Identification of Oligosaccharides in Human Milk Bound onto the Toxin A Carbohydrate Binding Site of Clostridium difficile

Thi Thanh Hanh  Nguyen; Jong Woon  Kim; Jun-Seong  Park; Kyeong Hwan  Hwang; Tae-Su  Jang; Chun-Hyung  Kim  and Doman  Kim

doi:10.4014/jmb.1509.09034

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Bioactive Compounds and Chemical Biology

Identification of Oligosaccharides in Human Milk Bound onto the Toxin A Carbohydrate Binding Site of Clostridium difficile

Thi Thanh Hanh Nguyen ¹, Jong Woon Kim ², Jun-Seong Park ³, Kyeong Hwan Hwang ³, Tae-Su Jang ¹, Chun-Hyung Kim ¹ and Doman Kim ^{1, 4*}

¹Institute of Food Industrialization, Institutes of Green Bio Science & Technology, Seoul National University, Pyeongchang 25354, Republic of Korea, ²Department of Obstetrics and Gynecology, Chonnam National University Medical School, Gwangju 61469, Republic of Korea, ³Amorepacific Corp. R&D Ctr., Gyeonggi-do 17074, Republic of Korea, ⁴Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang 25354, Republic of Korea

Received: September 11, 2015; Accepted: December 23, 2015

J. Microbiol. Biotechnol. 2016; 26(4): 659-665

Published April 28, 2016 https://doi.org/10.4014/jmb.1509.09034

Abstract

The oligosaccharides in human milk constitute a major innate immunological mechanism by which breastfed infants gain protection against infectious diarrhea. Clostridium difficile is the most important cause of nosocomial diarrhea, and the C-terminus of toxin A with its carbohydrate binding site, TcdA-f2, demonstrates specific abolishment of cytotoxicity and receptor binding activity upon diethylpyrocarbonate modification of the histidine residues in TcdA. TcdA-f2 was cloned and expressed in E. coli BL21 (DE3). A human milk oligosaccharide (HMO) mixture displayed binding with TcdA-f2 at 38.2 respond units (RU) at the concentration of 20 μg/ml, whereas the eight purified HMOs showed binding with the carbohydrate binding site of TcdA-f2 at 3.3 to 14 RU depending on their structures via a surface plasma resonance biosensor. Among them, Lacto-N-fucopentaose V (LNFPV) and Lacto-N-neohexaose (LNnH) demonstrated tight binding to TcdA-f2 with docking energy of −9.48 kcal/mol and −12.81 kcal/mol, respectively. It displayed numerous hydrogen bonding and hydrophobic interactions with amino acid residues of TcdA-f2.

Keywords

Clostridium difficile, human milk oligosaccharides, molecular docking, surface plasmon resonance, toxin A

