Mother Knows Best: Deciphering the Antibacterial Properties of

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Article Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Mother Knows Best: Deciphering the Antibacterial Properties of Human Milk Oligosaccharides Kelly M. Craft and Steven D. Townsend*

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Department of Chemistry, Vanderbilt University, 7330 Stevenson Science Center, Nashville, Tennessee 37235, United States CONSPECTUS: 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 (Gramnegative-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.

I. INTRODUCTION Glycobiology has emerged as one of the most influential subdisciplines of chemical biology.1−6 At its core, the objective of glycobiology research is to decipher the roles carbohydrates play in biological systems. Deciphering carbohydrate function, however, often poses a monumental challenge due to the complexity and diversity inherent to carbohydrate synthesis. Unlike nucleotides and proteins which are synthesized through reliably templated biosynthetic pathways, carbohydrate synthesis is not under tight genetic supervision. In other words, glycan structure is not genetically encoded. To add to this complexity, carbohydrate structure can vary immensely due to a vast array of monosaccharide building blocks which can vary in ring size, locations and types of glycosidic linkages, and modifications such as sulfonation and phosphorylation. Moreover, in contrast to amide and phosphodiester linkages, glycosidic bond formation generates a new stereocenter. Given the intricate nature of carbohydrate synthesis, it is perhaps © XXXX American Chemical Society

unsurprising that access to structurally defined molecules represents a major hurdle in glycobiology research. Similarly, it should not be surprising that without access to these molecules carbohydrate function is destined to remain broadly defined. Due to the unique obstacles of carbohydrate research, it is imperative that multiple disciplines come together in a cooperative manner to work toward the common goal of deciphering carbohydrate function. Without this cooperation, the prospect of addressing the most attractive and challenging problems in glycobiology is bleak. Our lab identifies as a biology-driven synthesis group, i.e., the biological problem drives innovation in chemistry.7−10 However, when we started in 2014, we found ourselves as a total synthesis group with no expertise in biology. While the idea of cooperative science permeates our culture, in reality, Received: December 12, 2018

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DOI: 10.1021/acs.accounts.8b00630 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Accounts of Chemical Research organizing a multidisciplinary team is difficult due to the communication gap between chemists and biologists and the differing ways these researchers often approach problems. For example, biologists excel at defining macromolecular function, yet the substrates they study often lack structural definition or homogeneity. Contrarily, chemists focus on comparatively narrow, albeit well-defined, structures but often overlook function. In the course of our studies, we consistently encountered these conflicting interests. While we could articulate the importance of our project to chemists, our colleagues in biology were often unenthusiastic. In fact, one of our current team members initially classified the underlying logic for the program as “an oversimplification.” Another member thought the topic was simply too difficult for a junior lab to undertake. Despite these criticisms, it was our hope that we could change their minds by passing on the bravado of our synthetic organic chemistry training. After all, as Prof. Danishefsky told one of us as a postdoc: “The idea that the only limitations to the power of synthesis to create highly complex targets arises f rom limitations in imagination and persistence is a particularly unique message which chemistry offers.” For the last 5 years, our team has been engaged in a program focused on the identification, synthesis, and biological evaluation of HMOs. The key resource which enabled initiation of this program was the Vanderbilt Pre3 Initiative, an interdisciplinary group of researchers with a shared interest in reducing the burden of adverse pregnancy outcomes and prematurity. At numerous Pre3 meetings, we engaged in spirited discussions with Prof. Jennifer Gaddy (a microbiologist with experience in bacterial pathogenesis, highresolution imaging, and animal modeling) and Prof. David Aronoff (a physician-scientist with expertise in clinical infectious disease). These discussions provided the inspiration we needed to move forward with a high-risk project wherein our organic synthesis expertise would not be involved until the second phase of the program. The details of this project are presented below.

Figure 1. Group B Streptococcus in the biofilm state. Scanning electron microscopy (SEM) images taken by the Gaddy and Townsend Research Groups.

