Effect of nonheme iron-containing ferritin Dpr in the stress response and virulence of pneumococci

Abstract

Streptococcus pneumoniae (pneumococcus) produces hydrogen peroxide as a by-product of metabolism and provides a competitive advantage against cocolonizing bacteria. As pneumococci do not produce catalase or an inducible regulator of hydrogen peroxide, the mechanism of resistance to hydrogen peroxide is unclear. A gene responsible for resistance to hydrogen peroxide and iron in other streptococci is that encoding nonheme iron-containing ferritin, dpr, but previous attempts to study this gene in pneumococcus by generating a dpr mutant were unsuccessful. In the current study, we found that dpr is in an operon with the downstream genes dhfr and clpX. We generated a dpr deletion mutant which displayed normal early-log-phase and mid-log-phase growth in bacteriologic medium but survived less well at stationary phase; the addition of catalase partially rescued the growth defect. We showed that the dpr mutant is significantly more sensitive to pH, heat, iron concentration, and oxidative stress due to hydrogen peroxide. Using a mouse model of colonization, we also showed that the dpr mutant displays a reduced ability to colonize and is more rapidly cleared from the nasopharynx. Our results thus suggest that Dpr is important for pneumococcal resistance to stress and for nasopharyngeal colonization.

FIG 1

Generation of dpr mutants. (A) Schematic diagram of the generation of the dpr mutants and revertants using a bicistronic (Janus) cassette. Black arrows indicate the position of primers for reverse transcription-PCR in Fig. 1B. (B) dpr, dhfr, and clpX are in a single transcript. RNA was extracted from pneumococcal TIGR4 and 603 strains, and cDNA was generated by reverse transcription. PCR products using cDNA (top) or RNA (bottom) were amplified using primers listed in Table 1. (C) PCR amplification of genomic DNA from revertant and KO strains using two outside primers. (D) Western blotting was performed to confirm the phenotypes of the dpr KO and revertant strains in the 603 strain background. Pneumolysin was used as a loading control.

FIG 2

Binding of iron to Dpr. Purified Dpr and control BSA proteins were added to PBS with or without 1 mM Fe²⁺ and incubated on ice for 30 min. Proteins were separated with nondenaturing gel. Iron-bound proteins were visualized with Ferene S (left), and protein bands were visualized with Coomassie brilliant blue (right).

FIG 3

Growth curve of dpr mutant and revertant strains. Bacteria were grown to an OD₆₀₀ of 0.5 and diluted in THY medium to start the culture. (A) OD₆₀₀ was monitored until growth of bacteria reached stationary phase, and the number of CFU at each time point was determined by serial dilution. (B) Optical density of the RT and KO strains in the presence (solid lines) or absence (dashed lines) of catalase (cat) after cells reached stationary phase. (C) Cell viability of RT and KO strains in the presence (solid lines) or absence (dashed lines) of catalase after cells reached stationary phase.

FIG 4

Sensitivity of the dpr deletion mutant to hydrogen peroxide, pH, and temperature stress conditions. (A) Bacteria were treated with different concentrations of H₂O₂ for 30 min. (B) Bacteria were treated at different pHs (pH 4 for 2 h or pH 11 for 75 min), subjected to elevated temperature (42°C for 2 h), or treated with 10 mM H₂O₂ for 2 h. Numbers of CFU were determined and normalized to the starting number of CFU. Open bar, KO strain; gray bar, RT strain. Results are from one experiment that is representative of at least three independent experiments.

FIG 5

Growth of the RT (A) and KO (B) strains in the presence of ferrous chloride. Bacteria were grown to mid-log phase and diluted in THY containing different concentrations of ferrous chloride. Background absorbance caused by the addition of ferrous chloride was subtracted.

FIG 6

Compared to the RT strain, the dpr mutant is more susceptible to killing by macrophages but not neutrophils. (A) A neutrophil surface killing assay was performed using a range of cell-to-bacterium ratios. Percentages of surviving bacteria of the RT and KO strains are shown. (B) The RT and KO strains were incubated with murine macrophages at an MOI of 20 for 30 min. acrophages were washed with fresh medium, and numbers of total adherent plus intracellular bacteria were determined by plating cell lysates. (C) Surviving intracellular bacteria were counted after gentamicin treatment to kill extracellular bacteria, sequential washes, and plating of cell lysates.

FIG 7

The ability of the dpr mutant to colonize the nasopharynx is severely impaired. (A) Groups of 5 or 10 mice were challenged intranasally with either the RT or KO strain, and their nasal colonization densities were determined at different time points. Colonization densities of the RT and KO strains at each time point were compared by the Mann-Whitney U test. (B) Competition between RT and KO strain in mice. Mice were intranasally challenged with 10⁷ CFU of a 1:1 ratio of RT and KO strains, and samples were taken at days 1, 3, and 7. Total numbers of CFU were determined by growth on BAP. The absolute quantity of strain-specific genomic DNA in a sample was determined using strain-specific primer sets and then used to calculate the ratio of the two strains in the NP. No KO strain was detected at any time point.