References

Bartlett JG. 2008. Historical perspectives on studies of Clostridium difficile and C. difficile infection. Clin. Infect. Dis. 46: S4-S11.
Black RE, Morris SS, Bryce J. 2003. Where and why are 10 million children dying every year? Lancet 361: 2226-2234.
Bode L. 2012. Human milk oligosaccharides: every baby needs a sugar mama. Glycobiology 22: 1147-1162.
Borgert CJ, Borgert SA, Findley KC. 2005. Synergism, antagonism, or additivity of dietary supplements: application of theory to case studies. Thromb. Res. 117: 123-132; discussion 145-151.
Chaturvedi P, Warren CD, RuizPalacios GM, Pickering LK, Newburg DS. 1997. Milk oligosaccharide profiles by reversedphase HPLC of their perbenzoylated derivatives. Anal. Biochem. 251: 89-97.
Coppa GV, Pierani P, Zampini L, Carloni I, Carlucci A, Gabrielli O. 1999. Oligosaccharides in human milk during different phases of lactation. Acta Paediatr. Suppl. 88: 89-94.
De Leoz MLA, Kalanetra KM, Bokulich NA, Strum JS, Underwood MA, German JB, et al. 2015. Human milk glycomics and gut microbial genomics in infant feces show a correlation between human milk oligosaccharides and gut microbiota. J. Proteome Res. 14: 491-502.
Dingle T, Wee S, Mulvey GL, Greco A, Kitova EN, Sun JX, et al. 2008. Functional properties of the carboxy-terminal host cell-binding domains of the two toxins, TcdA and TcdB, expressed by Clostridium difficile. Glycobiology 18: 698-706.
Duleba K, Pawlowska M, Wietlicka-Piszcz M. 2014. Clostridium difficile infection in children hospitalized due to diarrhea. Eur. J. Clin. Microbiol. Infect. Dis. 33: 201-209.
Egerer M, Giesemann T, Jank T, Satchell KJ, Aktories K. 2007. Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on cysteine protease activity. J. Biol. Chem. 282: 25314-25321.
El-Hawiet A, Kitova EN, Kitov PI, Eugenio L, Ng KK, Mulvey GL, et al. 2011. Binding of Clostridium difficile toxins to human milk oligosaccharides. Glycobiology 21: 1217-1227.
El-Hawiet A, Kitova EN, Klassen JS. 2015. Recognition of human milk oligosaccharides by bacterial exotoxins. Glycobiology 25: 845-854.
Greco A, Ho JG, Lin SJ, Palcic MM, Rupnik M, Ng KK. 2006. Carbohydrate recognition by Clostridium difficile toxin A. Nat. Struct. Mol. Biol. 13: 460-461.
Idota T, Kawakami H, Murakami Y, Sugawara M. 1995. Inhibition of cholera toxin by human milk fractions and sialyllactose. Biosci. Biotechnol. Biochem. 59: 417-419.
Jangi S, Lamont JT. 2010. Asymptomatic colonization by Clostridium difficile in infants: implications for disease in later life. J. Pediatr. Gastroenterol. Nutr. 51: 2-7.
Kobata A. 2010. Structures and application of oligosaccharides in human milk. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 86: 1-17.
Liu B, Newburg DS. 2013. Human milk glycoproteins protect infants against human pathogens. Breastfeed. Med. 8: 354-362.
Locascio RG, Ninonuevo MR, Freeman SL, Sela DA, Grimm R, Lebrilla CB, et al. 2007. Glycoprofiling of bifidobacterial consumption of human milk oligosaccharides demonstrates strain specific, preferential consumption of small chain glycans secreted in early human lactation. J. Agric. Food Chem. 55: 8914-8919.
Mitty RD, LaMont JT. 1994. Clostridium difficile diarrhea:pathogenesis, epidemiology, and treatment. Gastroenterologist 2: 61-69.
Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, Olson AJ. 1998. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J. Comput. Chem. 19: 1639-1662.
Morrow AL, Ruiz-Palacios GM, Jiang X, Newburg DS. 2005. Human-milk glycans that inhibit pathogen binding protect breast-feeding infants against infectious diarrhea. J. Nutr. 135: 1304-1307.
Newburg DS. 2009. Neonatal protection by an innate immune system of human milk consisting of oligosaccharides and glycans. J. Anim. Sci. 87(13 Suppl): 26-34.
Nguyen TT, Woo HJ, Kang HK, Nguyen VD, Kim YM, Kim DW, et al. 2012. Flavonoid-mediated inhibition of SARS coronavirus 3C-like protease expressed in Pichia pastoris. Biotechnol. Lett. 34: 831-838.
Nguyen TTH, Kang HK, Kim YM, Jang TS, Kim D. 2014. Inhibition effect of flavonoid compounds against neuraminidase expressed in Pichia pastoris. Biotechnol. Bioproc. Eng. 19: 70-75.
Nguyen TTH, Ryu HJ, Lee SH, Hwang S, Breton V, Rhee JH, Kim D. 2011. Virtual screening identification of novel severe acute respiratory syndrome 3C-like protease inhibitors and in vitro confirmation. Bioorg. Med. Chem. Lett. 21: 3088-3091.
Ninonuevo MR, Park Y, Yin HF, Zhang JH, Ward RE, Clowers BH, et al. 2006. A strategy for annotating the human milk glycome. J. Agric. Food Chem. 54: 7471-7480.
Ranalli A, Lucera L, Contento S. 2003. Antioxidizing potency of phenol compounds in olive oil mill wastewater. J. Agric. Food Chem. 51: 7636-7641.
Roberts AK, Shone CC. 2001. Modification of surface histidine residues abolishes the cytotoxic activity of Clostridium difficile toxin A. Toxicon 39: 325-333.
Sauerborn M, Leukel P, von Eichel-Streiber C. 1997. The Cterminal ligand-binding domain of Clostridium difficile toxin A (TcdA) abrogates TcdA-specific binding to cells and prevents mouse lethality. FEMS Microbiol. Lett. 155: 45-54.
Shang J, Piskarev VE, Xia M, Huang PW, Jiang X, Likhosherstov LM, et al. 2013. Identifying human milk glycans that inhibit norovirus binding using surface plasmon resonance. Glycobiology 23: 1491-1498.
Smilowitz JT, Lebrilla CB, Mills DA, German JB, Freeman SL. 2014. Breast milk oligosaccharides: structure-function relationships in the neonate. Annu. Rev. Nutr. 34: 143-169.
Thi THN, Moon YH, Ryu YB, Kim YM, Nam SH, Kim MS, et al. 2013. The influence of flavonoid compounds on the in vitro inhibition study of a human fibroblast collagenase catalytic domain expressed in E. coli. Enzyme Microb. Technol. 52: 26-31.
Totten SM, Zivkovic AM, Wu S, Ngyuen U, Freeman SL, Ruhaak LR, et al. 2012. Comprehensive profiles of human milk oligosaccharides yield highly sensitive and specific markers for determining secretor status in lactating mothers. J. Proteome Res. 11: 6124-6133.
Urashima T, Asakuma S, Leo F, Fukuda K, Messer M, Oftedal OT. 2012. The predominance of type I oligosaccharides is a feature specific to human breast milk. Adv. Nutr. 3: 473s-482s.
Wallace AC, Laskowski RA, Thornton JM. 1995. Ligplot - a program to generate schematic diagrams of protein ligand interactions. Protein Eng. 8: 127-134.
Wultanska D, Obuch-Woszczatynski P, Banaszkiewicz A, Radzikowski A, Pituch H, Mlynarczyk G. 2010. Prevalence of Clostridium difficile in the gastrointestinal tract of hospitalized children under two years of age. Med. Dosw. Mikrobiol. 62:77-84.
Zilberberg MD, Shorr AF, Kollef MH. 2008. Increase in adult Clostridium difficile-related hospitalizations and casefatality rate, United States, 2000-2005. Emerg. Infect. Dis. 14:929-931.