II. GROUP B STREPTOCOCCUS INFECTION Streptococcus agalactiae (Group B Strep; GBS) is a transient, encapsulated Gram-positive diplococcus that colonizes the gastrointestinal and genital tracts of ca. 50% of pregnant women at some point in their pregnancy (Figure 1).11,12 Interestingly, this bacterium actually colonizes around 30% of all people at any given time. Although GBS colonization is asymptomatic in healthy adults, it can pose a real threat to elderly and infant populations. Indeed, GBS is a major cause of chorioamnionitis, preterm birth, septicemia, pneumonia, and meningitis.13−15 Additionally, GBS is the leading cause of neonatal sepsis in the Americas, Australia, Europe, and Africa, and is responsible for up to 12% of stillbirths worldwide.16 Transmission from mother to child, also known as vertical transmission, occurs when GBS ascends from the vagina to the amniotic fluid after onset of labor or during passage through the birth canal. To prevent this transmission, the Centers for Disease Control (CDC) recommends the use of intrapartum antibiotic prophylaxis (IAP) for mothers who screen positive for GBS; women are tested for GBS late in their third trimester.17−19 Furthermore, the CDC has outlined a set of guidelines for selection of the optimal antibiotic to use for IAP (Figure 2). For GBS positive mothers, IAP is generally administered at the time of hospital admission and every 4 h after that until the infant is delivered.18 Interestingly, we have

Figure 2. Current CDC guidelines for intrapartum antibiotic prophylaxis (IAP) against Group B Streptococcus (GBS). IAP is highly effective at preventing early-onset GBS disease in infants born to women who are colonized by GBS.

not uncovered any studies which examine the minimally effective administration of IAP. Broadly speaking, GBS is susceptible to penicillin, ampicillin, extended-spectrum penicillins, cephalosporins, and vancomycin. As penicillin has a narrow spectrum of antimicrobial activity and a well understood safety profile for both mother and infant, penicillin is the antibiotic of choice for IAP.18 Unfortunately, β-lactam allergies complicate the antibiotic selection process as this allergy obviously precludes the use of penicillin. If a patient is at “low risk” for anaphylaxis due to penicillin or cephalosporin administration, cefazolin is offered. If the patient is at “high risk” for anaphylaxis, a non-β-lactam antibiotic must be used. Selection of an appropriate non-βlactam, however, is plagued by the fact that GBS is poorly susceptible to numerous non-β-lactam antibiotics.20 For example, ca. 30% of GBS isolates are resistant to erythromycin while 20% are resistant to clindamycin.18 For this reason, erythromycin is no longer recommended for prophylaxis, and clindamycin use depends on the results of GBS antibiotic B


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Accounts of Chemical Research

Figure 3. Human milk fractionation workflow.

similarity, HMOs can serve as soluble decoy ligands for pathogen surface receptor glycans and virulence agents. Based on the established patterns of activity, our program began by testing the hypothesis that HMOs inhibit pathogenic growth. The rate limiting step in testing this hypothesis was the acquisition of human milk. Indeed, human milk is one of the few items you cannot order from Aldrich. Fortunately, Prof. Jörn Weitkamp, a pediatrician at Vanderbilt University Medical Center, joined our team and was able to provide large quantities of milk that was generously donated by his patients. One factor that distinguished our program from others in the field was our decision not to pool milk samples from the outset. We hypothesized that assaying each sample independently would enable observation of phenotypic differences in samples, i.e., samples would vary in activity from mother to mother. Although partitioning milk into its macromolecular components may seem esoteric, the separation and purification process is actually well-established. Our procedure is illustrated in Figure 3. Briefly, fats are removed first via centrifugation. Proteins are then precipitated using ethanol. Next, the HMOcontaining supernatant is treated with β-galactosidase to hydrolyze lactose into its constituent glucose and galactose monomers. HMOs can then be purified (i.e., desalted and separated from glucose and galactose) using size exclusion chromatography. Following this purification step, HMO extracts are analyzed using nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HRMS). Together, these techniques verify extract purity and provide information about general HMO composition. Moreover, this information allows for classification of donors as Secretors or non-Secretors; Secretor status and HMO composition are both dependent on the types of glycosyltransferases an individual possesses. A Secretor is an individual whose blood type antigens are secreted into body fluids.

susceptibility tests. While clindamycin is the go-to drug for those at high-risk for β-lactam-induced anaphylaxis, if a GBS isolate is resistant to clindamycin, vancomycin is recommended. While IAP has dramatically reduced the GBS disease burden, it does have its shortcomings. First, although IAP is largely effective at preventing the development of early-onset disease in infants, it is not effective against late-onset disease.18 Second, there are concerns that IAP treatment may kill not only GBS but also benefical bacteria that reside in an infant’s gut.21 Indeed, collateral damage to beneficial species is a common side effect of antibiotic treatment.22−24 In infants, this damage would be particularly troubling as healthy gut development is a major contributor to infant health and proper development.25,26 Finally, as is evidenced by the resistance of GBS to antibiotics like erythromycin, resistance evolution resulting from mis- and overuse of antibiotics is an urgent concern. Given these shortcomings and concerns, there is a growing need to develop non-antibiotic-based defenses against GBS.

