High-Throughput Mutagenesis and Cross-Complementation Experiments Reveal Substrate Preference and Critical Residues of the Capsule Transporters in Streptococcus pneumoniae

MOP (Multidrug/Oligosaccharidyl-lipid/Polysaccharide) family transporters are found in almost all life forms. They are responsible for transporting lipid-linked precursors across the cell membrane to support the synthesis of various glycoconjugates. While significant progress has been made in elucidating their transport mechanism, how these transporters select their substrates remains unclear. Here, we systematically tested the MOP transporters in the Streptococcus pneumoniae capsule pathway for their ability to translocate noncognate capsule precursors. Sequence similarity cannot predict whether these transporters are interchangeable. We showed that subtle changes in the central aqueous cavity of the transporter are sufficient to accommodate a different cargo. These changes can occur naturally, suggesting a potential mechanism of expanding substrate selectivity. A directed evolution experiment was performed to identify gain-of-function variants that translocate a noncognate cargo. Coupled with a high throughput mutagenesis and sequencing (Mut-seq) experiment, residues that are functionally important for the capsule transporter were revealed. Lastly, we showed that the expression of a flippase that can transport unfinished precursors resulted in an increased susceptibility to bacitracin and mild cell shape defects, which may be a driving force to maintain transporter specificity. IMPORTANCE All licensed pneumococcal vaccines target the capsular polysaccharide (CPS). This layer is highly variable and is important for virulence in many bacterial pathogens. Most of the CPSs are produced by the Wzx/Wzy mechanism. In this pathway, CPS repeating units are synthesized in the cytoplasm, which must be flipped across the cytoplasmic membrane before polymerization. This step is mediated by the widely conserved MOP (Multidrug/Oligosaccharidyl-lipid/Polysaccharide) family transporters. Here, we systematically evaluated the interchangeability of these transporters and identified the residues important for substrate specificity and function. Understanding how CPS is synthesized will inform glycoengineering, vaccine development, and antimicrobial discovery.