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Research article

J. Microbiol. Biotechnol. 2016; 26(4): 659-665

Published online April 28, 2016 https://doi.org/10.4014/jmb.1509.09034

Identification of Oligosaccharides in Human Milk Bound onto the Toxin A Carbohydrate Binding Site of Clostridium difficile

Thi Thanh Hanh Nguyen ¹, Jong Woon Kim ², Jun-Seong Park ³, Kyeong Hwan Hwang ³, Tae-Su Jang ¹, Chun-Hyung Kim ¹ and Doman Kim ^{1, 4*}

¹Institute of Food Industrialization, Institutes of Green Bio Science & Technology, Seoul National University, Pyeongchang 25354, Republic of Korea, ²Department of Obstetrics and Gynecology, Chonnam National University Medical School, Gwangju 61469, Republic of Korea, ³Amorepacific Corp. R&D Ctr., Gyeonggi-do 17074, Republic of Korea, ⁴Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang 25354, Republic of Korea

Received: September 11, 2015; Accepted: December 23, 2015

Abstract

The oligosaccharides in human milk constitute a major innate immunological mechanism by
which breastfed infants gain protection against infectious diarrhea. Clostridium difficile is the
most important cause of nosocomial diarrhea, and the C-terminus of toxin A with its
carbohydrate binding site, TcdA-f2, demonstrates specific abolishment of cytotoxicity and
receptor binding activity upon diethylpyrocarbonate modification of the histidine residues in
TcdA. TcdA-f2 was cloned and expressed in E. coli BL21 (DE3). A human milk oligosaccharide
(HMO) mixture displayed binding with TcdA-f2 at 38.2 respond units (RU) at the
concentration of 20 μg/ml, whereas the eight purified HMOs showed binding with the
carbohydrate binding site of TcdA-f2 at 3.3 to 14 RU depending on their structures via a
surface plasma resonance biosensor. Among them, Lacto-N-fucopentaose V (LNFPV) and
Lacto-N-neohexaose (LNnH) demonstrated tight binding to TcdA-f2 with docking energy of
−9.48 kcal/mol and −12.81 kcal/mol, respectively. It displayed numerous hydrogen bonding
and hydrophobic interactions with amino acid residues of TcdA-f2.