III. ANTIBACTERIAL ACTIVITIES OF HETEROGENOUS HMO EXTRACTS Our current mechanistic understanding of how HMOs modulate bacterial behavior is limited to two areas: HMOs as prebiotics and HMOs as antiadhesive antimicrobials.27−35 The prebiotic function is attributable to the inability of infants to digest HMOs (infants are deficient in digestive glycosidases) but the ability of beneficial microorganisms in the infant’s gut to digest these complex sugars. HMOs pass intact to the large intestine where they are selectively fermented by the commensal flora, which in turn provides the commensals with a competitive advantage over pathogens. The antiadhesive function is largely attributable to structural similarities between HMOs and various cell surface glycans. Because of this C


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One of the more intriguing aspects of our initial studies was that each woman produced HMO mixtures of differing levels of activity. Initially, we hypothesized that these differences were related to Secretor status as Secretor status and HMO composition are closely related. However, assaying HMOs from 19 donors of varying blood groups against three strains of GBS revealed no relationship between biological activity and Secretor status. In fact, the data suggested that HMOs from Secretors and non-Secretors generally demonstrated comparable levels of biological activity.36 This result was admittedly perplexing as we expected the general differences in HMO composition between Secretors and non-Secretors to result in different HMO activity profiles for these two groups. Without validation for this initial hypothesis, we formulated a second hypothesis that women who are colonized by GBS produce more potent HMOs. Evaluation of this hypothesis is ongoing. Returning to the central hypothesis of our program, we moved to evaluate the antivirulence properties of HMOs, namely their ability to inhibit biofilm production; biofilm production contributes to a pathogen’s ability to survive in a hostile host environment. It is no coincidence that the ESKAPE pathogens, which are characterized by high levels of antimicrobial resistance, are also prolific biofilm producers.40 Thus, antibiofilm agents represent another desirable class of compounds. In order to test our hypothesis that HMOs inhibit biofilm production, a plate-based assay was once again employed. With this assay, bacterial growth and biofilm formation were quantitated using spectrophotometric readings at OD600 and OD560, respectively. More specifically, following initial biomass readings, the free-floating bacteria (not in the biofilm) were removed and the remaining adherent bacteria (in the biofilm) were stained with crystal violet. The ratio of biofilm/biomass (biofilm produced per bacterial cell) then gave us a quantitative measurement of biofilm production. Excitingly, we observed that HMOs possess antibiofilm activity against multiple Gram-positive species. We did not, however, observe any antibiofilm activity in the A. baumannii model (Table 1).36 In addition to quantitative effects, the qualitative effects of HMO supplementation on GBS biofilm production were evaluated using high-resolution scanning electron microscopy (SEM) to evaluate changes in biofilm size and architecture (Figure 4). In general, we observed that biofilms grown in the

With purified HMOs in hand, we were ready to test our central hypothesis that HMOs possess antimicrobial and antivirulence activities. Given the obvious relevance of GBS to infant health, we initially elected to screen for HMO antimicrobial activity against GBS. Antimicrobial activity was assessed by evaluating GBS growth and viability over 24 h using a plate-based assay. This assay allowed for quantification of bacterial growth via spectrophotometric readings at an optical density of 600 nm (OD600) and viability via enumeration of colony forming units (CFUs). To our delight, we discovered that HMOs possess potent bacteriostatic activity against GBS (Table 1).36,37 Buoyed by this finding, we set our sights on the current holey grail of pathogens: the ESKAPE pathogens. Table 1. HMO Antimicrobial and Antibiofilm Activity Against Various Bacterial Pathogens at 24 h maximum growth inhibition maximum biofilm inhibition

S. agalactiae

S. aureus

A. baumannii

89% 93%

none 60%

11% none

The ESKAPE pathogens are a collection of pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) that are aptly named for their ability to “escape” the action of antimicrobials.38−40 Thus, compounds which are active against this cohort of pathogens are extremely desirable. For our studies, we elected to screen against Staphylococcus aureus, a well-known Gram-positive pathogen, and Acinetobacter baumannii, an up-and-coming Gram-negative pathogen. Each of these microbes are significant to child health.35 Encouragingly, HMOs demonstrated antimicrobial activity against A. baumannii, though this activity was significantly diminished compared to the GBS model (Table 1). Somewhat surprisingly, HMOs had little to no effect on the growth of methicillin-resistant S. aureus (MRSA) (Table 1).36 As a final note, while these studies showed that HMOs possess antimicrobial activity, at the time, we did not understand the mechanism of action behind the observed activity. Ultimately, deciphering this mechanism would require additional experiments. These experiments are described below.

