Interrogation of Human Milk Oligosaccharide Fucosylation Patterns for

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Interrogation of Human Milk Oligosaccharide Fucosylation Patterns for Antimicrobial and Antibiofilm Trends in Group B Streptococcus Kelly M Craft, Harrison C Thomas, and Steven D. Townsend ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00234 • Publication Date (Web): 13 Oct 2018 Downloaded from http://pubs.acs.org on October 15, 2018

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ACS Infectious Diseases

Interrogation of Human Milk Oligosaccharide Fucosylation Patterns for Antimicrobial and Antibiofilm Trends in Group B Streptococcus Kelly M. Craft1, Harrison C. Thomas1, and Steven D. Townsend1,2,3* 1 Department

of Chemistry, Vanderbilt University, 7330 Stevenson Center, Nashville, Tennessee 37235,

United States 2 Institute

of Chemical Biology, Vanderbilt University, 896 Preston Research Building, Nashville,

Tennessee 37232, United States 3 Vanderbilt

Institute for Infection, Immunology, and Inflammation, Vanderbilt University, Medical

Center North A-5302, 1161 21st Ave South, Nashville, Tennessee 37232, United States *Corresponding author: [email protected] Supporting Information Placeholder For newborns, human milk oligosaccharides (HMOs) serve as an important source of protection against pathogenic Gram-positive and Gram-negative bacteria. HMOs most notably prevent infection by functionung as decoy receptors that bind pathogens to prevent cellular adhesion. HMOs also play a protective role by acting as prebiotics that selectively promote the growth of symbiotic gut bacteria over pathogenic species. Fucosylated HMOs in particular are well-known for their roles as both decoy receptors and prebiotics. Recently, we discovered that HMOs possess antimicrobial activity against Group B Streptococcus (GBS) by increasing cellular permeability. As HMO extracts from a single donor can contain over 100 different structures, however, studies using heterogenous HMO mixtures do not provide insight into the specific structural requirements needed to achieve antimicrobial activity. In this study, we 1 ACS Paragon Plus Environment

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address this void by completing a structure activity study on the antimicrobial and antibiofilm activities of 6 neutral, fucosylated and 5 neutral, nonfucosylated HMOs against GBS. We determined that while the presence of fucose alone does not correlate to antimicrobial activity, the location and degree of fucosylation does play a key role in HMO antimicrobial activity. Moreover, the antimicrobial and antibiofilm activities of single HMOs were found to be strain-specific. This further supports our vision of developing narrow-spectrum antibacterial agents against GBS. Keywords: human milk oligosaccharide, HMO, fucose, fucosylation, Group B Strep, GBS

Infection is characterized by a pathogenic microorganism invading a host and subsequently multiplying in association with the host's tissues. The capacity of a bacterium to cause disease reflects its relative pathogenicity. On this basis, bacteria can be organized into three groups. The first group are the primary pathogens. These pathogens are responsible for disease due to their presence inside a healthy host. For example, Camplyobacter spp is known to cause infectious diarrhea in healthy patients.1-2 Opportunistic pathogens on the other hand are pathogens that cause disease in a host that has a compromised defense system. Additionally, this class of pathogen can cause disease due to atypical access to the inside of a host. Acinetobacter baumannii is an example of an increasingly important opportunistic pathogen. A. baumannii has been shown to have high incidences of infection among immunocompromised hosts and patients in intensive care units.3-4 In contrast to primary and opportunistic pathogens, bacteria that rarely cause human disease fall into the final group, the non-pathogenic bacteria. Bifidobacteria, for example, are non-pathogenic gut microbes that not only fail to cause disease but are important for the development and maintenance of a healthy gut microbiome.5-6 While these bacterial classifications may appear concrete, there are certain bacteria which can occasionally transcend these groupings. For example, some bacterial species are generally non-pathogenic but can become opportunistic pathogens under the right conditions. Streptococcus agalactiae (Group B 2 ACS Paragon Plus Environment

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Streptococcus, GBS) is a prime example of this reality. GBS is an asymptomatic colonizer of the digestive and genitourinary tracts of healthy humans (non-pathogenic bacteria). However, in neonates and immunocompromised adults, GBS can cause invasive infections (opportunistic pathogen). GBS infection in infants is attributed mainly to transmission from infected mother to infant during labor and delivery. As a result of this vertical transmission, GBS is a primary cause of septicemia, meningitis, and substantial pregnancy-related neonate morbidity. While implementation of intrapartum antibiotic prophylaxis (IAP) during labor and delivery has resulted in a dramatic decrease in the incidence of early-onset neonatal GBS disease, IAP is unfortunately not effective at preventing late-onset GBS disease.7-9 A. S. agalactiae (GBS) (3 strains)

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Figure 1. Summary of previously disclosed antibacterial activities of heterogenous HMO extracts (A) Antimicrobial and antibiofilm activity of HMOs.10-11 (B) Maximum minimum inhibitory concentration (MIC) fold reductions when antibiotics are used in combination therapies with HMOs.12 Recently, we discovered that human milk oligosaccharides (HMOs) modulate growth and biofilm production for several bacterial pathogens (Figure 1A).10-11 Heterogenous HMOs isolated from 19 3 ACS Paragon Plus Environment


