Structural analysis of glycans and development of microarrays from Helcobacter pylori cell surface glycome

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Ano 2014

LISETE MACHADO E

SILVA

ANÁLISE ESTRUTURAL DE GLICOSÍDEOS E

DESENVOLVIMENTO DE MICROARRAYS DO

GLICOMA DA MEMBRANA DE HELICOBACTER

PYLORI

STRUCTURAL ANALYSIS OF GLYCANS AND

DEVELOPMENT OF MICROARRAYS FROM

HELICOBACTER PYLORI

CELL SURFACE

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Ano 2014

LISETE MACHADO E

SILVA

ANÁLISE ESTRUTURAL DE GLICOSÍDEOS E

DESENVOLVIMENTO DE MICROARRAYS DO

GLICOMA DA MEMBRANA DE HELICOBACTER

PYLORI

Tese apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Doutora em Bioquímica, realizada sob a orientação científica do Doutor Manuel António Coimbra Rodrigues da Silva, Professor Associado com Agregação do Departamento de Química da Universidade de Aveiro e co-orientação do Doutor José Alexandre Ferreira, Investigador de Pós-Doutoramento do Departamento de Química da Universidade de Aveiro e do Instituto Português de Oncologia (IPO) do Porto e da Doutora Maria Angelina de Sá Palma, Investigadora da FCT e do REQUIMTE do Departamento de Química da Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa (FCT-UNL).

Apoio financeiro da FCT e do FSE no âmbito do III Quadro Comunitário de Apoio.

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o júri

presidente Doutora Maria Ana Dias Monteiro Santos

Professora Catedrática da Universidade de Aveiro

Professor Ten Feizi

Professor and Director of the Glycosciences Laboratory, Department of Medicine, Faculty of Medicine, Imperial College London – United Kingdom

Doutor Celso Albuquerque Reis

Investigador Coordenador do IPATIMUP – Instituto de Patologia e Imunologia Molecular da Universidade do Porto

Doutora Amélia Pilar Grases Santos Silva Rauter

Professora Associada com Agregação da Faculdade de Ciências da Universidade de Lisboa

Doutor Manuel António Coimbra Rodrigues da Silva

Professor Associado com Agregação da Universidade de Aveiro (Orientador)

Doutor Manuel António da Silva Santos

Professor Associado com Agregação da Secção Autónoma de Ciências da Saúde da Universidade de Aveiro

Doutora Maria do Rosário Gonçalves Reis Marques Domingues

Professora Auxiliar com Agregação da Universidade de Aveiro

Doutora Maria Angelina de Sá Palma

Investigadora da FCT - Fundação para a Ciência e Tecnologia e do REQUIMTE, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa (Coorientadora)

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Acknowledgements Undertaking this thesis would not have been possible without the support of many people.

First of all, I would like to express my sincere gratitude to my supervisor Prof. Manuel António Coimbra for trusting and encouraging me to develop the present work. Thank you Professor for all of your transmitted scientific knowledge, for your patience, support, friendship, and most importantly, for your availability that was crucial to carry out this thesis. Thank you “scientific father”, for helping me to grow throughout these years. I must tell you that it was a pleasure for me working with you, and will always be.

To my co-supervisor Dr. José Alexandre Ferreira, my sincere thanks for presenting me a new world, the complex world of H. pylori, with which I enjoyed work. Also, thank you for your friendship and helpful discussions over these years.

To Dr. Angelina Palma, also my co-supervisor, many thanks for the endless support, mainly over the last and intense year and half. Thank you for showing me a tiny part of the applicability of carbohydrate microarrays, which gave me the opportunity of being introduced to another reality. Thank you for helping me in this work both at FCT-UNL and at Imperial College London, for your friendship and for the leisure moments we spent together in these amazing cities.

I would like to thank the QOPNA research unit from Department of Chemistry of the University of Aveiro (UA), for accepting me as a PhD student and providing all the conditions to carry this investigation; to Fundação para a Ciência e Tecnologia (FCT) for the grant (SFRH/BD/71455/2010) and for the

H.pylori´s project (EXPL/BBB-BQB/0750/2012).

My sincere thanks to those involved in H. pylori project: to Prof. Céu Figueiredo from IPATIMUP, Porto, for providing the clinical isolates and the reference H. pylori strain, as well as the human serum infected with this bacterium used in this investigation; to Dr. Rui Ferreira also from IPATIMUP, for teaching me how to grow H. pylori; to Dr. Nuno Guimarães from FEUP and IPATIMUP for kindly providing the non-infected human serum; and to Dr. Nuno Azevedo from FEUP for kindly providing the strain 2191.

To Prof. Sónia Mendo, from Department of Biology of UA, and especially to her PhD student Cátia Santos and to the lab technicians Mrs. Helena Dias and Mr. Armando Costa, my sincere gratitude for the guidance in growing the bacterial cells and for all the great moments that made this challenge more easily achieved.

I would like also to thank Prof. Rosário Domingues and my colleague Ana Moreira from Department of Chemistry of UA for helping me with all Mass Spectrometry experiments, for their availability and patience with my residual samples!

To all of my lab colleagues from UA, thank you for creating a friendly environment! A special thank goes to Rita Bastos for her friendship and company during many weekends that we spent working in the lab, to Magda Santos for helping me with GC-MS experiments, to Helena Teixeira for the efficiency in ordering materials and reagents and to the Post-docs Cláudia Nunes, Elisabete Coelho, Joana Simões and Cláudia Passos, for their

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I would like to express my sincere gratitude to Cláudia Passos, with whom I shared the house over these last 4 years. Thank you Cláudia for your hospitality, friendship, and excellent leisure moments we have spent together. I cannot forget Ângela Cunha, who also worked and lived with us during 2 years. Great times! Thank you Ângela for everything!

Part of this work was done in the FCT-UNL, Lisbon. Therefore, I would like to express my gratitude to Prof. Maria João Romão and colleagues, from Department of Chemistry, and to Prof. Isabel de Sá-Nogueira from Department of Life Sciences for accepting me in as visiting student in their labs. To Viviana Correia, Lia Godinho, Mário Ferreira, Raquel Portela and Damien Costa, thank you for sharing your knowledge in Microbial Genetics, for your friendship and for the amazing leisure times we spent together.

To André Pinheiro and Bruno Veigas, my housemates during my stay in Lisbon, thank you for your company and for good moments of fun.

This scientific journey ended in London, at the Glycosciences Laboratory, where I spent one month, under the supervision of Prof. Ten Feizi. Thank you Professor for this great opportunity, for your kindness and hospitality. Thank you for sharing your expertise and knowledge in Glycobiology. I am looking forward to learning much more with you. I also would like to acknowledge Dr. Wengang Chai, Dr. Yhibing Zhang, Dr. Robert Childs, Dr. Colin Herbert, Dr. Yan Liu, and the PhD students Zhen Li and Chao Gao for their dedication in helping me to enrich this work. Thank you!

O excelente desempenho desta tese também só foi possível devido à presença e apoio incondicional de amigos fora do mundo académico.

