Exploitation of Capsule Polymerases for Enzymatic Synthesis of Polysaccharide Antigens Used in Glycoconjugate Vaccines

Litschko, Christa; Budde, Insa; Berger, Monika; Fiebig, Timm

doi:10.1007/978-1-0716-0795-4_16

Cited by 6 publications

(7 citation statements)

References 26 publications

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“…3 ), it was generated in situ by Bcs1 and RpiA. Interestingly, CDP-ribitol was only used up in the presence of PRPP, with concomitant production of CMP and large polymeric products detectable by Alcian blue-stained PA gel 18 (Fig. 1d,f , reaction 6).…”

Section: Resultsmentioning

confidence: 95%

“…2 ) as indicated in b . Polymers were visualized using Alcian blue 18 . Constructs are numbered, as shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Polymerases assembling a heteropolymer, in which monosaccharides are bridged by phosphodiester linkages 29 , 34 , belong to a protein family called stealth 35 . Polyol-phosphate-containing polymers are assembled by TagF-like enzymes 15 , 18 , 27 , 30 . Structural data for capsule polymerases embedded in group 2 systems are limited to the chondroitin polymerase K4CP from E. coli K4 (ref.…”

Section: Discussionmentioning

confidence: 99%

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A multi-enzyme machine polymerizes the Haemophilus influenzae type b capsule

et al. 2023

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Bacterial capsules have critical roles in host-pathogen interactions. They provide a protective envelope against host recognition, leading to immune evasion and bacterial survival. Here we define the capsule biosynthesis pathway of Haemophilus influenzae serotype b (Hib), a Gram-negative bacterium that causes severe infections in infants and children. Reconstitution of this pathway enabled the fermentation-free production of Hib vaccine antigens starting from widely available precursors and detailed characterization of the enzymatic machinery. The X-ray crystal structure of the capsule polymerase Bcs3 reveals a multi-enzyme machine adopting a basket-like shape that creates a protected environment for the synthesis of the complex Hib polymer. This architecture is commonly exploited for surface glycan synthesis by both Gram-negative and Gram-positive pathogens. Supported by biochemical studies and comprehensive 2D nuclear magnetic resonance, our data explain how the ribofuranosyltransferase CriT, the phosphatase CrpP, the ribitol-phosphate transferase CroT and a polymer-binding domain function as a unique multi-enzyme assembly.

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Section: Resultsmentioning

confidence: 95%

“…2 ) as indicated in b . Polymers were visualized using Alcian blue 18 . Constructs are numbered, as shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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A multi-enzyme machine polymerizes the Haemophilus influenzae type b capsule

et al. 2023

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“…We furthermore used electron microscopy (EM) to evaluate whether pleD * induction may alter the bacterial surface topography and thus contribute to the observed auto‐aggregation. We also separated secreted polysaccharides on a high percentage PAGE and performed a combined Alcian blue and silver staining (Litschko et al, 2021) in order to evaluate whether differences in exopolysaccharide production might account for the observed auto‐aggregative behavior of the pleD* expressing PAO1 strain or might contribute to the physical hindrance of motility. Both non‐specific methods, EM and PAGE, did not allow the identification of altered surface topologies (Figure S1) or differences in exopolysaccharide production in PAO1 harboring the pleD* construct (Figure S2) even when the strains were grown to stationary phase and auto‐aggregation became even more prominent.…”

Section: Resultsmentioning

confidence: 99%

“…If indicated, samples were concentrated through lyophilization. Alcian blue silver‐stained PAGE was performed according to (Litschko et al, 2021). In brief, the supernatant was mixed 1:1 with loading buffer (2 M sucrose in 10× TBE) and separated on a 15% PAGE using 1x TBE (100 mM Tris, 10 mM boric acid, 2.5 mM EDTA) as mobile phase.…”

Section: Methodsmentioning

confidence: 99%

Pseudomonas aeruginosa post‐translational responses to elevated c‐di‐GMP levels

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et al. 2022

Molecular Microbiology

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C‐di‐GMP signaling can directly influence bacterial behavior by affecting the functionality of c‐di‐GMP‐binding proteins. In addition, c‐di‐GMP can exert a global effect on gene transcription or translation, for example, via riboswitches or by binding to transcription factors. In this study, we investigated the effects of changes in intracellular c‐di‐GMP levels on gene expression and protein production in the opportunistic pathogen Pseudomonas aeruginosa. We induced c‐di‐GMP production via an ectopically introduced diguanylate cyclase and recorded the transcriptional, translational as well as proteomic profile of the cells. We demonstrate that rising levels of c‐di‐GMP under growth conditions otherwise characterized by low c‐di‐GMP levels caused a switch to a non‐motile, auto‐aggregative P. aeruginosa phenotype. This phenotypic switch became apparent before any c‐di‐GMP‐dependent role on transcription, translation, or protein abundance was observed. Our results suggest that rising global c‐di‐GMP pools first affects the motility phenotype of P. aeruginosa by altering protein functionality and only then global gene transcription.

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