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patients exhibited antimicrobial and antibiofilm activity against GBS, antibiofilm activity against methicillin-resistant Staphylococcus aureus (MRSA), and antimicrobial activity against A. baumannii. Taken together, these results show the breadth of activity as HMOs were found to have antibacterial activity against both Gram-positive and Gram-negative pathogens. Furthermore, we discovered that HMO extracts potentiate the activity of aminoglycosides, macrolides tetracyclines, and lincosamides (intracellular targeting antibiotics) against GBS. HMOs, however, do not potentiate glycopeptides or -lactams (antibiotics that target cell wall synthesis) (Figure 1B).12 These findings are notable as GBS is not particularly sensitive to aminoglycosides, tetracyclines, and macrolides. Additionally, we observecd that HMOs potentiate aminoglycosides against A. baumannii and S. aureus. Based on these potentiation patterns, we hypothesized that HMOs increase cellular permeability. This hypothesis was validated using a live/dead assay which revealed that HMOs increase membrane permeability toward propidium iodide in the GBS model. While our studies thus far demonstrate the potential therapeutic utility of HMOs, the results lack a description of the antibacterial activities of single-entity HMOs. As biological function is intimately tied to HMO structure, we questioned which HMOs were most effective.13 Determining specific structureactivity relationships is difficult, though, as nearly 200 distinct HMOs have been identified, each incorporating up to 50 monosaccharide residues.14-16 Structurally, HMOs are composed of just five monosaccharide building blocks: glucose (Glc), N-acetylglucosamine (GlcNAc), galactose (Gal), fucose (Fuc), and N-acetylneuraminic acid (Neu5Ac) or sialic acid (Sia). Lactose (Gal-1,4Glc) forms the reducing end of all HMOs and can be elongated with “n” units of N-acetyllactosamine (Gal1,3GlcNAc). HMO chains are ultimately terminated by the addition of lacto-N-biose (Gal1,4GlcNAc). Additionally, lactose and the polylactosamine backbone can be sialylated via -2,3 or -2,6 linkages and/or fucosylated via -1,2, -1,3, or -1,4 linkages. Depending on the presence or lack of sialic acid, HMOs aredivided into two groups: neutral and acidic. Acidic or sialylated component represents 10-20% HMOs.14,

16

Previously, we observed that several 4

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sialylated HMOs possess antimicrobial activity. Moreover, we observed the activity was unique to larger HMOs such as LS-tetrasaccharide a (LST a), LS-tetrasaccharide c (LST c), disialyllacto-N-tetraose (DSLNT). No antimicrobial activity was observed for simple sialylated lactose derivatives such as 3’sialyllactose (3’-SL) and 6’-sialyllactose (6’-SL) (Figure 2).17

Figure 2. Results of previous study assessing antimicrobial activity of sialylated HMOs against S. agalactiae strains GB590 and GB2.17 (A) Lactose, 3’-sialyllactose (3’-SL), and 6’-sialyllactose (6’-SL) were found to possess no antimicrobial activity. (B) Lacto-N-tetraose (LNT) and its sialylated variants LS-tetrasaccharide a (LST a) and disialyllacto-N-tetraose (DSLNT) were all found to possess antimicrobial activity. (C) LS-tetrasaccharide c (LST c), a sialylated lacto-N-neotetraose (LNnT) variant, was also found to possess antimicrobial activity. Lactose, LNT, and LNnT residues are in black, sialic acid residues are highlighted in orange. In contrast to the acidic fraction, the neutral HMO fraction comprises around 80-90% of the total HMO concentration. Moreover, over half of these neutral HMOs are fucosylated.14,

18

Unlike HMO 5



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sialylation, the process of HMO fucosylation is well understood and has been clarified based on the involvement of blood group-synthesizing fucosyltransferases (FUTs); HMO fucosylation patterns mirror blood group characteristics. For example, FUT2 incorporates fucose via α-1,2 linkages to terminal Gal whereas FUT3 incorporates fucose via α-1,4 linkages to internal GlcNAc residues. Furthermore, the relative amounts of these enzymes determine fucosylated HMO concentrations.14,19-20 Fucosylated HMOs are well-known for their ability to protect infants from pathogenic colonization.13 This ability is due largely to the fact that fucosylated HMOs often share structural homology with host epithelial cell surface glycans. Thus, fucosylated HMOs can serve as soluble receptor analogs that compete for bacterial binding with intestinal mucosa. For example, 2’-fucosyllactose (2’-FL) has been shown to inhibit the binding of Campylobacter jejuni to intestinal cells as well as the binding of noroviruses to histo-blood group antigens (HBGAs).1-2, 21-22 2’-FL as well as 3-FL (3-fucosyllactose) has also been found to inhibit the adhesion of Pseudomonas aeruginosa to epithelial cells.23-24 Furthermore, 3-FL and DFL (difucosyllactose) were shown to inhibit the adhesion of Escherichia coli to epithelial cells.25-26 Given the protective roles of various fucosylated HMOs, we sought to determine whether these properties extended to GBS. Additionally, we sought to investigate whether the presence of fucose residue(s) was necessary for antibacterial activity. In the present study, we have used our partnership with industrial carbohydrate producer Glycom to evaluate the antibacterial activity of six single-entity neutral, fucosylated HMOs as well as five neutral, non-fucosylated HMOs against two strains of GBS (Figure 3).

