Universidade Estadual de Campinas Faculdade de Odontologia ... · É sobre escalar e sentir Que o...

Universidade Estadual de Campinas

Faculdade de Odontologia de Piracicaba

IRLAN DE ALMEIDA FREIRES

O PAPEL DA GLICOPROTEÍNA CNM DE STREPTOCOCCUS

MUTANS NA INVASÃO INTRACELULAR, ADESÃO A TECIDOS

CARDÍACOS HUMANOS E CARIOGENICIDADE IN VIVO

THE ROLE OF STREPTOCOCCUS MUTANS CNM IN

INTRACELLULAR INVASION, ADHESION TO HUMAN

CARDIAC TISSUES, AND CARIOGENICITY IN VIVO

PIRACICABA 2017

IRLAN DE ALMEIDA FREIRES

O PAPEL DA GLICOPROTEÍNA CNM DE STREPTOCOCCUS

MUTANS NA INVASÃO INTRACELULAR, ADESÃO A TECIDOS

CARDÍACOS HUMANOS E CARIOGENICIDADE IN VIVO

THE ROLE OF STREPTOCOCCUS MUTANS CNM IN

INTRACELLULAR INVASION, ADHESION TO HUMAN

CARDIAC TISSUES AND CARIOGENICITY IN VIVO

PIRACICABA 2017

Tese de Doutorado apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para obtenção do titulo de Doutor em Odontologia, na Área de Farmacologia, Anestesiologia e Terapêutica.

Thesis presented to the Piracicaba Dental

School of the University of Campinas in partial

fulfilment of the requirements for the degree of

Doctor in Dentistry, in Pharmacology,

Anesthesiology and Therapeutics area.

Orientador: Prof. Dr. Pedro Luiz Rosalen

Este exemplar corresponde à versão final da tese defendida por Irlan de Almeida Freires, e orientada por Pedro Luiz Rosalen.

DEDICATÓRIA

À DEUS.

Agradeço, em plenitude, pelas benções sobre mim derramadas, pela sapiência nas tomadas de

decisão e construção deste trabalho, e pelas pessoas maravilhosas que fizeram parte do meu

caminho. Agradeço, sobretudo, por compreender de forma edificante o intrigante elo entre

ciência e fé.

À MINHA AMADA MÃE (in memoriam).

Dedico-lhe a minha trajetória de vida. Espero ser reflexo do seu sorriso, dos ensinamentos e do

ser singelo que esteve comigo por 14 (lindos) anos. Eu não vivo sem você, eu vivo para você,

pois quando um amor é muito forte permanece vivo em suas lembranças.

AGRADECIMENTOS

À Universidade Estadual de Campinas (UNICAMP) e à Faculdade de Odontologia de

Piracicaba (FOP) pelo apoio institucional.

À Profª Drª Cinthia P. M. Tabchoury, coordenadora dos cursos de Pós-graduação da

FOP/UNICAMP e ao Prof. Dr. Marcelo de Castro Meneghim, coordenador do Programa

de Pós-Graduação em Odontologia da FOP/UNICAMP.

Ao meu orientador, Prof. Dr. Pedro Luiz Rosalen, pelo brilhante exemplo de vida pessoal

e profissional. Foram cinco anos frutíferos de convivência diária, um intenso período de

aprendizagem, construção e amadurecimento. Juntos, formamos uma verdadeira

equipe em prol da ciência e sociedade. Obrigado pelo legado de integridade,

compromisso, afinco e competência. Sempre fui admirador de sua capacidade crítica e

resolutiva, da inteligência emocional e da criatividade, e tantos outros atributos

essenciais para um pesquisador de sucesso. Agradeço o privilégio de sua orientação e,

acima de tudo, por tê-lo em minha história de vida como alguém inesquecível.

Aos professores da Área de Farmacologia, Anestesiologia, e Terapêutica, Profª Drª Maria

Cristina Volpato, Prof. Dr. Eduardo Dias de Andrade, Prof. Dr. Francisco Carlos Groppo e

Profª Drª Karina Cogo, pelos ensinamentos, convívio harmonioso e amizade.

Aos queridos Profs. Drs. Jacqueline Abranches e José Lemos (orientadores no exterior)

por terem me recebido em seu laboratório no Center for Oral Biology (COB), University

of Rochester Medical Center, Rochester, New York, EUA. O doutorado sanduíche junto a

pesquisadores tão renomados foi importantíssimo para consolidar o meu

desenvolvimento profissional e acadêmico e, não menos importante, crescimento como

ser humano. Acima de tudo, esta oportunidade me abriu portas promissoras para o

futuro próximo com este grupo de sucesso.

Ao grande colaborador do meu projeto na University of Rochester, Dr. Alejandro Áviles-

Reyes. Agradeço pela ajuda imprescindível na condução dos experimentos e análise dos

dados, alguém com quem aprendi muito ao longo de 12 meses. Sua proatividade,

capacidade crítica e competência são admiráveis.

Aos amigos do laboratório na University of Rochester, Cindy Góes Dodo, Cristina

Colomer-Winter, Jessica K. Kajfasz e seu marido Mark, James H. Miller, Kathleen Scott-

Anne (Kathy), Beatriz Bezerra e Robert Quivey (diretor do COB). Obrigado pelo

companheirismo, pela ajuda no laboratório e pela comunhão de momentos

inesquecíveis.

Ao Dr. William H. Bowen (in memoriam) por sua contribuição inestimável com tudo o

que sabemos sobre cárie. Tive a oportunidade incrível de ser treinado por ele para os

procedimentos de dessalivação de ratos. Seu legado é eterno e seus ensinamentos

sobrevivem ao tempo.

Aos colaboradores do meu projeto na FOP/UNICAMP, Prof. Dr. Marcelo Meneghim e,

em especial, a Emílio Prado, pela enorme ajuda e solicitude na coleta das amostras.

Também agradeço à Secretaria Municipal de Saúde do Município de Piracicaba e à

Unidade Básica de Saúde (UBS) 1º de Maio, na pessoa da Enfermeira Ana Paula, pelo

acolhimento da nossa equipe na UBS e por incentivar a nossa pesquisa.

Aos professores das bancas de qualificação primeira e segunda fases: Profª Drª Renata

Mattos-Granner, Prof. Dr. Fábio Mialhe, Profª Drª Marlise Klein, Profª Drª Cinthia P. M.

Tabchoury. Aos professores titulares da banca de defesa: Prof. Dr. Pedro Luiz Rosalen,

Profª Drª Janaína de Cássia Orlandi Sardi, Profª Drª Ana Paula Souza Pardo, Profª Drª

Maria Regina Lorenzetti Simionato e Profª Drª Luciana Kfouri Siriani. Aos professores

suplentes da banca de defesa: Profª Drª Maria Cristina Volpato, Profª Drª Carina Denny

e Prof. Dr. Marcos Guilherme da Cunha. Obrigado a todos pela criteriosa avaliação e

estimadas contribuições a fim de enriquecer o nosso trabalho.

À Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) por me conceder

uma bolsa de doutorado (2013/25080-7) e uma Bolsa Estágio em Pesquisa no Exterior

(BEPE) (2014/07231-0).

À Eliane Melo pela amizade de cinco anos. Sua presença faz a diferença no laboratório.

Obrigado por me ensinar a ser prático, a focar nos meus objetivos e a seguir em direção

ao que faz me feliz. Sentirei muita falta da sua companhia, das nossas risadas e

confidencialidades, mas estarei sempre disposto a voltar e lhe abraçar.

A José Carlos, pela ajuda imprescindível na realização do estudo animal e pela

convivência diária.

À Elisa dos Santos, que desde o raiar do dia estampa um sorriso contagiante. Obrigado

por sua boa vontade em ajudar ao próximo, bem como dedicação e competência.

Obrigado, também, por fazer o melhor café do mundo!

Aos meus amigos companheiros de jornada na FOP: Aline Castilho, Bruno Nani, Bruno

Muniz (Bigode), Bruno Bueno-Silva, Bruna Benso, Diego Romário, Luiz Eduardo, Livia

Galvão, Jonny Burga, Marcos Cunha e Marcelo Franchin. Agradeço em especial à Janaína

Sardi e Josy Goldoni, pessoas incríveis com quem tive o privilégio de conviver e aprender.

Obrigado pelo companheirismo e pela comunhão de momentos alegres e difíceis. Tive

uma relação muito peculiar com cada um de vocês ao longo deste período e estou

convicto de que semeamos uma amizade para toda a vida.

Aos alunos de iniciação científica, Gabriel Wilson e Pedro Andrade, por todo o apoio na

realização dos experimentos.

Aos técnicos do Biotério da FOP/UNICAMP, Wanderlei Francisco Vieira e Rafael Soares

de Sousa, pela colaboração e apoio na condução do experimento animal.

Ao Prof. Ricardo Dias de Castro, meu orientador na UFPB, pela prestimosa amizade e

parceria científica.

À Juliano Marçal Lopes, por dividir comigo a sua vida e agregar os seus sonhos aos meus.

Obrigado por descobrir o mundo ao meu lado e ser o melhor companheiro de viagens

que alguém pode ter. Obrigado pela paciência – quando tive que trabalhar aos fins de

semana ou quando fiquei 365 dias distante em busca do meu sonho. Obrigado por me

ensinar tantas coisas e, ainda, me presentear com a sua família.

À Lívia Araújo Alves, pela amizade de longa data, incentivo, pela força incrível que tem

dentro de si e irradia a todos que a conhecem.

Às minhas ‘mães’ Madrinha Nininha e Tia Edézia pelo carinho materno, pela

preocupação, pelo zelo, incentivo e orgulho.

Aos meus irmãos queridos, Tatiana, Itamara e Clóvis, pelo amor incondicional, que é

combustível para que eu possa seguir meus projetos.

À minha família, pela atuação basilar nas decisões mais importantes da minha vida.

Vocês me incentivaram a estar aqui e buscar sempre crescimento pessoal e profissional.

À família Elias Faustense: Tiana, Jandir, Jeisson e Jan. Obrigado por me acolherem como

um verdadeiro filho e por me mostrarem o valor da família.

Às famílias Piracicabanas: Nice, Marcelo, Cristina, Pedro, Marcelo Filho, Natália,

Guilherme, Vinicius, Sérgio, Marilena, Esther e Rafael. Obrigado pelo acolhimento e pelo

amor fraterno.

À minha amada terra Livramento, PB, onde cresci e, com simplicidade, aprendi a ser

feliz.

Enfim, sou profundamente grato a todos que, direta ou indiretamente, contribuíram

para a concretização deste ideal.

EPÍGRAFE

Não é sobre chegar no topo do mundo E saber que venceu

É sobre escalar e sentir Que o caminho te fortaleceu.

É saber se sentir infinito

Num universo tão vasto e bonito É saber sonhar

E, então, fazer valer a pena cada verso Daquele poema Sobre acreditar.

Trecho da Canção “Trem Bala”, de autoria de Ana Vilela

RESUMO

Um dos principais agentes etiológicos da cárie dentária, Streptococcus mutans, dispõe de

um sofisticado repertório de mecanismos de sobrevivência e virulência, pelos quais pode

causar doenças orais e extra-orais. A capacidade de S. mutans interagir com componentes

de matriz extracelular tecidual, tais como colágeno, permite a colonização de diferentes

tecidos do hospedeiro. Previamente, demonstramos que a proteína ligante de colágeno

Cnm (~120 kDa) promove adesão ávida de S. mutans à dentina radicular ex vivo e

aumenta cárie coronária in vivo. Todavia, a influência de Cnm sobre o desenvolvimento

de cárie radicular, onde o colágeno é abundante, permanece desconhecida. No Capítulo

I, utilizamos um modelo de ratos dessalivados sob alto desafio cariogênico para investigar

o papel de Cnm no desenvolvimento de cárie radicular. Ratos infectados com a cepa

OMZ175Δcnm (knockout) apresentaram menos cárie coronária com envolvimento inicial

de dentina quando comparados aos infectados com a cepa selvagem OMZ175 e a

complementada OMZ175Acnm::pcnm (p<0,05) após 5 semanas. No entanto, não foram

observadas diferenças significativas quanto ao desenvolvimento de cárie radicular.

