Mix-and-Match System for the Enzymatic Synthesis of Enantiopure Glycerol-3-Phosphate-Containing Capsule Polymer Backbones from Actinobacillus pleuropneumoniae , Neisseria meningitidis , and Bibersteinia trehalosi

Abstract

Capsule polymers are crucial virulence factors of pathogenic bacteria and are used as antigens in glycoconjugate vaccine formulations. Some Gram-negative pathogens express poly(glycosylglycerol phosphate) capsule polymers that resemble Gram-positive wall teichoic acids and are synthesized by TagF-like capsule polymerases. So far, the biotechnological use of these enzymes for vaccine developmental studies was restricted by the unavailability of enantiopure CDP-glycerol, one of the donor substrates required for polymer assembly. Here, we use CTP:glycerol-phosphate cytidylyltransferases (GCTs) and TagF-like polymerases to synthesize the poly(glycosylglycerol phosphate) capsule polymer backbones of the porcine pathogen Actinobacillus pleuropneumoniae, serotypes 3 and 7 (App3 and App7). GCT activity was confirmed by high-performance liquid chromatography, and polymers were analyzed using comprehensive nuclear magnetic resonance studies. Solid-phase synthesis protocols were established to allow potential scale-up of polymer production. In addition, one-pot reactions exploiting glycerol-kinase allowed us to start the reaction from inexpensive, widely available substrates. Finally, this study highlights that multidomain TagF-like polymerases can be transformed by mutagenesis of active site residues into single-action transferases, which in turn can act in trans to build-up structurally new polymers. Overall, our protocols provide enantiopure, nature-identical capsule polymer backbones from App2, App3, App7, App9, and App11, Neisseria meningitidis serogroup H, and Bibersteinia trehalosi serotypes T3 and T15. IMPORTANCE Economic synthesis platforms for the production of animal vaccines could help reduce the overuse and misuse of antibiotics in animal husbandry, which contributes greatly to the increase of antibiotic resistance. Here, we describe a highly versatile, easy-to-use mix-and-match toolbox for the generation of glycerol-phosphate-containing capsule polymers that can serve as antigens in glycoconjugate vaccines against Actinobacillus pleuropneumoniae and Bibersteinia trehalosi, two pathogens causing considerable economic loss in the swine, sheep, and cattle industries. We have established scalable protocols for the exploitation of a versatile enzymatic cascade with modular architecture, starting with the preparative-scale production of enantiopure CDP-glycerol, a precursor for a multitude of bacterial surface structures. Thereby, our approach not only allows the synthesis of capsule polymers but might also be exploitable for the (chemo)enzymatic synthesis of other glycerol-phosphate-containing structures such as Gram-positive wall teichoic acids or lipoteichoic acids.

A model industrial workhorse: Bacillus subtilis strain 168 and its genome after a quarter of a century

“…In B. subtilis this applies to 26 tRNA genes lacking a CCA‐3′OH terminus, including the single tRNA Cys gene (Campos Guillén et al, 2019 ). In addition to its role in coordinating synthesis of the bacterial proteome driven by ribosome synthesis (You et al, 2013 ), CTP is involved in membrane synthesis via the formation of CDP‐diglycerides and related metabolites (Centola et al, 2021 ), and in capsule biosynthesis (Li, Gale, et al, 2021 ; Litschko et al, 2021 ). This involvement in growth further extends to sporulation through the control of spore surface growth by glucose‐1‐phosphate cytidylyltransferase (Shuster et al, 2019 ).…”

Novel Multiplex PCR Assay and Its Application in Detecting Prevalence and Antibiotic Susceptibility of Porcine Respiratory Bacterial Pathogens in Guangxi, China

“…Porcine respiratory disease complex (PRDC) is the most common disease in pig farming; it causes growth retardation, poor feed conversion, and severe acute mortality, posing a very serious threat to the global pig industry ( 1 ). PRDC is frequently caused by multiple pathogenic microorganisms, involving several viral pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and swine influenza virus (SIV), and multiple bacterial pathogens such as Actinobacillus pleuropneumoniae , Glaesserella parasuis , Streptococcus suis , Pasteurella multocida , and Mycoplasma hyopneumoniae ( 2 – 9 ). The pathogens that cause PRDC are usually classified as primary and secondary pathogens.…”

“…Each pathogen can cause PRDC independently, and the clinical signs caused by different pathogen infections are very similar. Simultaneous infection with multiple pathogens can cause more severe symptoms and lesions and even result in higher mortality than single-pathogen infections ( 2 – 6 ). Viral pathogens are frequently considered the main factors which cause PRDC, but numerous bacteria also play important roles in the development of PRDC ( 9 – 14 ).…”

A Cell‐Free Multi‐enzyme Cascade Reaction for the Synthesis of CDP‐Glycerol

“…Unfortunately, this synthesis method is particularly laborious and had low yields of substrate conversion. In a later effort, the enzymatic synthesis using glycerolphosphate cytidylyltransferase (tagD) and glycerol kinase (glpK) was described [11a,13] . This enzymatic one‐step reaction synthesis of CDP‐glycerol starts from CTP and glycerol‐3‐phosphate, both of which are expensive at around € 999 per 50 g and around € 245 per 25 mg, respectively (Carbosynth, 2022).…”