High-Throughput Mutagenesis and Cross-Complementation Experiments Reveal Substrate Preference and Critical Residues of the Capsule Transporters in
            <i>Streptococcus pneumoniae</i>

Chua, Wan-Zhen; Maiwald, Matthias; Chew, Kean Lee; Lin, Raymond Tzer‐Pin; Zheng, Sanduo; Sham, Lok‐To

doi:10.1128/mbio.02615-21

Cited by 11 publications

(12 citation statements)

References 67 publications

(109 reference statements)

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“…The strains used in this study are listed in SI Appendix , Table S1 . Clinical isolates of S. pneumoniae expressing 84 CPS types were collected from hospitals and research institutes ( 69 ). Unless otherwise specified, S. pneumoniae was grown in brain heart infusion broth (BHI; ThermoFisher Scientific CM1135B) or on tryptic soy agar-II plates supplemented with 5% (v/v) sheep blood (blood agar; Biomed Diagnostics, 221261) at 37 °C in 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

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Influence of glycan structure on the colonization of Streptococcus pneumoniae on human respiratory epithelial cells

Chun

Tan

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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Virtually all living cells are encased in glycans. They perform key cellular functions such as immunomodulation and cell–cell recognition. Yet, how their composition and configuration affect their functions remains enigmatic. Here, we constructed isogenic capsule-switch mutants harboring 84 types of capsular polysaccharides (CPSs) in Streptococcus pneumoniae . This collection enables us to systematically measure the affinity of structurally related CPSs to primary human nasal and bronchial epithelial cells. Contrary to the paradigm, the surface charge does not appreciably affect epithelial cell binding. Factors that affect adhesion to respiratory cells include the number of rhamnose residues and the presence of human-like glycomotifs in CPS. Besides, pneumococcal colonization stimulated the production of interleukin 6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and monocyte chemoattractantprotein-1 (MCP-1) in nasal epithelial cells, which also appears to be dependent on the serotype. Together, our results reveal glycomotifs of surface polysaccharides that are likely to be important for colonization and survival in the human airway.

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Section: Methodsmentioning

confidence: 99%

“…S. pneumoniae strains were constructed by transforming genomic DNA, isothermal assembly products, or PCR amplicons after inducing natural competence ( 69 , 70 ). Primers used for the synthesis of PCR fragments are tabulated in SI Appendix , Table S2 .…”

Section: Methodsmentioning

confidence: 99%

Influence of glycan structure on the colonization of Streptococcus pneumoniae on human respiratory epithelial cells

Chun

Tan

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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“…Provided that the GT subcomplex co-evolves with CpsA and CpsJ, and CpsH maintains its interaction with CpsC, the recruitment mechanism will remain functional. Consistently, the amino acid sequence of CpsJ strongly correlates with the cargo it transports 69 . This observation suggests the GTs and CpsJ may co-evolve because the CPS precursors likely track with the GTs that produce them.…”

Section: Discussionmentioning

confidence: 62%

The divisome but not the elongasome organizes capsule synthesis in Streptococcus pneumoniae

et al. 2023

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The bacterial cell envelope consists of multiple layers, including the peptidoglycan cell wall, one or two membranes, and often an external layer composed of capsular polysaccharides (CPS) or other components. How the synthesis of all these layers is precisely coordinated remains unclear. Here, we identify a mechanism that coordinates the synthesis of CPS and peptidoglycan in Streptococcus pneumoniae. We show that CPS synthesis initiates from the division septum and propagates along the long axis of the cell, organized by the tyrosine kinase system CpsCD. CpsC and the rest of the CPS synthesis complex are recruited to the septum by proteins associated with the divisome (a complex involved in septal peptidoglycan synthesis) but not the elongasome (involved in peripheral peptidoglycan synthesis). Assembly of the CPS complex starts with CpsCD, then CpsA and CpsH, the glycosyltransferases, and finally CpsJ. Remarkably, targeting CpsC to the cell pole is sufficient to reposition CPS synthesis, leading to diplococci that lack CPS at the septum. We propose that septal CPS synthesis is important for chain formation and complement evasion, thereby promoting bacterial survival inside the host.

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“…2, 6A, and 7). The primary structure of HM3343 strongly suggests that it functions as a flippase to translocate CPS precursors across the inner membrane (14,16). However, it is not clear how HM3344 and HM3345 are involved in CPS synthesis based on in silico analysis.…”

Section: Discussionmentioning

confidence: 99%

Capsular polysaccharide-mediated protein loading onto extracellular membrane vesicles of a fish intestinal bacterium,Shewanella vesiculosaHM13

Kamasaka

Kawamoto

Tsudzuki

et al. 2023

Preprint

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Bacterial extracellular membrane vesicles (EMVs) play various physiologically important roles mediated by cargo proteins. However, our understanding of the molecular mechanism underlying cargo loading onto EMVs is limited. In this study, we analyzed the mechanism of cargo protein loading onto EMVs from a fish intestinal Gram-negative bacterium,Shewanella vesiculosaHM13. This strain secretes EMVs carrying a major cargo protein, P49. Near the P49 gene, we found genes having homology to genes involved in protein secretion and surface polysaccharide-chain synthesis. Among them, the deletion of genes encoding homologs of a flippase involved in bacterial extracellular polysaccharide synthesis (HM3343), phosphoethanolamine transferase (HM3344), and glycerophosphodiester phosphodiesterase (HM3345) resulted in the loss of capsular polysaccharide (CPS) of EMVs. We conducted anin vitroP49 loading assay onto P49-free EMVs to examine whether P49 was loaded onto the EMVs via its interaction with the CPS of the EMVs. We found that purified P49 was loaded onto EMVs harboring CPSin vitro, whereas it was not loaded onto EMVs from the mutants lacking CPS production due to the loss of HM3343, HM3344, and HM3345. Transmission electron microscopy of EMVs loaded with P49in vitroandin vivoshowed spherical nanoparticles around the EMVs, whereas such particles were not observed for EMVs without loaded P49, implying that P49 constitutes those particles on the surface of EMVs. These results indicate that P49 is loaded onto EMVs via its interaction with the CPS of EMVs.

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High-Throughput Mutagenesis and Cross-Complementation Experiments Reveal Substrate Preference and Critical Residues of the Capsule Transporters in Streptococcus pneumoniae

Abstract: All licensed pneumococcal vaccines target the capsular polysaccharide (CPS). This layer is highly variable and is important for virulence in many bacterial pathogens.

Cited by 11 publications

References 67 publications

Influence of glycan structure on the colonization of Streptococcus pneumoniae on human respiratory epithelial cells

Influence of glycan structure on the colonization of Streptococcus pneumoniae on human respiratory epithelial cells

The divisome but not the elongasome organizes capsule synthesis in Streptococcus pneumoniae

Capsular polysaccharide-mediated protein loading onto extracellular membrane vesicles of a fish intestinal bacterium,Shewanella vesiculosaHM13

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