Assim, quero expressar o meu sincero reconhecimento às minhas “compinchas” Juliana Pinto, Marta Costa e Raquel Carvalho por me terem acompanhado, sempre de perto, em mais uma etapa importante da minha vida. Obrigada pela vossa amizade, confiança e companheirismo de anos.

Ao Bruno Silva, por nunca me fazer esquecer da pylori! Ao Adriano Moreira e também ao Ricardo Sousa, por todas as discussões relacionadas com a área de investigação. Ao Hugo Batista, Bruno e Sandra Macedo, Marisa Quintas, Joana Pinto, Daniel Silva, José Gomes da Costa, Carmen Curralo, Miguel Macedo, Rómulo Silva, Patrícia Arrimar, Salomé Gaio, Hélio Dias, Licínia Sousa, Libânia Braga, Sérgio Soares, Pedro Santos, Patrícia Silva e João Fonseca. Ao Ricardo Silva e ao Márcio Cruz. Obrigada a todos vocês por fazerem parte da minha vida, por contribuírem para o meu bem-estar e por todas as experiências partilhadas ao longo destes anos.

À Joana Lia e à Joana Coelho, pela amizade que começou no tempo da Licenciatura, no Porto, e que se mantém até ao presente.

Ao Marco Monteiro pelos 16 anos de uma grande amizade. Obrigada Marco, por não falhares nenhuma etapa da minha vida.

À D. Paula, ao Sr. Lemos, à D. Augusta, ao Sr. Abreu e toda a restante equipa do Restaurante “Mar à Vista”, que foi durante muito tempo a minha 2ª casa. Obrigada pelo carinho, amizade e pela confiança depositada durante todos estes anos.

Por fim, um especial agradecimento à minha irmã, Marisa Silva, e aos meus pais, Manuel Luís Silva e Filomena Machado, a quem dedico esta tese. Obrigada pelo vosso carinho, boa disposição, constante presença, paciência e apoio incondicional. Obrigada pela qualidade de vida proporcionada, e por todo o esforço realizado durante anos que me permitiu chegar até aqui. Obrigada por acreditarem em mim e por tudo!

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palavras-chave Helicobacter pylori, lipopolissacarídeos, antigénios de Lewis, ribanas,

quito-oligossacarideos, análise estrutural, anticorpos, lectinas, microarrays de carboidratos, SDS-PAGE e Western blot.

resumo O Helicobacter pylori é uma bactéria patogénica que afeta mais de metade

da população mundial com doenças gastrointestinais e está associado ao cancro gástrico. A superfície celular do H. pylori é decorada com lipopolissacarídeos (LPSs), que são constituídos por três regiões distintas: uma região polissacarídica variável (“O-chain”), um “core” oligossacarídico estruturalmente conservado e o lípido A, que ancora o LPS à membrana celular. A “O-chain” apresenta estruturas oligossacarídicas únicas, tais como os antigénios de Lewis (Le), que são semelhantes às presentes na mucosa gástrica e que estão envolvidas na interação com o hospedeiro. Glucanas, heptoglicanas e ribanas também constituem o “core” dos LPSs do H. pylori. Glicanas do tipo amilose e mananas também são referidas como integrantes de estirpes de H. pylori, possivelmente co-expressas com os LPSs. A complexidade dos LPSs do H. pylori tem dificultado o estabelecimento de uma relação entre a estrutura e a função em interação com o hospedeiro, que é crucial para o desenvolvimento de vacinas. Os microarrays de carboidratos são ferramentas recentes no estudo de antigénios e de novos ligandos oligossacarídicos, e que têm revelado a sua função em interações patogénio-hospedeiro.

O trabalho desta tese teve como objetivos principais a análise estrutural de LPSs de estirpes do H. pylori isoladas de biópsias gástricas de Portugueses sintomáticos e a construção de um microarray de LPSs do H. pylori (H. pylori LPS microarray) para estudos funcionais de interação com proteínas.

Os LPSs foram extraídos da superfície celular de cinco isolados clínicos do

H. pylori e de 1 estirpe de referência (NCTC 26695) pelo método fenol/água,

fracionados por cromatografia de exclusão molecular e analisados por cromatografia em fase gasosa acoplada à espetrometria de massa. Os oligossacarídeos resultantes da hidrólise ácida parcial do LPS foram analisados por espetrometria de massa de electrospray. Para além de estruturas existentes no “core”, a análise estrutural, revelou a presença de antigénios de Le do tipo-2, Lex_{e Le}y_{, e de sequências de resíduos de}

N-acetil-lactosamina (LacNAc), tipicamente encontrados em H. pylori. A identificação de resíduos de glucose ligados na posição O-6 nos LPSs das estirpes 2191 e NCTC 26695, indicou a presença de _{6-glucanas.} Outros domínios, nomeadamente ribanas, compostos por resíduos de ribofuranose ligados em posição O-2, foram identificados nos LPSs da maioria dos isolados clínicos. A presença de uma galactana (O-3 galactose), recentemente identificada noutra estirpe do H. pylori, foi também identificada para a estirpe 14382. Nos LPSs das estirpes 2191 e CI-117, a elevada quantidade de resíduos de N-acetilglucosamina (GlcNAc) ligados na posição O-4, sugeriu a presença de glicosídeos semelhantes à quitina (_{4GlcNAc) que, ao nosso conhecimento,} ainda não foram descritos para o H. pylori.

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Para a construção do novo microarray, os LPSs analisados e os neoglicolípidos (NGLs) derivados de frações oligossacarídicas dos LPSs foram imobilizados não-covalentemente em suportes de nitrocelulose, juntamente

com NGLs com sequências oligossacarídicas definidas, LPSs de outras bactérias e outros polissacarídeos. O microarray foi avaliado com proteínas de especificidade conhecida, que incluíram anticorpos monoclonais (mAbs) específicos para os antigénios de Le e estruturas relacionadas, um módulo de ligação a carboidratos (CBM), lectinas de plantas e os recetores do sistema imunitário DC-SIGN e Dectina-1. A análise com estas proteínas forneceu informação complementar à análise estrutural, para além de ter sido útil para o controlo de qualidade do microarray.

A análise do microarray revelou a ocorrência de antigénios de Le do tipo-2, Lex e Ley, mas não do tipo-1, Lea e Leb, o que está de acordo com os resultados obtidos na análise estrutural. Os LPSs do H. pylori foram reconhecidos pela DC-SIGN, que é uma lectina conhecida por interagir com esta bactéria através dos antigénios de Le expressos nos seus LPSs. A lectina UEA-I, específica para _{-fucose, mostrou ser restrita para determinantes do} grupo sanguíneo H do tipo-2 e para os LPSs das estirpes CI-117 e 14382. A ocorrência de antigénios do tipo H-2 e do tipo H-1 nos LPSs destas estirpes foi corroborada utilizando mAbs específicos. Os antigénios do tipo H-1 já tinham sido identificados em H. pylori, mas os do tipo H-2 foram identificados pela primeira vez neste estudo. Os LPSs do H. pylori foram também reconhecidos por lectinas com especificidade para oligossacarídeos de quitina (_4-linked GlcNAc). A STL, que mostrou uma ligação restrita aos tri- e pentassacarídeos, reconheceu distintivamente o LPS da estirpe CI-117, que poderão ser internas, dada a ausência de ligação detectada pela lectina WGA, com especificidade para terminais não redutores _{4GlcNAc. A análise dos LPSs do H. pylori por} SDS-PAGE e Western blot com a STL apontou para a presença destas estruturas na “O-chain” deste LPS.