Interessantemente, os ratos infectados com OMZ175 ou OMZ175Δcnm::pcnm

apresentaram maior exposição radicular (até cerca de 18,5% mais elevada, P <0,05) do

que aqueles infectados com OMZ175Δcnm ou UA159 (cepa naturalmente negativa para

Cnm). Em síntese, este capítulo descreve a contribuição de Cnm no desenvolvimento da

cárie, particularmente nos estágios iniciais de lesão dentinária, e destaca seu potencial

impacto no desenvolvimento de doenças periodontais e destruição óssea alveolar, a ser

investigado em estudos futuros. Há também evidências de que Cnm desempenha um

papel importante, e ainda subestimado, no desenvolvimento de doenças cardiovasculares.

Contudo, ainda não se conhecem os mecanismos pelos quais patógenos orais Cnm+

colonizam e persistem em tecidos cardíacos humanos. No Capítulo II, utilizamos um

sistema de expressão heteróloga em Lactococcus lactis NZ9800 para investigar o papel

de Cnm na invasão intracelular, adesão a tecidos cardíacos ex vivo e desenvolvimento de

endocardite infecciosa (EI) in vivo. Semelhante a S. mutans, a expressão de Cnm em L.

lactis mediou ligação ao colágeno e laminina, invasão de células endoteliais da artéria

coronária humana e aumentou a virulência em larvas de Galleria mellonella (p<0,05).

Utilizando um modelo de colonização da valva aórtica humana ex vivo, demonstramos

que as cepas Cnm+ de S. mutans ou L. lactis colonizaram tecidos da valva aórtica de 10 a

100 vezes mais que as cepas Cnm- (p<0,001). Finalmente, um ensaio de EI em coelhos

revelou que a cepa Cnm+ de L. lactis foi recuperada de tecidos cardíacos com uma

frequência significativamente mais elevada (~ 70%, p=0,0001) em relação à cepa Cnm-.

Em síntese, este capítulo descreve que a presença de Cnm é suficiente para conferir

virulência a um organismo não patogênico e confirma a utilidade do sistema heterólogo

de L. lactis para caracterização adicional de fatores de virulência bacterianos.

Coletivamente, os nossos resultados fornecem evidência de que a ligação a componentes

da matriz extracelular tecidual mediada por Cnm é um atributo de virulência importante

de S. mutans para o desenvolvimento de doenças orais e sistêmicas.

Palavras-chave: Streptococcus mutans, adesinas bacterianas, cárie radicular, doenças

cardiovasculares, matriz extracelular.

ABSTRACT

One of the major etiological pathogens of dental caries, Streptococcus mutans, utilizes a

sophisticated repertoire of survival and virulence mechanisms, thereby potentially

causing oral and extra-oral diseases. The ability of S. mutans to interact with tissue

extracellular matrix components, such as collagen, enables colonization of different host

tissues. Previously, we demonstrated that the 120-kDa surface collagen-binding protein

Cnm promotes stringent adhesion of S. mutans to root dentin ex vivo and increases coronal

caries in vivo. Nevertheless, the impact of Cnm on root caries development, where

collagen is abundant, remains unknown. In Chapter I, we used a desalivated rat model

under a highly cariogenic challenge to investigate whether Cnm contributes to root caries

development. Rats infected with the cnm-knockout strain OMZ175Δcnm showed lower

levels of slight dentinal smooth-surface caries when compared to the wild type OMZ175

and complemented strain OMZ175Δcnm::pcnm (P<0.05) after 5 weeks. Nevertheless, no

significant differences were observed for root caries development. Surprisingly, rats

infected with OMZ175 or OMZ175Δcnm::pcnm showed increased root surface exposure

(up to ~18.5% higher, P<0.05) than those infected with OMZ175Δcnm or UA159

(naturally defective for Cnm). In summary, this chapter describes the contribution of Cnm

to caries development, particularly in early stages of lesion severity, and highlights its

potential impact on periodontal diseases and alveolar bone destruction. Cnm has also been

reported to play an important and underestimated role in the onset of cardiovascular

diseases. Yet, the mechanisms employed by Cnm+ oral pathogen to colonize and persist

in human heart tissues are still poorly understood. In Chapter II, we used a heterologous

expression system in Lactococcus lactis NZ9800 to investigate the role of Cnm in

intracellular invasion, adhesion to cardiac tissues ex vivo and development of infective

endocarditis (IE) in vivo. Similar to S. mutans, expression of Cnm in L. lactis enabled

robust binding to collagen and laminin, invasion of human coronary artery endothelial

cells and increased virulence in Galleria mellonella larvae (P<0.05). Using an ex vivo

human heart tissue colonization model, we showed that Cnm+ strains of either S. mutans

or L. lactis outcompete their Cnm- counterparts by 10-100 fold for tissue colonization

(P<0.001). Finally, a rabbit IE assay showed that the L. lactis Cnm+ strain was recovered

from heart tissues at significantly higher frequencies (~70%, p=0.0001) than the L. lactis

Cnm- strain. In summary, this chapter describes that Cnm is enough to confer virulence

to an otherwise non-pathogenic organism and confirms the usefulness of the L. lactis

heterologous system for further characterization of bacterial virulence factors.

Collectively, our results provide evidence that binding to extracellular matrix

components mediated by Cnm is an important virulence attribute of S. mutans to cause

oral and systemic diseases.

Keywords: Streptococcus mutans, bacterial adhesins, root caries, cardiovascular

diseases, extracellular matrix.

LISTA DE ABREVIATURAS E SIGLAS1

BHI, brain heart infusion;

CBPs, collagen-binding proteins;

CFU, colony-forming unit;

CI, competition/competitive index;

EB, electroporation buffer;

EBM, endothelial basal medium;

ECM, endothelial cell medium;

ECM, extracellular matrix;

EPS, extracellular polysaccharides

FBS, fetal bovine serum;

FIB-FE-SEM, focused ion beam field emission scanning electron microscope;

GM17, M17 broth supplemented with 0.5% glucose;

GM17-Erm or GM17-Chlor, M17 broth supplemented with 0.5% glucose plus

erythromycin or chloramphenicol (Chlor);

GM17S, GM-17 containing 1% glycine and 0.5 M sucrose;

GTF, glycosyltransferase

HBSS, Hank's balanced salt solution;

HCAEC, human coronary artery endothelial cells;

IE, infective endocarditis;

PVDF, polyvinylidene fluoride;

SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis;

SEM, scanning electron microscopy;

TSA, tryptic soy agar;

VS, viridans streptococci.

1 Abreviaturas correspondem à versão na língua inglesa.

SUMÁRIO

1 INTRODUÇÃO…………………………………………………………………….....................................15

2 ARTIGOS

2.1 Artigo: Streptococcus mutans Cnm contributes to caries development and

alveolar bone loss in desalivated rats…………………………………………………………………………….19

2.2 Artigo: Heterologous Expression of Streptococcus mutans Cnm in Lactococcus

lactis Promotes Intracellular Invasion, Adhesion to Human Cardiac Tissues and

Virulence 36

3 DISCUSSÃO 64

4 CONCLUSÃO...............................................................................................................................68

REFERÊNCIAS..................................................................................................................................69

ANEXOS............................................................................................................................................73

Anexo 1. Informação CPG/001/2015. Trata do Formato Alternativo das........... ....................73

Dissertações de Mestrado e Teses de Doutorado da UNICAMP.

Anexo 2. Artigo científico publicado referente ao Capítulo II.............. .....................................74

Anexo 3. Certificado de aprovação do Comitê de Ética no Uso de Animais................ ......76

CEUA/UNICAMP.

Anexo 4. Certificado de aprovação para atividades em contenção com......................... ....77

organismos geneticamente modificados e seus derivados (CIBio – FOP/UNICAMP)

15

INTRODUÇÃO

Streptococcus mutans é um dos principais microrganismos associados à

etiopatogenia da cárie dentária, a doença infecciosa mais prevalente em todo o mundo

(Kassebaum et al., 2015). É sabido que a virulência de S. mutans reside principalmente

em três atributos essenciais: (i) capacidade de aderir e formar biofilmes nas superfícies

dentárias pela produção de polissacarídeos extracelulares (PECs) via glicosiltransferases

(Gtfs); (ii) produção de grandes quantidades de ácidos orgânicos a partir de uma ampla

gama de carboidratos e sobrevivência em meio ácido (Banas, 2004); e (iii) tolerância a

estresses ambientais (Kajfasz et al., 2010; Galvão et al., 2015). A maioria dos estudos na

literatura têm sido conduzidos seguindo um modelo clássico, sacarose-dependente, para

compreender o desenvolvimento e a progressão da doença cárie. Entretanto, novos fatores

de virulência, não diretamente relacionados à produção de polímeros e quedas de pH,

também têm sido atribuídos à capacidade de S. mutans em causar doenças orais, como a

cárie dentária e, sobretudo, infecções sistêmicas (Nobbs et al., 2009; Abranches et al.,

2011; Avilés-Reyes et al., 2016), como será discutido mais adiante.

Diversas proteínas ancoradas ou associadas à superfície celular e capazes de se

ligar à componentes da matriz extracelular (MEC) do hospedeiro já foram descritas em

S. mutans (Nobbs et al., 2009), dentre as quais se destacam: SpaP, WapA, Cnm, Cbm,

SmFnB, entre outras (Avilés-Reyes et al., 2016). A produção de ácidos orgânicos por

bactérias cariogênicas, em consequência da fermentação de carboidratos da dieta,

associada ao acúmulo de biofilme oral podem desencadear o processo carioso, que se

inicia pela perda de componente mineral do esmalte dentário (Krzyściak et al., 2014). O

avanço do desequilíbrio bioquímico e posterior destruição do esmalte ou cemento expõem

estruturas colagenosas da dentina coronária ou radicular, respectivamente, as quais

constituem substratos altamente favoráveis à ligação ávida por adesinas bacterianas

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(Miller et al., 2015). Assim sendo, torna-se imprescindível compreender não apenas os

mecanismos dependentes de sacarose para o estabelecimento de S. mutans na cavidade

oral, mas, sobretudo, sua capacidade pouco conhecida de persistir no hospedeiro e invadir

tecidos por meio da aderência direta a proteínas da MEC ou outros componentes

celulares.

Cnm é uma glicoproteína ligadora de colágeno e laminina encontrada em cepas

invasivas de S. mutans (Nakano et al., 2010; Abranches et al., 2011). A proteína Cnm

tem aproximadamente 120 kDa e é composta de um domínio conservado de ligação ao

colágeno (domínio A) e um domínio único rico em treonina (domínio B) de função ainda

desconhecida, com ancoragem à parede celular por meio de um motif LPXTG (sortase

recognition sequence). Aproximadamente 10 a 20% dos isolados de S. mutans

apresentam o gene cnm, localizado entre os genes SMU.2067 and SMU.2069, o qual

codifica a proteína Cnm (Sato et al., 2004; Nomura et al., 2009; Nakano et al., 2010).

Um estudo recente do nosso grupo de pesquisa demonstrou que Cnm promove

adesão firme a tecidos da dentina coronária e radicular ex vivo bem como medeia a

invasão de S. mutans a fibroblastos gengivais (HGF-1) e queratinócitos do epitélio oral

(HOK) (Miller et al., 2015). O estudo também apontou o potencial cariogênico de cepas

Cnm+ em superfícies lisas e sulco de ratos, e demonstrou que a inativação do gene cnm

não alterou parâmetros relacionados à produção de PECs, aciduricidade e acidogênese do

microrganismo. Não obstante, a contribução de Cnm para o desenvolvimento de cárie de

raiz, onde há abundância de colágeno, bem como para a perda óssea adjacente, permanece

desconhecida.

