Identification of a novel sialic acid transporter in Haemophilus ducreyi

Abstract

Haemophilus ducreyi, the causative agent of chancroid, produces a lipooligosaccharide (LOS) which terminates in N-acetyllactosamine. This glycoform can be further extended by the addition of a single sialic acid residue to the terminal galactose moiety. H. ducreyi does not synthesize sialic acid, which must be acquired from the host during infection or from the culture medium when the bacteria are grown in vitro. However, H. ducreyi does not have genes that are highly homologous to the genes encoding known bacterial sialic acid transporters. In this study, we identified the sialic acid transporter by screening strains in a library of random transposon mutants for those mutants that were unable to add sialic acid to N-acetyllactosamine-containing LOS. Mutants that reacted with the monoclonal antibody 3F11, which recognizes the terminal lactosamine structure, and lacked reactivity with the lectin Maackia amurensis agglutinin, which recognizes alpha2,3-linked sialic acid, were further characterized to demonstrate that they produced a N-acetyllactosamine-containing LOS by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analyses. The genes interrupted in these mutants were mapped to a four-gene cluster with similarity to genes encoding bacterial ABC transporters. Uptake assays using radiolabeled sialic acid confirmed that the mutants were unable to transport sialic acid. This study is the first report of bacteria using an ABC transporter for sialic acid uptake.

FIG. 1.

LOS structure, nomenclature, and SDS-PAGE. (A) H. ducreyi strain 35000HP LOS structure and nomenclature (6). (B) Silver-stained SDS-PAGE gel of LOS isolated from 35000HP (lanes 1 and 10) and 35000HP-305, 35000HP-306, 35000HP-310, 35000HP-313, 35000HP-319, 35000HP-320, 35000HP-322, and the lst mutant 35000HP-RSM203 (lanes 2 to 9). The sialylated glycoform (A₅a₁) is absent from all of the LOS samples except the parent strain 35000HP (indicated by an arrow). In the absence of the A₅a₁ structure, the b branch structures (A₅b₁ and A₅b₂) are more readily observed. (C) Silver-stained SDS-PAGE gel of LOS isolated from 35000HP (lanes 1 and 2), lst mutant 35000HP-RSM203 (lanes 3 and 4), and 35000HP-310 (lanes 5 and 6). Lanes with a + are LOS samples which were treated with neuraminidase, lanes with a − are LOS samples which were not treated with neuraminidase. The neuraminidase treatment only affected the glycoforms present in the 35000HP LOS (sialylated glycoform indicated by an arrow); this treatment had no affect on the LOS from the lst mutant 35000HP-RSM203 or 35000HP-310. These data are representative of all the sialic acid transporter mutants presented in this study. The three core heptoses are l-glycero-d-manno-heptose; the branch heptose, in italics, is of the d-glycero-d-manno configuration.

FIG. 2.

Matrix-assisted laser desorption ionization-time of flight spectra of O-deacylated LOS isolated from 35000HP and 35000HP-306 grown on chocolate agar plates (A) and chocolate agar plates supplemented with 1 mM sialic acid (B). The spectra from the two strains are quite similar, except for the absence of the sialylated glycoform peaks, A₅a₁ and A₅a₁*, in the 35000HP-306 spectra. Additionally, the A₅b₁, A₅b₁*, and A₅b₂ peaks, corresponding to the addition of GlcNAc (without and with PEA) and lactosamine to the A₅ structure, respectively, seem more prevalent in the 35000HP-306 spectra. Corresponding with previous findings (50), the sialylated glycoform peak A₅a₁* is more abundant in the 35000HP sample grown on sialic acid-supplemented medium. The 35000HP-306 data are representative of all the sialic acid transporter mutants presented in this study.

FIG. 3.

Open reading frame map of the H. ducreyi sialic acid transporter (sat) region. The genes satABCD, corresponding to Hd1669 to -1672, respectfully, and their directions of transcription are shown. The site of transposon insertion in each mutant is indicated by the corresponding mutant strain name.

FIG. 4.

Sialic acid uptake assay. Bacteria were grown on plates and suspended to an optical density at 600 nm of 2. Bacteria were then incubated with [³H]NeuAc for the indicated time, filtered, washed, and counted. (A) A 15-min time course study was performed on the parent strain 35000HP. The plot is the mean of triplicate determinations. (B) A single 10-min time point determination of [³H]NeuAc uptake from (reading from left to right) the satB mutant 35000HP-306, the satD mutant 35000HP-313, the satA mutant 35000HP-319, the satC mutant 35000HP-322, and the parent strain 35000HP. The results are the means of triplicate determinations. Results shown are representative of at least two independent experiments.

FIG. 5.

Hypothetical model for sialic acid transport in H. ducreyi. Protein functions for the model were based on sequence comparisons with other ABC-transporter systems. (A) SatA in a closed conformation, with sialic acid bound, interacts with SatB and SatC to initiate transport and hydrolysis. (B) When SatA binds tightly with SatB/SatC, it may transition to an open conformation, which has decreased affinity for sialic acid, and SatB/SatC may reorient to expose a sugar binding site. (C) When ATP is hydrolyzed, sialic acid is transported, SatA is released, and SatB/SatC return to their original conformation.