Mother Knows Best: Deciphering the Antibacterial Properties of Human Milk Oligosaccharides

Abstract

This Account describes the risky proposition of organizing a multidisciplinary team to interrogate a challenging problem in chemical biology: characterizing how human milk, at the molecular level, protects infants from infectious diseases. At the outset, our initial hypothesis was that human milk oligosaccharides (HMOs) possess antimicrobial and antivirulence activities. Early on, we discovered that HMOs do indeed modulate bacterial growth and biofilm production for numerous bacterial pathogens. In light of this discovery, three priorities emerged for our program moving forward. The first was to decode the mode of action behind this activity. The second was to decipher the functional effects of HMO structural diversity as there are ca. 200 unique HMOs present in human milk. Finally, we set our sights on discovering novel uses for HMOs as we believed this would uniquely position our team to achieve a major breakthrough in human health and wellness. Through a combination of fractionation techniques, chemical synthesis, and industrial partnerships, we have determined the identities of several HMOs with potent antimicrobial activity against the important neonate pathogen Group B Streptococcus (Group B Strep; GBS). In addition to a structure-activity relationship (SAR) study, we observed that HMOs are effective adjuvants for intracellular-targeting antibiotics against GBS. This included two antibiotics that GBS has evolved resistance to. At their half maximal inhibitory concentration (IC₅₀), heterogeneous HMOs reduced the minimum inhibitory concentration (MIC) of select antibiotics by up to 32-fold. Similarly, we observed that HMOs potentiate the activity of polymyxin B (Gram-negative-selective antibiotic) against GBS (Gram-positive species). Based on these collective discoveries, we hypothesized that HMOs function by increasing bacterial cell permeability, which would be a novel mode of action for these molecules. This hypothesis was validated as HMOs were found to increase membrane permeability by around 30% compared to an untreated control. The question that remains is how exactly HMOs interact with bacterial membranes to induce permeability changes (i.e., through promiscuous insertion into the bilayer, engagement of proteins involved in membrane synthesis, or HMO-capsular polysaccharide interactions). Our immediate efforts in this regard are to apply chemoproteomics to identify the molecular target(s) of HMOs. These investigations are enabled through manipulation of HMOs produced via total synthesis or enzymatic and whole-cell microbial biotransformation.