No Capítulo I, utilizamos um modelo com ratos dessalivados sob alto desafio

cariogênico para investigar a contribuição da proteína Cnm de S. mutans para o

desenvolvimento e severidade de cárie e perda óssea alveolar in vivo.

17

O papel de Cnm no desenvolvimento de endocardite infecciosa

S. mutans dispõe de um sofisticado arsenal de mecanismos de sobrevivência e

virulência (Krzyściak et al., 2014), que permitem a este microganismo se estabelecer no

hospedeiro e favorecer o desenvolvimento de doenças orais, como a cárie dentária, e

doenças sistêmicas, como a endocardite infecciosa (EI) (Nakano et al., 2010a).

A EI é uma infecção grave do endocárdio, com incidência anual crescente

variando entre 2-7 casos/100.000 indivíduos em diferentes países (Yiu et al., 2007;

Fernández-Hidalgo & Almirante, 2012) e taxa de mortalidade entre 15 e 30% dos casos

(Murdoch et al., 2009), sendo, portanto, um grave problema de saúde pública mundial. A

patogênese do EI é caracterizada pela formação de vegetações no endocárdio resultantes

de um processo inflamatório. Essas vegetações consistem em um coágulo de fibrina

contendo plaquetas e células inflamatórias por entre as quais se aderem os

microrganismos (Shun et al., 2005). Estafilococos, enterococos e estreptococos do grupo

Viridans são os grupos microbianos mais frequentemente associados ao desenvolvimento

de EI em humanos (Werdan et al., 2014). Nesse cenário, S. mutans é responsável por

aproximadamente 20% dos casos de EI causada por microrganismos do grupo Viridans

(Banas, 2004; Que & Moreillon, 2011). Há relatos na literatura de que DNA genômico

de S. mutans foi detectado com alta frequência em lesões de valvas cardíacas e placas de

ateroma (Nakano et al., 2010). Entretanto, informações sobre os mecanismos moleculares

pelos quais esse patógeno oral sobrevive na corrente sanguínea e infecta os tecidos

cardíacos ainda são escassas.

O gene cnm foi encontrado com frequência nos sorotipos e, f e k, e raramente no

sorotipo c (Sato et al., 2004; Nomura et al., 2009; Nakano et al., 2010).

Interessantemente, os isolados de S. mutans que pertencem aos sorotipos e, f e k são raros

na cavidade oral (20%, 5% e 5%, respectivamente), mas comumente detectados em

18

pacientes com EI, aterosclerose e outras infecções sistêmicas (Nakano et al., 2010). Além

disto, estudos in vivo e transversais sugerem uma associação entre a presença de S. mutans

Cnm-positivo dos sorotipos e, f e k e patologias extra-orais, como acidente vascular

encefálico hemorrágico, micro-hemorragias cerebrais e esteatohepatite não alcoólica

(Nakano et al., 2011; Naka et al., 2014; Miyatani et al., 2015), indicando que Cnm pode

contribuir para a virulência e patogenicidade de S. mutans em infecções sistêmicas.

A bactéria Gram-positiva Lactococcus lactis é um microrganismo não patogênico

utilizado na indústria de laticínios para a produção de produtos lácteos fermentados

(Mierau & Kleerebezem, 2005). Em pesquisa, L. lactis tem sido utilizado na produção de

proteínas heterólogas (Morello et al., 2008) e vacinas (Wyszynska et al., 2015). A

presença de um baixo número de genes que codificam proteínas secretadas e ancoradas à

superfície torna L. lactis um hospedeiro desejável para a caracterização de fatores de

virulência de superfície de bactérias Gram-positivas patogênicas, visto que em outros

microrganismos há redundância de função de diversas proteínas de parede celular (Que

et al., 2005; Piroth et al., 2008; Danne et al., 2011).

No Capítulo II, utilizamos um sistema de expressão heteróloga em L. lactis para

investigar o papel de Cnm na invasão intracelular, adesão a tecidos cardíacos ex vivo e

desenvolvimento de endocardite infecciosa em modelo animal.

Coletivamente, os resultados desta tese fornecem evidência de que Cnm é um

importante fator de virulência de S. mutans, até então pouco conhecido, relacionado à

aderência, invasão e persistência deste patógeno no hospedeiro.

19

Capítulo I 1,2

Streptococcus mutans Cnm contributes to caries development and alveolar

bone loss in desalivated rats

Irlan Almeida Freiresa, Jacqueline Abranchesb, José A. Lemosb, Pedro Luiz Rosalena,*

a Department of Physiological Sciences, Piracicaba Dental School, University of Campinas,

Piracicaba, SP, Brazil. b Department of Oral Biology, University of Florida College of Dentistry, Gainesville, Florida,

USA.

*Corresponding author:

Pedro Luiz Rosalen

Department of Physiological Sciences, Piracicaba Dental School, University of Campinas

(UNICAMP), 13414-018, Piracicaba, SP, Brazil. Phone +55 19 2106-5208; fax: +55 19 2106-

5250, [email protected].

Keywords: dental caries; bacterial adhesins; collagen-binding proteins; extracellular

matrix components; alveolar bone loss.

1 Esta tese está apresentada no formato alternativo, conforme informação da CPG/001/2015 (Anexo 1).

2 Este manuscrito será submetido ao periódico Molecular Oral Microbiology (2015 Impact Factor:

3.061, 2016 Thomson Reuters, Journal Citation Reports).

mailto:[email protected]

20

SUMMARY

In addition to glucan-mediated binding, S. mutans utilizes sucrose-independent

mechanisms (e.g. expression of collagen-binding proteins - CBPs) to thrive and persist in

oral and extra-oral sites. Previously, we demonstrated that the CBP Cnm promotes

stringent adhesion of S. mutans to root dentinal tissues ex vivo. Here, we used a

desalivated rat model under a highly cariogenic challenge to investigate whether Cnm

contributes to the extension and severity of coronal and root caries in vivo. SPF female

Wistar rats were infected with Cnm+ or Cnm- strains. To stimulate root caries

development, animals were desalivated and fed diet 2000 (56% sucrose) plus 5% sucrose-

water ad libitum for 5 weeks. Rats infected with the cnm-knockout strain OMZ175Δcnm

showed significantly lower average levels of slight dentinal smooth-surface caries

(57.1±19.6) when compared to the wild type OMZ175 (79.7±17.7) and complemented

strain OMZ175Δcnm::pcnm (81.3±15.4) (P<0.05). Nevertheless, no significant

differences were observed between Cnm+ and Cnm- strains for root surface caries

(P>0.05). Surprisingly, rats infected with OMZ175 or OMZ175Δcnm::pcnm showed

increased root surface exposure, which reflects bone loss (up to ~18.5% higher, P<0.05),

than those infected with OMZ175Δcnm or UA159 (naturally Cnm defective strain). This

study reports on the contribution of S. mutans Cnm to caries development, particularly in

early stages of lesion progression, and highlights the potential impact of Cnm on alveolar

bone destruction in desalivated rats.

21

INTRODUCTION

One of the major etiological pathogens of dental caries, Streptococcus mutans,

utilizes a sophisticated repertoire of survival and virulence mechanisms, thereby

triggering the development and progression of oral (Krzyściak et al., 2014) and extra-oral

diseases (Nakano et al., 2010a; Freires et al., 2017). Most studies in the literature have

focused on classical sucrose-dependent pathways of S. mutans associated with caries

development, including glycosyltransferase (Gtf) activity (Bowen & Koo, 2011),

exopolysaccharide (EPS) synthesis (Ren et al., 2016), and high tolerance to pH

fluctuations as well as acid production (Kim & Burne, 2017). Nevertheless, recent

evidence has also shed light on some non-classical sucrose-independent mechanisms as

a key strategy used by S. mutans to thrive and persist either inside or outside the oral

cavity (Nobbs et al., 2009; Abranches et al,. 2011; Avilés-Reyes et al., 2016).

A number of cell wall-anchored or surface-associated adhesins able to bind

extracellular matrix (ECM) proteins have been described in S. mutans, including SpaP,

WapA, Cnm, Cbm, SmFnB, among others (Nobbs et al., 2009; Avilés-Reyes et al., 2016).

In the oral millieu, lactic acid production by cariogenic bacteria due to fermentation of

dietary carbohydrates associated with biofilm buildup may initiate the carious process

(Krzyściak et al., 2014). If continuously repeated, demineralization and further

destruction of enamel structures expose underlying collagenous tissues of the coronal or

root dentin, thereby creating a favorable substrate for bacterial adhesins. Hence, it

becomes critical to understand not only the glucan-related mechanisms for the

establishment of S. mutans in oral biofilms, but also its potential capacity to persist in and

invade tissues through a direct binding to ECM proteins or other host components.

Cnm is a 120-kDa cell-surface collagen- and laminin-binding protein found in

approximately 10-20% of S. mutans strains (Nakano et al., 2010). Several studies have

22

linked the presence of Cnm with extra-oral diseases, such as infective endocarditis

(Nakano et al., 2010b; Freires et al., 2017), haemorrhagic stroke (Nakano et al., 2011),

cerebral microbleeds (Miyatani et al., 2015) and non-alcoholic steatohepatitis (Naka et

al., 2014). However, the study of such a glycoprotein as a bolstering factor for the onset

of oral diseases, where S. mutans naturally occurs, is very recent. Approximately 20% of

S. mutans strains recovered from inflamed dental pulp were Cnm+, all of which being able

to adhere to human dental pulp fibroblasts (Nomura, Ogaya and Nakano, 2016). In

addition, we previously demonstrated that Cnm promotes stringent adhesion to dentinal

and root tissues ex vivo as well as to collagen-coated surfaces, and increases cariogenicity

in vivo (Miller et al., 2015). Nevertheless, the impact of Cnm on root caries development,

where collagen is abundant, remains unknown.

Rodent models have been considered the gold standard for the study of caries

development in vivo (Larson & Fitzgerald, 1964; Hana et al., 2017). Moreover, it is well

known that humans and rodents with greatly reduced salivary flow are more prone to

develop coronal and root surface caries than are those with intact salivary function

(Bowen, Madison and Pearson, 1988). The diminished salivary buffering capacity allied

to the cariogenic stress of a sucrose-rich diet promote a highly acidic plaque environment,

which impairs tooth remineralization, exposes adjacent collagenous tissues, and promotes

bone loss (Humphrey & Wiliamson, 2001). Here, we used a desalivated rat model under

a highly cariogenic challenge to investigate whether S. mutans Cnm contributes to the

extension and severity of coronal and root caries in vivo.

MATERIAL AND METHODS

Animals. Fifty-six specific pathogen-free (SPF) female Wistar rats aged 19 days were

purchased from the Multidisciplinary Center for Biological Research (CEMIB) at

23

University of Campinas, SP, Brazil. All procedures followed the ethical principles for the

use and care of animals. This study had prior approval of the Ethics Committee on Animal

Use (CEUA) and of the Institutional Biosafety Board due to the use of genetically

modified organisms (GMOs), University of Campinas, SP, Brazil, under protocols nº.

4232-1/2016 and A006/2016, respectively.

Bacterial strains and growth conditions. Wild type and defective strains and plasmids

used in this study originated from the study by Abranches et al. (2011). The S. mutans

strains UA159 (non-invasive, Cnm-, used as a reference due to its known cariogenicity),

OMZ175 (invasive, native Cnm+), OMZ175Δcnm (non-invasive, Cnm-) and

OMZ175Δcnm/pMSP3535::cnm (complemented, invasive, Cnm+) were routinely

cultured in brain heart infusion (BHI) (Difco®) medium at 37°C in a 5% CO2 atmosphere.

To enhance infectivity, the strains were previously grown for 72 h on vertically positioned

glass rods in the presence of BHI plus 2% sucrose, with fresh medium replacement once

a day. Then biofilm cells were scrapped from the rods and allowed to grow overnight in

fresh sucrose-free BHI at 37°C, 5% CO2, to serve as a starter culture.