Figure 4. Effect of HMO supplementation on GBS biofilm production. Comparison of GBS grown in THB + 1% glucose (left) and GBS grown in THB + 1% glucose further supplemented with HMOs (right) shows altered biofilm architecture and morphology due to HMO treatment. D


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Figure 5. Results of HMO/antibiotic combination treatments. Antibiotic structures and maximum significant MIC fold reductions for combination treatments are provided.

presence of HMOS were less diffuse, less voluminous, and lacking in nutrient channels. Moreover, we observed that

HMO supplementation could alter GBS morphology. Indeed, while GBS phenotypically forms long, organized chains, HMO E


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Figure 6. LIVE/DEAD BacLight assay schematic. This assay can be used to determine the extent to which a compound permeabilizes the bacterial membrane.

quantitatively access membrane permeability/integrity. SYTO 9 is able to pass through intact membranes and subsequently stain cells green while the larger PI can only pass through membranes with breached integrity (associated with dead cells) to stain cells red. As PI can quench the signal of SYTO 9, the ratio of SYTO 9 to PI signal, i.e. green to red, yields a measurement of live to dead cells or intact to nonintact cell membranes (Figure 6). Gratifyingly, the results of this assay indicated that HMOs increase GBS membrane permeability in a concentration-dependent manner (Table 2).41

supplementation caused a truncated chain phenotype and a condensed packing morphology within the biofilm. In addition to SEM, confocal laser scanning microscopy was used to investigate the structural and compositional features of GBS biofilms. Using this microscopic technique, we observed that HMO supplementation reduced biofilm thickness. Furthermore, we observed that oligosaccharides were localized at the top of the biofilms.37

IV. ADJUVANT ACTIVITY OF HETEROGENEOUS HMO EXTRACTS Building off the results of our previous studies, we hypothesized that HMOs could sensitize GBS to antimicrobial agents. Importantly, testing this hypothesis enables examination of the therapeutic utility of HMOs in combination therapies while simultaneously helping to decipher the mechanism(s) underlying HMO antibacterial activity. In a first-generation approach, we demonstrated that HMOs could potentiate the activity of polymyxin B against GBS (Figure 5).37 Polymyxins are cell membrane-targeting antibiotics that are effective against Gram-negative bacteria but are typically inactive against Gram-positive bacteria. This dichotomy has been attributed to the extra protection afforded to the cell membrane in Gram-positive bacteria by a thick peptidoglycan layer; in Gram-negative bacteria, the membrane is the outermost layer. Thus, we hypothesized that HMOs damage the outer peptidoglycan layer which subsequently increases polymyxin B’s access to the membrane and resultantly potentiates its activity. In a second-generation study, we observed that HMOs potentiate the activity of four classes of intracellular-targeting antibiotics: aminoglycosides, lincosamides, macrolides, and tetracyclines.41 Conversely, no HMO-mediated activity potentiation was observed for cell wall-targeting antibiotics including β-lactams, cephalosporins, carbapenems, and glycopeptides (Figure 5). In the GBS model, HMO combination therapies resulted in MIC reductions of up to 16-fold for clindamycin and gentamicin and up to 32-fold for erythromycin and minocycline. In the MRSA and A. baumannii models, aminoglycosides were the only antibiotics which experienced significant HMO-mediated activity potentiation. Specifically, we observed an 8-fold reduction for gentamicin against MRSA and 4-fold reductions for amikacin and tobramycin against A. baumannii. In light of these collective results, we hypothesized that HMOs act by increasing cellular permeability.

Table 2. Results of the LIVE/DEAD BacLight Assaya LIVE/DEAD cell ratio decrease from control HMO concn (mg/mL)

S. agalactiae strain GB590

S. agalactiae strain CNCTC 10/84

S. agalactiae strain GB2

20.5 10.25 5.25 2.56

28% 28% 27% 33%

28% 30% 43% 30%

30% 27% 23% 54%

a

Results after 24 h of growth in THB. Control = bacteria grown in absence of HMOs.