O microarray dos LPSs do H. pylori foi utilizado para a análise do soro de um indivíduo infetado com H. pylori (H. pylori+

CI-5), e mostrou um aumento na

detecção de IgGs para os LPSs de H. pylori no soro H. pylori+ quando comparado com o soro de um indivíduo não-infetado (H. pylori-). A resposta observada foi específica para o LPS do isolado da estirpe CI-5, causadora da infeção.

O trabalho desenvolvido nesta tese contribuiu para a extensão do conhecimento estrutural dos LPSs dos isolados clínicos do H. pylori. A construção do microarray dos LPSs de H. pylori permitiu o estudo de interações com proteínas do hospedeiro, e mostrou ser útil na análise serológica de indivíduos infetados com esta bactéria. Deste modo, espera-se que o uso destas técnicas complementares possa contribuir para uma melhor compreensão da complexidade molecular dos LPSs e do seu papel na patogenicidade.

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keywords Helicobacter pylori, lipopolysaccharides, Lewis antigens, ribans, chitin-like

glycans, structural analysis, carbohydrate microarrays, anticorpos, lectins, SDS-PAGE and Western blot.

abstract Helicobacter pylori is a bacterial pathogen that affects more than half of the

world’s population with gastro-intestinal diseases and is associated with gastric cancer. The cell surface of H. pylori is decorated with lipopolysaccharides (LPSs) composed of three distinct regions: a variable polysaccharide moiety (O-chain), a structurally conserved core oligosaccharide, and a lipid A region that anchors the LPS to the cell membrane. The O-chain of H. pylori LPS, exhibits unique oligosaccharide structures, such as Lewis (Le) antigens, similar to those present in the gastric mucosa and are involved in interactions with the host. Glucan, heptoglycan, and riban domains are present in the outer core region of some H. pylori LPSs. Amylose-like glycans and mannans are also constituents of some H. pylori strains, possibly co-expressed with LPSs. The complexity of H. pylori LPSs has hampered the establishment of accurate structure-function relationships in interactions with the host, and the design of carbohydrate-based therapeutics, such as vaccines. Carbohydrate microarrays are recent powerful and sensitive tools for studying carbohydrate antigens and, since their emergence, are providing insights into the function of carbohydrates and their involvement in pathogen-host interactions.

The major goals of this thesis were the structural analysis of LPSs from H.

pylori strains isolated from gastric biopsies of symptomatic Portuguese patients

and the construction of a novel pathogen carbohydrate microarray of these LPSs (H. pylori LPS microarray) for interaction studies with proteins.

LPSs were extracted from the cell surface of five H. pylori clinical isolates and one NCTC strain (26695) by phenol/water method, fractionated by size exclusion chromatography and analysed by gas chromatography coupled to mass spectrometry. The oligosaccharides released after mild acid treatment of the LPS were analysed by electrospray mass spectrometry. In addition to the conserved core oligosaccharide moieties, structural analyses revealed the presence of type-2 Lex_{and Le}y_{antigens and N-acetyllactosamine (LacNAc)}

sequences, typically found in H. pylori strains. Also, the presence of O-6 linked glucose residues, particularly in LPSs from strains 2191 and NCTC 26695, pointed out to the expression of a _{6-glucan. Other structural domains, namely} ribans, composed of O-2 linked ribofuranose residues were observed in the LPS of most of H. pylori clinical isolates. For the LPS from strain 14382, large amounts of O-3 linked galactose units, pointing to the occurrence of a galactan, a domain recently identified in the LPS of another H. pylori strain. A particular feature to the LPSs from strains 2191 and CI-117 was the detection of large amounts of O-4 linked N-acetylglucosamine (GlcNAc) residues, suggesting the presence of chitin-like glycans, which to our knowledge have not been described for H. pylori strains.

For the construction of the H. pylori LPS microarray, the structurally analysed LPSs, as well as LPS-derived oligosaccharide fractions, prepared as neoglycolipid (NGL) probes were noncovalently immobilized onto nitrocellulose-coated glass slides. These were printed together with NGLs of selected sequence defined oligosaccharides, bacterial LPSs and polysaccharides.

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binding proteins (CBPs) of known specificity. These included Le and blood group-related monoclonal antibodies (mAbs), plant lectins, a carbohydrate-binding module (CBM) and the mammalian immune receptors DC-SIGN and Dectin-1. The analysis of these CBPs provided new information that complemented the structural analyses and was valuable in the quality control of the constructed microarray.

Microarray analysis revealed the occurrence of type-2 Lex_{and Le}y_{, but not}

type-1 Lea or Leb antigens, supporting the results obtained in the structural analysis. Furthermore, the H. pylori LPSs were recognised by DC-SIGN, a mammalian lectin known to interact with this bacterium through fucosylated Le epitopes expressed in its LPSs. The _{-fucose-specific lectin UEA-I, showed} restricted binding to probes containing type-2 blood group H sequence and to the LPSs from strains CI-117 and 14382. The presence of type-2, as well H-type-1 in the LPSs from these strains, was confirmed using specific mAbs. Although H-type-1 determinant has been reported for H. pylori LPSs, this is the first report of the presence of H-type-2 determinant.

Microarray analysis also revealed that plant lectins known to bind _4-linked GlcNAc chitin oligosaccharide sequences bound H. pylori LPSs. STL, which exhibited restricted and strong binding to _{4GlcNAc tri- and pentasaccharides,} differentially recognised the LPS from the strain CI-117. The chitin sequences recognised in the LPS could be internal, as no binding was detected to this LPS with WGA, known to be specific for nonreducing terminal of _4GlcNAc sequence. Analyses of the H. pylori LPSs by SDS-PAGE and Western blot with STL provided further evidence for the presence of these novel domains in the O-chain region of this LPS.

H. pylori LPS microarray was also applied to analysis of two human sera.

The first was from a case infected with H. pylori (H. pylori+ CI-5) and the second

was from a non-infected control.The analysis revealed a higher IgG-reactivity towards H. pylori LPSs in the H. pylori+

serum, than the control serum. A

specific IgG response was observed to the LPS isolated from the CI-5 strain, which caused the infection.

The present thesis has contributed to extension of current knowledge on chemical structures of LPS from H. pylori clinical isolates. Furthermore, the H.

pylori LPS microarray constructed enabled the study of interactions with host

proteins and showed promise as a tool in serological studies of H. pylori-infected individuals. Thus, it is anticipated that the use of these complementary approaches may contribute to a better understanding of the molecular complexity of the LPSs and their role in pathogenesis.

