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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.

SUBMITTER: Budde I

PROVIDER: S-EPMC7186170 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A DNA region involved in Actinobacillus pleuropneumoniae serotype 5 capsular polysaccharide (CP) biosynthesis was identified and characterized by using a probe specific for the cpxD gene involved in CP export. The adjacent serotype 5-specific CP biosynthesis region was cloned from a 5.8-kb BamHI fragment and an 8.0-kb EcoRI fragment of strain J45 genomic DNA. DNA sequence analysis demonstrated that this region contained four complete open reading frames, cps5A, cps5B, cps5C, and cps5D. Cps5A, Cps5B, and Cps5C showed low homology with several bacterial glycosyltransferases involved in the biosynthesis of lipopolysaccharide or CP. However, Cps5D had high homology with KdsA proteins (3-deoxy-D-manno-2-octulosonic acid 8-phosphate synthetase) from other gram-negative bacteria. The G+C content of cps5ABC was substantially lower (28%) than that of cps5D and the rest of the A. pleuropneumoniae chromosome (42%). A 2.1-kb deletion spanning the cloned cps5ABC open reading frames was constructed and transferred into the J45 chromosome by homologous recombination with a kanamycin resistance cassette to produce mutant J45-100. Multiplex PCR confirmed the deletion in this region of J45-100 DNA. J45-100 did not produce intracellular or extracellular CP, indicating that cps5A, cps5B, and/or cps5C were involved in CP biosynthesis. However, biosynthesis of the Apx toxins, lipopolysaccharide, and membrane proteins was unaffected by the mutation. Besides lack of CP biosynthesis, and in contrast to J45, J45-100 grew faster, was sensitive to killing in precolostral calf serum, and was avirulent in pigs at an intratracheal challenge dose three times the 50% lethal dose (LD50) of strain J45. At six times the J45 LD50, J45-100 caused mild to moderate lung lesions but not death. Electroporation of cps5ABC into A. pleuropneumoniae serotype 1 strain 4074 generated strain 4074(pJMLCPS5), which expressed both serotype 1 and serotype 5 CP. However, serotype 1 capsule expression was diminished in 4074(pJMLCPS5) in comparison to 4074. The recombinant strain produced significantly less total CP (serotypes 1 and 5 CP combined) in log phase (P = 0.0012) but significantly more total CP in late stationary phase than 4074 (P < 0.0001). In addition, strain 4074(pJMLCPS5) caused less mortality and bacteremia in pigs and mice following respiratory challenge than strain 4074, indicating that virulence was affected by diminished capsule production. These results emphasize the importance of CP in the serum resistance and virulence of A. pleuropneumoniae.

Project description:The capsular polysaccharide (CP) of Actinobacillus pleuropneumoniae is required for virulence of the bacteria in swine. However, a molecular investigation of whether the type or quantity of CP affects A. pleuropneumoniae virulence has not been reported. To initiate this investigation, a DNA region downstream of conserved genes required for CP export in A. pleuropneumoniae serotype 1 was cloned and sequenced. Three open reading frames, designated cps1A, cps1B, and cps1C, were identified that had amino acid homology to bacterial carbohydrate biosynthesis genes. A kanamycin resistance cassette (Kan(r)) was inserted into a 750-bp deletion spanning cps1AB or into a 512-bp deletion in cps1B only, and the constructs were cloned in a suicide vector. The Kan(r) gene was then transferred into the chromosome of strain 4074 by homologous recombination to produce strain 4074Deltacps1N and strain 4074Deltacps1B, respectively. Strain 4074Deltacps1N produced no detectable CP, but strain 4074Deltacps1B made 15% of the serotype 1 CP made by the parent strain, 4074, as determined by enzyme-linked immunosorbent assay and precipitation of free CP. The cps1ABC genes of strain 4074 and the cps5ABC and cps5ABCDE genes of serotype 5a strain J45 were cloned into the shuttle vector pLS88 and electroporated into 4074Deltacps1N to produce 4074Deltacps1N(pABcps101), 4074Deltacps1N(pJMLcps53), and 4074Deltacps1N(pABcps55), respectively. Strain 4074Deltacps1N(pABcps101) produced about 33% of the serotype 1 CP produced by strain 4074. Strains 4074Deltacps1N(pJMLcps53) and 4074Deltacps1N(pABcps55) produced serotype 5a CP in similar quantity or in fourfold excess, respectively, to that produced by strain 4074. With intratracheal challenge in pigs at similar dosages, the order of virulence of strains producing serotype 1 CP (assessed by mortality, lung consolidation, hemorrhage, and fibrinous pleuritis) was the following: strain 4074 > strain 4074Deltacps1N(pABcps101) > or = strain 4074Deltacps1N > strain 4074Deltacps1B. Strain 4074Deltacps1N(pJMLcps53) was less virulent than strain 4074Deltacps1N(pABcps55). However, both strains produced serotype 5a CP in similar or greater quantities than was observed for production of serotype 1 CP by the parent strain, 4074, but were less virulent than the parent strain. Therefore, the amount of serotype 1 or 5a CP produced by isogenic strains of A. pleuropneumoniae correlated with the virulence of the bacteria in pigs. However, virulence was also influenced by the type of CP produced or by its mechanism of expression.

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

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