Experimental caries model. The contribution of Cnm to dental caries was assessed using

methods described elsewhere (Bowen, Pearson and Young, 1988), with modifications. At

the age of 19 days, pups were weaned and randomly assigned into four groups of 14

animals each, as follows: Group 1 (S. mutans UA159), Group 2 (S. mutans OMZ175),

Group 3 (S. mutans OMZ175Δcnm) and Group 4 (S. mutans

OMZ175Δcnm/pMSP3535::cnm). Pups were submitted to confirmatory screening for the

presence of specific pathogens and total cultivable bacteria. Then starter cells from a 72-

h rod-grown biofilm culture, as described above, were grown to exponential phase (~0.5

24

OD600nm) in BHI and swabbed onto all hard and soft surfaces of the oral cavity of the

animals (teeth, marginal gingiva, tongue, mouth floor, palate, and jugal mucosa) for 4

consecutive days (approximate volume of 120 µL per swab). The animals were screened

for strain implantation at the age of 25 days by plating oral swabs onto mitis salivarius

agar (Difco®) (MSA) plus 200 U bacitracin/liter (Sigma-Aldrich, St. Louis, USA) (MSB).

To bolster caries development, pups were anesthetized and submitted to desalivation

procedures at the age of 26 days. First, both parotid ducts were ligated by passing a #4

silk suture beneath the ducts. Then the submandibular and sublingual glands were excised

following blunt dissection through a midline incision. Wounds were approximated using

surgical clips (Clay Adams, Parsippany, NJ) and removed after a 4-day recovery period.

At this point, rats were housed in pairs in screen-bottomed cages and fed a high-sucrose

diet (Diet 2000, 56% sucrose) plus 5% sucrose water ad libitum for 5 weeks. All rats were

weighed weekly and monitored daily for behavioral and physical appearance alterations.

Microbiological analysis. After 5 weeks, the animals were killed. Their lower left jaws

were aseptically dissected, placed in sterile saline solution (0.9%, w/v) and sonicated

(three 10-s pulses at 5-s intervals, at 30 W; Vibracell, Sonics and Material Inc., Newtown,

CT). Then the solution was serially diluted and plated for CFUs (colony-forming units)

onto MSB for S. mutans counts and onto blood agar (5% sheep blood) for total bacterial

counts. The proportion of S. mutans over the total cultivable microbiota was calculated.

Coronal caries scoring. Smooth-surface caries were scored by a single calibrated blind

examiner, who showed excellent intra-rater agreement according to Intraclass Correlation

Coefficient (0.93). Scores were set based on their extension and severity (E, enamel

lesion; Ds, slight dentinal caries; Dm, moderate dentinal caries – 3/4 of the dentin

25

affected; Dx, extensive dentinal caries – all dentin affected), according to Larson’s

modification of Keyes’ system (Keyes, 1958; Larson, 1981).

Root surface exposure and root caries scoring. Root surface exposure, which indirectly

reflects alveolar bone loss, and root surface caries were estimated by a modification of

the Rosen et al.’s method (1981) described by Bowen, Pearson and Young (1988).

Briefly, roots were divided into 4 areas, except in the area of furcation, which was divided

into 2 sections. Caries and/or root exposure in each area was given a value of 1. Root

exposure was estimated by the distance from the cemento-enamel junction to the tip of

the alveolar bone. According to this method, each maxillary first molar could theoretically

have a maximum root surface score of 44. All values obtained were expressed as average

score (± standard deviation).

Statistical analysis. Coronal and root caries score and root exposure data were checked

for normality using Shapiro-Wilk test and submitted to one-way analysis of variance

(ANOVA) followed by Tukey-Kramer HSD (Honest Standard Deviation) test. Intra- and

inter-group pairwise comparisons of animal weight gains were performed using paired-t

test and ANOVA with Tukey’s post-hoc test, respectively. Statistical analysis was carried

out on GraphPad Prism 6.0 software (San Diego, California, USA), with a 5%

significance level.

RESULTS

All rats gained weight (P<0.001) and remained active throughout the duration of

the study. Consistent with this, the average weight gains did not differ among groups in

any of the 5 experimental weeks (P>0.05, Table S1, supplementary material).

26

Figure 1 shows the total cultivable microbiota and S. mutans counts after oral

infection of rats with the selected microorganisms. Overall, no significant difference was

observed in total flora (Figure 1A) and S. mutans (Figure 1B) carriage among groups

(P>0.05), except for rats infected with OMZ175Δcnm::pcnm, which showed lower total

flora counts than those infected with UA159 (P<0.01), although these numbers did not

differ from those of OMZ175 (P>0.05). Likewise, similar proportions of S. mutans per

total flora were found among the reference (UA159) and experimental groups (P>0.05),

revealing that all strains successfully implanted in the oral cavity of the rats.

Figure 1. Total cultivable microbiota (A) and Streptococcus mutans (B) recovered from

left jaws of animals 5 weeks after infection with UA159, OMZ175, OMZ175Δcnm or

OMZ175Δcnm::pcnm (complemented). (C) Proportion (%) of S. mutans per total

microbiota. The results represent the mean and standard deviations of one experiment

(n=14). Asterisks indicate statistically significant differences at 0.05 when compared with

UA159 (One-way analysis of variance with Tukey’s post-hoc).

Table 1 shows the amount of smooth-surface caries developed upon infection with

Cnm+ and Cnm- strains. In all cases, rats infected with OMZ175Δcnm showed lower

average levels of enamel (E) and dentinal (Ds, Dm, Dx) smooth-surface caries when

compared to OMZ175 or OMZ175Δcnm::pcnm, with statistical significance in slight

dentinal (Ds) lesions (P<0.05). Importantly, the reference strain UA159 produced a high

amount of coronal caries, with no significant difference from Cnm+-infected rats in (E)

27

and (Ds) lesions (P>0.05). These findings reveal that Cnm plays an important role in

coronal caries development, particularly in early stages of lesion progression.

Table 1. The influence of S. mutans Cnm on the development and severity of smooth-surface

caries in desalivated Wistar rats.

Strain Lesion

extension (E)

Lesion severity

Ds Dm Dx

UA159 114.7 (± 18.9)a 97.8 (± 20.7)a 69.2 (± 24.8)a 32.2 (± 28.7)a

OMZ175 104.0 (± 16.5)a 79.7 (± 17.7)a 33.5 (± 25.3)b 5.2 (± 10.0)b

OMZ175Δcnm 91.9 (± 28.8)a 57.1 (± 19.6)b 33.0 (± 22.2)b 2.6 (± 4.3)b

OMZ175Δcnm::pcnm 102.9 (± 17.5)a 81.3 (± 15.4)a 43.1 (± 22.0)b 8.6 (± 11.5)b The results are given in averages (± standard deviations). Different letters in the same column indicate statistically

significant differences at 0.05. Analysis of variance using Tukey–Kramer honest significant difference test for all

pairs was used to determine statistical significance. E, enamel; Ds, slight dentinal caries; Dm, moderate dentinal

caries; Dx, extensive dentinal caries.

Despite the potential role of Cnm in the progression of smooth-surface caries, no

significant differences were observed between Cnm+ and Cnm- strains for root surface

caries (P>0.05), as shown in Table 2. Nevertheless, rats infected with OMZ175 or

OMZ175Δcnm::pcnm surprisingly showed increased root surface exposure in most cases

(up to ~18.5%) than those infected with OMZ175Δcnm or UA159 (P<0.05). Interestingly,

Cnm+ strains promoted a significantly higher amount of mandibular bone destruction than

the reference strain UA159 (P<0.05).

Table 2. The influence of S. mutans Cnm on root surface exposure and root caries in desalivated Wistar rats.

Strain Root surface exposure Root surface caries

Mandible Maxilla Total Mandible Maxilla Total

UA159 96.7 (± 10.0)a 96.3 (± 12.4)a 96.5 (± 11.0)a 10.4 (± 9.0)a 5.6 (± 8.6)a 8.0 (± 8.9)a

OMZ175 116.3 (± 17.3)bc 106.4 (± 7.5)ab 111.3 (± 13.9)b 5.2 (± 8.5)a 0.2 (± 0.9)a 2.8 (± 6.4)ab

OMZ175Δcnm 94.7 (± 13.9)a 90.0 (± 12.1)c 92.4 (± 12.9)a 3.0 (± 5.3)a 0.1 (± 0.3)a 1.5 (± 3.9)b

OMZ175Δcnm::pcnm 103.3 (± 9.0)ac 97.3 (± 9.3)ac 100.3 (± 9.5)a 6.8 (± 6.6)a 2.0 (± 4.5)a 4.4 (± 6.0)ab

The results are given in averages (± standard deviations). Different letters in the same column indicate statistically

significant differences at 0.05. Analysis of variance using Tukey–Kramer honest significant difference test for all pairs

was used to determine statistical significance.

DISCUSSION

Dental caries remains one of the most costly and widespread chronic diseases

worldwide, with an enormous economic and social burden to healthcare systems

28

(Kassebaum et al., 2015). Despite the extensively studied mechanisms related to

etiopathogenesis, new virulence traits of S. mutans, including Cnm expression, have been

unraveled (Avilés-Reyes et al., 2014; Miller et al., 2015). Here, we studied for the first

time the contribution of S. mutans Cnm to root caries development and, potentially,

alveolar bone loss, indirectly measured by root surface exposure.

The caries model used in our study with desalivated Wistar rats revealed that the

presence of Cnm did not seem to be a prerequisite for oral colonization, since both Cnm+

and Cnm- strains successfully implanted in the oral cavity of the rats after 5 weeks.

However, these findings disagree with those reported by Miller et al. (2015), who used a

Sprague Dawley conventional (non-desalivated) caries model. The authors showed that

carriage of OMZ175Δcnm was significantly lower than that of the wild type OMZ175.

Several reasons may explain such divergence from our study: (i) the highly acidic

environment promoted by desalivation, which may have favored glucan-binding

mechanisms for oral colonization, given that S. mutans biofilms form more efficiently at

low pH (Li & Burne, 2001) and that there is no difference in EPS production between

wild type and Cnm defective strains (Miller et al., 2015); (ii) reduced salivary clearance

of the microorganisms; (iii) microbiome shifting caused by desalivation, which may have

facilitated oral colonization by OMZ175Δcnm; and, to a minor extension, (iv) the

constitutional variability of the animal species and their susceptibility to caries (Van Reen

et al., 1962). We note that the caries model used herein poses a substantially more

aggressive cariogenic challenge than that used by Miller et al. (2015), as our major aim

now was to produce carious lesions on the root surfaces.

As for smooth-surface caries, OMZ175 and OMZ175Δcnm::pcnm consistently

developed greater amounts of enamel and dentinal lesions than OMZ175Δcnm. The caries

scores reported herein are higher and more aggresive than those previously reported by

29

Miller et al. (2015), probably due to the diminished salivary function of the animals. All

groups in our study showed high Dm and Dx scores, which indicates severe coronal

destruction regardless of the presence of Cnm. It is likely, however, that an endpoint

shorter than five weeks could have revealed Cnm-related differences in advanced dentinal

lesions. The experimental period of five weeks was selected based on the study by Bowen,

Pearson and Young (1988), in which authors showed that a six-week challenge is very

destructive – and thus unnecessary and unethical – while a 3 to 4-week challenge is

insufficient to promote a fair amount of root caries in desalivated animals. It is also worth

noting that intrinsic host factors play a protective role against caries, for instance, saliva.

The desalivation procedure may alter some of its several functions, including innate (non-

immunoglobulin factos) and acquired (specific sIgA immunoglobulin) immunity,

buffering capacity, sugar clearance, among others, which directly make animals more

prone to caries (Leone & Oppenheim, 2001).

