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, 190 (1), 221-30

Growth of Streptococcus Pneumoniae on Human Glycoconjugates Is Dependent Upon the Sequential Activity of Bacterial Exoglycosidases

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Growth of Streptococcus Pneumoniae on Human Glycoconjugates Is Dependent Upon the Sequential Activity of Bacterial Exoglycosidases

Amanda M Burnaugh et al. J Bacteriol.

Abstract

In the human host, Streptococcus pneumoniae encounters a variety of glycoconjugates, including mucin, host defense molecules, and glycans associated with the epithelial surface. S. pneumoniae is known to encode a number of glycosidases that may modify these glycoconjugates in vivo. Three exoglycosidases, a neuraminidase (NanA), beta-galactosidase (BgaA), and N-acetylglucosaminidase (StrH), have been previously demonstrated to sequentially deglycosylate N-linked glycans on host defense molecules, which coat the pneumococcal surface in vivo. This cleavage is proposed to alter the clearance function of these molecules, allowing pneumococci to persist in the airway. However, we propose that the exoglycosidase-dependent liberation of monosaccharides from these glycoconjugates in close proximity to the pneumococcal surface provides S. pneumoniae with a convenient source of fermentable carbohydrate in vivo. In this study, we demonstrate that S. pneumoniae is able to utilize complex N-linked human glycoconjugates as a sole source of carbon to sustain growth and that efficient growth is dependent upon the sequential deglycosylation of the glycoconjugate substrate by pneumococcal exoglycosidases. In addition to demonstrating a role for NanA, BgaA, and StrH, we have identified a function for the second pneumococcal neuraminidase, NanB, in the deglycosylation of host glycoconjugates and have demonstrated that NanB activity can partially compensate for the loss or dysfunction of NanA. To date, all known functions of pneumococcal neuraminidase have been attributed to NanA. Thus, this study describes the first proposed role for NanB by which it may contribute to S. pneumoniae colonization and pathogenesis.

Figures

FIG. 1.
FIG. 1.
Demonstration that S. pneumoniae can utilize human AGP to sustain growth, an ability that is hypothesized to be dependent upon the cleavage and utilization of the complex N-linked glycan. (A) A schematic representation of the triantennary N-linked glycan structure of human AGP is shown: this antennary structure comprises approximately 38% of all glycans bound to AGP, with bi- and tetra-antennary chains accounting for 62% (38). Sugar residues are labeled above their corresponding symbols, and arrows indicate cleavage sites for pneumococcal neuraminidase (NanA), β-galactosidase (BgaA), and N-acetylglucosaminidase (StrH). Lines indicate linkages between sugar residues, and linkages are specified by characters found either above or below a given line. This N-linked glycan structure is commonly found in the human airway and has been identified on host defense molecules, which coat the pneumococcal surface in vivo (19). (B) Growth of S. pneumoniae strain 6A-T on semidefined C+Y medium supplemented with either 10 mM lactose and 10 mM sucrose, 5 mg of human AGP/ml, or no sugar as described in Materials and Methods. OD600 values are the means from three independent experiments each run in triplicate; 95% confidence intervals are represented by gray shading around each growth curve, indicating statistically significant differences in pneumococcal growth exhibited in each of the three media.
FIG. 2.
FIG. 2.
Demonstration that the growth of S. pneumoniae on human AGP is dependent upon exoglycosidase activity and that the extent of growth correlates to the ability of the strain to deglycosylate AGP. (A) Growth of wild-type and exoglycosidase mutants of S. pneumoniae strain 6A-T on semidefined C+Y medium supplemented with 5 mg of human AGP/ml. OD600 data for 6A-T on medium supplemented with either 10 mM lactose and 10 mM sucrose or no sugar are provided for reference. Growth was analyzed as described in the legend to Fig. 1. (B) Western blotting for human AGP. Samples were harvested from 96-well plates after a 30-h growth assay, electrophoresed on a 12.5% SDS-PAGE gel, and transferred onto an Immobilon-P membrane, and AGP was detected with monoclonal mouse anti-human AGP (primary antibody) and AP-conjugated donkey anti-mouse IgG (secondary antibody).
FIG. 3.
FIG. 3.
Demonstration that 6A-TΔnanA desialylates human AGP in a NanA-independent manner. Following the growth of bacteria on AGP for 30 h, 0.2-μg samples of AGP were electrophoresed on 12.5% SDS-PAGE gel and transferred onto an Immobilon-P membrane. SNA (A), MAA (B), and GNA (C) lectins were used to detect α2,6-linked sialic acid, α2,3-linked sialic acid, and terminal mannose, respectively.
FIG. 4.
FIG. 4.
Both NanA and NanB contribute to the growth of S. pneumoniae on human glycoconjugates. (A) Growth of wild-type 6A-T, 6A-TΔnanA, 6A-TΔnanB, and 6A-TΔnanAΔnanB on semidefined C+Y medium supplemented with 5 mg of AGP/ml. Growth curves of 6A-T on medium supplemented with either 10 mM lactose and 10 mM sucrose or no sugar are provided for reference. Growth was analyzed as described in the legend to Fig. 1. (B) Growth of 6A-TΔnanAΔnanBΔbgaAΔstrH on lactose and sucrose, AGP, and no-sugar media. OD600 data are the means from three independent experiments each run in triplicate. For clarity, 95% confidence intervals have been omitted from the data demonstrating bacterial growth on lactose and sucrose as well as on no sugar. Following the growth of bacteria on AGP for 30 h at 37°C, 0.2-μg samples of AGP were electrophoresed on 12.5% SDS-PAGE gel and transferred onto Immobilon-P membranes. (C) Western blotting for human AGP using monoclonal mouse anti-human AGP (primary antibody) and AP-conjugated donkey anti-mouse IgG (secondary antibody) for detection. (D) 6A-TΔnanAΔnanB does not desialylate AGP and is unable to expose detectable amounts of mannose. SNA (i) and GNA (ii) lectins were used to detect α2,6-linked sialic acid and terminal mannose, respectively.
FIG. 5.
FIG. 5.
Demonstration that both NanA and NanB but not NanC contribute significantly to the growth of TIGR4 on human AGP. (A) Growth of wild-type S. pneumoniae strain TIGR4, as well as single and double neuraminidase mutants, on semidefined C+Y medium supplemented with 5 mg of human AGP/ml. OD600 data for TIGR4 on medium supplemented with either 10 mM lactose and 10 mM sucrose or no sugar are provided for reference. OD600 data are representative of three independent experiments each run in triplicate. (B) Growth of TIGR4ΔnanAΔnanBΔbgaAΔstrH on lactose and sucrose, AGP, and no-sugar media. OD600 data are the means from three independent experiments each run in triplicate. For clarity, 95% confidence intervals have been omitted from the data demonstrating bacterial growth on lactose and sucrose as well as on no sugar.

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