Letters Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX
Nonmicrobicidal Small Molecule Inhibition of Polysaccharide Metabolism in Human Gut Microbes: A Potential Therapeutic Avenue Anthony D. Santilli,† Elizabeth M. Dawson,† Kristi J. Whitehead,*,‡ and Daniel C. Whitehead*,† †
Department of Chemistry, Clemson University, Clemson, South Carolina 29634, United States Department of Biological Sciences, Clemson University, Clemson, South Carolina 29634, United States
‡
S Supporting Information *
ABSTRACT: A new approach for the nonmicrobicidal phenotypic manipulation of prominent gastrointestinal microbes is presented. Low micromolar concentrations of a chemical probe, acarbose, can selectively inhibit the Starch Utilization System and ablate the ability of Bacteroides thetaiotaomicron and B. f ragilis strains to metabolize potato starch and pullulan. This strategy has potential therapeutic relevance for the selective modulation of the GI microbiota in a nonmicrobicidal manner.
T
most thoroughly characterized example of this system resides within Bacteroides thetaiotaomicron (Bt), a prominent member of the gut microbial community.48−51 The Sus in Bt consists of eight genes, susRABCDEFG, coding in part for a protein complex, SusCDEFG (Figure 1).49,51 These proteins are responsible for the cell-surface recognition, gylcosidic cleavage, and importation of starch metabolites.52,53 The Bt genome harbors 163 homologues of the SusC and SusD proteins for the
he gastrointestinal (GI) microbiota (i.e., the consortium of bacteria, fungi, viruses, and archaea resident in the human gut) leverages a wide range of influences on human health.1 Undesirable changes in the GI microbiota have been implicated in a large number of diseases including type I2−4 and type II5,6 diabetes mellitus, obesity,7−15 cancer,16−18 allergies and asthma,19−22 inflammatory bowel disease,23−25 irritable bowel syndrome,26−28 autism,29,30 eating disorders,31 and multiple sclerosis.32 The GI microbiota also play an important role in shaping host innate33 and adaptive immunity.34 Thus, a selective method for the modulation of its constituents could be a therapeutic boon.35−40 Probiotic (i.e., beneficial) microbial strains,41−43 prebiotic dietary supplements,42,43 and fecal microbiota transplant (FMT) therapy44 have been investigated previously for the modulation of the GI microbiota, but the chemistry community has been relatively slow to develop smallmolecule therapeutics to that end.40 The classical strategy for small-molecule therapy employs bactericidal or bacteriostatic antibiotics. This strategy is hindered by the propensity of microbes to evolve antibiotic resistance rapidly. Further, the adventitious invasion of pathogens after antibiotic treatment is well-documented.45−47 In the present study, we sought to validate a strategy whereby a small molecule probe might disrupt the ability of prominent gut microbial constituents to metabolize complex carbohydrates that are not typically utilized by the human host. We hypothesize that this type of inhibition might in turn reduce the ability of these microbes to forage nutrients in the competitive gut ecosystem, thus allowing for targeted shifts in the microbiota as a potential therapeutic strategy.40 We targeted the Starch Utilization System (Sus) of members of the Bacteroides genus as a potential druggable target. The © XXXX American Chemical Society
Figure 1. Starch utilization system (Sus) of Bacteroides thetaiotaomicron (adapted from ref 49). Received: April 2, 2018 Accepted: April 16, 2018 Published: April 16, 2018 A
DOI: 10.1021/acschembio.8b00309 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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ACS Chemical Biology
Figure 2. Human α-amylase inhibitors and polysaccharides evaluated in this study.