Other well known surface-associated adhesins of S. mutans have also been

implicated in coronal caries development in addition to Cnm. The 185-kDa protein SpaP,

also known as antigen I/II or P1, binds to components of the salivary pellicle, e.g.

agglutinins, and plays a key role in primary colonization of teeth, particularly under low

sucrose conditions. Deletion of spaP gene decreased the amount of enamel and dentinal

buccal, sulcal and proximal caries in Fischer rats (Crowley et al., 1999). WaP-A is another

wall-associated protein (47-kDa) found in S. mutans, which has been postulated to bind

collagen and contribute to caries development (Han, Zhang and Dao, 2006). Due to its

binding properties, WaP-A was considered as a potential target for a dental caries vaccine,

especially because it is widely conserved in human pathogenic strains of S. mutans (Han

& Dao, 2005).

30

Collagen type I constitutes the most abundant component of the dentin organic

matrix, accounting for 90% of the organic matter (Ho et al., 2007). Hence, the collagen-

binding capacity of S. mutans may confer enhanced capacity to colonize and impair root

surfaces (Han, Chang and Dao, 2006). Based on this and on the previously reported

stringent adhesion to dentinal and root tissues ex vivo (Miller et al., 2015), we

hypothesized that Cnm expression in S. mutans would bestow this microorganism an

increased ability to develop root lesions in vivo. Our data showed no significant difference

in Cnm+ and Cnm- strains as for the amount of mandibular and maxillary root lesions. On

the other hand, the presence of Cnm seemed to aggravate alveolar bone loss resulting

from the carious process, indirectly measured by root exposure. Cnm+ strains were found

to promote more root surface exposure than their Cnm- counterpart, particularly in the

mandible. Furthermore, in some Cnm+ samples of our study, root surface was highly

exposed even in the absence of lesions on the roots, which may indicate activation of an

inflammatory response leading to osteoclast activation and bone destruction

(Hajishengallis et al., 2016). Hence, Cnm-related host immune cell recruitment and

release of proinflammatory cytokines (e.g., IL-17, IL-1β, TNF-α), chemokines (e.g.,

CXCR2) and other signaling molecules (e.g., RANK/RANK-L) (Hajishengallis, 2014;

Hajishengallis et al., 2016) merit further research and are now being investigated by our

research group.

In summary, S. mutans Cnm contributes to further dentinal caries development on

smooth surfaces, but not on the roots, and seems to aggravate alveolar bone loss in

desalivated rats. Future research should focus on understanding the molecular

mechanisms of alveolar bone destruction upon infection with Cnm+ strains.

31

ACKNOWLEDGEMENTS

The authors would like to dedicate this study to Dr. William H. Bowen (in

memoriam) for his prestigious contribution and assistance with the desalivation training

procedures. This work was supported by São Paulo Research Foundation (FAPESP,

Brazil, grants no. 2014/07231-0 and no. 2013/25080-7) and by the National Council for

Scientific and Technological Development (CNPq, Brazil, grant no. 310522/2015-3).

32

SUPPLEMENTARY MATERIAL

Table S1. Average weight gains (± SD) of Wistar rats infected with Cnm+ and Cnm- strains and fed a highly cariogenic diet for five weeks.

Group Average weight gain in grams (± SD)

1st week 2nd week 3rd week 4th week 5th week p-value2

UA159 17.8 ± 11.6 20.2 ± 5.7 11.8 ± 9.7 31.1 ± 12.2 14.4 ± 6.6 p<0.0001

OMZ175 15.9 ± 7.8 21.4 ± 5.8 11.1 ± 5.5 32.2 ± 8.5 13.7 ± 7.3 p<0.0001

OMZ175Δcnm 15.7 ± 14.3 20.3 ± 11.0 13.9 ± 16.3 31.4 ± 16.6 13.8 ± 10.9 p<0.0001

OMZ175Δcnm::pcnm 17.8 ± 9.9 17.5 ± 6.4 14.1 ± 9.0 28.9 ± 11.4 14.1 ± 4.5 p<0.0001

p-value1 0.9349 0.5941 0.8743 0.9142 0.9957 1 Analysis of variance (ANOVA) with Tukey-Kramer test was used to determine statistical significance among groups each week. 2 Paired-t test was used to determine statistical significance of average weight in each group individually from baseline (1st week) to the endpoint

(5th week).

33

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mutans. Appl Environm Microbiol. 2017, doi: 10.1128/AEM.03274-16 [Epub

ahead of print].

Krzyściak W, Jurczak A, Kościelniak D, Bystrowska B, Skalniak A. The virulence

of Streptococcus mutans and the ability to form biofilms. Eur J Clin Microbiol

Infect Dis. 2014; 33:499-515. doi:10.1007/s10096-013-1993-7.

Larson RH, Fitzgerald RJ. Caries development in rats of different ages with controlled

flora. Arch Oral Biol. 1964; 9:705-12.

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Tanzer JM ed. Animals Models in Cariology. Washington, DC: Information

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Leone CW, Oppenheim FG. Physical and chemical aspects of saliva as indicators of risk

for dental caries in humans. J Dent Educ. 2001; 65:1054-62.

Li Y, Burne RA. Regulation of the gtfBC and ftf genes of Streptococcus mutans in

biofilms in response to pH and carbohydrate. Microbiology. 2001; 147:2841-8.

Miller JH, Aviles-Reyes A, Scott-Anne K, Gregoire S, Watson GE, Sampson E, et al. The

collagen binding protein Cnm contributes to oral colonization and cariogenicity of

Streptococcus mutans OMZ175. Infect Immun. 2015; 83:2001-10.

Miyatani F, Kuriyama N, Watanabe I, Nomura R, Nakano K, Matsui D, et al. Relationship

between Cnm-positive Streptococcus mutans and cerebral microbleeds in humans.

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Naka S, Nomura R, Takashima Y, Okawa R, Ooshima T, Nakano K. A specific

Streptococcus mutans strain aggravates non-alcoholic fatty liver disease. Oral Dis.

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Nakano K, Hokamura K, Taniguchi N, Wada K, Kudo C, Nomura R, et al. The collagen-

binding protein of Streptococcus mutans is involved in haemorrhagic stroke. Nat

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diseases—from molecular mechanisms to clinical cases: cell-surface structures of

novel serotype k Streptococcus mutans strains and their correlation to virulence. J

Pharmacol Sci. 2010a 113:120-125. Doi: 10.1254/jphs.09R24FM.

Nakano K, Nomura R, Taniguchi N, Lapirattanakul J, Kojima A, Naka S, et al. Molecular

characterization of Streptococcus mutans strains containing the cnm gene encoding

a collagen-binding adhesin. Arch Oral Biol. 2010b; 55:34-9.

Nobbs AH, Lamont RJ, Jenkinson HF. Streptococcus adherence and colonization.

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Nomura R, Ogaya Y, Nakano K. Contribution of the Collagen-Binding Proteins of

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36

Capítulo II 3,4

Heterologous Expression of Streptococcus mutans Cnm in Lactococcus

lactis Promotes Intracellular Invasion, Adhesion to Human Cardiac Tissues

and Virulence

Irlan A. Freiresa,b, Alejandro Avilés-Reyesc, Todd Kittend, P. J. Simpson-Haidarise,

Michael Swartzf, Peter A. Knightf, Pedro L. Rosalena, José A. Lemosc, Jacqueline

Abranchesc,*

a Department of Physiological Sciences, Piracicaba Dental School, University of Campinas,

Piracicaba, SP, Brazil. b Center for Oral Biology, University of Rochester Medical Center, Rochester, NY, USA. c Department of Oral Biology, University of Florida College of Dentistry, Gainesville, Florida,

USA. d Philips Institute for Oral Health Research, Virginia Commonwealth University, Richmond,

Virginia, USA e Department of Medicine/Hematology-Oncology Division and Department of Pathology and

Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA f Department of Surgery Cardiac Division, University of Rochester School of Medicine and

Dentistry, Rochester, New York, USA.

* Corresponding author

Department of Oral Biology, 1395 Center Drive, Box 100424, University of Florida

College of Dentistry, Gainesville, FL 32610

Tel: (362) 273-6672; Fax: (352) 273-8290

Email: [email protected]

Keywords: Collagen-binding protein, infective endocarditis, bacterial pathogenesis,

Streptococcus mutans, Lactococcus lactis.

3 Esta tese está apresentada no formato alternativo, conforme informação da CPG/001/2015 (Anexo 1).

4 Este manuscrito encontra-se publicado no periódico Virulence, 8(1) 2017 (2015 Impact Factor: 5.418,

2016 Thomson Reuters, Journal Citation Reports), conforme consta no Anexo 2. A formatação do

manuscrito e das referências segue os padrões da revista. O artigo publicado foi capa do vol 8, n.1, 2017

(issue cover) e também tema de um editorial escrito pela Dr. Angela Nobbs, University of Bristol, Bristol,

UK, conforme Anexo 2.1.

mailto:[email protected]

37

ABSTRACT

In S. mutans, the expression of the surface glycoprotein Cnm mediates binding to

extracellular matrix proteins, endothelial cell invasion and virulence in the Galleria

mellonella invertebrate model. To further characterize Cnm as a virulence factor, the cnm

gene from S. mutans strain OMZ175 was expressed in the non-pathogenic Lactococcus

lactis NZ9800 using a nisin-inducible system. Despite the absence of the machinery

necessary for Cnm glycosylation, Western blot and scanning electron microscopy

analyses demonstrated that Cnm was effectively expressed and translocated to the cell

wall of L. lactis. Similar to S. mutans, expression of Cnm in L. lactis enabled robust

binding to collagen and laminin, invasion of human coronary artery endothelial cells and

increased virulence in G. mellonella. Using an ex vivo human heart tissue colonization

model, we showed that Cnm-positive strains of either S. mutans or L. lactis outcompete

their Cnm-negative counterparts for tissue colonization. Finally, Cnm expression

facilitated L. lactis adhesion and colonization in a rabbit model of infective endocarditis.

Collectively, our results provide unequivocal evidence that binding to extracellular

matrices mediated by Cnm is an important virulence attribute of S. mutans and confirm

the usefulness of the L. lactis heterologous system for further characterization of bacterial

virulence factors.

INTRODUCTION

The association of Streptococcus mutans with dental caries, the most prevalent

and costly infectious disease worldwide, is well established.1, 2 The capacity of S. mutans

to form biofilms in the presence of sucrose, to produce organic acids upon fermentation

of dietary carbohydrates, and to tolerate large fluctuations in pH are considered the major

cariogenic traits of this bacterium.3, 4 However, the medical implications of S. mutans are

38

not limited to the oral cavity as this organism can cause extra-oral infections and may

play an important and underestimated role in the onset of cardiovascular diseases.5-9 This

facet of S. mutans pathogenesis is likely associated with its ability to interact with

extracellular matrix (ECM) components such as collagen, which enables colonization of

different host tissues.10, 11

Infective endocarditis (IE) is a life-threatening bacterial infection of the

endocardium with approximately 45,000 occurrences per year in the U.S.12 and mortality

rates around 15-20%.9, 13, 14 Along with staphylococci and enterococci, viridans

streptococci (VS) are a major bacterial group associated with IE12, and S. mutans is

estimated to account for ~ 20% of all VS-associated IE cases.9, 15 In addition, S. mutans

DNA has been detected at a high frequency in cardiovascular tissues from patients that

underwent valve replacement surgery and atherosclerotic plaque removal.16, 17 Yet, the

mechanisms employed by S. mutans to colonize and persist in human heart tissues are

still poorly understood.

