Human Milk Oligosaccharides Inhibit Group B Streptococcal Growth by Binding PcsB, an Essential Cell Wall Separation Protein

Human milk oligosaccharides (HMOs) are complex sugars in breast milk that protect babies by preventing harmful bacteria from colonizing the gut. Our team extended the study of HMOs beyond the neonatal gut and characterized their antimicrobial activity against group B Streptococcus (GBS), a diplococcus responsible for invasive perinatal infection. To date, the mechanism of action of this antimicrobial activity has remained obscure. To address this key gap, we employed untargeted proteomics, which revealed downregulation of PcsB, an essential murein hydrolase required for cell division. Following successful purification of the active domain of PcsB, we found that this protein domain restores GBS growth in the presence of HMOs, thereby validating PcsB as an HMO protein-interacting partner. In silico docking and molecular dynamics simulations predicted that two fucosylated HMOs, lacto-N-fucopentaose I (LNFPI) and lacto-N-fucopentaose III (LNFPIII), bind to PcsB. In silico predictions were validated using microscale thermophoresis assays, which reported dissociation constants of 5 ± 1 mM for LNFPI and 263 ± 72 μM for LNFPIII. Lastly, to test the hypothesis that HMOs may directly modulate the enzymatic activity of the CHAP domain, we employed a turbidimetric assay with commercial PG as the substrate. This assay provided further evidence that HMOs inhibit CHAP. Together, these data suggest that HMOs inhibit GBS growth by binding PcsB at its catalytic site, disturbing essential cell wall separation and division.