polysaccharides that can be metabolized by Bt only by engaging one of its Sus-like protein complexes suited for the recognition and cleavage of those particular polysaccharides.66−69 (Note: Bf cannot metabolize chondroitin sulfate or levan.) The bacteria were cultured in an incubating 96-well plate reader housed within an anaerobic chamber. The cultures were incubated at 37 °C for 48 h. The optical density measured at 600 nm (OD600) of the culture was monitored every 30 m after a brief shaking period. Growth curves of each organism grown in the presence of a drug candidate on a particular carbon source were generated and compared to growth curves from untreated control cultures. To ensure reproducibility, each assay was completed in triplicate and on two separate days. These initial screens revealed that acarbose (1) was able to inhibit the ability of both species to metabolize several carbon sources (Figure 3, see also Figures S2−S6 in the Supporting Information for details concerning the evaluation of compounds 2 and 3). As expected, the metabolism of glucose by both Bt and Bf was not significantly impacted by 1, as is evident by the clear overlay of the growth curves arising from treated and untreated cultures (Figure 3A and D). This result confirms that 1 does not exhibit microbicidal activity against these two species. In sharp contrast, when pullulan (6) was offered as the only available carbon source, Bt grew to 4.2 ± 0.4%, while the growth of Bf on pullulan was diminished to just 3.7 ± 0.3% of the control culture density in the presence of 100 μM of 1 (Figure 3B and E, respectively). Similarly potent inhibition was observed when the strains were cultured in the presence of potato starch (5). Bt growth was diminished to 0.9 ± 0.6% of the untreated culture (Figure
recognition and binding of other dietary and host-derived polysaccharides, highlighting their reliance on this strategy for energy harvest.48−51 Further, our initial focus on members of the Bacteroides genus was motivated by observations that a bloom in these bacteria appears to precede the initial stages of autoimmunity associated with type I diabetes in genetically at-risk patients.4,54−58 Finally, the Bacteroides genus is a prominent member of the Bacteroidetes phylum, which comprises on average 57% of the total gut microbial community in healthy humans.59,60 The Sus of the Bacteroides genus serves as a canonical model for the polysaccharide metabolism of the entire Bacteroidetes phylum.51 One of the principal components of the Bt Sus is an αamylase, SusG, that cleaves the glycoside linkages within starchbased polysaccharides. Bt mutants bearing a deletion of the SusG gene cannot metabolize starch.61 Thus, we surmised that a screen of known human α-amylase or α-glucosidase inhibitors might reveal an inhibitory probe capable of arresting the Sus in Bt and other Bacteroides species. The human α-glucosidase inhibitors acarbose (1), miglitol (2), and voglibose (3) (Figure 2) were evaluated for their potential to disrupt the starch utilization of Bt and B. f ragilis (Bf). Compounds 1−3 were initially evaluated at 100 μM against Bt and Bf cultured in minimal media containing glucose, potato starch (5), or pullulan (6). Both species can metabolize glucose without engaging the Sus machinery.62 In contrast, both species can metabolize potato starch and pullulan, a fungal starch product,63−65 only by action of the Sus.62 Chondroitin sulfate (7)66,67 and levan (8)68,69 are nonglucose based B
DOI: 10.1021/acschembio.8b00309 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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Figure 3. Growth curves in the presence of 100 μM acarbose. (A) B. thetaiotaomicron (Bt) grown in the presence (dashed orange line) and absence (solid black line) of 100 μM acarbose in glucose minimal media. (B) Bt grown in the presence and absence of 100 μM acarbose in pullulan minimal media. (C) Bt grown in the presence and absence of 100 μM acarbose in potato starch minimal media. (D) B. f ragilis (Bf) grown in the presence and absence of 100 μM acarbose in glucose minimal media. (E) Bf grown in the presence and absence of 100 μM acarbose in pullulan minimal media. (F) Bf grown in the presence and absence of 100 μM acarbose in potato starch minimal media.
Figure 4. Dose−response study. (A) % growth of B. thetaiotaomicron (Bt) in the presence of 0.1−100 μM acarbose in pullulan minimal media. (B) % growth of B. f ragilis (Bf) in the presence of 0.1−100 μM acarbose in pullulan minimal media. (C) % growth of Bt in the presence of 0.1−100 μM acarbose in potato starch minimal media. (D) % growth of Bf in the presence of 0.1−100 μM acarbose in potato starch minimal media.
C
DOI: 10.1021/acschembio.8b00309 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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ACS Chemical Biology
Figure 5. Selective targeting of Sus substrates. (A) B. f ragilis growth curves on potato starch minimal media in the presence of 100 μM acarbose (orange dashed line), 100 μM acarviosin (purple dotted line), and untreated control culture (black solid line). (B) B. f ragilis growth curves on pullulan minimal media in the presence of 100 μM acarbose, 100 μM acarviosin, and untreated control culture. (C) Dose−response study: B. f ragilis culture treated with 5−100 μM acarviosin in potato starch (black bars) or pullulan (gray bars) minimal media.