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Fig. 1.1 General architecture of H. pylori’s LPS. a) Structural domains present in biosynthetically complete LPS. b) S: smooth- form; SR: semi form; R: rough-form of LPS phenotypes………... 6 Fig. 1.2 Lewis blood group and related determinants found on the H. pylori LPS……….. 7 Fig. 1.3 Lipid A present in H. pylori. a) Major molecular structure of Lipid A; b) Minor

molecular structure of Lipid A……… 9 Fig. 1.4 The structure of the a) core OS, b) core OS / O-chain linkers and c) O-chain region

present in H. pylori……….. 11 Fig. 1.5 Variability of the expression of blood-group O-chain PS, heptoglycan and/or glucan

in LPS of H. pylori that share a common core OS……… 21 Fig. 1.6 Aldobiouronic domains and glucans in H. pylori……….... 26 Fig. 1.7 Structure of a trisaccharide repeating unit of terminal O-2 and O-2,6 Man present in

LPS of H. pylori mutant strain NCTC 116γ7……….. 27 Fig. 1.8 Structure of cell surface branched mannan present in H. pylori 968……….. 28 Fig. 1.9 Structure of the major glycoform from H. pylori strain NCTC 26695 HP0826::Kan,

containing a (1→6)--D-glucan linked to a (1→γ)--DD-heptan in the outer core…… 32 Fig. 1.10 Schematic representation of the carbohydrate microarray system……….. 37 Fig. 1.11 Principles of the preparation of neoglycolipid probes by reductive amination or

oxime-ligation for noncovalent immobilisation on solid matrices………. 39 Fig. 2.1 Overview of the methods used in this thesis………... 51 Fig. 2.2 General representation of sugar analysis on a polysaccharide composed of 4-linked

-Glc units………... 61 Fig. 2.3 General representation of linkage analysis on a polysaccharide composed of 4-linked

-Glc units………... 63 Fig. 2.4 The basic components of a mass spectrometer………... 64 Fig. 3.1 Sugar composition (relative %mol) of “crude” H. pylori glycan-rich aqueous extracts

obtained for each sample………. 85 Fig. 3.2 Chromatogram resulting from S-300 column calibration with standard dextrans (Blue

dextran, 150 kDa and 1β kDa) and glucose………. 86 Fig. 3.3 Sephacryl S-300 calibration curve used in this thesis to predict the molecular weight

of each purified H. pylori LPS……… 87 Fig. 3.4 Sephacryl S-300HR chromatographic profiles of the different isolated H. pylori LPSs

obtained after sequential treatment with DNAse, RNAse and proteinase K……… 88 Fig. 3.5 Glucose standard curve performed with four different Glc concentrations………….... 91

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(FA_S300) carried out on a DB-γ5ms column………... 93 Fig. 3.8 GC-MS chromatogram of glycosidic linkage analysis of purified LPS from H. pylori

strain CI-117 (FA_S300) carried out on a DB-1ms column………... 94 Fig. 3.9 GC-MS chromatogram of glycosidic linkage analysis of purified LPS from H. pylori

CI-117 (FA_S300) carried out on a DB-1ms column. Mass spectra and the corresponding MS fragmentation pattern of two particular linkages (2-D-Ribf and

2-DD-Hep)………... 95 Fig. 3.10 MALDI-TOF mass spectrum of alditol acetates of LPS from H. pylori strain

β191………... 101 Fig. 3.11 Chromatographic profiles of the different H. pylori LPSs obtained by SEC carried out

on a polyacrylamide Bio-Gel P-2 column after partial acid hydrolysis with 50 mM TFA performed in different temperature and time conditions……… 103 Fig. 3.12 ESI-LIT-MS2 spectrum of the ion at m/z 536 assigned to the inner core structure

[Hep(AEP)-Kdo-H2O+H]+ resulting from partial acid hydrolysis……….. 113

Fig. 3.13 ESI-LIT-MSn spectra: (A) ESI-MS2 spectrum of the ion at m/z 653, assigned to the outer core structure [Hex-Hex-Hex-Hex-Hex-H2O+Na]

+

resulting from partial acid hydrolysis; (B) ESI-MS3 spectrum of the ion at m/z 491 from the ion at m/z 653; (C) ESI-MS4 spectrum of the ion at m/z 347 from the ion at m/z 491 derived from the ion

at m/z 65γ……… 114

Fig. 3.14 ESI-LIT-MSn spectra: (A) ESI-MS2 spectrum of the ion at m/z 967, assigned to the outer core structure [Rib-Rib-Gal-Glc-Hep-Fuc-H2O+Na]+, resulting from partial

acid hydrolysis of LPS from CI-5 strain; (B) ESI-MS3 spectrum of the ion at m/z 703 from the ion at m/z 967; (C) ESI-MS4 spectrum of the ion at m/z 541 from the ion at

m/z 703 derived from the ion at m/z 967………. 116 Fig. 3.15 ESI-LIT-MSn spectra: (A) ESI-MS2 spectrum of the ion at m/z 1038, assigned to the

outer core structure [GlcNAc-Fuc-Hep-Glc-Gal-Rib-H2O+Na] +

, resulting from partial acid hydrolysis of LPS from CI-5 strain; (B) ESI-MS3 spectrum of the ion at

m/z 817 derived from the ion at m/z 10γ8………... 117 Fig. 3.16 ESI-LIT-MSn spectra: (A) ESI-MS2 spectrum of the ion at m/z 906, assigned to the

outer core structure [GlcNAc-Fuc-Hep-Glc-Gal-H2O+Na]+, resulting from partial

acid hydrolysis of LPS from CI-5 strain; (B) ESI-MS3 spectrum of the ion at m/z 685 derived from the ion at m/z 906………... 118 Fig. 3.17 ESI-LIT-MSn spectra: (A) ESI-MS2 spectrum of the ion at m/z 772, assigned to

[GlcNAc-Rib-Rib-Rib-Rib-H2O+Na]+, resulting from partial acid hydrolysis of LPS

from CI-5 strain; (B) ESI-MS3 spectrum of the ion at m/z 569 derived from the ion at

m/z 77β……… 120

Fig. 3.18 ESI-LIT-MS2 spectrum of the ion at m/z 366, assigned to [LacNAc-H2O+H]+,

resulting from partial acid hydrolysis of LPS from 14255 and CI-5 strains……….. 121

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xix Fig. 3.19 ESI-LIT-MSn spectra: (A) ESI-MS2 spectrum of the ion at m/z 771, assigned to

[LacNAc-LacNAc-H2O+Na]+, resulting from partial acid hydrolysis of LPS from

CI-5 strain; (B) ESI-MS3 spectrum of the ion at m/z 591 derived from the ion at m/z 771; (C) ESI-MS4 spectrum of the ion at m/z 388 from the ion at m/z 591, derived from the ion at m/z 771……….. 123 Fig. 3.20 (A) ESI-MS2 spectrum of the ion at m/z 569, assigned to

[GlcNAc-LacNAc-H2O+H]+, resulting from partial acid hydrolysis of LPS from CI-5 strain; (B)

