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...

Pyruvate oxidase is a key function in the metabolism and lifestyle of many lactic acid bacteria and its activity depends on the presence of environmental oxygen. In Streptococcus pneumoniae the protein has been suggested to play a major role in metabolism and has been implicated in virulence, oxidative stress survival and death in stationary phase. Under semi-aerobic conditions, transcriptomic and metabolite profiling analysis of a spxB mutant grown on glucose showed minor changes compared to the wild type, apart from the significant induction of two operons involved in carbohydrate uptake and processing. This induction leads to a change in the sugar utilization capabilities of the bacterium, as indicated by the analysis of the growth profiles of the D39 parent and spxB mutant on alternative carbohydrates. Metabolic analysis and growth experiments showed that inactivation of SpxB has no effect on the glucose fermentation pattern, except under aerobic conditions. More importantly, we show that mutation of spxB results in the production of increased amounts of capsule, the major virulence factor of S. pneumoniae. Part of this increase can be attributed to induction of capsule operon (cps) transcription. Therefore, we propose that S. pneumoniae utilizes pyruvate oxidase as an indirect sensor of the oxygenation of the environment, resulting in the adaption of its nutritional capability and the amount of capsule to survive in the host.

Streptococcus pneumoniae is one of the major causative agents of pneumonia, sepsis, meningitis and other morbidities. In spite of its heavy disease burden, surprisingly little is known about the mechanisms involved in the switch of life style, from commensal colonizer of the nasopharynx to invasive pathogen. In vitro experiments, and mouse models have shown that S. pneumoniae can be internalized by host cells, which coupled with intracellular vesicle transport through the cells, i.e. transcytosis, is suggested to be the first step of invasive disease. To further dissect the process of S. pneumoniae internalization, we chemically inhibited discrete parts of the cellular uptake system. We show that this invasion of the host cells was facilitated via both clathrin- and caveolae-mediated endocytosis. After internalization we demonstrated that the bulk of the internalized S. pneumoniae was killed in the lysosome. Interestingly, inhibition of the lysosome altered transcytosis dynamics as it resulted in an increase in the transport of the internalized bacteria out of the cells via the basal side. These results show that uptake of S. pneumoniae into host cells occurs via multiple pathways, as opposed to the often proposed view of invasion being dependent on specific, and singular receptor-mediated endocytosis. This indicates that the endothelium not only has a critical role as a physical barrier against S. pneumoniae in the blood stream, but also in degrading S. pneumonia cells that have adhered to, and invaded the endothelial cells.