3C), while Bf only grew to 0.4 ± 0.3% of the untreated control culture (Figure 3F). These data clearly demonstrate the ability of 1 to arrest polysaccharide metabolism mediated by the Sus in these two Bacteroides species. Acarbose treatment did not appreciably affect the growth of the two strains on the disaccharide maltose (a transcriptional inducer for the Sus70,71), nor did it inhibit the growth of Bt on chondroitin sulfate (7) or levan (8), complex polysaccharides requiring different Sus-like enzyme clusters (see Supporting Information for details).66−69 These results highlight that the observed inhibition with 1 is likely specific to the Sus system while other Sus-like clusters remain unaffected.66−69 In order to confirm the observed inhibition in the growth curve analyses, and to demonstrate that treated cultures contained viable cells after acarbose exposure, cell counts were generared for Bt and Bf cultures grown in glucose, pullulan, and potato starch minimal media both with and without 100 μM acarbose treatment (see Supporting Information for further details). We then evaluated the concentration dependence on the observed inhibitory effects. Bt and Bf cultures were grown in the presence of pullulan (Figure 4A and B) and potato starch (Figure 4C and D) as the sole carbon source in the presence of 0.1−100 μM 1. On pullulan, Bt growth was 4.1 ± 0.5% (i.e., compared to untreated control cultures), 5.3 ± 0.1%, 21.4 ± 15.2%, 36.2 ± 23.9, and 70 ± 21.1%, for 100, 50, 10, 5, and 1 μM concentrations of 1, respectively (Figure 4A). These data correspond to approximately 96%, 95%, 79%, 64%, and 30% growth inhibition, respectively. At 0.5 and 0.1 μM concentrations of 1, the growth of Bt was not appreciably affected. Bf cultures grew on pullulan minimal media to 4.1 ± 0.1%, 3.8 ± 0.1%, 11.0 ± 6%, and 42.5 ± 10.7% compared to untreated control cultures in the presence of 100, 50, 10, and 5 μM concentrations of acarbose, respectively (Figure 4B). These data correspond to approximately 96%, 96%, 89%, and 57% growth inhibition, respectively. Growth inhibition was not apparent as the concentration of acarbose was decreased further to 0.1−1 μM. When Bt was cultured on potato starch (Figure 4C), the treated cultures grew to 0.4 ± 0.0%, 0.5 ± 0.1%, 10.8 ± 4.9%, and 38.6 ± 2.7% compared to untreated control cultures, corresponding to approximately 99%, 99%, 89%, and 61% growth inhibition for 100, 50, 10, and 5 μM concentrations of 1, respectively. Lower concentrations of 1 (i.e., 0.1−1 μM) did not appreciably affect the growth of Bt on potato starch minimal media (i.e., less than 12% growth inhibition). Finally, when Bf was grown in minimal media containing potato starch, inhibitory effects were apparent at concentrations as low as 0.5 μM acarbose (i.e., 42% growth
inhibition), with greater than 90% inhibition apparent at concentrations as low as 5 μM (Figure 4D). We surmise that the primary cause of the observed inhibitory effects might arise from the binding of acarbose into the active site of the cell-surface glycolytic enzyme of the suite, SusG (see Figure 1). In fact, SusG has been crystallized with an acarbose metabolite bound within its active site.72 Nevertheless, the eight proteins of the Sus suite work in concert to harness, cleave, and import starch substrates, so it is possible that acarbose may also play secondary inhibitory roles by engaging with other constituents of the Sus. For instance, acarbose might also interact with the other biding sites on SusG72 or interfere with the surface recognition (i.e., SusD)50,73,75 or secondary binding proteins (i.e., SusE and SusF)74−76 of the system, thus precluding efficient recognition or binding to the polysaccharide substrate. We next turned to the evaluation of a structural truncation of 1, acarviosin (4; Figure 2), at 100 μM on Bf grown on potato starch and pullulan. This molecule represents the core structure of 1 but lacks the maltose unit at the reducing end of the parent probe. Figure 5A highlights a representative growth curve for Bf grown on potato starch in the presence of acarviosin (dotted purple line) overlaid with curves of an untreated culture (solid black