Making the Enterobacterial Common Antigen Glycan and Measuring Its Substrate Sequestration

Abstract

The enterobacterial common antigen (ECA), a three-sugar repeat unit polysaccharide produced by Enterobacteriaceae family members, impacts bacterial outer membrane permeability, and its biosynthesis affects the glycan landscape of the organism. ECA synthesis impacts the production of other polysaccharides by reducing the availability of shared substrates, the most notable of which is the 55-carbon polyisoprenoid bactoprenyl phosphate (BP), which serves as a carrier for the production of numerous bacterial glycans including ECA, peptidoglycan, O-antigen, and more. Here, using a combination of in vitro enzymatic synthesis and liquid chromatography–mass spectrometry (LC-MS) analysis of bacterial lysates, we provide biochemical evidence for the effect on endogenous polyisoprenoid pools from cell culture that arises from glycan pathway disruption. In this work, we have cloned and expressed each gene involved in ECA repeat unit biosynthesis and reconstituted the pathway in vitro, providing LC-MS characterized standards for the investigation of cellular glycan-linked intermediates and BP. We then generated ECA deficient mutants in genes associated with production of the polysaccharide, which we suspected would accumulate materials identical to our standards. We found that indeed accumulated products from these cells were indistinguishable from our enzymatically prepared standards, and moreover we observed a concomitant decrease in cellular BP levels with each mutant. This work provides the first direct biochemical evidence for the sequestration of BP upon the genetic disruption of glycan biosynthesis pathways in bacteria. This work also provides methods for the direct assessment of both the ECA glycan, and a new understanding of the dynamic interdependence of the bacterial polysaccharide repertoire.

ElyC and Cyclic Enterobacterial Common Antigen Regulate Synthesis of Phosphoglyceride-Linked Enterobacterial Common Antigen

“…A number of studies have shown that disruption of intermediate steps in ECA biogenesis leads to isoprenoid carrier stress and peptidoglycan synthesis defects due to the accumulation of ECA synthesis intermediates on Und-P ( 26 – 30 ). In fact, a recent study has biochemically confirmed the accumulation of these intermediates ( 25 ). Similar defects have been observed with the disruption of O-antigen or colanic acid biosynthesis ( 60 , 83 ).…”

“…The ECA and peptidoglycan pathways compete for Und-P and UDP-GlcNAc as depicted in Fig. S1 in the supplemental material ( 25 , 60 , 61 ). Although deletion of wecA causes generally mild phenotypes, deletion of later genes in ECA biosynthesis (e.g., wecB , wecG , wecF , or wzxE ) causes the accumulation of ECA intermediates sequestering Und-P, disrupting peptidoglycan biosynthesis and resulting in increased permeability defects, cell shape defects, and envelope stress response activation ( 26 – 30 , 62 , 63 ).…”

“…(i) The ECA species observed in the immunoblot assay appears to be the major form of ECA present in the cells. To our knowledge, polymerized ECA attached to Und-P has never been detected when Und-P-linked ECA biosynthesis intermediates are investigated ( 25 , 45 ). In fact, loss of the ability to make a full subunit of ECA was required to initially demonstrate that ECA is synthesized on Und-P ( 45 ).…”

“…In this case, our data would suggest that the Und-PP released by the polymerization of ECA combined with new synthesis of Und-P is sufficient for peptidoglycan synthesis. Recent advances in the biochemical analysis of ECA biosynthesis intermediates ( 25 ) will make this an interesting area for future investigation.…”

“…S1 in the supplemental material). In many Enterobacterales , deleting the first gene in ECA biosynthesis, wecA , not only prevents ECA biosynthesis but also prevents O-antigen biosynthesis and increases precursor availability for peptidoglycan biosynthesis ( 22 – 25 ). Deletion of downstream genes in ECA biosynthesis, such as wecE or wecF , leads to accumulation of intermediates in ECA biosynthesis, interfering with peptidoglycan biosynthesis, altering cell shape, increasing envelope permeability, and activating envelope stress response systems ( 26 – 30 ).…”