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Functional Structure/Activity Relationships
How fine structural differences of xylo-oligosaccharides and arabinoxylooligosaccharides regulate differential growth of Bacteroides species Mihiri Mendis, Eric C. Martens, and Senay Senay Simsek J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01263 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018
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Journal of Agricultural and Food Chemistry
Title: How fine structural differences of xylo-oligosaccharides and arabinoxylo-oligosaccharides regulate differential growth of Bacteroides species
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Mihiri Mendisa, Eric C. Martensb, and Senay Simseka#
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a
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ND, 58108-6050
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b
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Arbor, MI
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#
Department of Plant Sciences, North Dakota State University, PO Box 5050, Dept 7670 Fargo,
Department of Microbiology and Immunology, University of Michigan Medical School, Ann
Corresponding Author: Senay Simsek
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Corresponding author. Phone: (701) 231-7737. Fax: (701) 231-8474.
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E-mail:
[email protected] 12
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Abstract
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Xylo-oligosaccharides (XOS) and arabinoxylo-oligosaccharides (AXOS) could be used
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to selectively favor growth of certain gut bacterial groups. The objective of this research was to
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understand how the structural differences of XOS and AXOS influenced the growth of
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Bacteroides species commonly found in the intestine. We report that the specific structural
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details of XOS and AXOS dictate the differential growth of Bacteroides species in the intestine.
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We also investigated the expression of two polysaccharide utilization loci (PULs) in a strain of
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B. ovatus upon growth on AXOS using three different susC transcripts as sentinel reporter genes.
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23- α-L-arabinofuranosyl-xylotriose (A4) was shown to upregulate small xylan PUL gene
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expression while 23, 33-di- α-L-arabinofuranosyl-xylotriose (A6) decreased expression of this
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PUL. These results reveal new details about the potentially very specific structure-function
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relationship of XOS and AXOS that could be used in targeted alteration of the microbial
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population in the gut through dietary interventions to maintain health.
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Key words: Bacteroides species, polysaccharide utilization loci (PUL) gene expression,
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Journal of Agricultural and Food Chemistry
Introduction The intestine is an important organ that consists of an extensive surface area and permits
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vital interactions with the external world, including with the gut microbiota.1 The microbiota
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refers to “the microbial life forms within a given habitat or host.”2 The gut microbiota exerts a
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significant impact on host physiology, impacting the control of energy homeostasis, the immune
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system, digestion and vitamin synthesis1 and inhibition of pathogen colonization.3 More so, the
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involvement of gut microbiota in conditions such as depression4 and autism5 has also been
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suggested. A potential role between the microbiome and Parkinson’s (PD) disease6 and multiple
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sclerosis (MS)7 has also been indicated.
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Studies of healthy adult gut microbiota have shown that it is composed primarily of
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members of a few bacterial phyla, with the Bacteroidetes and Firmicutes being most numerous.8
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The Bacteroidetes generally encode more carbohydrate-active enzymes (CAZymes) than other
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phyla such as Firmicutes, indicating their enhanced capacity to utilize wide range of
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polysaccharide substrates.9 The members of the genus Bacteroides are adept at utilizing plant
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and host derived polysaccharides10 These Bacteroides are rich in CAZymes involved in the
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acquisition and metabolism of various glycosides including glycoside hydrolases and
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polysaccharide lyases which are organized into polysaccharide utilization loci (PULs) that are
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distributed throughout the genome.10
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Different bacteria have preferential growth on different substrates. It was observed that
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different Bacteroides species preferred different substrates depending on the structural and
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chemical complexity of the polysaccharide substrate.11 This indicates that just as dietary
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polysaccharides have the potential to alter the growth of specific bacteria, these polysaccharides
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can be utilized for selective targeting of specific bacteria. Xylan is a polysaccharide that is found
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in many plant materials. Xylans consists of a back bone of β-(1 → 4)-linked xylopyranose units
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onto which 4-O-methylglucuronic acid (MeGlcA) groups, O-acetyl groups or other sugars such
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as arabinose can be substituted at C (O)-2 and/or C (O)-3 positions.12 Ferulic acid can be found
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attached to the C(O)-5 position of these arabinose13 Arabinoxylans are xylans with arabinose
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substitution at C-2 and/or C-3 positions of the xylan backbone. Arabinoxylans are prevalent in
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many cereal grains: wheat (5.5-7.2%), barley (3.9-5.4%), maize (1-2%), rice (2-3%).14
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Arabinoxylans can be hydrolyzed into xylo-oligosaccharides (XOS) and arabinoxylo-
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oligosaccharides (AXOS).
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Genus Bacteroides contain the most expanded glycolytic gene repertoires that target
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xylan degradation.15 Previous research has shown closely related Bacteroides species to utilize
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xylan in different ways.11 Rose, et al. 16 indicated differences among the fermentation profiles for
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AX from different cereals (maize, rice and wheat), which consists of different structural features.
