An enzyme-based protocol for cell-free synthesis of nature-identical capsular oligosaccharides from Actinobacillus pleuropneumoniae serotype 1

Abstract

Actinobacillus pleuropneumoniae (App) is the etiological agent of acute porcine pneumonia and responsible for severe economic losses worldwide. The capsule polymer of App serotype 1 (App1) consists of [4)-GlcNAc-β(1,6)-Gal-α-1-(PO4-] repeating units that are O-acetylated at O-6 of the GlcNAc. It is a major virulence factor and was used in previous studies in the successful generation of an experimental glycoconjugate vaccine. However, the application of glycoconjugate vaccines in the animal health sector is limited, presumably because of the high costs associated with harvesting the polymer from pathogen culture. Consequently, here we exploited the capsule polymerase Cps1B of App1 as an in vitro synthesis tool and an alternative for capsule polymer provision. Cps1B consists of two catalytic domains, as well as a domain rich in tetratricopeptide repeats (TPRs). We compared the elongation mechanism of Cps1B with that of a ΔTPR truncation (Cps1B-ΔTPR). Interestingly, the product profiles displayed by Cps1B suggested processive elongation of the nascent polymer, whereas Cps1B-ΔTPR appeared to work in a more distributive manner. The dispersity of the synthesized products could be reduced by generating single-action transferases and immobilizing them on individual columns, separating the two catalytic activities. Furthermore, we identified the O-acetyltransferase Cps1D of App1 and used it to modify the polymers produced by Cps1B. Two-dimensional NMR analyses of the products revealed O-acetylation levels identical to those of polymer harvested from App1 culture supernatants. In conclusion, we have established a protocol for the pathogen-free in vitro synthesis of tailored, nature-identical App1 capsule polymers.

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

“…3d ). As expected, the observed polymers are slightly shorter in the presence of acceptor, indicating specific elongation and a certain control over the product length, which would be beneficial for the biotechnological use of the enzyme 29 , 30 (Extended Data Fig. 8 ).…”

“…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.…”

“…The developed hydrolysis protocols yielded oligomers with free reducing ends that can be coupled through reductive amination and have the right size (for example, 10–25 repeating units) for vaccine development studies 7 . The single action transferases developed herein might even allow the step-wise build-up of the polymer according to established protocols 30 . The structural and biochemical characterization of Bcs3, the identification of homologs in both Gram-negative and Gram-positive pathogens and the development of fermentation-free synthesis protocols represent important milestones toward understanding, manipulating and exploiting multi-enzyme machines that will allow new access to structurally complex surface glycans.…”

The two-component system CpxA/CpxR is critical for full virulence in Actinobacillus pleuropneumoniae

“…Previous studies have found that CpxAR is required for virulence in A. pleuropneumoniae (Li et al, 2018;Yan et al, 2020). However, the crucial role of CpxAR in the pathogenesis CPS is one of the major virulence factors of A. pleuropneumoniae, which can protect the bacteria from the host's immune response, such as phagocytic uptake and complement-mediated bacteriolysis (Budde et al, 2020). The thickness of the capsule is related to the virulence of A. pleuropneumoniae, and generally, the thicker the capsule, the more virulent the strain (Dubreuil et al, 2000).…”

Structural and mechanistic basis of capsule O-acetylation in Neisseria meningitidis serogroup A

“…Serogroup A oligosaccharides of defined DP were produced by partial hydrolysis of the phosphodiester bond, starting from enzymatically synthesized, non- O -acetylated serogroup A polymer 17 . The polymer was incubated in sodium acetate buffer (50 mM sodium acetate, pH 4.8) for 6 h at 73 °C, yielding oligosaccharides that carry a phosphomonoester at the non-reducing end 19 , 52 , which could be removed by calf intestinal alkaline phosphatase 16 , 53 . Obtained oligosaccharides were purified by AEC on an ÄKTA FPLC system (GE Healthcare) equipped with a MonoQ HR 5/5 column (GE Healthcare).…”