Characterisation of anhydro-sialic acid transporters from mucosa-associated bacteria

Abstract Sialic acid (Sia) transporters are critical to the capacity of host-associated bacteria to utilise Sia for growth and/or cell surface modification. While N-acetyl-neuraminic acid (Neu5Ac)-specific transporters have been studied extensively, little is known on transporters dedicated to anhydro-Sia forms such as 2,7-anhydro-Neu5Ac (2,7-AN) or 2,3-dehydro-2-deoxy-Neu5Ac (Neu5Ac2en). Here, we used a Sia-transport-null strain of Escherichia coli to investigate the function of members of anhydro-Sia transporter families previously identified by computational studies. First, we showed that the transporter NanG, from the Glycoside-Pentoside-Hexuronide:cation symporter family, is a specific 2,7-AN transporter, and identified by mutagenesis a crucial functional residue within the putative substrate-binding site. We then demonstrated that NanX transporters, of the Major Facilitator Superfamily, also only transport 2,7-AN and not Neu5Ac2en nor Neu5Ac. Finally, we provided evidence that SiaX transporters, of the Sodium-Solute Symporter superfamily, are promiscuous Neu5Ac/Neu5Ac2en transporters able to acquire either substrate equally well. The characterisation of anhydro-Sia transporters expands our current understanding of prokaryotic Sia metabolism within host-associated microbial communities.

The pKD13-type template plasmid, pES134, was made by Gibson assembly of PCR products ES184+ES185 for the marker cassette, amplified from the synthetic construct pES101 (GenScript), and ES186+ES187 for the backbone, amplified from pKD13.pES134 reproduces the design of pKD13 [2] and may be used in the same way to introduce unmarked in-frame deletions in the E. coli chromosome [3] without illegitimate recombination with pre-engineered FRT scars because of FRT-F3 orthogonality [4].pES134 is available from Addgene (127551).Constructs pDRT1, pDRT2, pDRT7, pES21, pSEV21, and pSEV33, all isogenic derivatives of pWKS30 [6] carrying different sialic acid transporter genes under lac promoter control, reproduce the design of pES1G, pES41, and pES156, described previously [5,7,8], and were made in an equivalent manner by restriction-ligation of PCR products amplified from genomic DNAs (Tables 1 and S1).pDRT4 was made by Gibson assembly of PCR products ESN257+ESN607 for nanG (entire coding sequence minus stop codon), amplified from pDRT1, and ESN255+ESN606 for the backbone, amplified from pSEV7 (pSEV7 is a derivative of pWKS30 coding for C-terminal TEV site and His6 tag adding sequence -ENLYFQGLEHHHHHH to the last residue of the encoded transporter; Eunice Lee and Emmanuele Severi, unpublished).pDRT5 was made analogously, with the nanG-[D40A] insert amplified as two overlapping PCR products (ESN257+ESN612 and ESN613+ESN607).All constructs were verified by sequencing using oligos ESN102 and ESN103 [9].
Cell fractionation and western blotting.Transformed TRXC2 strains were precultured as described for the growth experiments, refreshed to an initial OD600 of 0.1 in M9Amp supplemented with 0.1 mM IPTG and 2 mg ml -1 glucose, and grown to ca 1 OD600.Cells were then harvested and subjected to cellular fractionation as described in [9] to separate the membrane fraction, samples of which were then probed with anti-His antibody (Invitrogen) as per the same reference.