The S. mutans surface protein Cnm is a collagen- and laminin- binding

glycoprotein shown to mediate adhesion to collagen and laminin, invasion of endothelial

cells, and virulence in an invertebrate model.18-20 Epidemiological studies have shown

that cnm is present in approximately 15% of S. mutans isolates, being found at higher

frequency among serotype e, f and k strains but rarely present in serotype c strains.18, 21-23

Interestingly, isolates of S. mutans that belong to serotypes e, f and k are rare in the oral

cavity but commonly detected in patients with IE, atherosclerosis and other systemic

infections.1, 22 Moreover, in vivo and cross-sectional studies suggest an association

between S. mutans serotypes e, f and k expressing Cnm and extra-oral pathologies such

as haemorrhagic stroke, cerebral microbleeds and non-alcoholic steatohepatitis, 5, 6, 24

39

indicating that Cnm may contribute to the pathogenic potential of S. mutans during

systemic infections

The Gram-positive bacterium Lactococcus lactis is a non-pathogenic organism

used in the dairy industry for the production of fermented milk products.25 In addition, L.

lactis is used for the production of heterologous proteins,26 and in live mucosal vaccines.27

The presence of secretory and protein anchoring enzymes (e.g. SecA and SrtA) combined

with the low number of genes encoding secreted and surface-anchored proteins makes L.

lactis a desirable host for the characterization of surface virulence factors of pathogenic

Gram-positive bacteria.26, 28-30 For example, systemic or mucosal delivery of a

recombinant L. lactis strain expressing the Streptococcus agalactiae pilus 1 genes was

shown to protect mice from subsequent challenges with S. agalactiae.31 Other bacterial

proteins successfully expressed in L. lactis include Staphylococcus aureus clumping

factor A (ClfA) and fibronectin-binding protein A (FnBPA),30 Staphylococcus

epidermidis SdrF32, and Listeria monocytogenes LnlA.33 In each of these studies,

heterologous expression of these foreign factors was sufficient to increase the pathogenic

potential of this otherwise harmless microorganism. Here, the contribution of Cnm to

bacterial virulence was further investigated using the L. lactis heterologous expression

system. Our results provide unequivocal evidence that Cnm is a major virulence factor of

S. mutans that contributes to systemic infections.

RESULTS

Optimization of Cnm expression in L. lactis

After successful electroporation of pMSP3535::cnm into L. lactis NZ9800, the

concentration of nisin that conferred optimal expression of Cnm without causing growth

inhibitory effects was determined. The growth of L. lactis was only noticeably affected

at 25 ng ml-1 nisin or higher concentrations (data not shown). Western blot analysis

40

showed a concentration-dependent induction of Cnm from pMSP3535::cnm by nisin with

strong induction observed at 10 ng ml-1 and above (Fig. 1A). Non-induced cultures of L.

lactis pMSP3535::cnm showed basal expression of Cnm, which is in line with previous

reports demonstrating that the pMSP3535 system is leaky.34, 39 Immunofluorescence

labeling of intact cells confirmed expression and surface localization of Cnm (Fig. 1B).

Based on these results, we chose 10 ng ml-1 nisin for all subsequent experiments as it

yielded robust protein production without interfering with cell growth.

Figure 1. Expression of Cnm from pMSP3535::cnm in recombinant L. lactis. (A)

Western blot analysis of protein lysates was performed with a polyclonal antibody

specific to Cnm. A nisin concentration-dependent induction of Cnm was observed from

pMSP3535::cnm, and the uninduced culture showed basal protein expression. (B)

Immunofluorescence labeling analysis revealing Cnm expression on the surface of L.

lactis.

41

Cnm+ L. lactis binds to ECM and invades HCAEC

The ability of the recombinant L. lactis Cnm+ strain to bind to collagen and

laminin in vitro was tested. When compared to the parent strain NZ9800 hosting the

empty vector, expression of Cnm from pMSP3535::cnm led to robust binding to both

collagen and laminin (Fig. 2A and B, respectively). In accordance with our expression

analysis (Fig. 1), uninduced pMSP3535::cnm produced low levels of Cnm and therefore

enabled the recombinant L. lactis strain to bind to both ECM proteins, albeit at lower

levels than nisin-induced cells (P < 0.05). To further demonstrate that the ability to bind

to collagen is mediated by Cnm, we performed the collagen-binding assay in the presence

of anti-rCnmA antibody. As expected, co-incubation of L. lactis cells expressing Cnm

with anti-rCnmA antibodies led to significant inhibition of collagen binding activity

compared to L. lactis incubated with pre-immune serum (Fig. 2C). In agreement with

increased collagen and laminin binding activities observed for nisin-induced cells,

expression of Cnm enabled L. lactis to invade HCAEC (Fig. 2D). While ECM binding

was observed in uninduced cultures, HCAEC invasion levels were negligible in the

absence of nisin (P > 0.05).

Expression of Cnm mediates adherence to aortic valve tissues ex vivo

To determine if Cnm bestows an enhanced heart tissue binding capacity to either

S. mutans or L. lactis ex vivo, competition binding assays were performed using freshly

extirpated human aortic valve tissues. In agreement with the ECM-binding in vitro data,

the S. mutans OMZ175 and nisin-induced Cnm+ L. lactis strains outcompeted their Cnm-

counterparts by approximately 10 and 100-fold, respectively (Fig. 3). Scanning electron

microscopy of tissues infected for 90 min with monocultures of L. lactis (Fig. 4) or S.

mutans (data not shown) confirmed that only Cnm+ strains could effectively bind to the

42

collagenous fibrils of damaged valve tissues (Figs. 4A and 4C). Notably, Cnm+ strains

could also adhere to tissue areas where no apparent damage was observed (Fig. 4B).

Figure 2. Cnm promotes binding to ECM proteins and invasion of endothelial cells.

(A and B) Binding of bacterial cells to extracellular matrix proteins: (A) collagen type I

and (B) laminin. (C) Inhibition of binding to collagen type I by anti-rCnmA antibody

(1:20). (D) HCAEC invasion assay showing the normalized percentage of invasion for

each strain after 3 h of co-incubation (*P < 0.05, **P < 0.01, ***P < 0.0001, ANOVA

followed by Tukey’s post-test). The S. mutans OMZ175 (Cnm+) strain was used as a

positive control in all assays. The symbols “+” and “–“ in the graphs denote induction or

no induction with nisin, respectively.

43

Figure 3. Expression of Cnm mediates bacterial adherence to aortic valve sections.

A competition assay was developed to test the contribution of Cnm toward bacterial

adherence to human aortic valves ex vivo. A 1:1 mixed infection of the valve sections was

allowed for 90 min using Cnm+ and Cnm- strains. The competitive index (CI) was

calculated based on the initial inocula, where a value of “=1” represents no difference and

“>1” represents preferred binding by the numerator strain.

44

Figure 4. Cnm mediates binding to the collagenous fibrils of damaged and

apparently undamaged infected human valve sections. SEM analysis of valve sections

incubated with bacterial monocultures showed Cnm+ L. lactis binding to bundles of

collagenous fibrils on the rough edges of the valves whereas no Cnm- L. lactis were found

attached to this area (A). Cnm+ L. lactis could also adhere to tissue areas where no

apparent damage was observed in contrast with their Cnm- counterparts (B).

45

Figure 4. (C) Computer-colored SEM photomicrograph (5,000x) showing Cnm+

bacterial cells (green) attached to the collagenous fibrils (light brown) of damaged human

aortic valve sections ex vivo.

Cnm contributes to virulence in Galleria mellonella

The ability of the L. lactis strains to kill the larvae of G. mellonella, a model of

systemic infection was assessed. As shown in Figure 5, the mortality rates of G.

mellonella were significantly higher in larvae infected with the Cnm+ strain compared to

those infected with the wild-type strain harboring the empty vector control (P < 0.05).

Remarkably when considering the extremely low virulence potential of L. lactis, the

recombinant Cnm+ strain showed a similar pattern of killing when compared to the Cnm+

S. mutans OMZ175 strain.

46

Figure 5. Cnm contributes to virulence in the G. mellonella systemic infection model.

The Kaplan-Meier curves indicate the percent survival at selected intervals of larvae

infected with S. mutans OMZ175 and L. lactis strains for 96 h. The mortality rates of G.

mellonella were significantly higher in larvae infected with Cnm+ L. lactis compared to

those infected with the wild-type NZ9800 harboring the empty vector control (pMSP3535)

(P < 0.05).

Cnm contributes to endocardium adherence in vivo

A rabbit model of IE 38 was employed to examine the effect of Cnm expression

on L. lactis adherence to endocardium and vegetations. In one experiment, a mixed

inoculum of NZ9800 (pCIE) and NZ9800 (pMSP3535::cnm) was injected into rabbits

intravenously 1 h after surgery. Two hours later, the rabbits were euthanized and the

hearts removed. This time was chosen so as to allow enough time for clearance of bacteria

from the bloodstream,40 but not so long as to have excessive loss of Cnm due to growth

without nisin. Catheters were clamped into place during necropsy to preserve their

positions. At this early stage, vegetations were not visible, and the entire endocardial

surface in apparent contact with the catheter was excised and homogenized. Following

sonication, both the mixed inoculum and the homogenates were plated on selective media

47

to determine strain ratios. In the second experiment, animals were inoculated 2 days after

surgery. Vegetations, which were visible in all animals, were removed and homogenized.

Because vegetations are composed primarily of platelets and fibrin rather than collagen,41

the endocardium beneath each vegetation was also collected, homogenized, and plated

separately.

As shown in Figure 6, the CI value of the cells harvested from the endocardium

were similar in animals inoculated 1 hour or 2 days after catheterization (geometric mean

= 1.68 and 1.66, respectively), indicating a ~67% increase in infectivity of the Cnm-

expressing strain relative to the control strain. These values were significantly greater

from 1.0 — the value indicating no change in infectivity — whether the two experiments

were analyzed separately or together. In contrast, there was no significant difference in

the CI value for bacteria recovered from vegetations.

Figure 6. Cnm contributes to bacterial endocardium adherence in vivo. Rabbits were

co-inoculated with Cnm-expressing and control L. lactis strains 1 hour or 2 days after

insertion of an intracardiac catheter as indicated and then sacrificed 2 hours later. Bacteria

were recovered from endocardial surfaces (End.), and in the 2-day samples, from adjacent

48

vegetations (Veg.). CI values (for L. lactis Cnm+ over L. lactis Cnm-) are indicated for

each sample, with mean CIs indicated by a horizontal line. Means significantly different

from 1.0 (dashed line) are indicated: *P < 0.05; ***P < 0.001. Like symbols in the 2-day

experiment indicate samples recovered from the same animal. Red circle, sample from

which one of the two strains was not recovered (L. lactis Cnm-); the limit of detection

was used for the calculation.

DISCUSSION

The presence of Cnm has been correlated with an increased ability of S. mutans

to invade and persist intracellularly, and to cause disease.6, 18, 20, 21, 42, 43 In the present

study, by expressing Cnm in the non-pathogenic L. lactis host we provided unequivocal

evidence that Cnm contributes to bacterial virulence.

Various properties of L. lactis have justified its attractiveness for heterologous

expression of surface proteins. For instance, the fermentative metabolism of L. lactis is

relatively simple and well known, the genomes of various strains are available and known

to encode a very low number of genes encoding secreted and surface-anchored proteins.25,

44, 45 Here, we showed that Cnm expression increased the ability of L. lactis to bind to

type I collagen and laminin in vitro, adhere to aortic valve tissues ex vivo, and colonize

endocardial surfaces in vivo. Of note, type I collagen is the most abundant ECM

component in human46, 47 and rabbit48 aortic valves. Although ex vivo experiments cannot

reproduce the immunologic responses and constant shear force of blood flow, our

findings highlighted the contribution of Cnm in binding of organisms to collagen-rich

tissues in both humans and rabbits.