VI. HMO STRUCTURE ACTIVITY RELATIONSHIPS At this stage in the program, we had disclosed that HMOs possess antibacterial activity against multiple pathogens and that this activity was attributable to alteration of bacterial membrane permeability. That said, we still lacked knowledge about the activities of individual HMOs. To tease out this information, our original plan was to fractionate whole HMO extracts into smaller mixtures based on mass using size exclusion chromatography. We could then test each mixture independently in the hopes of narrowing down the list of potential active compounds. This information would then be used to guide synthetic efforts toward single-entity HMOs. Following successful synthesis, the individual compounds could be tested for activity to give the desired SARs. Though viable, it remained that this plan would require a significant commitment of time and resources. Fortunately, a bit of luck came our way in the form of a generous donor. Glycom, a Danish biotech company, provided us with numerous HMOs that are both cost and, some may argue, synthetically prohibitive. Glycom is dedicated to the scientific and clinical development of HMOs for a broad range of applications and, as such, are wonderful collaborators for our program. With their gift, we were able to assay 17 HMOs against GBS (Figure 7). The results are summarized in Table 3.7,42,43 While varying levels of activity were observed for the compounds, several general trends emerged. First, while numerous single compounds were potent antimicrobials, overall, no single compound was as effective of an

V. HMO ANTIMICROBIAL MECHANISM OF ACTION To evaluate whether HMO antimicrobial activity was associated with changes in bacterial cell membrane permeability, we employed the LIVE/DEAD BacLight assay. This assay uses two stains, SYTO 9 and propidium iodine (PI), to F


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Figure 7. Geometric structural representations for HMOs evaluated for antimicrobial and antibiofilm activity against GBS. (A) Structures of HMO core carbohydrate structures. (B) Structures of lactose-based HMOs. (C) Structures of lacto-N-tetraose-based HMOs. (D) Structures of lacto-Nneotetraose-based HMOs.

activity (similar to heterogeneous HMO mixtures produced by patients), our future efforts are focused on interrogating synergy between different HMOs as this may be critical to determining which women produce the most potent sets of molecules.

antimicrobial as the heterogeneous HMO extract. Second, the vast majority of HMOs did not reduce biofilm production. Indeed, some compounds even increased biofilm production. Importantly, this increase is hypothesized to be a result of a high level of antimicrobial activity. Third, we discovered that different GBS strains had different susceptibilities to HMO supplementation, i.e. HMO antimicrobial activity was straindependent. This finding was particularly exciting given the increasing interest in developing narrow-spectrum antimicrobials, i.e., compounds that act on a very small subset of pathogens. Finally, as is characteristic of HMO antimicrobial activity in vivo, we found that small differences in HMO structure could drastically alter antimicrobial activity. For example, while 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL) were generally devoid of activity, difucosyllactose (DFL) possessed strong antimicrobial activity (Figure 7). Given that single entity HMOs displayed highly variable

VII. CONCLUSION In 2018, world health officials were perplexed by US opposition to a breastfeeding resolution. Given the lack of empathy toward and understanding of breastfeeding (for both mother and child), it is more important than ever to assemble interdisciplinary teams of chemists, biologists, and physician scientists to decipher, characterize, and ultimately unleash the therapeutic potential of human milk. Only interdisciplinary teams feature the specialized expertise, diverse methodologies, and theoretical approaches required to raise awareness of the applicability of human milk to address clinical problems. G


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Accounts of Chemical Research Table 3. Summary of Antimicrobial and Antibiofilm Activities of HMOs Against GBSa HMO lactose (Lac) 2′-fucosyllactose (2′-FL) 3-fucosyllactose (3-FL) difucosyllactose (DFL) lacto-N-triose II (LNT II) 3′-sialyllactose (3′-SL) 6′-sialyllactose (6′-SL) lacto-N-tetraose (LNT) lacto-N-fucopentaose I (LNFP I) lacto-N-fucopentaose II (LNFP II) LS-tetrasaccharide a (LST a) disialyllacto-N-tetraose (DSLNT) lacto-N-neotetraose (LNnT) lacto-N-fucopentaose III (LNFP III) lacto-N-neohexaose (LNnH) para-lacto-N-neohexaose (para-LNnH) LS-tetrasaccharide c (LST c) heterogeneous HMO extract

avg growth reductionb

avg viability reductionb

3% 8% 15% 51% 54% 13% 18% 24% 1% 31% 38% 28% 42% 26%

0% 0% 0% 17% 12% 0% 0% 11% 24% 15% 23% 18% 13% 14%

48% 23%

12% 9%

15% 82%

16% 23%

avg biofilm reductionc

avg growth reductionb

avg viability reductionb

0% 0% 4% 0%d 0%d 0% 13% 0%d 35% 0% 0%d 0% 0%d 0%

0% 9% 0% 0% 22% 0% 0% 0% 0% 0% 42% 18% 5% 0%

2% 9% 4% 11% 8% 5% 4% 0% 10% 9% 25% 21% 4% 9%

0% 0% 0% 3% 0% 10% 9% 28% 0% 0% 0%d 0% 13% 6%

0%d 0%

39% 0%

15% 8%

0% 13%

35% 73%

18% 24%

0% N/A

0% N/A

avg biofilm reductionc

a

Strongest activity is bolded. bAverage over 24 h of growth. cAverage at 24 h of growth. dSignificantly increased biofilm formation.