ESI-MS2 spectrum of the ion at m/z 569, assigned to [Rib4+Na]+, obtained from partial

acid hydrolysis of LPS from 14β55 strain………... 124 Fig. 3.21 ESI-LIT-MS2 spectra of fragmentation of type-2 Lewisx antigen in protonated and

sodiated forms. (A) ESI-MS2 spectrum of the ion at m/z 512, assigned to [Lex -H2O+H]

+

, resulting from partial acid hydrolysis of LPS from CI-5 strain; (B) ESI-MS2 spectrum of the ion at m/z 552, assigned to [Lex+Na]+, derived from partial acid hydrolysis of LPS from NCTC 26695 strain……… 126 Fig. 3.22 ESI-LIT-MSn spectra of fragmentation of di- Lewisx antigen structures. (A) ESI-MS2

spectrum of the ion at m/z 1063, assigned to [Lex-Lex-H2O+Na]+ ; (B) ESI-MS3

spectrum of the ion at m/z 917, assigned to [Lex-LacNAc-H2O+Na]+, derived from

the ion at m/z 1063; (C) ESI-MS4 spectrum of the ion at m/z 771, assigned to [LacNAc-LacNAc-H2O+Na]

+

, from the ion at m/z 917, derived from the ion at m/z 1063; (D) ESI-MS5 spectrum of the ion at m/z 388, assigned to [LacNAc-H2O+Na]+,

from the ion at m/z 771, derived from the ion at m/z 917, originated from the ion at

m/z106γ……….. 128

Fig. 3.23 Analysis of the reaction products of the conjugation of LPS oligosaccharide fractions from (A) H. pylori strains NCTC 26695 and (B) CI-5 to the ADHP amino lipid (AD). 130 Fig. 3.24 Spectrum profile of ADHP standard solutions at 10, 5 and 1 pmol/L used to

estimate the amount of NCTC 26695 F2-AD and CI-5 F2-AD……… 131 Fig. 3.25 Negative ion MALDI-TOF spectrum of the ADHP-NGL of the sample CI-5 F2-AD... 132 Fig. 3.26 MALDI-TOF-MS2 spectrum of fragmentation of the ion at m/z 1420, resulting from

the NGL product F2-AD of strain CI-5………... 132 Fig. 3.27 (A) MALDI-TOF-MS2 spectrum of fragmentation of the ion at m/z 1465, resulting

from the NGL product F2-AD of strain CI-5. (B) MALDI-TOF-MS3 spectrum of the ion at m/z 1319, derived from the ion at m/z 1465……….. 133 Fig. 3.28 H. pylori LPS microarray imaging before and after protein overlay. (A) Microarray

scanner images of a whole nitrocellulose-coated glass slide, showing the Cy3 fluorescence of arrayed spots; (B) single experiments (nitrocellulose coated pads), showing the Alexa-Fluor 647 fluorescence signals obtained with different proteins / human serum………... 135 Fig. 3.29 H. pylori LPS microarray analysis with Lewis-specific mAbs and with the

mammalian immune receptor DC-SIGN (Dendritic cell-specific ICAM-3-grabbing non-integrin)……… 139 Fig. 3.30 H. pylori LPS microarray analysis with the fucose-specific lectins AAL (Aleuria

aurantia lectin) and UEA-I (Ulex europaeus agglutinin-I), anti-blood group H and type-1 Lacto-N-tetraose (LNT) monoclonal antibodies……….. 143

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agglutinin)………... 146 Fig. 3.32 H. pylori LPS microarray analysis with glucose- and mannose-specific proteins…….. 148 Fig. 3.33 Application of the H. pylori LPS microarray to analysis of two human sera: the first

was from a case infected with H. pylori (CI-5) and the second was from a non-infected control ………... 150 Fig. 3.34 SDS-PAGE profile of LPS fractions derived from H. pylori strains after visualisation

by silver staining………. 152 Fig. 3.35 Western blot analysis of LPS fractions derived from H. pylori strains NCTC 26695

and CI-117 using the STL plant lectin and the anti-LexmAb……… 155 Supplem.

Fig. 1

Microarray analysis of the detection systems used for each protein as a quality control of the microarray set.……….. 187

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LIST OF TABLES

Table 1.1 Classification of H. pylori LPS into Glycotype families……… 24 Table 2.1 Strains used in this study………. 52 Table 2.2 Identification of H. pylori cultures grown in UA on Columbia agar supplemented

with 5% blood horse based on its morphology and biochemical tests……….. 53 Table 2.3 Summary of the aqueous glycan-rich extracts isolated from cell surface of H. pylori

strains………... 54 Table 2.4 Partial acid hydrolysis conditions performed to some purified H. pylori

LPSs……… 58 Table 2.5 Proteins used towards validation H. pylori LPS microarray platform, and their

reported specificities……… 73 Table 2.6 Experimental conditions used in this thesis for microarrays binding assays with

different proteins………. 75 Table 2.7 Conditions for the Western blot analysis using the plant lectin STL and the anti-Lex

monoclonal antibody………... 79 Table 3.1 Amount of bacterial biomass resulting from H. pylori growth on solid media (120

culture plates)……….. 83 Table 3.2 Summary of the aqueous glycan-rich extracts isolated from cell surface of H. pylori

strains……….. 84 Table 3.3 Prediction of the molecular weight of the purified H. pylori

LPSs………... 89 Table 3.4 Sugar amounts of the interested purified fraction, FA_S-300, of each H. pylori

sample expressed as equivalents of Glc……….. 91 Table 3.5 Relative sugar composition of the different H. pylori LPSs resulted from SEC

fractionation carried out on a Sephacryl S-300 HR column (FA_S300) after enzymatic hydrolysis with DNAse, RNAse, proteinase K……….. 93 Table 3.6 Glycosidic linkage profile of the different H. pylori LPSs resulted from SEC

fractionation carried on a Sephacryl S-300 HR (FA_S300) after enzymatic hydrolysis with DNAse, RNAse, proteinase K………. 96 Table 3.7 Glycosidic linkage profile of the different H. pylori LPSs resulted from SEC

fractionation carried on a Polyacrylamide Bio-Gel P-2 after partial acid hydrolysis with TFA: (A) FA_P2, 50 mM TFA, 65 ºC, 1 h of LPS from the strain 2191; (B) FA_P2, 50 mM TFA, 65 ºC, 1 h of LPS from the strain NCTC 26695; (C1-C3) is referred to CI-5 strain: (C1) FA_P2, 50 mM TFA, 100 ºC, 1 h; (C1a) F32_P2, 50 mM TFA, 100 ºC, 1 h; (C2) FA_P2, 50 mM TFA, 65 ºC, 1 h; (C3) FA_P2, 50 mM TFA, 65 ºC, β h………. 105 Table 3.8 Identified ions in the ESI-LIT-MS of the oligosaccharides (FB and FCfrom Bio-Gel

P-2 column) obtained from partial acid hydrolysis with 50 mM TFA, 65 ºC, 1 h of FA_S300 of H. pylori strain β191.………... 108

