Deletion of a cation transporter promotes lysis in Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae is a significant human pathogen which causes respiratory and serious invasive diseases. Mg(2+) is essential for life, and its concentration varies throughout the human body. Magnesium uptake plays an important role in the virulence of many bacterial pathogens. To study the Mg(2+) uptake of S. pneumoniae strain D39, a mutant was generated in SPD1383, a P-type ATPase with homology to the Salmonella Mg(2+) transporter MgtA, which has also been shown to be a Ca(2+) exporter in strain TIGR4. Under low-Ca(2+) conditions, mutation led to a growth defect in complex medium and the gene was nearly essential for growth under low-Mg(2+) conditions. Addition of Mg(2+) restored the normal growth of the mutant in all cases, but the addition of other divalent cations had no effect. Addition of Ca(2+), Mn(2+), and Zn(2+) in the presence of high Mg(2+) concentrations inhibited restoration of growth. The mutant was unable to proliferate in blood, which was also alleviated by the addition of Mg(2+). The protein was located in the membrane and produced in various S. pneumoniae strains and pathogenic streptococcal species. Surprisingly, mutation of the gene led to an elevated toxicity for endothelial cells. This was caused by an increased amount of pneumolysin in the medium, mediated by elevated lysis of the mutant. Thus, in this study, we uncovered a role for SPD1383 in Mg(2+) uptake and hypothesize that the protein is a Mg(2+/)Ca(2+) antiporter. Furthermore, a disturbance in Mg(2+) homeostasis seems to promote lysis of S. pneumoniae.

Fig. 1.

MgtA is the main Mg²⁺ importer in S. pneumoniae under normal growth conditions and is specific for Mg²⁺. (A) Growth of R6 (black squares) in GM17 compared to growth of R6 ΔmgtA in GM17 (black circles). GM17 contains 5 mM MgCl₂ (white circles), 5 mM MgSO₄ (light-gray circles), 5 mM MnCl₂ (black triangles), and 5 mM CaCl₂ (gray triangles). Results are the means of results from duplicate experiments plus standard deviations (SD) and are representative of at least two other experiments. (B) The mutant is unable to grow in CDM; growth can be restored by Mg²⁺ but not by Ca²⁺. Growth of D39 in CDM (black squares) and of D39 ΔmgtA in CDM (black circles), in CDM plus 5 mM MgCl₂ (white circles), in CDM plus 5 mM CaCl₂ (black triangles), and in CDM plus 5 mM MgCl₂ and 5 mM CaCl₂ (white triangles). Results are the means ± SD of results from three experiments. (C) The mutant is unable to grow in CDM, which can be restored by heterologous expression of the mgtA gene. The growth of the following strains in CDM containing 2 ng/ml desalted nisin was measured: D39 nisRK (black squares), D39 nisRK ΔmgtA (white circles) D39 nisRK ΔmgtA(pNZ8048) (gray circles), and D39 nisRK ΔmgtA(pNZmgtA) (black circles). Results are the means ± SD of results from three experiments. (D) The growth of the mutant in CDM with 10 mM MgCl₂ is inhibited by the addition of Mn²⁺ and Zn²⁺. Growth of D39 ΔmgtA in CDM with 0.5 μM MgCl₂ (dark-gray circles), 10 mM MgCl₂ (white circles), 10 mM MgCl₂ plus 2 mM MnSO₄ (white diamonds), 10 mM MgCl₂ plus 500 μM ZnCl₂ (black diamonds), 10 mM MgCl₂ plus 50 μM CoCl₂ (black squares), and 10 mM MgCl₂ plus 100 μM NiCl₂ (black triangle). Results are representative of three independent experiments.

Fig. 2.

The MgtA mutant is unable to proliferate in sheep blood, which is alleviated by the addition of Mg²⁺. Strains were inoculated into sheep blood, and samples were taken at various time points to determine the amount of bacteria. Growth of D39 (black diamonds) and D39 ΔmgtA (black circles) in blood and of D39 ΔmgtA in blood with 10 mM Mg²⁺ (white circles) and 10 mM Ca²⁺ (gray circles). *, below the detection limit of 1.7 E01 CFU ml⁻¹. Results shown are the means of results from two experiments + SD and are representative of another 4 independent experiments.

Fig. 3.

MgtA is a membrane protein, part of the core genome of S. pneumoniae, and conserved in pathogenic Streptococcus species. (A) Generation of polyclonal antibodies against MgtA. Western blot analysis of total protein samples of D39 and D39 ΔmgtA with antiserum raised in rabbits inoculated with purified MgtA-His protein. A specific band of around 100 kDa is present in the wild type (wt) but not in the mutant (ΔmgtA). (B) MgtA is a membrane protein. Western blot analysis using the anti-MgtA serum of the supernatant (sup), cytoplasm (cyto), and membrane (memb) fractions of strain D39 grown in GM17. A specific band of around 100 kDa is present in the membrane fraction. (C) MgtA is expressed in various S. pneumoniae strains. Western blot analysis using the anti-MgtA antiserum of total protein samples of strains D39, G54, 23F, 670-6B, and TIGR4. (D) Guide tree of Clustal W analysis of the amino acid sequences of MgtA homologues from sequenced S. pneumoniae strains and other (pathogenic) streptococcal species. (E) MgtA key epitopes are conserved, and the protein seems to be expressed in various pathogenic streptococcal species. Western blot analysis using the anti-MgtA antiserum of total protein of strain D39, two Streptococcus mitis strains, one Streptococcus sanguis strain, three S. pyogenes strains, and three Streptococcus agalactiae clinical isolates. (F) Production of MgtA in D39 grown in CDM and GM17 with the addition of various concentrations of Mg²⁺ and Ca²⁺. −, no addition; +Mg, with 10 mM MgCl₂ added; +Ca, with 5 mM CaCl₂ added.

Fig. 4.

Deletion of mgtA leads to an increase in pneumolysin release into the environment. (A) The mgtA mutant has an increased toxicity to HBMEC. Confluent HBMEC layers were incubated with approximately 1 × 10⁷ CFU of D39, D39 ΔmgtA, and D39 ΔmgtA Δply. The pictures are taken after 2 h of incubation; at the start of the experiment, the cells in each well looked similar (data not shown). Incubation with D39 ΔmgtA led to increased toxicity compared to that of the wild type, as is evidenced by the shrinking and rounding of the HBMEC and the disruption of the monolayer. This was not observed in the D39 ΔmgtA Δply mutant. (B) Increased pneumolysin release in the medium by the mutant. The D39 (W) and D39 ΔmgtA (M) strains were grown in GM17 and supernatant (sup), and cells were harvested at the exponential and stationary phases. Samples were subjected to Western blot analysis using anti-Ply and a cross-reactive S. aureus anti-TrxA antiserum, and signals on the Western blot were quantified using ImageJ.