line) and a culture treated with 100 μM 1 (dashed orange line). In this scenario, 4 returned nearly identical inhibitory effects as observed previously with 1. In sharp contrast, when offered pullulan, significant growth inhibition of Bf was apparent in the presence of 1 (dashed orange line) as expected, but 100 μM concentrations of 4 elicited no inhibitory effect (dotted purple line). This result highlights the ability to not only target one specific Starch Utilization System in the Bacteroides but also to selectively ablate the ability of the bacteria to metabolize a single metabolic target of that particular system in preference to another. Similarly selective inhibition of potato starch metabolism in preference to pullulan metabolism was apparent with Bt upon treatment with 4 (see Figure S9, Supporting Information). Finally, we evaluated the concentration dependence on the selective inhibition of potato starch metabolism by Bf induced by 4 (Figure 5C). The inhibitory effects of 4 were apparent at concentrations as low as 50 μM (∼95% growth inhibition, black bars). Note that the ability of Bf to metabolize pullulan was unaffected (gray bars). In order to probe the selectivity of acarbose inhibition, we next assessed its effect on other bacteria resident in the human gut. The Firmicutes are the other major phylum that populates the human gut, comprising approximately 40% of the normal gut microbiota.59,60 Certain members of the Firmicutes phylum are capable of metabolizing starch, but they employ different D
DOI: 10.1021/acschembio.8b00309 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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Figure 6. Growth curves in the presence of 100 μM acarbose. (A) R. bromii grown in the presence (dashed orange line) and absence (solid black line) of 100 μM acarbose in fructose RUM media. (B) R. bromii grown in the presence and absence of 100 μM acarbose in maltose RUM media. (C) R. bromii grown in the presence and absence of 100 μM acarbose in pullulan RUM media. (D) R. bromii grown in the presence and absence of 100 μM acarbose in potato starch RUM media.
metabolize starch. These two bacterial strains were unaffected by 100 μM acarbose treatment (see Supporting Information for details). Taken together, these results suggest that the inhibition of the Sus in members of the Bacteroides genus may be selective and could suggest a strategy for the targeted manipulation of the gastrointestinal microbiota for therapeutic gain. Indeed, acarbose exhibits excellent selectivity for inhibiting the metabolism of potato starch (cf. Figures 3C and F with Figure 6D). While the observation that R. bromii appears to be somewhat sensitive to acarbose when grown on pullulan suggests an erosion in selectivity, this observation is mitigated by the fact that pullulan (an occasional food additive) is comparatively much less frequently encountered in the human diet than potato starch.63−65 In conclusion, we have laid the groundwork for further investigation of a new strategy for the targeted manipulation of certain members of the GI microbiota in a nonmicrobicidal manner by modulating their capacity to metabolize certain polysaccharides that are commonly encountered in the human gut. Specifically, we have demonstrated that the small molecule probe, acarbose (1), is capable of selectively arresting the Sus in two prominent members of the GI microbiota, Bacteroides thetaiotaomicron and Bacteroides f ragilis. We have demonstrated that structural manipulation of the probe scaffold can impart further polysaccharide-specific selectivity in the observed inhibitory effects. Finally, our data suggest that the inhibitor probe is selective for the inhibition of starch metabolism of members of the Bacteroides genus in preference to a Firmicute keystone starch utilizer in the gut, Ruminococcus bromii. Likewise, the inhibitor has no effect on the growth of other important gut microbes. We predict that it is possible that the selective inhibition of polysaccharide metabolism could sufficiently reduce the competitive advantage of members of the Bacteroides genus, or more broadly the Bacteroidetes phylum, in order to effect lasting changes in the composition of the GI microbiota in a therapeutically advantageous manner. Current efforts in our laboratories are geared toward four main objectives: (1) to uncover the specific targets within the Sus that are inhibited by 1 and 4, (2) to demonstrate the ability of this strategy to effect changes in the GI microbiota in relevant
