Structural and biochemical characterization of the broad substrate specificity of Bacteroides thetaiotaomicron commensal sialidase

Park, Kwang‐Hyun; Kim, Mingyu; Ahn, Hee-Jeong; Lee, Daehan; Kim, Jin Hyo; Kim, Young-Wan; Woo, Eui-Jeon

doi:10.1016/j.bbapap.2013.04.028

Cited by 40 publications

(31 citation statements)

References 44 publications

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“…To measure sialidase enzyme activity, cell-free lysates from fecal samples were incubated with the artificial substrate 2-O-(p-Nitrophenyl)-tropheacetylneuraminic acid (pNP-SA), which is hydrolyzed by sialidases, and the subsequent release of the pNP moiety was monitored over time by LC-MS. pNP-SA was provided in 10-fold molar excess to its K m (K m = 0.11 mM) (Park et al, 2013) in order to ensure direct proportionality between the calculated initial reaction velocity and the sialidase concentration in each sample (Figure S6C). Prior to aTC administration, fecal samples from mice carrying Bt RS show no detectable sialidase activity, indicating tight repression of the enzyme and the absence of pNP in the gut (Figure 7B and Figure S6E).…”

Section: Resultsmentioning

confidence: 99%

Engineered Regulatory Systems Modulate Gene Expression of Human Commensals in the Gut

Lim

Zimmermann

Barry

et al. 2017

Cell

168

182

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Summary The gut microbiota is implicated in numerous aspects of health and disease, but dissecting these connections is challenging because genetic tools for gut anaerobes are limited. Inducible promoters are particularly valuable tools, because these platforms allow real-time analysis of the contribution of microbiome gene products to community assembly, host physiology, and disease. We developed a panel of tunable expression platforms for the prominent genus Bacteroides in which gene expression is controlled by a synthetic inducer. In the absence of inducer, promoter activity is fully repressed; addition of inducer rapidly increases gene expression by 4 to 5 orders of magnitude. Because the inducer is absent in mice and their diets, Bacteroides gene expression inside the gut can be modulated by providing the inducer in drinking water. We use this system to measure the dynamic relationship between commensal sialidase activity and liberation of mucosal sialic acid, a receptor and nutrient for pathogens.

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Section: Resultsmentioning

confidence: 99%

Engineered Regulatory Systems Modulate Gene Expression of Human Commensals in the Gut

Lim

Zimmermann

Barry

et al. 2017

Cell

168

182

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“…Members of the genus Bacteroides are an important contributor of bacterial sialidases (13, 18, 31, 39, 40) and sialic acid O -acetyl esterases (18, 39) relevant to the colonic niche. Thus, sialidases from Bacteroides fragilis (WP_010992682) and Bacteroides thetaiotaomicron (WP_008766031) were cloned, expressed in E. coli , and purified for use in our in vitro models of cooperative metabolism.…”

Section: Resultsmentioning

confidence: 99%

“…1 C ). We used well described sialidases produced by symbionts, commensals, and pathogens, including 1) the prevalent gut symbiont B. thetaiotaomicron (40), 2) the common human commensal and opportunistic pathogen B. fragilis (48), and 3) the water-borne bacterial intestinal pathogen Vibrio cholerae (49). The results of these experiments illustrate that sialidases known to be present within the context of the healthy gut and during infection can enable the use of sialic acids by other members of the community.…”

Section: Resultsmentioning

confidence: 99%

The sialate O-acetylesterase EstA from gut Bacteroidetes species enables sialidase-mediated cross-species foraging of 9-O-acetylated sialoglycans

