Biosynthetic Assembly of the Bacteroides fragilis Capsular Polysaccharide A Precursor Bactoprenyl Diphosphate-Linked Acetamido-4-amino-6-deoxygalactopyranose

Abstract

The sugar capsule Capsular Polysaccharide A (CPSA), which coats the surface of the mammalian symbiont Bacteroides fragilis, is a key mediator of mammalian immune system development. In addition, this sugar polymer has shown therapeutic potential in animal models of Multiple Sclerosis and other autoimmune disorders. The structure of the CPSA polymer includes a rare stereoconfiguration sugar acetamido-4-amino-6-deoxy-galactopyranose (AADGal) that we propose is the first sugar linked to a bactoprenyl diphosphate scaffold in the production of CPSA. In this report we have utilized a heterologous system to reconstitute bactoprenyl-diphosphate-linked AADGal production. Construction of this system included a previously reported Campylobacter jejuni dehydratase, PglF, coupled to a B. fragilis encoded aminotransferase (WcfR) and initiating hexose-1-phosphate transferase (WcfS). Function of the aminotransferase was confirmed by capillary electrophoresis and a novel high performance liquid chromatography (HPLC) method. Production of the rare uridine diphosphate (UDP)-AADGal was confirmed through a series of 1D and 2D nuclear magnetic resonance experiments and high resolution mass spectrometry (HR-MS). A spectroscopically unique analogue of bactoprenyl phosphate was utilized to characterize the transfer reaction catalyzed by WcfS, and allowed HPLC based isolation of the isoprenoid-linked sugar product. Importantly, the entire heterologous system was utilized in a single pot reaction to biosynthesize the bactoprenyl-linked sugar. This work provides the first critical step in the in vitro reconstitution of CPSA biosynthesis.

Tuning the Production of Variable Length, Fluorescent Polyisoprenoids Using Surfactant-Controlled Enzymatic Synthesis

“…17–19,26 Reaction mixtures were prepared in a volume of 200 μ L, which contained 17.2 μ M UppS Bf , 10 mM 2CNA-GPP, and 75 mM IPP in OTG at 20 × CMC or DDM at 100 × CMC. The OTG reaction was quenched after 1.5 h with n -propanol, while the DDM reaction was allowed to continue for several hours.…”

Functional identification of a galactosyltransferase critical to Bacteroides fragilis Capsular Polysaccharide A biosynthesis

“…The first sugar appended to BP 1 is N-acetyl-4-amino-6-deoxylgalactopyranose (AADGal) in a reaction catalyzed by the membrane-localized protein WcfS, to afford a bactoprenyl diphosphate-linked AADGal (BPP-AADGal 2 ). ( 14, 15 ) Each of the remaining sugars are expected to then be incorporated one at a time by the presumed glycosyltransferases WcfQ, WcfN and WcfP to provide Galf(1→3)-β-GalNAc(1→3)-β-4,6(Pyr)Gal-(1→3)-α-AADGal(1→PP-bactoprenyl 3 . ( 16 ) The Wzy polymerase then is expected to link the tetrasaccharide repeat unit from the AADGal to the GalNAc residue as many as 120 times to give the capsular polymer.…”

“…The role of the initiating hexose-1-phosphate transferase, WcfS, in CPSA repeat unit biosynthesis was biochemically elucidated utilizing isoprenoid analogues that enhanced the ability to track the production of bactoprenyl phosphate-linked reaction products (Figure 2). ( 14 ) These analogues were derived from a chemically synthesized 4-nitroaniline or 2-amideaniline geranyl diphosphate (2AA-GPP 4a, NAGPP 4b ) that was then elongated by the enzyme Undecaprenyl Pyrophosphate Synthase (UPPS) in the presence of isopentenyl diphosphate (IPP, 5 ). ( 15, 18 ) The long chain variable length isoprenoid product ( 6a-b ) of these reactions were then cleaved with a commercially available Alkaline Phosphatase to provide monophosphate substrate for WcfS (2AA-B(n)P, 7a , 4NA-B(n)P, 7b ).…”

“…Each of the isoprenoid analogues were substrates for WcfS and the transfer of AADGal-phosphate was observed in a straight-forward reverse phase HPLC assay. ( 14, 15 ) …”

Inhibition of phospho-MurNAc-pentapeptide translocase (MraY) by nucleoside natural product antibiotics, bacteriophage ϕX174 lysis protein E, and cationic antibacterial peptides

“…Campbell et al have shown that inhibition of TarO with tunicamycin sensitises methicillin-resistant S. aureus strains to beta-lactam antibiotics, implying a fundamental connection between teichoic acid biosynthesis and peptidoglycan assembly in S. aureus [17]. Other homologues in this phospho-sugar transferase family include a glucose-1-phosphate transferase WcaJ involved in colonic acid capsule biosynthesis in E. coli and C. crescentus [18], and transferase WcfS involved in B. fragilis capsular polysaccharide biosynthesis [19].…”