A Conserved UDP - glucose Dehydrogenase Outside the hasABC Operon 1 Contributes to Capsule Biogenesis in Group A Streptococcus

22 Group A Streptococcus (GAS) is a human-specific bacterial pathogen responsible for serious 23 morbidity and mortality worldwide. The hyaluronic acid (HA) capsule of GAS is a major 24 virulence factor, contributing to bloodstream survival through resistance to neutrophil and 25 antimicrobial peptide killing, and in vivo pathogenicity. Capsule biosynthesis has been 26 exclusively attributed to the ubiquitous hasABC hyaluronan synthase operon, which is highly 27 conserved across GAS serotypes. Previous reports indicate that hasA, encoding hyaluronan 28 synthase, and hasB, encoding UDP-glucose 6-dehydrogenase, are essential for capsule 29 production in GAS. Here, we report that precise allelic exchange mutagenesis of hasB in GAS 30 strain 5448, a representative of the globally disseminated M1T1 serotype, did not abolish HA 31 capsule synthesis. In silico whole-genome screening identified a putative HasB paralog, 32 designated HasB2, with 45% amino acid identity to HasB at a distant location in the GAS 33 chromosome. In vitro enzymatic assays demonstrated that recombinant HasB2 is a functional 34 UDP-glucose 6-dehydrogenase enzyme. Mutagenesis of hasB2 alone slightly decreased capsule 35 abundance; however, a ΔhasBΔhasB2 double mutant became completely acapsular. We conclude 36 that HasB is not essential for M1T1 GAS capsule biogenesis due to the presence of a newly 37 identified HasB paralog HasB2, which most likely resulted from gene duplication. The 38 identification of redundant UDP-glucose 6-dehydrogenases underscores the importance of HA 39 capsule expression for M1T1 GAS pathogenicity and survival in the human host. 40 41 42