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Xu 17 concluded that specific molecular regions of dietary fibers differentiate gut bacteria.
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Depending on the structural complexity of the XOS and AXOS, different Bacteroides species
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could respond to these substrates differently. Understanding how these structurally different
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oligosaccharides influence the growth of commensal bowel bacteria could lead to manipulation
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of the gut microbiome through introduction of XOS and AXOS to correct dysbiosis. Thus, the
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objective of this research was to understand how the structural differences of XOS and AXOS
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impacted the growth of Bacteroides species commonly found in the intestine.
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Materials and Methods
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Materials
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The bacterial fermentation experiments were carried out using six publicly available Bacteroides strains belonging to five different species. The strains were: Bacteroides
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cellulosilyticus DSM 14838, Bacteroides ovatus ATCC 8483, Bacteroides ovatus 3-1-23 ,
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Bacteroides eggerthii DSM 20697, Bacteroides intestinalis DSM 17393, Bacteroides
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xylanisolvens XB1A , Bacteroides thetaiotaomicron ATCC 29148 (VPI 5482). All strains were
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originally isolated from human colon or fecal samples, representative of the most commonly
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encountered xylanolytic organisms in the gut.
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All the carbohydrate substrates were purchased from Megazyme. These included five
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XOS samples: 1,4-β-D-xylobiose (X2), 1,4-β-D-xylotriose (X3), 1,4-β-D-xylotetraose (X4), 1,4-
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β-D-xylopentaose (X5), 1,4-β-D-xylohexaose (X6) and 7 AXOS samples: 1,5-α-L-arabinobiose
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(A2), 32- α-L-arabinofuranosyl-xylobiose (A3), 23- α-L-arabinofuranosyl-xylotriose (A4), 33- α-
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L-arabinofuranosyl-xylotetraose (A5), 23, 33-di- α-L-arabinofuranosyl-xylotriose (A6), 23- α-L-
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plus-33- α-L-Arabinofuranosyl-xylotriose (A7), 23, 33-di- α-L-arabinofuranosyl-xylotetraose
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(A8), 23- α-L-plus-33- α-L-arabinofuranosyl-xylotetraose (A9) and three arabinoxylan
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polysaccharides: low molecular weight AX (AX L), medium molecular weight AX (AX M),
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high molecular weight AX (AX H), monosaccharides: xylose (Xyl), arabinose (Ara), glucose
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(Glu).
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Pure strain growth experiments
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The bacterial growth experiments were carried out according to the methods described by
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Martens, et al. 18 Each carbohydrate substrate was prepare in to stock solutions (10 mg/mL)
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using millipore water. Each substrate solution was autoclaved at 121 ºC for 20 min. Substrate
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solution (30 µL) was pipetted into one of the designated wells on a 24 × 16 plate assigning three
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wells for each substrate per bacterial strain. Each bacterial strain inoculum was added to each
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well (30 µL), diluting the final substrate concentration to 5 mg/mL.
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The bacterial inocula were prepared as follows: each bacterium was inoculated into
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custom chopped meat media using sterile wood sticks from glycerol stocks from freezer. These
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cultures for assay inoculations were grown for 24 h at 37 0C under an anaerobic atmosphere of
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10% H2, 5% CO2, and 85% N2 using an anaerobic chamber (Coy manufacturing, Grass Lake,
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MI).
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Bacteroides minimal media was prepared using minimal media19 supplemented with
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additional compounds.20 In detail Bacteroides minimal media was prepared by adding together
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10 mL Bacteroides salts solution (KH2PO4 (544 g), NaCl (35 g), (NH4)2SO4 (45 g) dissolved in
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1L), 100 µL each of Vitamin K3 (1 mg/mL in ethanol), FeSO4 (0.4 mg/mL in 10 mM HCl),
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MgCl2 (0.1M in water), CaCl2 (0.8% (w/v) in water), Histidine/Hematin solution (1.9 mM
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Hematin in 0.2M Histidine solution), 50 µL of vitamin B12 stock (0.01 mg/mL in water), 100 mg
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L-cysteine, and 1 mL each of Balch’s vitamin mixture, trace mineral solution, Purine and
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Pyrimidine solution, and amino acid solution20 and bringing the volume to 50 mL with distilled
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water (pH 7.2) and filter sterilizing the media.
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The culture (1 mL) was drawn into 1.5 mL centrifuge tube and was centrifuged at 10 ×
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1000g for 1 min. The supernatant was discarded and Bacteroides minimal medium (1 mL) was
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added and the bacterial pellet was re-suspended in it. This was again centrifuged (10 × 1000g for
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1 min) to wash the bacteria. This step was repeated another time. After discarding the second
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washing, the bacterial pellet was re-suspended in 1 mL of Bacteroides minimal medium. This
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was the washed culture. Washed culture was pipetted into Bacteroides minimal medium to
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prepare the “diluted bacterial culture” (1:50 ratio, OD