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Figure 3. Structures of neutral HMOs assayed for antibacterial activity against S. agalactiae strains GB590 and GB2 (A) Structures of common HMO cores lactose, lacto-N-tetraose (LNT), lacto-Nneotetraose (LNnT). (B) Structures of fucosylated HMOs 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3FL), difucosyllactose (DFL), lacto-N-fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), and lacto-N-fucopentaose III (LNFP III). (C) Structures of neutral HMOs lacto-N-triose II (LNT II), paralacto-N-neohexaose (para-LNnH), lacto-N-hexaose (LNnH). HMO core structures are in black, fucose residues are highlighted in red, non-fucosylated elongations of HMO core structures are highlighted in blue. Results and Discussion Previous work from our laboratory has shown that the antibacterial effects of heterogeneous HMOs on GBS are strain-specific.10, 12 Thus, in the present study, we again elected to screen against multiple strains of GBS in order to determine whether antibacterial effects of homogeneous HMOs are also strainspecific. Specifically, we chose to screen against S. agalactiae strains GB590 and GB2. Importantly, these 7 ACS Paragon Plus Environment


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two strains are of differing serotypes; GB590 is a serotype III strain while GB2 is a serotype Ia strain. GBS strains can be categorized as one of ten serotypes (Ia, Ib, II-IX) based on bacterial capsular polysaccharides (Figure S1).27 Serotype III strains (such as GB590) are responsible for the highest incidences of infection.28 Serotype Ia strains (GB2) are also key players in the global GBS burden. Serotypes Ia, Ib, II, III, and V account for more than 85% of global invasive GBS disease occurrences.28 In addition to its human health relevance, GB2 was previously found to be the strain most susceptible to HMOs.10 Antimicrobial activity was assessed by examining GBS growth and viability in Todd-Hewitt broth (THB) over 24 h. Growth was quantitated via spectrophotometric readings at OD600 while cellular viability was evaluated through serial dilution of bacterial cultures and plating onto blood agar plates followed by enumeration of colony forming units (CFUs) the following day. GBS was grown in either THB alone or THB supplemented with ca. 5 mg/mL HMO. This concentration was selected for several reasons. First, in a previous study, we found the IC50 values of heterogenous HMO extracts against GB590 and GB2 to be around 5 mg/mL. Thus, dosing HMOs at 5 mg/mL would allow us to observe potential antibacterial activities without obliterating bacterial growth. Furthermore, this concentration is physiologically relevant as HMOs are typically found in milk between 5-25 mg/mL. Perhaps most importantly, given the “irreplaceable” nature of the HMOs used in this study, testing at the low end of physiological concentration allowed us to more thoroughly evaluate antibacterial activity. In addition to the six fucosylated HMOs, an evaluation of lactose, LNT, and LNnT is presented. Lactose serves as the core structure for 2’-FL, 3-FL, and DFL. Similarly, LNT is common to LNFP I and II while LNnT is the core of LNFP III. Inclusion of lactose, LNT, and LNnT therefore allows for determination of whether antibacterial activity is contingent on the presence of fucose residues. In addition to LNT and LNnT, three additional neutral, nonfucosylated HMOs were evaluated: lacto-N-triose II (LNT II), lacto-N-neohexaose (LNnH), and para-lacto-N-neohexaose (para-LNnH). Finally, as multiple laboratories, including our own, have found that heterogenous HMO mixtures possess stronger 8 ACS Paragon Plus Environment

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antimicrobial activity than single HMOs, we also elected to assay against an HMO mixture composed of whole HMO extracts from multiple donor milk samples.29 This accommodates evaluating whether any single HMO is more effective than a mixture. The antimicrobial activities of HMOs against GB590, as determined by changes in GBS growth and viability compared to GBS grown in media, are shown in Figure 4. While numerous single HMOs were found to significantly reduce bacterial growth and viability, no single HMO had as profound an effect as the HMO mixture. In terms of growth, the mixture was able to decrease growth by over 80% for the entirety of the first 8h. Impressively, by 24 h, bacterial growth had rebounded to 70% that of GBS grown in media. Similar trends were observed for bacterial viability. Namely, the mixture significantly reduced viability for the first 8 h with reductions reaching almost 40%. As was seen with GBS growth, by 24 h, cellular viability had begun to resemble that of the GBS grown in media. The ability of the bacteria to recover after being challenged with HMOs was not, however, unexpected. Indeed, we and others have found that HMOs act as bacteriostatic agents as opposed to bactericidal agents when dosed at the low end of physiological levels.11-12, 30 A.

