Carbonic Anhydrase Is Essential forStreptococcus pneumoniaeGrowth 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 (⌬pca), 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.The Gram-positive bacterium Streptococcus pneumoniae, or pneumococcus, is a human respiratory tract pathogen that contributes significantly to global mortality and morbidity. In addition, it is an important asymptomatic colonizer of the human nasopharynx, with carriage rates around 10% in adults and over 40% in children (6). Pneumococcal colonization and infection are closely linked, but knowledge of the factors that contribute to transmission, carriage, disease, and transition from carriage to disease is still limited. Research on components that physically contribute to host-pathogen interactions, such as capsular polysaccharides, adhesins, and toxins, has provided valuable insights into the process of pneumococcal pathogenesis (20). In contrast, the influence of environmental factors on pneumococcal growth and survival remains fairly unexplored.S. pneumoniae needs to adapt to various aerobic and anaerobic conditions, reflecting the different niches it occupies during transmission, colonization, and invasive disease. During niche transition, oxygen (O 2 ) levels change considerably. Levels of O 2 are 21% in ambient air, decrease to 10 to 15% in the alveoli of the lungs, and are about 5% in resting cells. In O 2 -rich conditions, S. pneumoniae expresses pyruvate oxidase (SpxB), which generates acetyl-phosphate as a source of ATP and hydrogen peroxide (H 2 O 2 ) fo...

Nontypeable Haemophilus influenzae Carbonic Anhydrase Is Important for Environmental and Intracellular Survival

“…To link the growth defect of the NTHi ⌬can strain with one or more of these biosynthetic pathways, we complemented NTHi ⌬can cultures with various metabolic intermediates. In contrast to our previous findings for S. pneumoniae carbonic anhydrase mutants (25), addition of different sources of saturated and unsaturated fatty acids (i.e., Tween 20, Tween 40, or Tween 80, oleic acid, or palmitic acid) did not complement the growth deficiency of the Rd ⌬can strain under CO 2 -poor conditions ( Fig. 3A and B).…”

“…Despite this knowledge, the biological role of HICA has not been studied so far. Our experiments clearly reveal an essential role for HICA in NTHi growth and survival under CO 2 -poor conditions, which is in line with the function of carbonic anhydrase in environmental survival of various other microorganisms (24,25,(38)(39)(40)(41)(42)(43). Carbonic anhydrases equilibrate intracellular levels of CO 2 and HCO 3 Ϫ , and in a CO 2 -poor environment this activity prevents the passive diffusion of endogenous CO 2 from cells to meet metabolic needs.…”

“…Bacterial virulence and CO 2 are linked in various bacterial species although the mechanisms are often unclear. Previously, we showed decreased intracellular survival of S. pneumoniae in the mouse monocyte cell line J774 (25). Therefore, we assessed the ability of the NTHi 86-028NP wild-type and ⌬can strains to adhere, invade, and survive in the human monocyte cell line THP-1.…”

“…Carbonic anhydrases are enzymes that catalyze the reversible hydration of CO 2 to bicarbonate (CO 2 ϩ H 2 O ↔ HCO 3 Ϫ ϩ H ϩ ) and are therefore often linked to HCO 3 Ϫ -dependent biosynthesis pathways for nucleic acids, several amino acids, and fatty acids (24,25). To link the growth defect of the NTHi ⌬can strain with one or more of these biosynthetic pathways, we complemented NTHi ⌬can cultures with various metabolic intermediates.…”

Growth Phase-Dependent Modulation of Rgg Binding Specificity in Streptococcus pyogenes

“…Rgg also bound to the noncoding DNA upstream of a gene encoding carbonic anhydrase (Table 3), which catalyzes the reversible hydration of CO 2 to bicarbonate (HCO 3 Ϫ ). Carbonic anhydrase is essential for the growth and survival of Streptococcus pneumoniae in an ambient air environment (10). A previous transcriptome analysis also showed changes in carbonic anhydrase transcripts between the wild type and the rgg mutant, suggesting that Rgg directly regulates its expression (26).…”