General Utilization of Fluorescent Polyisoprenoids with Sugar Selective Phosphoglycosyltransferases

Abstract

The protective surfaces of bacteria are comprised of polysaccharides and are involved in host invasion and colonization, host immune system evasion, as well as antibacterial resistance. A major barrier to our fundamental understanding of these complex surface polysaccharides lies in the tremendous diversity in glycan composition among bacterial species. The polyisoprenoid bactoprenyl phosphate (or undecaprenyl phosphate) is an essential lipid carrier necessary for early stages of glycopolymer assembly. Because of the ubiquity of bactoprenyl phosphate in these critical processes, molecular probes appended to this lipid carrier simplify identification of enzymatic roles during polysaccharide bioassembly. A limited number of these probes exist in the literature or have been assessed with such pathways, and the limits of their use are not currently known. Herein, we devise an efficient method for producing fluorescently modified bactoprenyl probes. We further expand our previous efforts utilizing 2-nitrileaniline, and additionally prepare nitrobenzoxadizol tagged bactoprenyl phosphate for the first time. We then assess enzyme promiscuity of these two probes utilizing four well characterized initiating phosphoglycosyltransferases: CPS2E (Streptococcus pneumoniae), WbaP (Salmonella enterica), WecA (Escherichia coli) and WecP (Aeromonas hydrophilia). Both probes serve as substrates for these enzymes and could be readily used to investigate a wide range of bacterial glycoassembly pathways. Interestingly, we have also identified unique solubility requirements for the

Tracking Colanic Acid Repeat Unit Formation from Stepwise Biosynthesis Inactivation in Escherichia coli

“…To do this, we used the same set of proteins described previously to prepare the pentasaccharide on native bactoprenyl phosphate. 11 A two-step procedure was used to prepare polyisoprenoid diphosphates with UppS and the native substrate, farnesyl diphosphate. Subsequent extraction and acid phosphatase treatment then generated the corresponding BP.…”

“…9−11 For instance, the initiating phosphoglycosyltransferase WbaP from Salmonella enterica transfers phospho-glucose, a nonnative substrate, in vitro but does not extensively catalyze this reaction in vivo. 11,12 Membrane-associated proteins are notoriously difficult to express and purify for several reasons that include unstable plasmids, toxic proteins, low yields, and inclusion body formation (i.e., misfolded proteins). 13,14 Similarly, obtaining naturally occurring lipid-linked intermediates from live cells is challenging due to competition for BP and the lack of methods available to detect these materials.…”

“…Various superfluous artifacts can arise during in vitro bioassays. The relaxed substrate specificity of glycans has been observed with some glycosyltransferases. − For instance, the initiating phosphoglycosyltransferase WbaP from Salmonella enterica transfers phospho-glucose, a non-native substrate, in vitro but does not extensively catalyze this reaction in vivo . , Membrane-associated proteins are notoriously difficult to express and purify for several reasons that include unstable plasmids, toxic proteins, low yields, and inclusion body formation (i.e., misfolded proteins). , Similarly, obtaining naturally occurring lipid-linked intermediates from live cells is challenging due to competition for BP and the lack of methods available to detect these materials . Some essential cell envelope intermediates are present in detectable quantities, but it is not known if non-essential surface glycans like colanic acid are also sequentially accumulated. , Increasing the level of capsule production by modifying regulatory genes has been shown to enhance mucoid phenotypes associated with colanic acid production. ,− However, genetic disruption of exopolysaccharide biosynthesis negatively affects cell physiology. , This is explained by sequestration of BP in the form of overaccumulation of dead-end intermediates and also the reduction of free BP for peptidoglycan cell wall synthesis, an essential component of the cell envelope.…”

Chemical Biology Tools for Modulating and Visualizing Gram-Negative Bacterial Surface Polysaccharides

“…Several lipid-based probes have been developed to examine the specificity of phosphoglycosyl transferases, such as WecA, and to evaluate the importance of lipid structure on subsequent steps of O-antigen biosynthesis (Figure B). Notably, Troutman and co-workers have recently developed fluorescent polyisoprenoids 27 and 28 to serve as reporters in the synthesis of glycolipids, including capsular and O-polysaccharides. − 2-Nitrileanilinobactoprenyl phosphate ( 28 ) was used to identify the functional roles of key proteins in the biosynthesis of the ECA repeat unit by facilitating HPLC-based analysis of complex intermediates . Additionally, 28 was able to support the synthesis of the repeat unit of Bacteroides fragilis capsular polysaccharide A, as well as part of the E. coli exopolysaccharide colanic acid, both of which rely on Und-P but use alternate transferases to WecA. , While lipid-based probes have not yet been reported to function in cells, we anticipate that they will facilitate methods to visualize lipid-linked intermediates in reconstituted membranes.…”

“…This work indicates that different lipids may be used to study various stages of surface polysaccharide synthesis, which may be useful for experiments aimed at accumulating particular intermediates, such as O-units, prior to polymerization by Wzy. Additionally, since the native Und-P carrier has limited aqueous solubility, the ability to use shortened lipid probes facilitates robust biochemical assays, as demonstrated by the utilization of shortened fluorescent lipid analogs by Troutman and co-workers. − …”