Control of inflammation is critical for therapy of infectious diseases. Pathogen-associated and/or danger-associated molecular patterns (PAMPs and DAMPs, respectively) are the two major inducers of inflammation. Because the CD24-Siglec G/10 interactions selectively repress inflammatory response to DAMPs, microbial disruption of the negative regulation would provide a general mechanism to exacerbate inflammation. Here we show that the sialic acid-based pattern recognitions of CD24 by Siglec G/10 are targeted by sialidases in polybacterial sepsis. Sialidase inhibitors protect mice against sepsis by a CD24-Siglecg-dependent mechanism, whereas a targeted mutation of either CD24 or Siglecg exacerbates sepsis. Bacterial sialidase and host CD24 and Siglecg genes interact to determine pathogen virulence. Our data demonstrate a critical role for disrupting sialic acid-based pattern recognitions in microbial virulence and suggest a therapeutic approach to dampen harmful inflammatory response during infection.

The carbohydrate-rich coating of human tissues and cells provide a first point of contact for colonizing and invading bacteria. Commensurate with N-glycosylation being an abundant form of protein glycosylation that has critical functional roles in the host, some host-adapted bacteria possess the machinery to process N-linked glycans. The human pathogen Streptococcus pneumoniae depolymerizes complex N-glycans with enzymes that sequentially trim a complex N-glycan down to the Man3GlcNAc2 core prior to the release of the glycan from the protein by endo-β-N-acetylglucosaminidase (EndoD), which cleaves between the two GlcNAc residues. Here we examine the capacity of S. pneumoniae to process high-mannose N-glycans and transport the products. Through biochemical and structural analyses we demonstrate that S. pneumoniae also possesses an α-(1,2)-mannosidase (SpGH92). This enzyme has the ability to trim the terminal α-(1,2)-linked mannose residues of high-mannose N-glycans to generate Man5GlcNAc2. Through this activity SpGH92 is able to produce a substrate for EndoD, which is not active on high-mannose glycans with α-(1,2)-linked mannose residues. Binding studies and X-ray crystallography show that NgtS, the solute binding protein of an ABC transporter (ABCNG), is able to bind Man5GlcNAc, a product of EndoD activity, with high affinity. Finally, we evaluated the contribution of EndoD and ABCNG to growth of S. pneumoniae on a model N-glycosylated glycoprotein, and the contribution of these enzymes and SpGH92 to virulence in a mouse model. We found that both EndoD and ABCNG contribute to growth of S. pneumoniae, but that only SpGH92 and EndoD contribute to virulence. Therefore, N-glycan processing, but not transport of the released glycan, is required for full virulence in S. pneumoniae. To conclude, we synthesize our findings into a model of N-glycan processing by S. pneumoniae in which both complex and high-mannose N-glycans are targeted, and in which the two arms of this degradation pathway converge at ABCNG.

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.

ABSTRACTThe mechanisms by whichStreptococcus pneumoniaeobtains 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 anhyl-dependent manner. A phosphoenolpyruvate-dependent phosphotransferase system (PTS) and an unsaturated glucuronyl hydrolase (Ugl) encoded downstream ofhylare 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 vivoexperiments support the hypothesis thatS. pneumoniaeutilizes 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 acidin vivoand provide mechanistic details of growth on this glycosaminoglycan.

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.