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Table 3.10 Identified ions in the ESI-LIT-MS of the oligosaccharides (FB, FC, FD and FEfrom Bio-Gel P-2 column) obtained from partial acid hydrolysis with 50 mM TFA, 65 ºC, 1 h of FA_S300 of H. pylori strain NCTC β6695. ……… 109 Table 3.11 Identified ions in the ESI-LIT-MS of the oligosaccharides (FB, F32, F34 and F47

from Bio-Gel P-2 column) obtained from partial acid hydrolysis with 50 mM TFA, 100 ºC, 1 h of FA_S300 of H. pylori strain CI-5. ……… 110 Table 3.12 Identified ions in the ESI-LIT-MS of the oligosaccharides (FB and FCfrom Bio-Gel

P-2 column) obtained from partial acid hydrolysis with 50 mM TFA, 65 ºC, 1 h of FA_S300 of H. pylori strain CI-5……… 111 Table 3.13 ESI-LIT-MS of the oligosaccharides (FB, FC and FD from Bio-Gel P-2 column)

obtained from partial acid hydrolysis with 50 mM TFA, 65 ºC, 2 h of the non-hydrolysed fraction, FA_P2 (50 mM, 65 ºC, 1 h), of H. pylori strain CI-5……… 112 Table 3.14 List of probes included in the H. pylori LPS microarray used for microarray data

analysis……… 136 Supplem.

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______________________________ a

McNaught, A. D. (1997). Nomenclature of carbohydrates. Carbohydrate Research, 297, 1-92 xxiii

Chemical structures of monosaccharides present in H. pylori cell surface glycans mentioned in this thesis

Symbol notation proposed by the Consortium for Functional Glycomics (CFG): http://glycomics.scripps.edu/coreD/PGAnomenclature.pdf

Throughout this thesis, oligo- and polysaccharide sequences were written according to the most recent version of carbohydrates nomenclature, established by IUPACa. However, extended or condensed/short forms were used in the sections concerned with structural analysis and microarrays, respectively. This was, thereby, deliberated in order to avoid changes in the carbohydrate structure´s representation typically used in those complementary research fields.

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xxiv Lea Leb Led (H-type-1) Type-1 Blood group A Type-1 Blood group B-like Type-2 chain LacNAc Lex Sialyl-Lex Ley H-type-2 Type-2 Blood group B-like

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CHAPTER 1

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3

1. Introduction

1.1 General insights into H. pylori

Helicobacter pylori is a microaerophilic Gram-negative bacterium that colonizes the

human gastric mucosa. It is one of the most widespread bacterial pathogens with a prevalence of up to 90% in developing countries and a worldwide infection rate of around 50%1. The prevalence of infection in developing countries is linked to a low socio-economic status, poor sanitation and gastro-oral, fecal-oral, and oral-oral routes, as well as iatrogenic transmission are currently considered the main vectors of dissemination1, 2.

H. pylori was first isolated from the human stomach 30 years ago. It was correlated

with gastric diseases by Robin Warren and Barry Marshall3, 4, a discovery that resulted in dramatic changes in the field of gastroenterology. H. pylori is currently recognised as the main etiologic agent of gastric malignancies such as gastritis, gastric atrophy, and peptic and duodenal ulcers5. Furthermore, infection has been directly associated with an increased risk of developing gastric cancer6, 7, which led the International Agency for Cancer Research, a part of the World Health Organization (WHO), to classify this bacterium as a type I (definitive) human carcinogen8. However, as of yet, eradication has been based on antibiotic regimes whose efficiency has been suffering considerable pitfalls as a result of increased resistance9, demanding for new therapeutics. The vast majority of humans acquire H. pylori in childhood and, once established, the bacteria can persist for life in the human stomach if left untreated10. Although only 30% of infected individuals are clinically symptomatic, the presence of H. pylori is always associated with active inflammation of the gastric mucosa11. It has been estimated that 10-15% of these individuals develop peptic ulcers and 1-3% will develop gastric carcinoma. Also, it has been reported that H. pylori increases the relative risk for gastric tumours by at least six-fold12.

1.2 The relevance of carbohydrates in host-H. pylori interactions

The surface of all living cells is decorated by carbohydrates (also designated as glycans). These are present in mammalian cells, mainly as glycoconjugates (glycoproteins, glycolipids, glycosaminoglycans and proteoglycans), and are also part of bacterial, fungal and plant polysaccharides13. Carbohydrates are involved in many biological events (e.g. cell-cell communication, adhesion and signaling mechanisms) through interactions with

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4

the corresponding carbohydrate-binding proteins (CBPs), including lectins and antibodies that recognise them as ligands13. Carbohydrates also play a critical role in host-pathogen interactions14. On one hand, many mammalian receptors for pathogens are glycoconjugates, which often contribute to their host specificity or tropism. On the other hand, pathogen carbohydrates may serve as antigens to several receptors of the immune system, which develop immune responses against the pathogen14. Changes in carbohydrate structures are associated with carcinogenesis15, virus infection16 and other diseases17.

Particularly in host-H. pylori interactions, carbohydrates play an active role in the mediation of colonization and modulation of immune responses18, 19, thus contributing to maintain the active infection20-23. For example, the adherence of the bacteria to the gastric epithelial cells, which is determinant for initial and long-time persistence in the human stomach, is mediated by bacterial outer membrane adhesins targeting host glycans20, 24, 25. The adhesin BabA was shown to recognise Lewisb (Leb) antigen, which is a carbohydrate antigen present in the gastric mucosa of secretor individuals26-29. Other adhesins, such as SabA, binds to sialylated antigens, which are carbohydrates that are usually found up-regulated in inflamed gastric tissues30, 31. In addition to their contribution to gastric colonization, H. pylori outer membrane adhesins are thought to be determinant in the colonization of other human body reservoirs such as the oral cavity32, 33, and may contribute to continuous re-infection and the failure of eradication interventions.

Like other Gram-negative bacteria, H. pylori cell surface outer membrane is mainly composed of lipopolysaccharides (LPSs) which are pathogenic and virulence markers that contribute to the severity and chronicity of infection22, 23, 34-36. Over the past twenty years, structural studies have unveiled the complex structural organization of the LPS and the molecular subtleties resulting from H. pylori inter-strain variation. These studies have highlighted that H. pylori LPSs show marked molecular mimicry of host blood group related Le antigens, a group of carbohydrate structures also expressed by the human gastric epithelium23. This is currently regarded as a determinant of the survival of the bacteria, whereas the molecular mimicry of the Lewis antigens provides a ‘camouflage’ for the bacteria in order to escape the host immune response22. In addition, several studies have pointed out that, upon prolonged infection, Le expression can trigger autoimmune responses that can contribute to pathologic outcomes37, 38. Other structural domains, such as heptoglycans39 and glucans have been described in particular H. pylori serotypes.

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5 Moreover, recently identified structures on H. pylori include mannans40 that are thought to be targets for mannose-binding lectin (MBL), as well as amylose-rich glycans exhibiting acidic domains41. These findings raise new questions about the biologic role of such carbohydrates and their contribution to pathogenesis. Furthermore, they constitute new putative targets for the development of carbohydrate-based vaccines directed to H. pylori.

