Pneumococcal neuraminidase is a key enzyme for sequential deglycosylation of host glycans, and plays an important role in host survival, colonization, and pathogenesis of infections caused by Streptococcus pneumoniae. One of the factors that can affect the activity of neuraminidase is the amount and position of acetylation present in its substrate sialic acid. We hypothesised that pneumococcal esterases potentiate neuraminidase activity by removing acetylation from sialic acid, and that will have a major effect on pneumococcal survival on mucin, colonization, and virulence. These hypotheses were tested using isogenic mutants and recombinant esterases in microbiological, biochemical and in vivo assays. We found that pneumococcal esterase activity is encoded by at least four genes, SPD_0534 (EstA) was found to be responsible for the main esterase activity, and the pneumococcal esterases are specific for short acyl chains. Assay of esterase activity by using natural substrates showed that both the Axe and EstA esterases could use acetylated xylan and Bovine Sub-maxillary Mucin (BSM), a highly acetylated substrate, but only EstA was active against tributyrin (triglyceride). Incubation of BSM with either Axe or EstA led to the acetate release in a time and concentration dependent manner, and pre-treatment of BSM with either enzyme increased sialic acid release on subsequent exposure to neuraminidase A. qRT-PCR results showed that the expression level of estA and axe increased when exposed to BSM and in respiratory tissues. Mutation of estA alone or in combination with nanA (codes for neuraminidase A), or the replacement of its putative serine active site to alanine, reduced the pneumococcal ability to utilise BSM as a sole carbon source, sialic acid release, colonization, and virulence in a mouse model of pneumococcal pneumonia.

Catabolism of galactose by Streptococcus pneumoniae alters the microbe’s metabolism from homolactic to mixed acid fermentation, and this shift is linked to the microbe’s virulence. However, the genetic basis of this switch is unknown. Pyruvate formate lyase (PFL) is a crucial enzyme for mixed acid fermentation. Functional PFL requires the activities of two enzymes: pyruvate formate lyase activating enzyme (coded by pflA) and pyruvate formate lyase (coded by pflB). To understand the genetic basis of mixed acid fermentation, transcriptional regulation of pflA and pflB was studied. By microarray analysis of ΔpflB, differential regulation of several transcriptional regulators were identified, and CcpA, and GlnR’s role in active PFL synthesis was studied in detail as these regulators directly interact with the putative promoters of both pflA and pflB, their mutation attenuated pneumococcal growth, and their expression was induced on host-derived sugars, indicating that these regulators have a role in sugar metabolism, and multiple regulators are involved in active PFL synthesis. We also found that the influence of each regulator on pflA and pflB expression was distinct in terms of activation and repression, and environmental condition. These results show that active PFL synthesis is finely tuned, and feed-back inhibition and activation are involved.

For the generation of energy, the important human pathogen Streptococcus pneumoniae relies on host-derived sugars, including ␤-glucoside analogs. The catabolism of these nutrients involves the action of 6-phospho-␤-glucosidase to convert them into usable monosaccharaides. In this study, we characterized a 6-phospho-␤-glucosidase (BglA3) encoded by SPD_0247. We found that this enzyme has a cell membrane localization and is active only against a phosphorylated substrate. A mutated pneumococcal ⌬SPD0247 strain had reduced 6-phospho-glucosidase activity and was attenuated in growth on cellobiose and hyaluronic acid compared to the growth of wild-type D39. ⌬SPD0247-infected mice survived significantly longer than the wild-type-infected cohort, and the colony counts of the mutant were lower than those of the wild type in the lungs. The expression of SPD_0247 in S. pneumoniae harvested from infected tissues was significantly increased relative to its expression in vitro on glucose. Additionally, ⌬SPD0247 is severely impaired in its attachment to an abiotic surface. These results indicate the importance of ␤-glucoside metabolism in pneumococcal survival and virulence. Streptococcus pneumoniae is a frequent occupant of the human nasopharynx, where it resides without causing symptoms (1). Conversely, the bacterium also is a major human pathogen, being a leading cause of bacterial pneumonia, otitis media, meningitis, and septicemia (1). The increasing trend of antibiotic resistance and the shortcomings of existing vaccines mean that a better understanding of the pathogenesis of pneumococcal diseases is required.Almost one-third of the transporters in the pneumococcal genome are dedicated to sugars (2), stressing the important role that these carbon sources play in the ability of pneumococci to survive in the host. The pneumococcus has been shown to ferment 32 different sugars in vitro, including various hexoses, ␣-and ␤-galactosides, and glucosides, as well as polysaccharides (3, 4). However, the concentrations of readily available simple carbohydrates in the respiratory tract are low (5); thus, the pneumococcus relies on complex host glycans for growth in vivo (4). Central to this is the ability to sequentially deglycosylate host glycoproteins (6).Mammalian extracellular matrix is rich in glycosaminoglycans (GAGs; e.g., hyaluronic acid), which contain ␤-linked disaccharide repeating units (7,8). The degradation of GAGs leads to the generation of structural analogues of cellobiose or N,N=-diacetylchitobiose [(GlcNAc) 2 ] and other ␤-linked disaccharides (9). Disaccharides such as these can be transported into the bacterial cell through phosphoenolpyruvate-dependent phosphotransferase systems (PTS), which transport and phosphorylate sugars simultaneously (10). The phosphorylated disaccharides then are hydrolyzed by cytoplasmic phospho-␤-glucosidases (EC 3.2.1.86) or phospho-␤-galactosidases (EC 3.2.1.85) that usually do not have hydrolytic activity toward nonphosphorylated substrates. The resulting glucose and glucose 6-pho...