While the first chapter of our program has concluded, several goals are driving the second chapter. First, while we have uncovered the mechanism of action of HMO antimicrobial activity, we have yet to discover specific cellular targets of HMOs. To address this void, we are working to synthesize the first HMO-based chemoproteomic tools to identify interacting partners of HMOs. Second, while IAP is a success story for modern obstetrics, it remains that antibiotic treatment can cause collateral damage to the established flora of the mom and developing flora of the child. Collateral damage to host symbiotes is a common problem with antibiotic treatments. The extent of damage due to IAP, however, is not well-known. Thus, we are highly interested in uncovering the secondary effects of IAP and leveraging HMOs to prevent destruction of symbiotes during antibiotic exposure.

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mentorship of Prof. Amanda Bryant-Freidrich in 2005. He completed Ph.D. studies at Vanderbilt University with Prof. Gary A. Sulikowski where he worked on the total synthesis of Bielschowskysin. From there, he moved to the laboratory of Prof. Samuel J. Danishefsky at Memorial Sloan-Kettering Cancer Center and Columbia University where he worked primarily on glycoprotein synthesis and Diels−Alder methodology. Since returning to Vanderbilt in 2014, his laboratory has been dedicated to studies in organic chemistry, glycobiology, and infectious diseases.

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ACKNOWLEDGMENTS The research described above was completed by the planning and experimentation of several graduate and undergraduate student colleagues. Their intellectual excellence, professionalism, and selflessness will always be inspiring. Prof. Jennifer Gaddy is acknowledged for her support and Prof. Christian Melander is acknolwedged for his mentorship. The program was enabled by the kind donation of human milk from a large number of women from across the United States. Their generosity and sacrifice is overwhelming. Financial support for our program in human milk science is provided by Vanderbilt University, the Vanderbilt Microbiome Initiative, the Vanderbilt Pre3 Initiative, a Deans Faculty Fellowship, Glycosyn, Friesland Campina, and the National Science Foundation (CAREER Award to SDT: CHE-1847804). K.M.C. was supported by the Vanderbilt Chemical Biology Interface (CBI) training program (T32 GM065086), the Vanderbilt Pre3 Initiative (travel grant), and the Mitchum E. Warren, Jr. Graduate Research Fellowship. Additional support from Glycom, Prolacta Biosciences, and Medolac is gratefully acknowledged.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Kelly M. Craft: 0000-0001-9753-2801 Steven D. Townsend: 0000-0001-5362-7235 Notes

The authors declare no competing financial interest. Biographies Kelly M. Craft was born in Atlanta in 1992. She received a B.S. in Chemistry from Birmingham-Southern College under the mentorship of Prof. David A. Schedler in 2014. She is currently completing her Ph.D. studies with Prof. Steven D. Townsend at Vanderbilt University. Her graduate studies have primarily focused on the synthesis and biological evaluation of human milk oligosaccharides. She will begin a postdoctoral fellowship with Prof. Andrew G. Myers at Harvard University in the Summer of 2019.

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REFERENCES

(1) Wang, L. X.; Davis, B. G. Realizing the Promise of Chemical Glycobiology. Chem. Sci. 2013, 4, 3381−3394. (2) Dwek, R. A. Glycobiology: Toward Understanding the Function of Sugars. Chem. Rev. 1996, 96, 683−720.

Steven D. Townsend was born in Detroit in 1983. He received a B.S. with Honors in Chemistry from Oakland University under the H