enzymatic machinery for that purpose. Specifically, while certain Firmicutes possess various amylases and pullulanases, they lack the homologues of the Sus present in the Bacteroides genus.77−81 It was our hope that the enzymes utilized by the Firmicutes to digest starches would be distinct enough from the Bacteroides Sus that they would be unaffected by treatment with acarbose. The common human gut microbe Ruminococcus bromii was chosen as a representative organism from the Firmicutes phylum due to it being a known “keystone” starch utilizer whose carbohydrate-degrading enzymes have been wellcharacterized.77−79 For R. bromii with acarbose, growth was investigated in RUM media (see Supporting Information) supplemented with either fructose, maltose, pullulan, or potato starch. Pullulan and potato starch both engage the starch metabolizing enzymes present in R. bromii, while fructose and maltose are metabolized without using these enzymes. (Note: Fructose was used as a monosaccharide control instead of glucose since R. bromii cannot metabolize the latter effectively.) R. bromii growth was probed using growth curves generated from 72 h cultures grown in a 96-well plate reader housed within an anaerobic chamber, similar to the experiments conducted for Bt and Bf. Each assay was completed in triplicate and repeated three times on separate days. R. bromii growth on fructose was not hindered by 100 μM acarbose, as can be seen in the clean overlay of the treated and untreated cultures in Figure 6A. Thus, similar to the results with Bt and Bf in glucose (cf. Figure 3A and D), acarbose has little effect on the growth of R. bromii in the presence of a monosaccharide. Growth of R. bromii on the disaccharide maltose was blunted initially, but the treated culture eventually reached approximately the same OD600 as the untreated culture by the end of the experiment (Figure 6B). R. bromii growth on pullulan was moderately inhibited by treatment with 100 μM acarbose, only growing to 53.8 ± 3.0% of the untreated cultures on pullulan (Figure 6C). Finally, R. bromii growth on potato starch was essentially unaffected by acarbose treatment (Figure 6D). We also evaluated the effects of acarbose treatment on two common gut microbes, Escherichia coli and Lactobacillus reuteri, to assess its effects on gut microbes that do not E
DOI: 10.1021/acschembio.8b00309 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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et al. (2012) A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature 490, 55−60. (6) Tilg, H., and Moschen, A. R. (2014) Microbiota and diabetes: an evolving relationship. Gut 63, 1513−1521. (7) Backhed, F., Ding, H., Wang, T., Hooper, L. V., Koh, G. Y., Nagy, A., Semenkovich, C. F., and Gordon, J. I. (2004) The gut microbiota as an environmental factor that regulates fat storage. Proc. Natl. Acad. Sci. U. S. A. 101, 15718−15723. (8) Turnbaugh, P. J., Ley, R. E., Mahowald, M. A., Magrini, V., Mardis, E. R., and Gordon, J. I. (2006) An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027−1031. (9) Turnbaugh, P. J., Hamady, M., Yatsunenko, T., Cantarel, B. L., Duncan, A., Ley, R. E., Sogin, M. L., Jones, W. J., Roe, B. A., Affourtit, J. P., Egholm, M., Henrissat, B., Heath, A. C., Knight, R., and Gordon, J. I. (2009) A core gut microbiome in obese and lean twins. Nature 457, 480−484. (10) Zhang, H., DiBaise, J. K., Zuccolo, A., Kudrna, D., Braidotti, M., Yu, Y., Parameswaran, P., Crowell, M. D., Wing, R., Rittmann, B. E., and Krajmalnik-Brown, R. (2009) Human gut microbiota in obesity and after gastric bypass. Proc. Natl. Acad. Sci. U. S. A. 106, 2365−2370. (11) Ley, R. E. (2010) Obesity and the human microbiome. Curr. Opin. Gastroenterol. 