Carbonic anhydrase is essential for Streptococcus pneumoniae growth in environmental ambient air

Abstract

The respiratory tract pathogen Streptococcus pneumoniae needs to adapt to the different levels of carbon dioxide (CO(2)) it encounters during transmission, colonization, and infection. Since CO(2) is important for various cellular processes, factors that allow optimal CO(2) sequestering are likely to be important for pneumococcal growth and survival. In this study, we showed that the putative pneumococcal carbonic anhydrase (PCA) is essential for in vitro growth of S. pneumoniae under the CO(2)-poor conditions found in environmental ambient air. Enzymatic analysis showed that PCA catalyzes the reversible hydration of CO(2) to bicarbonate (HCO(3)(-)), an essential step to prevent the cellular release of CO(2). The addition of unsaturated fatty acids (UFAs) reversed the CO(2)-dependent in vitro growth inhibition of S. pneumoniae strains lacking the pca gene (Deltapca), indicating that PCA-mediated CO(2) fixation is at least associated with HCO(3)(-)-dependent de novo biosynthesis of UFAs. Besides being necessary for growth in environmental ambient conditions, PCA-mediated CO(2) fixation pathways appear to be required for intracellular survival in host cells. This effect was especially pronounced during invasion of human brain microvascular endothelial cells (HBMEC) and uptake by murine J774 macrophage cells but not during interaction of S. pneumoniae with Detroit 562 pharyngeal epithelial cells. Finally, the highly conserved pca gene was found to be invariably present in both CO(2)-independent and naturally circulating CO(2)-dependent strains, suggesting a conserved essential role for PCA and PCA-mediated CO(2) fixation pathways for pneumococcal growth and survival.

FIG. 1.

Disruption of the pca gene in S. pneumoniae leads to CO₂-dependent growth inhibition. (A) Growth characteristics of the S. pneumoniae R6, D39, and TIGR4 wild-type and Δpca strains in CO₂-rich and CO₂-poor GM17 broth medium. (B) Growth of the S. pneumoniae R6bga::nisRK Δpca strain harboring either pNG8048E (empty vector), pUO1 (ecca), or pUO3 (pca) in CO₂-poor GM17 broth medium without (−nis) and with (+nis) 20 ng/ml nisin. Growth of the pneumococcal cultures was monitored by recording the OD₆₂₀. All curves in the graph present the averages of the results of three independent growth experiments.

FIG. 2.

Enzymatic activity and inhibition studies of recombinant GST-PCA. (A) The CA activity of GST-PCA (100 μg/ml) was measured by the changing-pH/dye indicator method at pH 7.5 and pH 8.4. (B) Inhibitory effect of the sulfonamides AZA (100 μM) and EZA (100 μM) on the CA activity of GST-PCA at pH 8.4. Under all conditions tested, hCAII (0.5 μg/ml) and nonenzymatic reactions were included as positive and negative controls, respectively. The curves for the nonenzymatic control reactions of the inhibition study overlapped with the curves for hCAII with an inhibitor and for the clarity of the graph were not displayed. All curves in the graphs present the averages of the results of three independent CA activity assays. Abs., absorbance.

FIG. 3.

Bicarbonate and oleic acid revert the CO₂ dependence of Δpca strains. (A) Growth of the S. pneumoniae TIGR4Δpca strain in CO₂-poor GM17 broth medium supplemented with NaHCO₃ (10 mM), adenine (200 μg/ml), uracil (200 μg/ml), arginine (200 μg/ml), aspartic acid (200 μg/ml), palmitic acid (0.01 mM in 0.1% Tween 40), or oleic acid (0.01 mM in 0.1% Tween 40). (B) Growth of the S. pneumoniae TIGR4Δpca strain in CO₂-poor GM17 broth medium with 0.1% Tween 20, Tween 40, or Tween 80. The growth of all pneumococcal broth cultures was monitored by recording the OD₆₂₀. All curves in the graph present the averages of the results of three independent growth experiments.

FIG. 4.

Scavengers for endogenous ROS delay the CO₂-dependent growth defect of Δpca strains. (A) Growth of the the R6 and TIGR4 S. pneumoniae Δpca strains in CO₂-poor GM17 broth medium without (−sal) or with 5 mM (+sal) sodium salicylate. (B) Growth of the S. pneumoniae R6 and TIGR4Δpca and ΔpcaΔspxB strains in CO₂-poor GM17 broth medium without (−cat) or with (+cat) 10,000 U/ml of catalase. The growth of all pneumococcal broth cultures was monitored by recording the OD₆₂₀. All curves in the graphs present the results of a single experiment that are characteristic of those for three independent growth experiments.

FIG. 5.

PCA is required for invasion and intracellular survival in host cells. (A and B) In vitro adherence of the TIGR4ΔcpsΔpca strain to Detroit 562 cells and HBMECs (A) and binding of the D39ΔcpsΔpca strain by J774 cells (B). The relative adherence and binding efficiencies were correlated to those of the TIGR4Δcps and D39Δcps strains, respectively. (C and D) Invasive properties of the TIGR4ΔcpsΔpca strain toward Detroit 562 cells and HBMECs (C) and uptake of the D39ΔcpsΔpca strain by J774 cells (D). The relative invasion and uptake efficiencies were correlated to the number of viable intracellular cells of the TIGR4Δcps and D39Δcps strains, respectively. (E and F) Intracellular survival kinetics of the TIGR4Δcps and TIGR4ΔcpsΔpca strains in Detroit 562 cells (E) and HBMECs (F). (G) Phagocytic killing of the D39Δcps and D39ΔcpsΔpca strains in J774 cells. Intracellular survival and phagocytic killing were correlated to viable-bacteria counts at time zero. *, statistically significant differences (P < 0.05).