Structural comparison between SlNanG and StMelB
An the AlphaFold2 model of SlNanG was generated via the Colaboratory online tool [10] and the "rank_001" model was used to search the entire PDB database [11] using the DALI server [12]; both online tools were used with default parameters.The top two hits were the two experimental structures of sugar-bound Salmonella typhimurium LT2 MelB [13], namely entries 7L16 (MelB bound to dodecyl 6-O-alpha-D-galactopyranosyl-beta-D-glucopyranoside; 20% identity, 3 Å rmsd across 440 pairs) and 7L17 (MelB bound to 4-nitrophenyl alpha-D-galactopyranoside; 20% identity, 2.9 rmsd across 439 pairs).To identify a potential substrate-binding site within SlNanG, we superimposed the AlphaFold2 model of SlNanG with entry 7L17 using ChimeraX [14], which was also used to visualise the resulting overlay.  1) were introduced into TRXC2 and tested for their ability to complement the growth of this strain on different Sias (all used at 1 mg ml -1 ≈ 3.5 mM) as sole carbon source.A: Neu5Ac; B: Neu5Ac2en; C: 2,7-AN; D: maltose.Light green: EcnanX; dark green: STM1132 (nanX from S. typhimurium LT2); yellow: EcnanT (positive control for growth on Neu5Ac, negative control for growth on 2,7-AN).Data are the average from triplicate sets ± SD.STM1132 codes for a transporter 57% identical to EcNanX [15].  1) were introduced into TRXC2 and tested for their ability to complement the growth of this strain on different Sias (all used at 1 mg ml -1 ≈ 3.5 mM) as sole carbon source.A: Neu5Ac; B: Neu5Ac2en; C: 2,7-AN; D: maltose.Light green: EcnanX; dark green: STM1132 (nanX from S. typhimurium LT2); yellow: EcnanT (positive control for growth on Neu5Ac, negative control for growth on 2,7-AN).Data are the average from triplicate sets ± SD.

Figure S3 .
Figure S3.Identification of SlNanG D40 as a functional residue for 2,7-AN uptake.A. Overlay between the AlphaFold2-predicted structure of SlNanG (cyan) and the experimental structure (PDB 7L17) of the melibiose transporter MelB (pink) from Salmonella typhimurium (note that AlphaFold2 predicts SlNanG to have 12 transmembrane helices, and not 11 as previously reported[16]).View from the extracytoplasmic side of the transporters into their substrate-binding cavities.Inset: zoom-in on MelB D19 showing the H-bonds with the co-crystallised ligand (the melibiose analogue, 4-nitrophenyl -Dgalactopyranoside) and pinpointing the equivalence of this residue with SlNanG D40; B: Espript[17] alignment (first ~ 60 residues only) of all NanG transporters of the ST8 family[16], with the asterisk highlighting D40 of SlNanG; C: anti-His-tag immunoblot of membrane fractions from E. coli TRXC2 expressing either SlNanG-His6 (from pDRT4), the D40A mutant of the same variant (from pDRT5), or no heterologous transporter at all (from pWKS30, "vector").Notably, SlNanG migrates as a considerably smaller species (37 rather than the predicted 55 kDa), as seen for other membrane proteins[18].

Figure S3 .
Figure S3.Identification of SlNanG D40 as a functional residue for 2,7-AN uptake.A. Overlay between the AlphaFold2-predicted structure of SlNanG (cyan) and the experimental structure (PDB 7L17) of the melibiose transporter MelB (pink) from Salmonella typhimurium (note that AlphaFold2 predicts SlNanG to have 12 transmembrane helices, and not 11 as previously reported[15]).View from the extracytoplasmic side of the transporters into their substrate-binding cavities.Inset: zoom-in on MelB D19 showing the H-bonds with the co-crystallised ligand (the melibiose analogue, 4-nitrophenyl a-Dgalactopyranoside) and pinpointing the equivalence of this residue with SlNanG D40; B: Espript[16] alignment (first ~ 60 residues only) of all NanG transporters of the ST8 family[15], with the asterisk highlighting D40 of SlNanG; C: anti-His-tag immunoblot of membrane fractions from E. coli TRXC2 expressing either SlNanG-His 6 (from pDRT4), the D40A mutant of the same variant (from pDRT5), or no heterologous transporter at all (from pWKS30, "vector").Notably, SlNanG migrates as a considerably smaller species (37 rather than the predicted 55 kDa), as seen for other membrane proteins[17].

NanX from Salmonella typhimurium LT2 is a 2,7-AN-specific transporter. Plasmids
BamHI, and BsaI and EspI sites releasing compatible ends with the former two) are underlined.Start and stop codons of Sia transporter genes are in bold and in italics, respectively.The mutagenic codon for SlnanG[D40A] is highlighted in grey.Please note that oligo ESN618, used to amplify STM1132 (nanX) from S. typhimurium LT2, anneals downstream of that gene into the 5´ end of STM1133 (nanY/nanOx) adding an in-frame stop codon.