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Figure 4. Effects of single-entity, neutral HMOs at 5 mg/mL concentration on the growth and viability of GB590 in Todd-Hewitt Broth. Growth was measured via OD600 readings taken at 0, 2, 4, 6, 8, and 24 h. Mean OD600 for each HMO source and time point is indicated by the corresponding symbols. Viability was assessed via enumeration of CFU/mL performed at 0, 2, 4, 6, 8, and 24 h. Log10CFU/mL for each HMO source and time point is designated by the corresponding symbols. (A) Growth of GB590 (OD600) in the presence of neutral, fucosylated HMOs and an HMO mixture. (B) Viability of GB590 (CFU/mL) corresponding to the OD values graphed in Figure 5A. (C) Growth of GB590 (OD600) in the presence of neutral, nonfucosylated HMOs, lactose, and an HMO mixture. (D) Viability of GB590 (CFU/mL) corresponding to the OD values graphed in Figure 5C. Data displayed represent the mean OD600 or log10CFU/mL ± SEM of at least three independent experiments, each with three technical replicates. **** represents p < 0.0001 by two-way ANOVA with posthoc Dunnett’s multiple comparison test comparing the growth and viability of GBS in each HMO supplementation condition to the growth and viability of GBS in media alone. For single-HMOs, the presence or absence of fucose on a molecule was not a predictor of antimicrobial activity against GB590. For example, LNFP II and LNFP III displayed similar levels and patterns of growth and viability depressions as their nonfucosylated counterparts LNT and LNnT, respectively. Similarly, aside from significant growth reductions at 6 h, 2’-FL and 3-FL showed no more antimicrobial activity than lactose (which was devoid of any activity). Furthermore, the nonfucosylated HMOs LNT II and LNnH showed fairly similar levels of antimicrobial activity to LNFP II and LNFP III. In fact, these nonfucosylated compounds even tended to suppress growth to a greater extent on average than LNFP II and III. While the absence of fucose did not correlate to lessened antimicrobial activity, the location and number of fucose residues did appear to have an effect on HMO antimicrobial activity. For example, while LNFP II significantly reduced GB590 growth between 6 and 24 h, LNFP I did not significantly reduce growth at any point in the 24 h time frame; LNFP I and II are each monofucosylated LNT derivatives that differ only in the location of the fucose residue (Figure 3). Interestingly, while LNFP I did not significantly decrease growth, it did reduce bacterial viability between 2 and 24 h with reductions reaching as high as 30%. Additionally, it was the only HMO source to significantly decrease viability at 24 h. Conversely, LNFP II significantly decreased viability between 2 and 6 h. It is important to note, however, that between 2 and 6 h, both fucosylated LNT derivatives showed comparable reductions in viability. 10 ACS Paragon Plus Environment

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Comparison of the effects of DFL on GB590 growth and viability with those of 2’-FL and 3-FL further highlighted the importance of location and number of fucose residues. As mentioned, aside from significant growth reductions at 6 h, 2’-FL and 3-FL were devoid of antimicrobial activity. DFL, however, significantly reduced growth between 4 and 24 h and significantly reduced viability at 2 and 6 h; growth reductions ranged from around 30-50% while viability reductions were around 20%. As shown in Figure 3, DFL incorporates the functional aspects of both 2’-FL and 3-FL, i.e. DFL features -1,2 linked fucose residues at both C2’ and C3. Based on these results, it appears as though both fucose residues are necessary for the antimicrobial activity of fucosylated lactose derivatives against GB590. For the activities of nonfucosylated HMOs against GB590, a few notable trends emerged. First, LNT and LNnT, structural isomers differing only by the glycosidic linkage between the terminal galactose and subterminal glucosamine (Figure 3), had fairly similar levels and patterns of antimicrobial activity. Conversely, LNnH and para-LNnH, structural isomers differing only by the location of one Nacetyllactosamine unit (Figure 3), did show noticeable differences in antimicrobial activity. Although neither compound was able to significantly reduce viability past 4 h, LNnH did significantly reduce growth between 4 and 24 h while para-LNnH failed to significantly reduce growth at any point. Moving from GB590, the antimicrobial activities of HMOs against GB2 are shown in Figure 5. The HMO mixture was the most operative antimicrobial agent against GB2 with growth reductions reaching 95% and viability reductions up to 36%. In terms of single compound effects, as expected based on previous studies, the effects of homogenous HMOs were indeed found to be strain-specific. For example, although LNT and LNnT significantly reduced GB590 growth and viability at several time points, neither of these compounds had any effect on the growth or viability of GB2. Similarly, LNFP I, II, and III all saw reduced antimicrobial activity against GB2. In fact, generally speaking, GB2 was found to be less susceptible to individual HMO supplementation than GB590. Indeed, LNT II and LNnH were the only single compounds to significantly reduce growth during the 24 h growth window. Interestingly, LNFP III



was even found to significantly increase growth from 6 to 8 h. Impressively and uniquely, LNnH decreased growth from 4 to 24 h with reductions ranging on average around 20-50%. A.