1.3 Overview of H. pylori LPS molecular architecture

The LPSs expressed by H. pylori are vital both to the structural and functional integrity of the membrane18, 42. The structural and serological studies undertaken by Monteiro et al.23, 37, 43-50 have shown that H. pylori exhibit a classical Gram-negative LPS structure comprising three distinct structural domains: a polysaccharide (PS) known as O-chain (O-PS), an oligosaccharide moiety (OS) termed Core, and a fatty-acid rich moiety named Lipid A that anchors the macromolecule to the lipid bilayer (LPS: O-chain→Core→Lipid A~cell) (Fig. 1.1 a).

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6

Fig. 1.1 General architecture of H. pylori’s LPS. a) Structural domains present in biosynthetically complete

LPS. b) S: smooth- form; SR: semi rough-form; R: rough-form of LPS phenotypes. Abbreviations: GlcNAc,

N-acetylglucosamine; AEP, 2-aminoethylphosphate; P, phosphate group; Kdo, 3-deoxy-D-manno-octulosonic acid; Hep, heptose; Fuc, fucose; Gal, galactose; Glc, glucose. The monosaccharide residues are depicted as symbolic representation according to the Consortium for Functional Glycomics (CFG) guidelines (http://glycomics.scripps.edu/coreD/PGAnomenclature.pdf).

In H. pylori LPS, the conserved structure of the core contrasts with the considerable phenotypic diversity exhibited by the O-chains23. A unique and widespread characteristic of H. pylori O-chains is that they display several blood group determinants, such as Lewis (Le) determinants, typically expressed on the surface of human cells, particularly in the gastric pit. The majority of the strains studied so far exhibit internal Lex with terminal Lex or Ley units, but some strains can also co-express units of Lea 48, 51, Leb51, Lec 50, sialyl-Lex (sLex)26, and H-1 antigens (Led) 47, as well as A and B-like blood group antigens (Fig. 1.2).

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7

Fig. 1.2 Lewis blood group and related determinants found on the H. pylori LPS - The monosaccharide residues are depicted as symbolic representation according to the Consortium for Functional Glycomics (CFG) guidelines (http://glycomics.scripps.edu/coreD/PGAnomenclature.pdf).

A number of H. pylori strains additionally produce structural domains, attached to the core, in the form of a glucan and/or heptoglycans, contributing to antigenic diversity52,

53

.

LPS phenotypic diversity is also observed in subpopulations within a bacterial community, normally resulting from variable size O-antigen chains, differentiated levels of Le glycosylation23, and the expression of glucan/heptoglycan domains. In particular, the Le O-chain phenotype can vary within a population both in the gastric mucosa as well as generated by in vitro sub-culturing and in vivo transfer to a new animal host23, 54. This differentiated Le expression within the same population is mostly explained by strong phase variation (antigenic variation) in response to different environmental conditions, a phenomenon commonly observed in microorganisms that is characterised by the on/off switching of the expression of cell surface epitopes55, 56. It is thought that this mechanism can provide H. pylori the means to adapt to specific gastric niches as well as to regulate the host inflammatory responses over the course of infection. Hence, diversification in the LPS molecule might be crucial both in the initial colonization of a new niche as well as the establishment of persistent infection54, 55. Nilsson et al.55 have shown that intra-individual isolates varied in Lewis antigen expression although the LPS diversity was relatively stable within each individual over time. Furthermore, considerable diversity in the levels of glycosylation and in the sizes of fucosylated O-antigen chains both within and between individuals was observed. These observations further reinforced that different LPS variants exist in the colonizing H. pylori population and that LPS present dynamic phenotypes that adapt to changes in the gastric environment and provide a means to regulate the inflammatory response of the host during disease progression54. LPS also vary in relation

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to the length of the O-chain. Namely, LPS with high molecular weight (Mw) structures (Mw > 10 kDa) are commonly referred as smooth-type LPS57 (S-LPS, Fig. 1.1 b). The microorganisms that produce this type of structures are recognised in solid plaques as glistening, rounded and smooth surface colonies. The S-LPS are characteristic of clinical isolates recovered from fresh biopsies. In fact, the predominant observation of S-LPS in H.

pylori colonizing human gastric mucosa makes it regard as the favoured phenotype in

vivo55. Still, within the same population, other phenotypes can coexist, namely LPS exhibiting oligomeric O-chains, presenting a less smooth, more granular and flattened surface. These LPS phenotypes are termed semi-rough (SR-LPS, Fig. 1.1 b). Certain strains or subpopulations carry mutations in the otherwise well-conserved rfb locus which contains a selection of genes involved in O-chain synthesis and attachment, thus resulting in cells with a dominant rough LPS (R-LPS) phenotype (Fig. 1.1 b). The absence of O-chains is also observable in cells resulting from long term subcultivation in solid medium. However, in liquid medium, the phase shift from S- to R-LPS phenotypes can be reversed, irrespective of the medium composition56. These observations suggest that changes can be induced in the LPS expression by growth conditions in vitro, with major implications in the biological studies on LPS58.

1.3.1 Lipid A and other cell surface glycolipids

The lipid moiety of the LPS, termed lipid A, anchors the glycosidic domain to the bacterial cell through hydrophobic and electrostatic forces59. Structural investigations using chemical and spectroscopy methods have shown that this is the most conserved region of the LPS and exhibits a molecular architecture similar to other bacteria60. Namely, it consists of a partially acylated, hydrophilic (1→6)-linked -D-glucosamine disaccharide,

[-D-GlcpN-(1→6)--D-GlcpN]23, 57, 61, 62. However, unlike other Gram-negative bacteria of the Enterobacteriaceae family, H. pylori lipid A shows significantly lower endotoxic activity due to fine structural differences57, 63-65.

Among H. pylori lipid A predominates structures composed of a GlcN -(1→6) disaccharide 1-phosphate or 1-(2-aminoethyl)phosphate backbone that is tetraacylated with (R)-3-hydroxyoctadecanoic acid, (R)-3-hydroxyhexadecanoic acid, and (R)-3-(octadecanoyloxy)octadecanoic acid at the 2-, 3- and 2'-positions, respectively62 (Fig. 1.3

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9 groups. In addition, S-LPS express lower amounts of hexaacyl lipid A resulting from the esterfication of the 3' position with 3-(dodecanoyloxy)hexadecanoic acid or (R)-3(tetradecanoyloxy) hexadecanoic acid (Fig. 1.3 b). In addition, an unusual heptaacyl form of lipid A, strikingly marked by the presence of palmitate structures, was recently observed in the H. pylori 26695 HP1191::Kan mutant strain, although in relatively low abundance66.

The underphosphorylation and the expression of lengthy fatty acids, in particular the 3-hydroxy fatty acids, differentiate H. pylori lipid A from that of other bacterial species, and are most likely responsible by its significantly lower endotoxic and immunological activity62, 65, 67. In agreement with these considerations, studies have demonstrated the importance of the phosphate groups and number of fatty acyl chains for cytokine induction via TLR-463, 68.

The low endotoxicity of H. pylori lipid A is thought to enhance the “camouflage” provided by Le antigen capping and thus may contribute to maintain an active and long-lasting infection.