26, 5−11. (12) Clarke, S. F., Murphy, E. F., Nilaweera, K., Ross, P. R., Shanahan, F., O’Toole, P. W., and Cotter, P. D. (2012) The gut microbiota and its relationship to diet and obesity: new insights. Gut Microbes 3, 186−202. (13) Delzenne, N. M., and Cani, P. D. (2011) Interaction between obesity and the gut microbiota: relevance in nutrition. Annu. Rev. Nutr. 31, 15−31. (14) Angelakis, E., Armougom, F., Million, M., and Raoult, D. (2012) The relationship between gut microbiota and weight gain in humans. Future Microbiol. 7, 91−109. (15) Arora, T., and Backhed, F. (2016) The gut microbiota and metabolic disease: current understanding and future perspectives. J. Intern. Med. 280, 339−349. (16) Louis, P., Hold, G. L., and Flint, H. J. (2014) The gut microbiota, bacterial metabolites and colorectal cancer. Nat. Rev. Microbiol. 12, 661−672. (17) Sears, C. L., and Garrett, W. S. (2014) Microbes, microbiota, and colon cancer. Cell Host Microbe 15, 317−328. (18) Schwabe, R. F., and Jobin, C. (2013) The microbiome and cancer. Nat. Rev. Cancer 13, 800−812. (19) Rachid, R., and Chatila, T. A. (2016) The role of the gut microbiota in food allergy. Curr. Opin. Pediatr. 28, 748−753. (20) Arrieta, M.-C., and Finlay, B. (2014) The intestinal microbiota and allergic asthma. J. Infect. 69, S53−S55. (21) Hanski, I., von Hertzen, L., Fyhrquist, N., Koskinen, K., Torppa, K., Laatikainen, T., Karisola, P., Auvinen, P., Paulin, L., Makela, M. J., Vartiainen, E., Kosunen, T. U., Alenius, H., and Haahtela, T. (2012) Environmental biodiversity, human microbiota, and allergy are interrelated. Proc. Natl. Acad. Sci. U. S. A. 109, 8334−8339. (22) Penders, J., Gerhold, K., Thijs, C., Zimmermann, K., Wahn, U., Lau, S., and Hamelmann, E. (2014) New insights into the hygiene hypothesis in allergic diseases: mediation of sibling and birth mode effects by the gut microbiota. Gut Microbes 5, 239−244. (23) Ananthakrishnan, A. N. (2015) Epidemiology and risk factors for IBD. Nat. Rev. Gastroenterol. Hepatol. 12, 205−217. (24) Nagalingam, N. A., and Lynch, S. V. (2012) Role of the Microbiota in Inflammatory Bowel Diseases. Inflamm. Bowel Dis. 18, 968−984. (25) Packey, C. D., and Sartor, R. B. (2009) Commensal bacteria, traditional and opportunistic pathogens, dysbiosis and bacterial killing in inflammatory bowel diseases. Curr. Opin. Infect. Dis. 22, 292−301. (26) Collins, S. M. (2014) A role for the gut microbiota in IBS. Nat. Rev. Gastroenterol. Hepatol. 11, 497−505. (27) Ohman, L., Tornblom, H., and Simren, M. (2015) Crosstalk at the mucosal border: importance of the gut microenvironment in IBS. Nat. Rev. Gastroenterol. Hepatol. 12, 36−49.
animal models, (3) to explore the applicability of this strategy to other members of the GI microbiota, and (4) to discover other molecular scaffolds that are capable of inhibiting the Bacteroides starch utilization system with enhanced potency and selectivity. We are actively pursuing all of these objectives, and the results of these studies will be reported in due course.
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METHODS
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ASSOCIATED CONTENT
Full details for all experimental methods and additional supporting data (i.e., Figures S1−S10 and accompanying narrative) are provided in the Supporting Information. S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.8b00309. Figures S1−S10 and all other experimental protocols (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Kristi J. Whitehead: 0000-0001-7113-8726 Daniel C. Whitehead: 0000-0001-6881-2628 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the JDRF (Project 1-INO-2015131-A-N), the Clemson University Creative Inquiry Program, and the Clemson University Departments of Chemistry and Biological Sciences for financial support. We also thank E. Martens, N. Koropatkin, and N. Pudlo (University of Michigan) for gifts of the bacterial strains, growth protocols, and many helpful discussions and suggestions.
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REFERENCES
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DOI: 10.1021/acschembio.8b00309 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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