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Figure 5. Effects of single-entity, neutral HMOs dosed at ca. 5 mg/mL on the growth and viability of GB2 in Todd-Hewitt Broth. Growth was measured via OD600 readings taken at 0, 2, 4, 6, 8, and 24 h. Mean OD600 for each HMO source and time point is indicated by the respective symbols. Viability was assessed via enumeration of CFU/mL performed at 0, 2, 4, 6, 8, and 24 h. Log10CFU/mL for each HMO source and time point is indicated by the respective symbols. (A) Growth of GB2 (OD600) in the presence of neutral, fucosylated HMOs and an HMO mixture. (B) Viability of GB2 (CFU/mL) corresponding to the OD values graphed in Figure 6A. (C) Growth of GB2 (OD600) in the presence of neutral, nonfucosylated HMOs, lactose, and an HMO mixture. (D) Viability of GB2 (CFU/mL) corresponding to the OD values graphed in Figure 6C. Data displayed represent the mean OD600 or log10CFU/mL ± SEM of at least three independent experiments, each with three technical replicates. **** represents p < 0.0001 by two-way ANOVA with posthoc Dunnett’s multiple comparison test comparing the growth and viability of GBS in each HMO supplementation condition to the growth and viability of GBS in media alone. While bacterial growth was largely unaffected by homogeneous HMO supplementation, numerous compounds did significantly decrease bacterial viability. As with growth, LNnH was the most effective compound at reducing cellular viability. LNnH decreased GB2 viability by around 15% over the entire 24 h period. Aside from 3-FL, all fucosylated compounds significantly reduced viability over several hours. Additionally, while LNT and LNnT did not reduce viability at any point, LNT II, LNnH, and para12 ACS Paragon Plus Environment

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LNnH did significantly reduce cellular viability over several hours. Although several compounds significantly decreased viability, it is important to note that the magnitudes of these reductions were universally smaller than those observed for GB590. This trend also holds true for the magnitudes of growth reductions seen for the two strains. Comparison of the antimicrobial activities of lactose, LNT, and LNnT against GB2 with their respective fucosylated derivatives shows that fucose generally appears to increase antimicrobial activity in this class of compounds, although in some cases, such as with LNFP III, the improvements in antimicrobial activity are minimal. It is also important to note that LNT II, which is a lactose-derived HMO that lacks a fucose residue, was also found to significantly reduce GB2 growth and viability. Nevertheless, although the presence of fucose was generally significant, it is not clear what effect fucose location and number of residues has on antimicrobial activity. As a final evaluation of HMO antibacterial activity, we evaluated the effects of individual HMOs on GBS biofilm formation. Biofilm production was assessed after 24 h using a plate-based assay that enables quantification of growth through spectrophotometric readings at OD600 followed by crystal violet staining and subsequent spectrophotometric reading at OD560 to measure biofilm production. To account for any associated antimicrobial activity, results are communicated as a ratio of biofilm to biomass. Biofilm production levels for GB590 and GB2 when treated with various single-entity HMOs relative to biofilm formation for GBS590 and GB2 grown in media alone are shown in Figure 6. No HMOs were found to decrease biofilm formation for either GBS strain. However, DFL, LNnT, LNT II, and LNnH did significantly increase biofilm formation in GB590.


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Relative GB2 biofilm production (OD560/OD600)

A. Relative GB590 biofilm production (OD560/OD600)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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HMOs

Figure 6. Effects of single-entity, neutral HMOs dosed at ca. 5 mg/mL on GBS biofilm production after 24 h of growth. (A) Biofilm production, denoted by the ratio of biofilm/biomass (OD560/OD600), by GB590 in the presence of single-entity HMOs relative to biofilm production in Todd-Hewitt Broth alone. Data displayed represent the relative mean biofilm/biomass ratio ± SEM of at least three independent experiments, each with three technical replicates, wherein biofilm production of GB590 in media alone is assigned a value of 100%. **** represents p < 0.0001 by one-way ANOVA, F = 9.811 with posthoc Dunnett’s multiple comparison test comparing biofilm production of GB590 in each HMO supplementation condition to biofilm production of GB590 in media alone. (B) Biofilm production, denoted by the ratio of biofilm/biomass (OD560/OD600), by GB2 in the presence of homogeneous HMOs relative to biofilm production in Todd-Hewitt Broth alone. Data displayed represent the relative mean biofilm/biomass ratio ± SEM of at least three independent experiments, each with three technical replicates, wherein biofilm production of GB2 in media alone is assigned a value of 100%. ** represents p < 0.005 by one-way ANOVA, F = 2.527 with posthoc Dunnett’s multiple comparison test comparing biofilm production of GB2 in each HMO supplementation condition to biofilm production of GB2 in media alone. Mean GBS biofilm production levels in media alone are marked with a dotted line. The promotion of biofilm production by these four compounds is not entirely unexpected as these four compounds showed some of largest and most prolonged growth repressions. Biofilm production is a wellknown bacterial strategy to protect against the action of antimicrobial agents. Indeed, the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) which are which are notorious for their abilities to resist antimicrobial action are also prolific biofilm producers.31-32 Based on our observations, we hypothesize that when challenged by strong antimicrobial agents, GBS increases biofilm production to evade antimicrobial action. A summary of the antimicrobial and antibiofilm activities of all single-entity HMO and lactose are provided in Table 1. Determinations of “strong,” “weak,” or “no” antimicrobial activity for each compound were determined using the following criteria: compounds that caused significant reductions in 14 ACS Paragon Plus Environment