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Accounts of Chemical Research (3) Boons, G. J.; Wu, P. Chemical Glycobiology. Glycobiology 2016, 26, 788. (4) Kiessling, L. L.; Splain, R. A. Chemical approaches to glycobiology. Annu. Rev. Biochem. 2010, 79, 619−653. (5) Seeberger, P. H. Chemical glycobiology: why now? Nat. Chem. Biol. 2009, 5, 368−372. (6) Bertozzi, C. R.; Kiessling, L. L. Chemical glycobiology. Science 2001, 291, 2357−2364. (7) Craft, K. M.; Townsend, S. D. Synthesis of lacto-N-tetraose. Carbohydr. Res. 2017, 440−441, 43−50. (8) Keith, D. J.; Townsend, S. D. Direct, microwave-assisted substitution of anomeric nitrate-esters. Carbohydr. Res. 2017, 442, 20−24. (9) Guan, Y.; Townsend, S. D. Metal-Free Synthesis of Unsymmetrical Organoselenides and Selenoglycosides. Org. Lett. 2017, 19, 5252−5255. (10) Keith, D. J.; Marasligiller, S. A.; Sasse, A. W.; Townsend, S. D. One-pot Microwave-assisted Conversion of Anomeric Nitrate-esters to Trichloroacetimidates. J. Visualized Exp. 2018, No. e56610. (11) Stoll, B. J.; Hansen, N. I.; Sanchez, P. J.; Faix, R. G.; Poindexter, B. B.; Van Meurs, K. P.; Bizzarro, M. J.; Goldberg, R. N.; Frantz, I. D., 3rd; Hale, E. C.; Shankaran, S.; Kennedy, K.; Carlo, W. A.; Watterberg, K. L.; Bell, E. F.; Walsh, M. C.; Schibler, K.; Laptook, A. R.; Shane, A. L.; Schrag, S. J.; Das, A.; Higgins, R. D.; Eunice Kennedy Shriver National Institute of Child, H.; Human Development Neonatal Research, N. Early onset neonatal sepsis: the burden of group B Streptococcal and E. coli disease continues. Pediatrics 2011, 127, 817−826. (12) Steer, A. C.; Lamagni, T.; Curtis, N.; Carapetis, J. R. Invasive group a streptococcal disease: epidemiology, pathogenesis and management. Drugs 2012, 72, 1213−1227. (13) Eickhoff, T. C.; Klein, J. O.; Daly, A. K.; Ingall, D.; Finland, M. Neonatal Sepsis and Other Infections Due to Group B BetaHemolytic Streptococci. N. Engl. J. Med. 1964, 271, 1221−1228. (14) Franciosi, R. A.; Knostman, J. D.; Zimmerman, R. A. Group B streptococcal neonatal and infant infections. J. Pediatr. 1973, 82, 707− 718. (15) Horvath, B.; Lakatos, F.; Toth, C.; Bodecs, T.; Bodis, J. Silent chorioamnionitis and associated pregnancy outcomes: a review of clinical data gathered over a 16-year period. J. Perinat. Med. 2014, 42, 441−447. (16) Nan, C.; Dangor, Z.; Cutland, C. L.; Edwards, M. S.; Madhi, S. A.; Cunnington, M. C. Maternal group B Streptococcus-related stillbirth: a systematic review. BJOG 2015, 122, 1437−1445. (17) Schrag, S. J. The past and future of perinatal group B streptococcal disease prevention. Clin. Infect. Dis. 2004, 39, 1136− 1138. (18) Cagno, C. K.; Pettit, J. M.; Weiss, B. D. Prevention of Perinatal Group B Streptococcal Disease Revised Guidelines from CDC. Am. Fam Physician 2010, 86, 59−65. (19) Schrag, S. J.; Verani, J. R. Intrapartum antibiotic prophylaxis for the prevention of perinatal group B streptococcal disease: experience in the United States and implications for a potential group B streptococcal vaccine. Vaccine 2013, 31 (Suppl 4), D20−D26. (20) Shore, E. M.; Yudin, M. H. Choice of antibiotic for group B streptococcus in women in labour based on antibiotic sensitivity testing. J. Obstet Gynaecol Can. 2012, 34, 230−235. (21) Gibbs, R. S.; Schrag, S.; Schuchat, A. Perinatal infections due to group B streptococci. Obstet. Gynecol. 2004, 104, 1062−1076. (22) Tanaka, S.; Kobayashi, T.; Songjinda, P.; Tateyama, A.; Tsubouchi, M.; Kiyohara, C.; Shirakawa, T.; Sonomoto, K.; Nakayama, J. Influence of antibiotic exposure in the early postnatal period on the development of intestinal microbiota. FEMS Immunol. Med. Microbiol. 2009, 56, 80−87. (23) Maxson, T.; Mitchell, D. A. Targeted Treatment for Bacterial Infections: Prospects for Pathogen-Specific Antibiotics Coupled with Rapid Diagnostics. Tetrahedron 2016, 72, 3609−3624. (24) Bizzarro, M. J.; Dembry, L. M.; Baltimore, R. S.; Gallagher, P. G. Changing patterns in neonatal Escherichia coli sepsis and