Keywords: Clostridium difficile, human milk oligosaccharides, molecular docking, surface plasmon resonance, toxin A

Abstract

References

Bartlett JG. 2008. Historical perspectives on studies of Clostridium difficile and C. difficile infection. Clin. Infect. Dis. 46: S4-S11.
Black RE, Morris SS, Bryce J. 2003. Where and why are 10 million children dying every year? Lancet 361: 2226-2234.
Bode L. 2012. Human milk oligosaccharides: every baby needs a sugar mama. Glycobiology 22: 1147-1162.
Borgert CJ, Borgert SA, Findley KC. 2005. Synergism, antagonism, or additivity of dietary supplements: application of theory to case studies. Thromb. Res. 117: 123-132; discussion 145-151.
Chaturvedi P, Warren CD, RuizPalacios GM, Pickering LK, Newburg DS. 1997. Milk oligosaccharide profiles by reversedphase HPLC of their perbenzoylated derivatives. Anal. Biochem. 251: 89-97.
Coppa GV, Pierani P, Zampini L, Carloni I, Carlucci A, Gabrielli O. 1999. Oligosaccharides in human milk during different phases of lactation. Acta Paediatr. Suppl. 88: 89-94.
De Leoz MLA, Kalanetra KM, Bokulich NA, Strum JS, Underwood MA, German JB, et al. 2015. Human milk glycomics and gut microbial genomics in infant feces show a correlation between human milk oligosaccharides and gut microbiota. J. Proteome Res. 14: 491-502.
Dingle T, Wee S, Mulvey GL, Greco A, Kitova EN, Sun JX, et al. 2008. Functional properties of the carboxy-terminal host cell-binding domains of the two toxins, TcdA and TcdB, expressed by Clostridium difficile. Glycobiology 18: 698-706.
Duleba K, Pawlowska M, Wietlicka-Piszcz M. 2014. Clostridium difficile infection in children hospitalized due to diarrhea. Eur. J. Clin. Microbiol. Infect. Dis. 33: 201-209.
Egerer M, Giesemann T, Jank T, Satchell KJ, Aktories K. 2007. Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on cysteine protease activity. J. Biol. Chem. 282: 25314-25321.
El-Hawiet A, Kitova EN, Kitov PI, Eugenio L, Ng KK, Mulvey GL, et al. 2011. Binding of Clostridium difficile toxins to human milk oligosaccharides. Glycobiology 21: 1217-1227.
El-Hawiet A, Kitova EN, Klassen JS. 2015. Recognition of human milk oligosaccharides by bacterial exotoxins. Glycobiology 25: 845-854.
Greco A, Ho JG, Lin SJ, Palcic MM, Rupnik M, Ng KK. 2006. Carbohydrate recognition by Clostridium difficile toxin A. Nat. Struct. Mol. Biol. 13: 460-461.
Idota T, Kawakami H, Murakami Y, Sugawara M. 1995. Inhibition of cholera toxin by human milk fractions and sialyllactose. Biosci. Biotechnol. Biochem. 59: 417-419.
Jangi S, Lamont JT. 2010. Asymptomatic colonization by Clostridium difficile in infants: implications for disease in later life. J. Pediatr. Gastroenterol. Nutr. 51: 2-7.
Kobata A. 2010. Structures and application of oligosaccharides in human milk. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 86: 1-17.
Liu B, Newburg DS. 2013. Human milk glycoproteins protect infants against human pathogens. Breastfeed. Med. 8: 354-362.
Locascio RG, Ninonuevo MR, Freeman SL, Sela DA, Grimm R, Lebrilla CB, et al. 2007. Glycoprofiling of bifidobacterial consumption of human milk oligosaccharides demonstrates strain specific, preferential consumption of small chain glycans secreted in early human lactation. J. Agric. Food Chem. 55: 8914-8919.
Mitty RD, LaMont JT. 1994. Clostridium difficile diarrhea:pathogenesis, epidemiology, and treatment. Gastroenterologist 2: 61-69.
Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, Olson AJ. 1998. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J. Comput. Chem. 19: 1639-1662.