Fig. 1.3 Lipid A present in H. pylori. a) Major molecular structure of lipid A; b) Minor molecular structure of lipid A (adapted from 69).

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1.3.2. The core

The core OS region is covalently attached to the O-6 linked nonreducing GlcN unit of lipid A via an acid-sensitive ketosidic linkage from 3-deoxy-D-manno-octulosonic acid

(Kdo) residue.

In H. pylori, the core has been mostly observed as a conserved non-repetitive region formed by a linear phosphorylated (by a monoester phosphate, P, or by a 2-aminoethylphosphate, AEP) backbone, α-D-Glcp-(1→γ)-α-D-Glcp-(1→4)- -D

-Galp-(1→7)-DαDHepp-(1→β)-LαDHepp (1→γ)[P/AEP →7]-LαDHepp-(1→5)-Kdo. The core is

normally extended throughout a 2-linked side-branch in the distal 7-linked DDHepp residue of the backbone glycan by an additional DD-Hepp residue43, 46, 47, 51 (Fig. 1.4 a).

The 7-linked position of this DαDHepp side-branch has been considered the bridging point between the core and the O-chain moiety (Fig. 1.4 b) The pointed out O-7 Hep linker can be further extended by 2-, 3- and/or 6-linked -DD-Hep residues23. Recently, two studies

have also reported that the bridging between the O-chain and the core can occur via an →7)-DαDHep-(1→3)--L-Fuc--GlcNAc-(1→ (Fig. 1.4 b)70, 71.

A recent structural re-investigation of the LPS from H. pylori Sydney strain (SS1), a mouse-adapted strain frequently used in animal studies of pathogenesis also revealed new structural features70. It was observed that H. pylori strain SS1, as well as its isogenic mutant SS1 HP0826::Kan, which has truncated LPS lacking O-chain polysaccharide following the disruption of 1,4-galactosyltransferase (encoded by HP0826 gene72), contained a linear pentasaccharide →4)--D-Gal(1→3)--D-Glc(1→7)-α-DD-Hep-(1

→3)--L-Fuc-(1→3)--D-GlcNAc-(1→ linking a conserved inner core region and the O-chain.

This pentasaccharide is then terminated in a linear chain composed of 2-linked -ribofuranosyl residues (riban) that constitute the attachment point for a O-chain polysaccharide70 (Fig. 1.4 b).

More recently, both H. pylori strains SS1 and SS1 HP0826::Kan were used to investigate the role of the O-chain polysaccharide in the colonization and pathology in Mongolia gerbils, another rodent H. pylori model73. It was demonstrated that both strains were able to colonize the gerbils stomach, resulting in chronic gastritis. Interestingly, colonization with SS1 HP0826::Kan led to structural changes in the LPS profile, determined by sugar and methylation analysis. Particularly, it was detected the presence of a novel homopolymer containing 3-linked galactose units (D-galactan), as well as an

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11 extended polymer of D-riban, composed of 2-linked -ribofuranose residues73, which

seems to be longer than the one previously described70. Although the precise LPS structure has not been yet determined, these results suggest that H. pylori uses a variety of mechanisms to promote colonization of the host and gastric pathology, including biosynthesis of de novo structures. Furthermore, contrarily to that observed in mice72, 74, H.

pylori-induced gastritis in gerbils seems to be O-chain-independent73.

The presence of pentoses, even though arabinose (Ara) but not Rib, has also been described in LPS purified fractions by Ferreira et al.40, 58. In addition to 2-linked residues, the authors also refer the presence of 5- and 3,5-linked Ara.

Fig. 1.4 The structure of the a) core OS, b) core OS / O-chain linkers and c) O-chain region present in H.

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1.3.3 The O-chain

Contrarily to the core, the O-chain presents considerable structural variability23. In fact, in the early stages of H. pylori research, Mills et al.53 developed a serotyping system based on different antigenicity of LPS molecules due to structural variations in the O-antigenic chains. Based on these observations, six distinct H. pylori serotypes (O:1 to O:6) were defined. Subsequently, detailed chemical and enzymatic-based Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR) studies23 complemented by serology75 have demonstrated that H. pylori O-chains express structures homologous to mammalian histo-blood group antigens, in particular Le determinants, the reason why they are often referred as “Le O-chains”. These studies also revealed that some strains often express elongated glucan and heptan domains bridging the core and Le O-chains. As reviewed by Monteiro23, the majority of the O-chains are composed of partially fucosylated43, 46, 47, glucosylated76, or galactosylated47, 77 type-2 N-acetyllactosamine backbone, -[→γ)--D-Gal-(1→4)--D

-GlcNAc-(1→], LacNAc, of various lengths, which may or may not be terminated at the nonreducing end by Le blood group determinants, in mimicry of human cell surface glycoconjugates. Molecular mimicry was also characteristic of the H. mustelae LPS, a colonizer of the gastric mucosa of ferrets, that expresses type-1 blood group A antigen [

-D-GalpNAc-(1→3)-[-L-Fuc-(1→2)]--D-Galp-(1→3)--D-GlcpNAc]78,79, denoting

similar strategies among Helicobacter spp.

Much structural variability has also been observed upon the analysis of strains from different geographical regions43, 46, 47, 49, 51. In particular, strains originating from North American and European populations predominantly express inner-chain type-2 Lex, [-D

-Galp-(1→4)-[-L-Fucp-(1→3)]--D-GlcpNAc], and terminated with Lex and Ley, [-L -Fucp-(1→2)--D-Galp-(1→4)-[-L-Fucp-(1→3)]--D-GlcpNAc], antigens in monomeric and/or polymeric forms43, 46, 47, 49. Frequently, incomplete Lewis antigens are observed implying that the building of Lewis determinants in H. pylori takes place through a sugar-by-sugar addition and not by a block-by-block synthesis, as observed for other enteric bacteria49. Even though the expression of oligomeric Le determinants is common among some human cells, namely granulocytes, their abundant expression is limited to malignant cells. Nevertheless, Le epitopes are expressed in the gastric tissue reinforcing the hypothesis of molecular mimicry.

Structural analysis of glycans and development of microarrays from Helcobacter pylori cell surface glycome

LISETE MACHADO E

SILVA

ANÁLISE ESTRUTURAL DE GLICOSÍDEOS E

DESENVOLVIMENTO DE MICROARRAYS DO

GLICOMA DA MEMBRANA DE HELICOBACTER

PYLORI

STRUCTURAL ANALYSIS OF GLYCANS AND

DEVELOPMENT OF MICROARRAYS FROM

HELICOBACTER PYLORI

CELL SURFACE

LISETE MACHADO E

SILVA

ANÁLISE ESTRUTURAL DE GLICOSÍDEOS E

DESENVOLVIMENTO DE MICROARRAYS DO

GLICOMA DA MEMBRANA DE HELICOBACTER

PYLORI

CONTENTS

CHAPTER 1

1. Introduction

1.1 General insights into H. pylori

1.2 The relevance of carbohydrates in host-H. pylori interactions

1.3 Overview of H. pylori LPS molecular architecture