ampicillin resistance in the era of intrapartum antibiotic prophylaxis. Pediatrics 2008, 121, 689−696. (25) Craft, K. M.; Townsend, S. D. The Human Milk Glycome as a Defense Against Infectious Diseases: Rationale, Challenges, and Opportunities. ACS Infect. Dis. 2018, 4, 77−83. (26) Ackerman, D. L.; Craft, K. M.; Townsend, S. D. Infant food applications of complex carbohydrates: Structure, synthesis, and function. Carbohydr. Res. 2017, 437, 16−27. (27) Garrido, D.; Dallas, D. C.; Mills, D. A. Consumption of human milk glycoconjugates by infant-associated bifidobacteria: mechanisms and implications. Microbiology 2013, 159, 649−664. (28) Chichlowski, M.; De Lartigue, G.; German, J. B.; Raybould, H. E.; Mills, D. A. Bifidobacteria isolated from infants and cultured on human milk oligosaccharides affect intestinal epithelial function. J. Pediatr. Gastroenterol. Nutr. 2012, 55, 321−327. (29) Zivkovic, A. M.; German, J. B.; Lebrilla, C. B.; Mills, D. A. Human milk glycobiome and its impact on the infant gastrointestinal microbiota. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (Suppl 1), 4653− 4658. (30) Marcobal, A.; Barboza, M.; Froehlich, J. W.; Block, D. E.; German, J. B.; Lebrilla, C. B.; Mills, D. A. Consumption of human milk oligosaccharides by gut-related microbes. J. Agric. Food Chem. 2010, 58, 5334−5340. (31) Newburg, D. S.; Grave, G. Recent advances in human milk glycobiology. Pediatr. Res. 2014, 75, 675−679. (32) Newburg, D. S. Glycobiology of human milk. Biochemistry (Moscow) 2013, 78, 771−785. (33) Morrow, A. L.; Ruiz-Palacios, G. M.; Jiang, X.; Newburg, D. S. Human-milk glycans that inhibit pathogen binding protect breastfeeding infants against infectious diarrhea. J. Nutr. 2005, 135, 1304− 1307. (34) Newburg, D. S. Human milk glycoconjugates that inhibit pathogens. Curr. Med. Chem. 1999, 6, 117−127. (35) Craft, K. M.; Townsend, S. D. The Human Milk Glycome as a Defense Against Infectious Diseases: Rationale, Challenges, and Opportunities. ACS Infect. Dis. 2018, 4, 77−83. (36) Ackerman, D. L.; Craft, K. M.; Doster, R. S.; Weitkamp, J. H.; Aronoff, D. M.; Gaddy, J. A.; Townsend, S. D. Antimicrobial and Antibiofilm Activity of Human Milk Oligosaccharides against Streptococcus agalactiae, Staphylococcus aureus, and Acinetobacter baumannii. ACS Infect. Dis. 2018, 4, 315−324. (37) Ackerman, D. L.; Doster, R. S.; Weitkamp, J. H.; Aronoff, D. M.; Gaddy, J. A.; Townsend, S. D. Human Milk Oligosaccharides Exhibit Antimicrobial and Antibiofilm Properties against Group B Streptococcus. ACS Infect. Dis. 2017, 3, 595−605. (38) Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E.; Gilbert, D.; Rice, L. B.; Scheld, M.; Spellberg, B.; Bartlett, J. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin. Infect. Dis. 2009, 48, 1−12. (39) Pendleton, J. N.; Gorman, S. P.; Gilmore, B. F. Clinical relevance of the ESKAPE pathogens. Expert Rev. Anti-Infect. Ther. 2013, 11, 297−308. (40) Santajit, S.; Indrawattana, N. Mechanisms of Antimicrobial Resistance in ESKAPE Pathogens. BioMed Res. Int. 2016, 2016, 2475067. (41) Craft, K. M.; Gaddy, J. A.; Townsend, S. D. Human Milk Oligosaccharides (HMOs) Sensitize Group B Streptococcus to Clindamycin, Erythromycin, Gentamicin, and Minocycline on a Strain Specific Basis. ACS Chem. Biol. 2018, 13, 2020−2026. (42) Craft, K. M.; Thomas, H. C.; Townsend, S. D. Sialylated variants of lacto-N-tetraose exhibit antimicrobial activity against Group B Streptococcus. Org. Biomol. Chem. 2019, DOI: 10.1039/ C8OB02080A. (43) Craft, K. M.; Thomas, H. C.; Townsend, S. D. Interrogation of Human Milk Oligosaccharide Fucosylation Patterns for Antimicrobial and Antibiofilm Trends in Group B Streptococcus. ACS Infect. Dis. 2018, 4, 1755−1765.

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