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growth and/or viability over at least 3 time points were classified as having “strong” activity; compounds that caused significant reductions in growth and/or viability over 2 time points were classified as having “weak” activity; compounds that caused no significant reductions in growth and/or viability over any time point or only caused significant reductions at one time point were classified as having “no” antimicrobial activity. Determinations of “strong,” “weak,” or “no” antibiofilm activity were determined using the following criteria: compounds that caused significant reductions in biofilm formation were classified as having “strong” antibiofilm activity; compounds that did not cause significant reductions but caused average reductions greater than 25% were classified as having “weak” antibiofilm activity; compounds that caused reductions less than 25% or increased biofilm production were classified as having “no” antibiofilm activity. Table 1. Summary of antibacterial effects of fucosylated and nonfucosylated, neutral HMOs against GBS S. agalactiae strain GB590

S. agalactiae strain GB2

human milk oligosaccharide

antimicrobial activity

antibiofilm activity

antimicrobial activity

antibiofilm activity

lactose

No

No

No

No

lacto-N-tetraose (LNT)

Strong

No

No

Weak

lacto-N-neotetraose (LNnT)

Strong

No*

No

No

2’-fucosyllactose (2’-FL)

No

No

Weak

No

3-fucosyllactose (3-FL)

No

No

No

No

difucosyllactose (DFL)

Strong

No*

Weak

No

lacto-N-fucopentaose I (LNFP I)

Strong

Weak

Weak

No

lacto-N-fucopentaose II (LNFP II)

Strong

No

Weak

No

lacto-N-fucopentaose III (LNFP III)

Strong

No

Weak

No

lacto-N-triose II (LNT II)

Strong

No*

Weak

No

para-lacto-N-neohexaose (paraLNnH)

No

No

Weak

No

lacto-N-hexaose (LNnH)

Strong

No*

Strong

No

*significantly increased biofilm production



Page 16 of 28

Given the strong antimicrobial activities of several single-entity HMOs against GBS, the last experiment described in this study focuses on testing the hypothesis that challenging GBS with an HMO mixture consisting of the most active HMOs would yield antimicrobial activity equal or superior to whole HMO extracts. As HMO antimicrobial activity was found to be strain-specific, different combinations of HMOs were used for GB590 and GB2. For GB590, the HMO mixture consisting of the most active compounds, denoted as GB590 HMO mixture in Figure S2 and S4, consisted of the following HMOs in equal quantities: LNFP I, LNFP II, LNFP III, LNnT, and LST a (Figure 7A). LST a was shown in a previous study to significantly decrease both GB590 growth and viability.17 For GB2, the HMO mixture consisting of the most active compounds, denoted as GB2 HMO mixture in Figure S3 and S4, contained the following HMOs in equal quantities: DSLNT, LST a, LST c, LNT II, and DFL (Figure 7B). DSLNT, LST a, and LST c were previously shown to possess strong antimicrobial activity against GB2.17


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Figure 8. Structures of HMOs used in custom HMO mixtures (A) Structures of HMOs assayed against S. agalactiae strain GB590. (B) Structures of HMOs assayed against S. agalactiae strain GB2. HMO core structures are shown in black, fucose residues are highlighted in red, sialic acid residues are highlighted in orange, non-fucosylated elongations of HMO core structures are highlighted in blue. In addition to the mixtures consisting of the five most potent antimicrobial HMOs for each GBS strain, we also elected to screen an HMO mixture that attempted to mimic the general composition of whole HMO extracts; this mixture is denoted as HMO extract mimic in Figures S2-4.16, 18, 33 Specifically, our goal was to duplicate both the makeup and concentration of the various classes of compounds present in HMO extracts, i.e. acidic/sialylated; neutral, fucosylated; and neutral, non-fucosylated. While whole HMO extracts can contain over 100 different HMO structures, the HMO extract mimic contained only 10 HMOs (see Supporting Information). In the assays, each custom HMO mixture was dosed at ca. 5 mg/mL. 17 ACS Paragon Plus Environment