Robinson

Lewis

2017

Journal of Biological Chemistry

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The gut harbors many symbiotic, commensal, and pathogenic microbes that break down and metabolize host carbohydrates. Sialic acids are prominent outermost carbohydrates on host glycoproteins called mucins and protect underlying glycan chains from enzymatic degradation. Sialidases produced by some members of the colonic microbiota can promote the expansion of several potential pathogens (e.g. Clostridium difficile, Salmonella, and Escherichia coli) that do not produce sialidases. O-Acetyl ester modifications of sialic acids help resist the action of many sialidases and are present at high levels in the mammalian colon. However, some gut bacteria, in turn, produce sialylate-O-acetylesterases to remove them. Here, we investigated O-acetyl ester removal and sialic acid degradation by Bacteroidetes sialate-O-acetylesterases and sialidases, respectively, and subsequent utilization of host sialic acids by both commensal and pathogenic E. coli strains. In vitro foraging studies demonstrated that sialidase-dependent E. coli growth on mucin is enabled by Bacteroides EstA, a sialate O-acetylesterase acting on glycosidically linked sialylate-O-acetylesterase substrates, particularly at neutral pH. Biochemical studies suggested that spontaneous migration of O-acetyl esters on the sialic acid side chain, which can occur at colonic pH, may serve as a switch controlling EstA-assisted sialic acid liberation. Specifically, EstA did not act on O-acetyl esters in their initial 7-position. However, following migration to the 9-position, glycans with O-acetyl esters became susceptible to the sequential actions of bacterial esterases and sialidases. We conclude that EstA specifically unlocks the nutritive potential of 9-O-acetylated mucus sialic acids for foraging by bacteria that otherwise are prevented from accessing this carbon source.

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“…The activity of sialidases increases upon removal of O-acetyl groups from sialic acids by sialate O-acetylesterases (Phansopa et al, 2015). B. thetaiotaomicron, for example, represents a commensal of the gut, that produces a number of glycoside hydrolases such as the B. thetaiotaomicron sialidase BTSA (Park et al, 2013), enabling this microorganism to release terminally bound sialic acids from mucin.…”

Section: Introductionmentioning

confidence: 99%

De-O-Acetylation of mucin-derived sialic acids by recombinant NanS-p esterases of Escherichia coli O157:H7 strain EDL933

Feuerbaum

Saile

Pohlentz

et al. 2018

International Journal of Medical Microbiology

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Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL933 encodes the single chromosomal 9-O-acetylesterase NanS, and several copies of prophage-encoded 9-O-acetylesterases (NanS-p). These enzymes have recently been shown to cleave 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac 2 ) to yield de-O-acetylated Neu5Ac, the latter of which may serve as a carbon and/or nitrogen source.In the current study, we investigated the NanS-and NanS-p-mediated digestion of synthetic O-acetylated neuraminic acids and bovine submaxillary glands mucin (BSM)-derived O-acetylneuraminic acids by high-performance thin-layer chromatography (HPTLC) and nano electrospray ionization mass spectrometry (nanoESI MS). Initial HPTLC analyses showed the expected activity of NanS and NanS-p variants for Neu5,9Ac 2 . However, all tested enzymes were unable to de-O-acetylate 5-N-acetyl-4-O-acetylneuraminic acid (Neu5,4 Ac 2 ) in our test system. The nanoESI MS analysis of neuraminic acids after treatment of BSM with NanS-p gave evidence that NanS-p variants of EHEC O157:H7 strain EDL933 cleave off O-acetyl groups from mono-, di-, and tri-O-acetylated Neu5Ac and N-glycolylneuraminic acid (Neu5Gc), regardless of the carbon positions C7, C8 or C9 of the acetate esters. This enzyme activity leads to neuraminidase-accessible Neu5Ac and Neu5Gc on mucin glycans.Moreover, we could demonstrate by HPTLC analyses that recombinant Bacteroides thetaiotaomicron sialidase (BTSA-His) was able to cleave Neu5Ac and Neu5,9Ac 2 from BSM and that the combination of BTSA-His with both NanS-His and NanS-p-His derivatives enhanced the release of de-O-acetylated core Neu5Ac and Neu5Gc from mammalian mucin O-glycans.Growth experiments with EHEC wildtype strain EDL933, its nanS and nanS/nanS-p1a-p7 mutant and exogenous BTSA-His in BSM demonstrated that the presence of BTSA-His enhanced growth of EDL933 and the nanS deletion mutant but not the nanS/nanS-p1a-p7 mutant.Thus, we hypothesize that the expression of sialic acid O-acetylesterases with a broad specificity could be an advantage in competition with the gut microbiota for nutrients and facilitate EHEC colonization in the human large intestine.

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Structural and biochemical characterization of the broad substrate specificity of Bacteroides thetaiotaomicron commensal sialidase

Cited by 40 publications

References 44 publications

Engineered Regulatory Systems Modulate Gene Expression of Human Commensals in the Gut

Engineered Regulatory Systems Modulate Gene Expression of Human Commensals in the Gut

The sialate O-acetylesterase EstA from gut Bacteroidetes species enables sialidase-mediated cross-species foraging of 9-O-acetylated sialoglycans

De-O-Acetylation of mucin-derived sialic acids by recombinant NanS-p esterases of Escherichia coli O157:H7 strain EDL933

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