Dataset Information

Molecular Characterization of N-glycan Degradation and Transport in Streptococcus pneumoniae and Its Contribution to Virulence.

ABSTRACT: 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-?-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 ?-(1,2)-mannosidase (SpGH92). This enzyme has the ability to trim the terminal ?-(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 ?-(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.

SUBMITTER: Robb M

PROVIDER: S-EPMC5215778 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Publications

Molecular Characterization of N-glycan Degradation and Transport in Streptococcus pneumoniae and Its Contribution to Virulence.

Robb Melissa M Hobbs Joanne K JK Woodiga Shireen A SA Shapiro-Ward Sarah S Suits Michael D L MD McGregor Nicholas N Brumer Harry H Yesilkaya Hasan H King Samantha J SJ Boraston Alisdair B AB

PLoS pathogens 20170105 1

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 prio ...[more]

PMID: 28056108

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Project description:The opportunistic pathogen Streptococcus pneumoniae (pneumococcus) is a human nasopharyngeal commensal, and host N-glycan metabolism promotes its colonization and invasion. It has been reported that glucose represses, while fetuin, a glycoconjugated model protein, induces, the genes involved in N-glycan degradation through the two-component system TCS07. However, the mechanisms of glucose repression and TCS07 induction remain unknown. Previously, we found that the pneumococcal aquaglyceroporin Pn-AqpC facilitates oxygen uptake, thereby contributing to the antioxidant potential and virulence. In this study, through Tandem Mass Tag (TMT) quantitative proteomics, we found that the deletion of Pn-aqpC caused a marked upregulation of 11 proteins involved in N-glycan degradation in glucose-grown pneumococcus R6. Both quantitative RT-PCR and GFP fluorescence reporters revealed that the upregulation of N-glycan genes was completely dependent on response regulator (RR) 07, but not on the histidine kinase HK07 of TCS07 or the phosphoryl-receiving aspartate residue of RR07 in ΔPn-aqpC, indicating that RR07 was activated in an HK07-independent manner when Pn-AqpC was absent. The deletion of Pn-aqpC also enhanced the expression of pyruvate formate lyase and increased formate production, probably due to reduced cellular oxygen content, indicating that a shunt of glucose catabolism to mixed acid fermentation occurs. Notably, formate induced the N-glycan degradation genes in glucose-grown R6, but the deletion of rr07 abolished this induction, indicating that formate activates RR07. However, the induction of N-glycan degradation proteins reduced the intraspecies competition of R6 in glucose. Therefore, although N-glycan degradation promotes pneumococcal pathogenesis, the glucose metabolites-based RR07 regulation reported here is of importance for balancing growth fitness and the pathogenicity of pneumococcus. IMPORTANCE Pneumococcus, a human opportunistic pathogen, is capable of metabolizing host complex N-glycans. N-glycan degradation promotes pneumococcus colonization in the nasopharynx as well as invasion into deeper tissues, thus significantly contributing to pathogenesis. It is known that the two-component system 07 induces the N-glycan metabolizing genes; however, how TCS07 is activated remains unknown. This study reveals that formate, the anaerobic fermentation metabolite of pneumococcus, is a novel activator of the response regulator (RR) 07. Although the high expression of N-glycan degradation genes promotes pneumococcal colonization in the nasopharynx and pathogenesis, this reduces pneumococcal growth fitness in glucose as indicated in this work. Notably, the presence of Pn-AqpC, an oxygen-transporting aquaglyceroporin, enables pneumococcus to maintain glucose homolactic acid fermentation, thus reducing formate production, maintaining RR07 inactivation, and controlling N-glycan degrading genes at a non-induced status. Thus, this study highlights a novel fermentation metabolism pattern linking TCS-regulated carbohydrate utilization strategies as a trade-off between the fitness and the pathogenicity of pneumococcus.

Dataset Information

Molecular Characterization of N-glycan Degradation and Transport in Streptococcus pneumoniae and Its Contribution to Virulence.

Publications

Molecular Characterization of N-glycan Degradation and Transport in Streptococcus pneumoniae and Its Contribution to Virulence.

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