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Ultimately, while each of the mixtures was capable of significantly reducing GB590 and/or GB2 growth and viability, none of the custom mixtures showed comparable antimicrobial activity to that of the whole HMO extract (Figures S2 and S3). Additionally, the custom mixtures possessed similar levels and patterns of activity to one another. Interestingly, against GB2, the HMO extract mimic and the GB2specific HMO mixture (GB2 HMO mixture) actually significantly increased GB2 viability starting at 6 h. In addition to antimicrobial activity, we also evaluated the effects of the custom HMO mixtures on GBS biofilm formation (Figure S4). For both GB590 and GB2, the HMO extract mimic was the only mixture capable of significantly decreasing biofilm production. On average, this mixture decreased GBS biofilm formation by over 50%. Given the similar magnitudes and patterns of antimicrobial activity for the HMO extract mimic and the GBS strain-specific HMO mixtures, it is intriguing that only the HMO extract mimic reduced biofilm formation as none of the HMOs singularly possess this activity. In fact, several HMOs increased biofilm formation against GB590. Elucidation of the mechanism of this interesting dichotomy is ongoing.

Conclusion In this study, a first in its class examination of the structure-activity relationship of fucosylated HMOs and antibacterial effects against GBS is described. While numerous fucosylated and nonfucosylated HMOs possessed similarly strong antimicrobial activity against S. agalactiae strain GB590, against S. agalactiae strain GB2, lacto-N-hexaose (LNnH) was the sole HMO to exhibit strong antimicrobial activity (Table 1). No HMO was found to significantly decrease biofilm production in either strain. Importantly, we observed that the presence of fucose residue(s) on an HMO core scaffold does not necessarily correlate to antimicrobial or antibiofilm activity. The location and degree of fucosylation does, however, appear to have a profound effect. Interestingly, the activity of single-entity HMOs against GBS is also strain-specific. As previously mentioned, serotypes are groups within a single species of 18 ACS Paragon Plus Environment

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microorganism, such as bacteria or viruses, that share homology in their cell surface oligosaccharides. The antigenicity and immunogenicity of capsular antigens have been identified as a major virulence factor of GBS and are associated with antimicrobial resistance patterns. In fact, the first line of defense for many pathogens is this physical layer of carbohydrates as it offers protection against a wide range of environmental pressures such as the host immune system and antibiotics. As we previously found that HMO extracts exert antimicrobial activity against GBS by increasing membrane permeability, the next phase of our program is aimed at identifying how differences in capsular polysaccharide structure influence membrane permeability. A proposed mechanism that agrees with the mechanistic data obtained thus far is that specific HMOs, due to their structural homology with capsular polysaccharides, are promiscuously incorporated into the membrane to increase permeability. As we have now identified specific HMOs that possess strong anti-GBS activity, the goal of future studies is to use chemical synthesis to convert these specific pharmacophores into tool compounds that can be used to identify specific HMO cellular targets. Results in this regard will be reported in due course.

Experimental Materials 2'-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose III (LNFP III), lacto-N-neohexaose (LNnH), and para-lacto-N-hexaose (para-LNnH) were generously donated by Glycom. Lacto-N-triose II (LNT II) and lacto-N-neotetraose (LNnT) were obtained from Carbosynth. D-lactose monohydrate was purchased from Sigma Aldrich. Lacto-N-tetraose was obtained through chemical synthesis34 and from Carbosynth. HMO isolation Human milk was obtained from 21 healthy, lactating women between 3 days and 3 months postnatal and stored between −80 and −20 °C. Deidentified milk was provided by Dr. Jörn-Hendrik Weitkamp from the 19 ACS Paragon Plus Environment


Page 20 of 28

Vanderbilt Department of Pediatrics, under a collection procedure approved by the Vanderbilt University Institutional Review Board (IRB#100897) and Medolac. Milk samples were thawed and centrifuged for 45 min. After centrifugation, the top lipid layer was discarded. The proteins were removed by diluting the supernatant with 1:1 v/v 180 or 200 proof ethanol, chilling the sample, and centrifuging for 45 min. The HMO- containing supernatant was concentrated in vacuo, dissolved in phosphate buffer (pH 6.5, 0.2 M), and heated to 37 °C.35-36 β-galactosidase from Kluyveromyces lactis was added, and the reaction stirred until lactose hydrolysis was complete as determined by TLC analysis. The reaction mixture was diluted with 1:0.5 v/v 180 or 200 proof ethanol, chilled, and centrifuged for 30 min. The supernatant was concentrated in vacuo, and the remaining salts, glucose, and galactose separated from the HMOs using P-2 Gel (H2O elutant). The HMOs were dried by lyophilization. HMO extracts were pooled to create a cocktail.12 Bacterial strains and culture conditions Clinical strains of GBS (GB590 and GB2) were provided by Dr. Shannon Manning at Michigan State University. Strains were grown on blood agar plates at 37 °C in ambient air overnight, subcultured into 5 mL of Todd-Hewitt broth (THB), and incubated with shaking at 180 RPM at 37 °C overnight. After incubation, bacterial density was quantified through absorbance readings at 600 nm (OD600) using a Promega GloMax-Multi Detection System plate reader. Bacterial numbers were determined using the predetermined coefficient of 1 OD600= 109 CFU/mL.