Recently, our group reported that Cnm is a glycoprotein and identified the

glycosylation machinery (Pgf) responsible for its post-translational modification. In

49

SDS-PAGE, the native Cnm migrates at approximately 120 kDa whereas the variant from

the deletion of the pgfS gene was found to migrate at ~ 90 kDa.19 When analyzed by

Western blot, the Cnm variant produced by L. lactis was found to migrate at ~ 90 kDa,

the same size as the unglycosylated version of Cnm produced in S. mutans.19 Moreover,

like the unglycosylated Cnm produced by the pgfS strain in S. mutans, the Cnm

expressed by L. lactis was highly susceptible to proteinase K degradation (data not

shown).19 These findings are supported by in silico analysis that indicated the absence of

Pgf homologs in the available genomes of L. lactis. Most importantly, the lack of Cnm

glycosylation may explain the lesser effect of Cnm expression on adhesion to endocardial

tissue in vivo compared to in vitro or ex vivo. Whether this difference is due to the

considerable shear forces and host proteases present in the in vivo model, or to some other

factor(s) remains to be determined. Expression of Cnm had no effect on colonization of

preexisting vegetations, which is not surprising considering these vegetations are

composed primarily of platelets and fibrin.41 Nevertheless, adhesins for collagen and

other matrix proteins have been identified previously as virulence factors for IE.29, 49, 50

As with these other adhesins, Cnm may allow adherence to damaged endocardium prior

to vegetation formation, thereby providing another route for infection in IE.51 The

increased adherence to endocardium observed is also consistent with previous reports of

association of Cbm-expressing strains, with both IE and non-IE cardiovascular diseases.7,

52

Collectively, our results clearly show that expression of Cnm alone confers

virulence to an otherwise non-pathogenic bacterium, and promotes bacterial adherence to

cardiac tissue in vivo and ex vivo. Thus, Cnm could be considered a target for the

development of antimicrobial strategies to mitigate virulence of invasive S. mutans strains.

50

MATERIAL AND METHODS

Bacterial strains and growth conditions

All strains used in this study are listed in Table 1. The L. lactis strains were

routinely cultured aerobically at 30°C in M17 broth (Difco) supplemented with 0.5% (w/v)

glucose (GM17). When necessary, 10 µg ml-1 erythromycin or 15 µg ml-1

chloramphenicol (Sigma-Aldrich) was added to the growth media (GM17-Erm or GM17-

Chlor, respectively). The S. mutans strains were routinely cultured in brain heart infusion

(BHI) medium at 37°C in a 5% CO2 atmosphere.

Table 1. Strains used in this study.

Strain Relevant

phenotype Reference

Streptococcus mutans

OMZ175 Cnm+ Lab collection

OMZ175 (pBGE) Cnm+ Ermr (21)

∆cnm Cnm– Kanr (21)

Lactococcus lactis subsp. lactis

NZ9800 WT, Cnm– Lab collection

NZ9800 (pMSP3535) Cnm– Ermr This study

NZ9800 (pMSP3535::cnm) Cnm+ Ermr This study

NZ9800 (pCIE) Cnm–, Chlorr This study

Note: WT (wild type); Kan (kanamycin); Erm (erythromycin); Chlor

(chloramphenicol).

Expression of Cnm in L. lactis

The nisin-inducible plasmid pMSP3535 34 harboring the cnm gene

(pMSP3535::cnm) from S. mutans OMZ175,21 was used to express Cnm in L. lactis. The

pMSP3535::cnm plasmid as well as the empty pMSP3535 vector 34 and the pCIE

plasmids (Dunny et al., unpublished) were electroporated into L. lactis NZ9800 as

detailed elsewhere 35 with some modifications. The pCIE plasmid, conferring resistance

to chloramphenicol, was generated from shuttle plasmid pCI13340 36 and introduced into

L. lactis for selection purposes in competition assays. Briefly, cells were grown overnight

51

at 30oC in M17 broth supplemented with 0.5% glucose (GM-17) and sub-cultured (1:100)

in fresh GM-17 containing 1% glycine and 0.5 M sucrose (GM17S). The culture was

grown to OD600 of 0.2, centrifuged at 5,000 rpm for 10 min at 4oC and washed 3 times

with cold electroporation buffer (EB) (0.5 M sucrose, 1 mM MgCl2, 7 mM K2HPO4-

KH2PO4 [pH 7.4]). Electrocompetent cells were resuspended in EB, quickly frozen on

dry ice/ethanol bath and stored at -80oC until use. A micro-pulser electroporation

apparatus (Bio-Rad Laboratories) (0.1cm cuvette, 1.4 kV, 600 Ω, 10 µF) was used to

electroporate the desired plasmids into L. lactis. After electroporation, cells were

incubated in GM17S for 3 h at 30oC and spread on GM17 plates containing the desired

antibiotic. Resulting colonies were screened for the presence of the desired plasmid by

PCR. For induction of Cnm, cells were grown overnight in GM17-Erm in the presence of

increasing concentrations of nisin (Sigma-Aldrich). The production and cell surface

localization of Cnm was confirmed by Western blotting and by immunofluorescence

labeling as detailed below.

Western Blot (WB) analysis

Overnight cultures of S. mutans OMZ175 and recombinant L. lactis strains were

used to prepare protein lysates by homogenization with 0.1 mm glass beads using a bead

beater (Biospec). Protein lysates were separated on a 10% SDS-PAGE and transferred to

a polyvinylidene fluoride (PVDF) membrane (Millipore) using standard protocols. The

presence of Cnm was detected using rabbit anti-rCnmA polyclonal antibody 19 diluted

1:2000 in 1 x phosphate buffered saline (PBS) with 0.1% Tween 20 pH 7.2 and anti-IgG

(goat anti-rabbit) horseradish peroxidase-coupled antibody diluted 1:10000 (Sigma-

Aldrich). Membranes were developed using an enhanced chemiluminescent detection kit

(GE Life Sciences).

52

Immunofluorescence labeling of Cnm

Cultures of recombinant L. lactis and S. mutans OMZ175 were used for cellular

localization of Cnm. Cells grown to mid-exponential phase were washed in PBS and

incubated in the presence of anti-rCnmA antibody (1:100) followed by anti-rabbit Alexa-

488 conjugate antibody (1:100) with three washes between each antibody exposure. Cell-

antibody complexes were visualized using an Olympus BX41 fluorescent microscope and

images were captured with the QCapture Software.

Binding to extracellular matrix (ECM) proteins

Collagen and laminin binding assays were performed as previously described.20,

21 Briefly, 96-well plates were coated with 40 µg ml−1 type I collagen from rat tail (Sigma-

Aldrich), or 50 µg ml−1 mouse laminin (Becton-Dickinson) for 18 h at 4oC. Individual

wells were washed and blocked with 5% bovine serum albumin for 1 h at 37oC. Overnight

bacterial cultures were washed twice in PBS and 100 μl aliquots of each bacterial

suspension added to the wells, followed by incubation for 3 h at 37oC. Loosely bound

bacteria were removed by washing with PBS and adhered cells were stained with 0.05%

crystal violet solution and then quantified at 575 nm. Experiments were also performed

with bacteria pre-incubated with anti-rCnmA antibody (1:20 dilution in PBS) for 30 min

before incubation with collagen. To confirm the requirement of Cnm in collagen binding,

experiments were performed as described above but bacteria were pre-incubated with

anti-rCnmA or pre-immune serum (1:100) for 30 min before exposure to collagen.19

Invasion of human coronary artery endothelial cells (HCAEC)

Cultivation of primary HCAECs and invasion assays were performed as

previously described.18, 21 The HCAEC monolayer was incubated with 1 ml of 2% fetal

bovine serum (FBS)-endothelial basal medium (EBM-2) containing 1-2 x 107 CFU m-1

53

of S. mutans OMZ175 or ∆cnm, or recombinant L. lactis strains for 2 h at a multiplicity

of infection of 100:1, followed by 3 h of incubation in 2% FBS-EBM-2 medium

containing 300 μg ml−1 gentamicin and 50 μg ml−1 penicillin G to kill extracellular

bacteria. After incubation with antibiotics, HCAEC were lysed with 1 ml of sterile water

and the mixture of lysed HCAEC and bacteria serially diluted and plated onto tryptic soy

agar (TSA) plates to determine the number of intracellular bacteria. The percentage of

invasion for each strain was calculated based on the total colony forming units (CFU)

number present in the initial inoculum and the number of intracellular bacteria recovered

from HCAEC lysates.

Adherence to human cardiac valve sections ex vivo

The ability of S. mutans and L. lactis strains to bind to human aortic valve tissues

ex vivo was assessed by adapting a protocol originally described for porcine heart

tissues.37 Human aortic valve tissues were obtained from patients undergoing valve

replacement surgery due to calcific stenosis (Institutional Review Board protocol no.

RSRB00053016, University of Rochester). Valvular tissues with no signs of calcification,

bleeding or inflammation were selected and 3-mm diameter valve sections were obtained

using a biopsy punch tool (Acuderm). Valve sections were incubated overnight in 12-

well plates with endothelial cell medium (ECM) supplemented with 5% FBS, 1000 mg

L-1 hydrocortisone, 12 mg L-1 bovine brain extract, 0.1% human recombinant epidermal

growth factor (Lonza) plus 300 µg ml-1 gentamicin. On the following day, sections were

washed twice with Hank's Balanced Salt Solution (HBSS, Corning Inc.) to remove

residual antibiotic and then transferred to a new plate. A competition assay was then

performed to determine the capacity of Cnm+ strains to compete with Cnm- strains. For

this, valve sections were infected with approximately 5 x 107 CFU ml-1 of a 1:1

suspension of Cnm+ and Cnm- strains bearing distinct antibiotic markers for CFU

54

determination of each strain. Plates were incubated for 90 min at 37°C (for S. mutans) or

30°C (for L. lactis) with gentle rocking. Valve sections were transferred to sterile

centrifuge tubes containing 1 ml of HBSS and vigorously agitated three times for 5

seconds using a vortex to remove loosely bound bacteria from the tissues. Washed valve

sections were homogenized in 500 µl PBS for 2 min using a motorized pestle (Fisher

Scientific), serially diluted and plated onto TSA plates with the corresponding antibiotics.

The competitive index (CI) was calculated based on the CFUs present in the initial

inoculum and the final enumeration of bacteria recovered from the tissue.

Scanning Electron Microscopy (SEM) analysis of valve sections

To visualize bacterial attachment to heart valve sections, tissue samples were

prepared and incubated in the presence of 5 x 107 CFU ml-1 S. mutans or L. lactis

monocultures as described in the previous section. Valve samples were washed and fixed

with 4% paraformaldehyde, 2.5% glutaraldehyde and 0.1 M sodium cacodylate for 2 days

at 4oC, with slow rocking. The samples were analyzed using a Zeiss-Auriga focused ion

beam field emission scanning electron microscope (FIB-FE-SEM) and the images

captured using the attached Gatan Erlangshen digital camera at the University of

Rochester Medical Center Electron Microscopy Core Facility.

Galleria mellonella infection

The systemic infection of G. mellonella has been previously detailed elsewhere.19,

21 Briefly, 5 µl aliquots containing 1-1.5x106 CFU of S. mutans OMZ175 (positive

control), or recombinant L. lactis were injected in the larvae hemocoel via the last left

proleg. Larvae injected with heat-killed bacteria (45 min, 80°C) were used as negative

55

controls. After injection, larvae were kept in the dark at 37°C, and survival was

periodically monitored for up to 4 days.

In vivo model of infective endocarditis in rabbits

The rabbit model of IE employed for this study was modified from a previously

used protocol.38 Specific-pathogen-free, male New Zealand White Rabbits 2 to 3 kg in

size were purchased from Robinson Services Inc. After acclimation, rabbits were injected

with acepromazine as a sedative, a cocktail of ketamine, xylazine, and buprenorphine for

general anesthesia, glycopyrrolate for control of respiratory secretions, and bupivacaine

at the incision site. An incision was then made in the neck region, and a 19-gauge catheter

(Intracath, BD Bioscience) was inserted through the right internal carotid artery,

contacting or passing through the aortic valve to cause minor damage. The catheter was

tied off, trimmed, and sutured into place, and the incision closed. Buprenorphine SR Lab

was used for post-surgical analgesia. The day prior to inoculation, L. lactis strains were

cultured overnight in GM17 and antibiotics as described above with 10 ng ml-1 nisin.

