The sialic acid transporter NanT is necessary and sufficient for uptake of 3‐deoxy‐d‐manno‐oct‐2‐ulosonic acid (Kdo) and its azido analog in Escherichia coli

Abstract

3-Deoxy-d-manno-oct-2-ulosonic acid (Kdo) is an essential component of lipopolysaccharides (LPS) in the Gram-negative bacterial outer membrane. Metabolic labeling of Escherichia coli LPS with 8-azido-3,8-dideoxy-d-manno-oct-2-ulosonic acid (Kdo-N ) has been reported but is inefficient. For optimization, it is important to understand how exogenous Kdo-N enters the cytoplasm. Based on similarities between Kdo and sialic acids, we proposed and verified that the sialic acid transporter NanT imports exogenous Kdo-N into E. coli. We demonstrated that E. coli ΔnanT were not labeled with Kdo-N , while expression of NanT in the ΔnanT mutant restored Kdo-N incorporation. Induced NanT expression in a strain lacking Kdo biosynthesis led to higher exogenous Kdo incorporation and restoration of full-length core-LPS, suggesting that NanT also transports Kdo. While Kdo-N incorporation was observed in strains having NanT, it was not detected in Pseudomonas aeruginosa and Acinetobacter baumannii, which lack nanT. However, heterologous expression of E. coli NanT in P. aeruginosa enabled Kdo-N incorporation and labeling, though this led to abnormal morphology and growth arrest. NanT seems to define which bacteria can be labeled with Kdo-N , provides opportunities to enhance Kdo-N labeling efficiency and spectrum, and raises the possibility of Kdo biosynthetic bypass where exogenous Kdo is present, perhaps even in vivo.

Genomic Characterization of Arcobacter butzleri Isolated From Shellfish: Novel Insight Into Antibiotic Resistance and Virulence Determinants

“…In both genomes, we identified orthologues of waa C and waa F genes, which are described as virulence determinant in A. thereius (Rovetto et al, 2017), but also in Pseudomonas aeruginosa , E. coli , Klebsiella pneumonia , and other Campylobacteraceae (Oldfield et al, 2002; DeLucia et al, 2011; Nilsson et al, 2018). waaF codes for a predicted ADP-heptose–LPS heptosyltransferase ( Ab 55 D3M61_00085; Ab 6V D3M75_11180), involved in the biosynthesis of lipooligosaccharide (LOS); waaC codes for a lipopolysaccharide heptosyltransferase ( Ab 55 D3M61_00195; Ab 6V D3M75_05185), which catalyzes the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds.…”

Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes

“…In order for Cho and its analogs to be incorporated, they need to enter the cytoplasm, where PL biosynthesis takes place. The absence of a transporter has been reported as problematic for the metabolic labeling of LPS with a 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) analog in some strains (31). Therefore, identification of the AECho transport mechanism(s) would allow determination of which bacteria might be able to uptake AECho and be metabolically labeled.…”

“…However, further studies are required to evaluate the impact of BetT and ProU on the uptake of AECho to fully understand whether the absence of AECho uptake systems may limit the utility in other strains. In addition, because mM concentrations of AECho were necessary to see efficient PC labeling in lptD4213 pcs, the identification of the transport mechanism might reduce the concentration/time needed for optimal labeling, much as the identification of the Kdo uptake receptor NanT allowed enhanced LPS metabolic labeling with a Kdo analog (31). As PC is produced in 15% of all bacteria, including pathogens like P. aeruginosa or L. pneumophila (34), those strains that naturally express Pcs should be able to synthesize AEPC if AECho can be readily taken up by the cells.…”

“…Because metabolic labeling has been used to incorporate synthetic analogs into different bacterial biomolecules, e.g. LPS (28)(29)(30)(31)(32), we chose to investigate whether metabolic labels could be incorporated into E. coli PLs and whether they would be labeled by fluorescent "click" reagents in cells with intact OM asymmetry or in cells with compromised OM. The most common PLs in the E. coli membrane are phosphatidylethanolamine (PE, ~80%), phosphatidylglycerol (PG, ~15%), and cardiolipin (CL, ~5%) (33).…”

Metabolic Labeling of Bacterial Glycans

“…60,61 Entry becomes even more challenging for charged sugars, sugar nucleotides and phosphorylated derivatives unless a specific pathway can be diverted for probe transport. 62 Some studies have employed partial permeabilization of the bacterial membranes to allow probe entry 63 ; − avoiding location of the bioorthogonal chemical function on a position which would preclude normal continuation of biosynthesis;…”