Streptococcus pneumoniae can utilize multiple sources of hyaluronic acid for growth.

The mechanisms by which Streptococcus pneumoniae obtains carbohydrates for growth during airway colonization remain to be elucidated. The low concentration of free carbohydrates in the normal human airway suggests that pneumococci must utilize complex glycan structures for growth. The glycosaminoglycan hyaluronic acid is present on the apical surface of airway epithelial cells. As pneumococci express a hyaluronate lyase (Hyl) that cleaves hyaluronic acid into disaccharides, we hypothesized that during colonization pneumococci utilize the released carbohydrates for growth. Hyaluronic acid supported significant pneumococcal growth in an hyl-dependent manner. A phosphoenolpyruvate-dependent phosphotransferase system (PTS) and an unsaturated glucuronyl hydrolase (Ugl) encoded downstream of hyl are also essential for growth on hyaluronic acid. This genomic arrangement is present in several other organisms, suggesting conservation of the utilization mechanism between species. In vivo experiments support the hypothesis that S. pneumoniae utilizes hyaluronic acid as a carbon source during colonization. We also demonstrate that pneumococci can utilize the hyaluronic acid capsule of other bacterial species for growth, suggesting an alternative carbohydrate source for pneumococcal growth. Together, these data support a novel function for pneumococcal degradation of hyaluronic acid in vivo and provide mechanistic details of growth on this glycosaminoglycan.

Identification of an ATPase, MsmK, which energizes multiple carbohydrate ABC transporters in Streptococcus pneumoniae.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia and results in over 1 million deaths each year worldwide. Asymptomatic colonization of the airway precedes disease, and acquisition of carbohydrates from the host environment is necessary for bacterial survival. We previously demonstrated that S. pneumoniae cleaves sialic acid from human glycoconjugates to be used as a carbohydrate source. The satABC genes are required for growth and import of sialic acid. The satABC genes are predicted to encode components of an ABC transporter but not the ATPases essential to energize transport. As this subunit is essential, an ATPase must be encoded elsewhere in the genome. We identified msmK as a candidate based on similarity to other known carbohydrate ATPases. Recombinant MsmK hydrolyzed ATP, revealing that MsmK is an ATPase. An msmK mutant was reduced in growth on and transport of sialic acid, demonstrating that MsmK is the ATPase energizing the sialic acid transporter. In addition to satABC, S. pneumoniae contains five other loci that are predicted to encode CUT1 family carbohydrate ABC transporter components; each of these lacks a predicted ATPase. Data indicate that msmK is also required for growth on raffinose and maltotetraose, which are the substrates of two other characterized carbohydrate ABC transporters. Furthermore, an msmK mutant was reduced in airway colonization. Together, these data imply that in vivo, MsmK energizes multiple carbohydrate transporters in S. pneumoniae. This is the first demonstration of a shared ATPase in a pathogenic bacterium.

Sialic acid transport contributes to pneumococcal colonization.

Streptococcus pneumoniae is a major cause of pneumonia and meningitis. Airway colonization is a necessary precursor to disease, but little is known about how the bacteria establish and maintain colonization. Carbohydrates are required as a carbon source for pneumococcal growth and, therefore, for colonization. Free carbohydrates are not readily available in the naso-oropharynx; however, N- and O-linked glycans are common in the airway. Sialic acid is the most common terminal modification on N- and O-linked glycans and is likely encountered frequently by S. pneumoniae in the airway. Here we demonstrate that sialic acid supports pneumococcal growth when provided as a sole carbon source. Growth on sialic acid requires import into the bacterium. Three genetic regions have been proposed to encode pneumococcal sialic acid transporters: one sodium solute symporter and two ATP binding cassette (ABC) transporters. Data demonstrate that one of these, satABC, is required for transport of sialic acid. A satABC mutant displayed significantly reduced growth on both sialic acid and the human glycoprotein alpha-1. The importance of satABC for growth on human glycoprotein suggests that sialic acid transport may be important in vivo. Indeed, the satABC mutant was significantly reduced in colonization of the murine upper respiratory tract. This work demonstrates that S. pneumoniae is able to use sialic acid as a sole carbon source and that utilization of sialic acid is likely important during pneumococcal colonization.

Identification of a pneumococcal glycosidase that modifies O-linked glycans.

Colonization of the airway by Streptococcus pneumoniae is typically asymptomatic; however, progression of bacteria beyond the oronasopharynx can cause diseases including otitis media and pneumonia. The mechanisms by which S. pneumoniae establishes and maintains colonization remain poorly understood. Both N-linked and O-linked glycans are abundant in the airway. Our previous research demonstrated that S. pneumoniae can sequentially deglycosylate N-linked glycans and suggested that this modification of sugar structures may aid in colonization. There is published evidence that S. pneumoniae expresses a secreted O-glycosidase that cleaves galactose beta1-3 N-acetylgalactosamine (Galbeta1-3GalNAc) from core-1 O-linked glycans; however, the biological function of this enzyme has not previously been determined. We established that the activity is not secreted but is instead surface associated in a sortase-dependent manner. Genome analysis revealed an open reading frame predicted to encode a sortase-dependent surface protein with sequence similarity to the O-glycosidase of Bifidobacterium longum. Deletion of this pneumococcal open reading frame confirmed that this gene encodes an O-glycosidase. Experiments using a model glycoconjugate demonstrated that this O-glycosidase, together with the neuraminidase NanA, is required for S. pneumoniae to cleave sialylated core-1 O-linked glycans. The ability of the O-glycosidase mutant to cleave this glycan structure was restored by both genetic complementation and the addition of O-glycosidase. The mutant showed a reduction in adherence to human airway epithelial cells and a significantly decreased ability to colonize the upper respiratory tract, suggesting that cleavage of core-1 O-linked glycans enhances the ability of S. pneumoniae to colonize the human airway.