-regulated sugar transporters.

Figure 7

Hemoglobin facilitates Spn growth on a human glycoprotein. (A) Schematic representation of the human α − 1-acid glycoprotein (AGP), as described in. Arrows indicate the cleavage sites of the Spn enzymes, neuraminidase (NanA), galactosidase (BgaA), and N-acetylglucosaminidase (StrH). Fresh medium was inoculated with D39 grown on BAPs (18 h, starting O.D.₆₀₀ = 0.05). (B) Shown is Spn growth in sugar-free CAT medium (no glucose added), or sugar-free CAT with 5 mg/ml AGP, and/or 20 µM hemoglobin (+ Hb). (C) The same as in (A), only that 20 µM human serum albumin (+ HSA) was added instead of hemoglobin. The data are representative of three independent experiments performed in triplicates; error bars indicate SD.

Figure 8

The heme/hemoglobin binding protein, Spbhp-37, plays a role in mediating the positive impact of hemoglobin in Spn. Fresh medium was inoculated with D39 (blue) and the isogenic Δspbhp-37 strain (orange) grown on BAPs (18 h, starting O.D.600 = 0.05). Shown is growth in (A) THYB with 3 mM 2, 2′-Dipyridyl (DP), THYB with DP and 10 μM heme (triangles)  or 20 μM hemoglobin (circles), or (B) THYB (empty symbols) or THYB with 20 µM hemoglobin (full symbol). The data are representative of three independent experiments performed in triplicates; error bars indicate SD. (C) Total intracellular iron content (ppm) determined by ICP-MS in D39 wild type and Spbhp-37 mutant cultures samples (normalized to optical density) grown in THYB with 20 µM hemoglobin. The data represents the average of three independent biological replicates; error bars indicate SD. (D) Fold change in gene expression in the wild type and Δspbhp-37 strains 2 h post hemoglobin treatment relative to the control (saline) as determined by qRT-PCR. The experiments were performed in duplicates with at least two biological replicates. The replicates data are shown as the mean ± SD. The asterisks denote statistical significance, P ≤ 0.05 (WT vs. MT, Student's t-test).