Morrow AL, Ruiz-Palacios GM, Jiang X, Newburg DS. 2005. Human-milk glycans that inhibit pathogen binding protect breast-feeding infants against infectious diarrhea. J. Nutr. 135: 1304-1307.
Newburg DS. 2009. Neonatal protection by an innate immune system of human milk consisting of oligosaccharides and glycans. J. Anim. Sci. 87(13 Suppl): 26-34.
Nguyen TT, Woo HJ, Kang HK, Nguyen VD, Kim YM, Kim DW, et al. 2012. Flavonoid-mediated inhibition of SARS coronavirus 3C-like protease expressed in Pichia pastoris. Biotechnol. Lett. 34: 831-838.
Nguyen TTH, Kang HK, Kim YM, Jang TS, Kim D. 2014. Inhibition effect of flavonoid compounds against neuraminidase expressed in Pichia pastoris. Biotechnol. Bioproc. Eng. 19: 70-75.
Nguyen TTH, Ryu HJ, Lee SH, Hwang S, Breton V, Rhee JH, Kim D. 2011. Virtual screening identification of novel severe acute respiratory syndrome 3C-like protease inhibitors and in vitro confirmation. Bioorg. Med. Chem. Lett. 21: 3088-3091.
Ninonuevo MR, Park Y, Yin HF, Zhang JH, Ward RE, Clowers BH, et al. 2006. A strategy for annotating the human milk glycome. J. Agric. Food Chem. 54: 7471-7480.
Ranalli A, Lucera L, Contento S. 2003. Antioxidizing potency of phenol compounds in olive oil mill wastewater. J. Agric. Food Chem. 51: 7636-7641.
Roberts AK, Shone CC. 2001. Modification of surface histidine residues abolishes the cytotoxic activity of Clostridium difficile toxin A. Toxicon 39: 325-333.
Sauerborn M, Leukel P, von Eichel-Streiber C. 1997. The Cterminal ligand-binding domain of Clostridium difficile toxin A (TcdA) abrogates TcdA-specific binding to cells and prevents mouse lethality. FEMS Microbiol. Lett. 155: 45-54.
Shang J, Piskarev VE, Xia M, Huang PW, Jiang X, Likhosherstov LM, et al. 2013. Identifying human milk glycans that inhibit norovirus binding using surface plasmon resonance. Glycobiology 23: 1491-1498.
Smilowitz JT, Lebrilla CB, Mills DA, German JB, Freeman SL. 2014. Breast milk oligosaccharides: structure-function relationships in the neonate. Annu. Rev. Nutr. 34: 143-169.
Thi THN, Moon YH, Ryu YB, Kim YM, Nam SH, Kim MS, et al. 2013. The influence of flavonoid compounds on the in vitro inhibition study of a human fibroblast collagenase catalytic domain expressed in E. coli. Enzyme Microb. Technol. 52: 26-31.
Totten SM, Zivkovic AM, Wu S, Ngyuen U, Freeman SL, Ruhaak LR, et al. 2012. Comprehensive profiles of human milk oligosaccharides yield highly sensitive and specific markers for determining secretor status in lactating mothers. J. Proteome Res. 11: 6124-6133.
Urashima T, Asakuma S, Leo F, Fukuda K, Messer M, Oftedal OT. 2012. The predominance of type I oligosaccharides is a feature specific to human breast milk. Adv. Nutr. 3: 473s-482s.
Wallace AC, Laskowski RA, Thornton JM. 1995. Ligplot - a program to generate schematic diagrams of protein ligand interactions. Protein Eng. 8: 127-134.
Wultanska D, Obuch-Woszczatynski P, Banaszkiewicz A, Radzikowski A, Pituch H, Mlynarczyk G. 2010. Prevalence of Clostridium difficile in the gastrointestinal tract of hospitalized children under two years of age. Med. Dosw. Mikrobiol. 62:77-84.
Zilberberg MD, Shorr AF, Kollef MH. 2008. Increase in adult Clostridium difficile-related hospitalizations and casefatality rate, United States, 2000-2005. Emerg. Infect. Dis. 14:929-931.

Articles Service

Research article

Identification of Oligosaccharides in Human Milk Bound onto the Toxin A Carbohydrate Binding Site of Clostridium difficile

Abstract

Keywords

References

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Article

Research article

Identification of Oligosaccharides in Human Milk Bound onto the Toxin A Carbohydrate Binding Site of Clostridium difficile

Abstract

References