Bacterial growth and viability assays Test strains were grown as described above and used to inoculate fresh THB or THB supplemented with ca. 5 mg/mL HMO. Inoculation was completed at a multiplicity of infection (MOI) of 106 colony forming units per 200 L of growth medium in 96 well tissue culture treated, sterile polystyrene plates (Corning, Inc.). Cultures were grown under static conditions at 37 oC in ambient air. Growth was quantified through


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spectrophotometric readings at OD600. Viability was assessed through serial dilution and plating onto blood agar plates followed by quantification of viable CFU/mL. Bacterial biofilm assay Test strains were grown as described and used to inoculate fresh THB or THB supplemented with ca. 5 mg/mL HMO. Inoculation was performed at a multiplicity of infection (MOI) of 106 colony forming units per 200 L of growth medium in 96 well tissue culture treated, sterile polystyrene plates (Corning, Inc.). Cultures were incubated under static conditions at 37 oC in ambient air for 24 h. Following spectrophotometric reading at OD600, culture media was removed and the wells were gently washed once with phosphate-buffered saline (PBS, pH 7.4) to removed non-adherent cells. Adherent cells were stained with a 10% crystal violet solution for 10 min. Excess stain was discarded and the wells gently washed with PBS followed by drying at room temperature for 30 minutes. The crystal violet stain was solubilized with an 80% ethanol/20% acetone solution and biofilm formation quantified through spectrophotometric reading at OD560.10 Statistical analysis All data shown signify 3 independent experiments each with 3 technical replicates. Data is expressed as the mean ± SEM. Statistical analyses were performed in GraphPad Prism Software v. 7.0c. Statistical significance for growth and viability were determined using two-way ANOVA with posthoc Dunnett’s multiple comparison test comparing growth and viability in the presence of HMOs to growth and viability in media. Statistical significance for biofilm production was determined using one-way ANOVA with posthoc Dunnett’s multiple comparison test comparing biofilm production in the presence of HMOs to biofilm production in media.

Conflicts of interest The authors declare no conflicts.



Page 22 of 28

Acknowledgements This work was supported by Vanderbilt University, the Vanderbilt Microbiome Initiative (VMI), the Department of Pediatrics at Vanderbilt University Medical Center, and Prolacta Biosciences for S.D.T. 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. H.C.T was supported by the Curb Scholars Program. Prof. Jennifer Gaddy is acknowledged for use of her facilities. The authors would like to acknowledge Prof. Shannon Manning at Michigan State University for generously providing clinical strains of GBS (GBS590 and GBS2). Ryan Doster is acknowledged for his assistance with viability assays. Jacky Lu is acknowledged for his assistance with bacterial culturing. Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: GBS type Ia and III serotype capsular polysaccharide (CPS) repeating unit structures; Composition of HMO extract mimic mixture; Growth and viability curves for S. agalactiae strain GB590 treated with HMO mixtures; Growth and viability curves for S. agalactiae strain GB2 treated with HMO mixtures; Relative biofilm formation of S. agalactiae strains GB590 and GB2 treated with HMO mixtures; Mass Spectral Analysis for 2’-FL, 3-FL, DFL, LNFP I, LNFP II, LNFP III, LNT, LNnT, LNnH, para-LNnH, and LNT II.

Author Information *Corresponding author: [email protected] 22 ACS Paragon Plus Environment

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Abbreviations HMOs, Human milk oligosaccharides; GBS, Group B Streptococcus; CFUs, colony forming units; LNT, lacto-N-tetraose; LNnT, lacto-N-neotetraose; 2’-FL, 2’-fucosyllactose; 3-FL, 3-fucosyllactose; DFL, difucosyllactose; LNFP I, lacto-N-fucopentaose I; LNFP II, lacto-N-fucopentaose II; LNFP III, lacto-Nfucopentaose III; LNT II, lacto-N-triose II; para-LNnH, para-lacto-N-neohexaose; LNnH, lacto-Nhexaose; glc, glucose; GlcNAc, N-acetylglucosamine; gal, galactose; fuc, fucose; Neu5Ac, Nacetylneuraminic acid; sia, sialic acid; LST a, LS-tetrasaccharide a; DSLNT, disialyllacto-N-tetraose; LNnT, lacto-N-neotetraose; LST c, LS-tetrasaccharide c; 3’-SL, 3’-sialyllactose; 6’-SL, 6’-sialyllactose; FUTs, fucosyltransferases; MIC, minimal inhibitory concentration.

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No HMO

9

LNT II

LNFP I

LNFP II

LNnT

***

LNnT

LNT II DFL

Neutral Human Milk Oligosaccharides (HMOs)

Log10 CFU/mL

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Page 28 of 28

LNFP II

Media LNnT

*

6

****

***

LNT II

**** ****

*

****

**** 3

0

4

8

12

16

20

Time (h)

Group B Streptococcus

Reduced bacterial viability


24

DFL

DFL

LNFP I

LNFP II

LNFP I

Interrogation of Human Milk Oligosaccharide Fucosylation Patterns for

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