A conserved UDP-glucose dehydrogenase encoded outside the hasABC operon contributes to capsule biogenesis in group A Streptococcus

Abstract

Group A Streptococcus (GAS) is a human-specific bacterial pathogen responsible for serious morbidity and mortality worldwide. The hyaluronic acid (HA) capsule of GAS is a major virulence factor, contributing to bloodstream survival through resistance to neutrophil and antimicrobial peptide killing and to in vivo pathogenicity. Capsule biosynthesis has been exclusively attributed to the ubiquitous hasABC hyaluronan synthase operon, which is highly conserved across GAS serotypes. Previous reports indicate that hasA, encoding hyaluronan synthase, and hasB, encoding UDP-glucose 6-dehydrogenase, are essential for capsule production in GAS. Here, we report that precise allelic exchange mutagenesis of hasB in GAS strain 5448, a representative of the globally disseminated M1T1 serotype, did not abolish HA capsule synthesis. In silico whole-genome screening identified a putative HasB paralog, designated HasB2, with 45% amino acid identity to HasB at a distant location in the GAS chromosome. In vitro enzymatic assays demonstrated that recombinant HasB2 is a functional UDP-glucose 6-dehydrogenase enzyme. Mutagenesis of hasB2 alone slightly decreased capsule abundance; however, a ΔhasB ΔhasB2 double mutant became completely acapsular. We conclude that HasB is not essential for M1T1 GAS capsule biogenesis due to the presence of a newly identified HasB paralog, HasB2, which most likely resulted from gene duplication. The identification of redundant UDP-glucose 6-dehydrogenases underscores the importance of HA capsule expression for M1T1 GAS pathogenicity and survival in the human host.

Fig 1

HasB is not essential for capsule production. (A) Quantitation of capsule expression of mid-log-phase M1T1 GAS WT and single hasA and hasB mutant strains. Data denote the means ± standard errors of the means of pooled data from two independent experiments performed in duplicate. Asterisks indicate values that are statistically significantly different (***, P < 0.001). bd, below detection. (B) Genomic organization of the has operon and hasB2 gene, which encodes a putative HasB paralog in M1T1 GAS. Schematic representations of the genomic regions are not drawn to scale. (C) A maximum-likelihood phylogram of HasB2 and a representative set of its homologs indicate a clear distinction between HasB and HasB2. Branch support values are indicated. The plasmid-encoded HasB homolog in Bacillus anthracis seems to be equally distant to both paralogs.

Fig 2

Alignment and 3D overlay of HasB and HasB2. (A) ClustalW alignment of HasB (GenBank GeneID 3571024) and its paralog HasB2 (GenBank GeneID 3572465) from serotype M1T1 GAS strain MGAS5005 (43). HasB2 is 45.3% identical to HasB at the amino acid level. Asterisks denote identical residues; periods indicate similar residues. Residues implicated in catalysis (yellow), NAD(H) hydrogen bonding (green), UDP-sugar binding (blue), and the dimer partner (red) are highlighted. The gray amino acid is implicated in catalysis and NAD(H) hydrogen bonding, while the orange amino acid is implicated in UDP-sugar binding (8). (B) HasB2 is predicted to have structural similarity to HasB. Superposition of HasB2 on the resolved 3D structure of HasB (8) was performed with Cn3D software (46). Sequence conservation is shown as follows: identical residues, red; similar or nonidentical residues, blue). Amino acid residues in the catalytic domain (T118, E141, E145, K204 N208, C260, and D264 of HasB) are indicated in yellow, beta-sheets are in purple, and alpha helices are in green (based on Campbell et al. [8]).

Fig 3

HasB2 is a functional UDP-glucose 6-dehydrogenase. Whole-cell lysates containing recombinant HasB and HasB2 were positive for UDP-glucose 6-dehydrogenase activity, compared to the plasmid-only control, as measured spectrophotometrically by the reduction of NAD at 340 nm. Data represent the means ± standard errors of the means of pooled data from two independent experiments performed in triplicate. *, P < 0.05; ***, P < 0.001.

Fig 4

HasB2 contributes to GAS capsule biogenesis. (A) Quantitation of capsule expression in the WT, mutant, and revertant strains propagated to mid-log phase using ELISA (A) and flow cytometric analysis (B) using hyaluronan-binding protein (HABP). ELISA data represent the means ± standard deviations of pooled normalized data from two independent experiments performed in duplicate. Flow cytometry data represent means ± standard deviations of two independent experiments. Asterisks indicate values that are statistically significantly different: *, P < 0.05; ***, P < 0.001. bd, below detection.