Twenty hours later, the cultures were diluted 10-fold into pre-warmed media of the same

composition and cultured another 3 h. Cells were harvested by centrifugation, washed

twice with cold PBS, diluted in PBS, and combined to achieve a concentration of 3 – 5 x

108 CFU ml-1 for each strain. One hour or 2 days after the surgeries, rabbits were

inoculated with 0.5 ml of bacterial cells via a peripheral ear vein. Remaining cells were

sonicated for 1.5 min at 50% power in a Biologics Model 150 sonicator (Biologic Inc.)

to break up clumps and chains and plated with an Eddy Jet spiral plater (Neutec Group

Inc.) on GM17 plates containing erythromycin or chloramphenicol to establish the ratio

of the two strains in the inoculum. Two hours after inoculation, each rabbit was

euthanized with intravenous Euthasol (Virbac) and was then checked for proper

56

placement of the catheter tip in the valve region or beneath it in the left ventricle. Any

rabbit in which the catheter was not correctly situated was excluded from the study. For

rabbits inoculated on the day of surgery, the aortic valves and surrounding endocardium

were removed and homogenized in 5 ml PBS using an electric homogenizer (Omni

International). For rabbits inoculated 2 days post-surgery, visible vegetations and the

endocardium underlying each vegetation were harvested separately and homogenized in

2 ml PBS each using a disposable 15-ml tissue grinder. In all cases, homogenates were

sonicated and plated as above for enumeration.

Statistical analysis

All results represent a minimum of three independent experiments. Descriptive

and inferential statistics were used to analyze the data on Graphpad Prism version 5.0.

One-way ANOVA followed by Tukey’s post-test was used to analyze the significance of

binding and invasion, with type I error set as 0.05. Analysis of log10-transformed

competitive indices was performed using one-way ANOVA with Bonferroni post hoc

comparisons to determine substrate preferences among strain pairs. For the G. mellonella

studies, Kaplan-Meier killing curves were plotted and estimation of differences in

survival was compared using the log-rank test. Log10-transformed competitive indices

were compared to 0 using a one-sample T test for rabbit studies.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgements

This work was supported by the National Institute of Dental and Craniofacial

Research R01 DE022559 to JL and JA, National Heart, Lung, and Blood Institute F31

57

HL124952 to AAR, São Paulo Research Foundation (FAPESP, Brazil, grants no.

2014/07231-0 and no. 2013/25080-7) to IAF, and the National Council for Scientific and

Technological Development (CNPq, Brazil, grant no. 308644/2011-5) to PLR. The

authors thank Dr. Gary Dunny (University of Minnesota) for kindly providing L. lactis

NZ9800 and the pCIE plasmid.

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64

DISCUSSÃO

A habilidade de S. mutans interagir com componentes da MEC por meio da

expressão de proteínas ligantes de colágeno, como Cnm, confere a este patógeno oral uma

capacidade aumentada para o desenvolvimento de cárie (Miller et al., 2015). Além disto,

devido à ampla distribuição e abundância de colágeno no corpo humano, outros sítios

onde S. mutans não ocorre naturalmente também encontram-se susceptíveis à infecção

por cepas invasivas (Avilés-Reyes et al., 2016). No presente estudo, nós utilizamos

abordagens orais (Capítulo I) e extra-orais (Capítulo II) para elucidar o papel desta

glicoproteína no desenvolvimento e progressão de doenças humanas, como a cárie e a EI.

Estas doenças geram alto ônus econômico direto e indireto, por exemplo, a cárie dentária

é responsável por um gasto anual de cerca de 90 bilhões de dólares (Listl et al., 2015) e

doenças cardiovasculares de 863 bilhões de dólares (World Heart Federation, 2010), bem

como elevada taxa de morbidade e/ou mortalidade em nível global (Werdan et al., 2014).

No Capítulo I, demonstramos que a presença de Cnm está associada a um

incremento de lesões iniciais de cárie em dentina coronária, corroborando relatos

anteriores (Miller et al., 2015). Em um período experimental de cinco semanas, todos os

grupos apresentaram altos escores de lesões avançadas em dentina (Dm e Dx), o que

indica a ocorrência de destruição coronária severa e independente da presença de Cnm.

Entretanto, é possível que diferenças relacionadas ao potencial cariogênico de Cnm em

lesões avançadas tenham ocorrido em um menor tempo experimental, mas não foram

detectadas devido ao avançar da doença nos demais grupos como resultado da

dessalivação. A escolha pelo período experimental de 5 semanas baseou-se no estudo de

Bowen, Pearson e Young (1988), no qual os autores concluíram que um período de 5

semanas de desafio cariogênico é ideal, visto que um desafio de 6 semanas é bastante

65

destrutivo – e, portanto, desnecessário – enquanto que um período de 3 a 4 semanas é

insuficiente para o desenvolvimento apropriado de cárie de raiz nos animais dessalivados.

Considerando que a matriz orgânica dentinária é composta por 90% de colágeno

tipo I, a capacidade de se ligar a este substrato favorece a colonização e desenvolvimento

de cárie na superfície radicular (Han, Chang and Dao, 2006; Ho et al., 2007). Em um

estudo prévio (Miller et al., 2015), demonstrou-se que OMZ175 apresenta uma

capacidade aumentada de se ligar à dentina radicular ex vivo quando comparada à cepa

com deleção do gene cnm. Com base no exposto, hipotetizamos que a expressão de Cnm

poderia contribuir para o desenvolvimento de cárie radicular in vivo. Nenhuma diferença

significante foi verificada entre cepas Cnm+ e Cnm-, incluindo a cepa referência UA159

(Cnm-), no que concerne ao desenvolvimento de cárie radicular na mandíbula e maxila.

Todavia, observamos que OMZ175 e OMZ175Δcnm::pcnm promoveram perda óssea

alveolar expressiva, até 18.5% maior do que a cepa knockout OMZ175Δcnm,

especialmente na região mandibular. Do ponto de vista clínico, este percentual de perda

óssea é significativo (Lima et al., 2000) e deve ser melhor investigado para

compreendermos a patogenicidade conferida pela expressão de Cnm não apenas na

progressão da cárie, mas também em doenças periodontais, pela perda de suporte ósseo.

A literatura aponta que a interação de S. mutans com o hospedeiro pode interferir

com a função imunológica, desencadear a produção de citocinas proinflamatórias e,

consequentemente, retardar o reparo e a homeostase teciduais (Nagata, Toleto e Oho,

2011). Em algumas amostras do nosso estudo, a superfície radicular encontrava-se

altamente exposta, mesmo na ausência de lesões extensas de cárie na raiz, o que pode

indicar a ativação de vias inflamatórias que promovem ativação osteoclastogênica e

destruição óssea (Hajishengallis et al., 2016). Portanto, estudos futuros devem investigar

a influência de microrganismos expressando Cnm sobre o recrutamento de células imunes

66

do hospedeiro, liberação de citocinas (ex.: TNF-α , IL-1β) e quimiocinas (CXCR2)

proinflamatórias, assim como outros mediadores de reabsorção alveolar (ex.:

RANK/RANK-L) (Hajishengallis, 2014; Hajishengallis et al., 2016).

A presença de Cnm também tem sido fortemente correlacionada à maior

capacidade de S. mutans em invadir células endoteliais do hospedeiro e persistir no

citoplasma (Abranches et al., 2011). No Capítulo II, nós expressamos Cnm em um

microrganismo não patogênico, L. lactis, e demonstramos evidência inequívoca de que

esta glicoproteína contribui para a virulência bacteriana, visto que L. lactis recombinante

mostrou-se consistentemente invasivo e patogênico.

Diversas propriedades tornam L. lactis um hospedeiro (host) atrativo para a

expressão heteróloga de proteínas de superfície. Por exemplo, o metabolismo de L. lactis

é relativamente simples e bem conhecido (Kunji, Slotboom e Poolman, 2003; Mierau &

Kleerebezem, 2005); e o genoma de cepas desta espécie já foi sequenciado e aponta para

um baixo número de genes que codificam secreção de proteínas e ancoragem à superfície

(Wegmann et al., 2007). Neste estudo, nós demonstramos que a expressão de Cnm

aumentou a habilidade de L. lactis em se ligar ao colágeno tipo I e à laminina in vitro,

bem como aderir à valva aórtica humana ex vivo e, interessantemente, colonizar

superfícies do endocárdio in vivo. É importante notar que o colágeno tipo I é o

componente da MEC mais abundante nas valvas cardíacas humanas (Latif et al., 2005;

Rodriguez et al., 2014) e de coelhos (Hinton et al., 2006). Muito embora os experimentos

ex vivo não consigam reproduzir a resposta imunológica e forças mecânicas do fluxo

sanguíneo, os nossos resultados apontam para uma forte aderência de organismos Cnm+

com tecidos ricos em colágeno, tanto em humanos quanto em coelhos, condição que

predipõe a etiopatogenia de distúrbios cardiovasculares (Hinton et al., 2006).

67

A expressão de Cnm não afetou a colonização de vegetações preexistentes, o que

era esperado, visto que vegetações desta natureza são compostas primariamente por

plaquetas e fibrina (Durack, 1975; Werdan et al., 2014). Entretanto, Cnm pode favorecer

a aderência ao endocárdio danificado anteriormente à formação da vegetação,

propiciando, assim, uma outra rota para EI (Bashore, Cabell and Fowler, 2006).

Em síntese, os nossos resultados mostram que a expressão de Cnm conferiu

virulência a um microrganismo não patogênico, promoveu aderência bacteriana à tecidos

cardíacos in vivo e ex vivo, e aumentou a cariogenicidade e absorção óssea em modelo

com ratos dessalivados. Os nossos achados abrem caminhos inovadores para o

desenvolvimento de terapias alvo-específicas contra doenças que ainda geram um

considerável impacto social e econômico, como a cárie dentária e a endocardite

infecciosa.

68

CONCLUSÃO

1) A expressão de Cnm em S. mutans contribuiu para o desenvolvimento de cárie de

superfície lisa em um modelo animal dessalivado sob alto desafio cariogênico, mas

não incrementou o desenvolvimento de cárie de raiz após cinco semanas. Entretanto,

a presença de Cnm foi relacionada a uma maior perda óssea alveolar nos animais

dessalivados.

2) A expressão heteróloga de Cnm em Lactococcus lactis permitiu concluir que esta

glicoproteina confere capacidade aumentada de ligação bacteriana ao colágeno e à

laminina (componentes da matrix extracelular), invasão intracelular, aderência à

valva aórtica humana ex vivo e, finalmente, desenvolvimento de endocardite

infecciosa in vivo. Além disto, o presente estudo confirma a efetividade do sistema

heterólogo L. lactis para caracterização de fatores de virulência de superfície em

bactérias Gram-positivas.

Com base no exposto, a proteína ligante de colágeno Cnm pode ser considerada

um alvo em potencial para o desenvolvimento de novas terapias antimicrobianas que

visam mitigar a colonização e infecção de valvas cardíacas por cepas invasivas e, na

cavidade oral, onde S. mutans ocorre naturalmente, controlar o desenvolvimento e

progressão de cárie dentária por mecanismos alternativos aos disponíveis atualmente.

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79: 2277-84.

5 De acordo com as normas da UNICAMP/FOP, baseadas na padronização do International Committee of

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ANEXOS

Anexo 1. Informação CPG/001/2015. Trata do Formato Alternativo (Art 2º) das

Dissertações de Mestrado e Teses de Doutorado da UNICAMP.

INFORMAÇÃO CCPG/001/2015

Substitui Informação CCPG/002/2013

E altera a redação da versão aprovada pela CCPG em 17/06/2015).

(Nova redação dos itens; 2, 3 e 4, Inciso I, do Art. 1º)

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Anexo 2. Artigo científico publicado referente ao Capítulo II.

Por questões de direitos autoriais da revista, o artigo final publicado na íntegra não será

inserido nesta tese, mas será disponibilizado aos componentes da banca, e pode ser

acessado no link: Virulence 8(1), 2017:

http://www.tandfonline.com/eprint/3UvVRJurMhk7XbvuHyIN/full.

(2015 Impact Factor: 5.418, 2016 Thomson Reuters, Journal Citation Reports)

http://www.tandfonline.com/eprint/3UvVRJurMhk7XbvuHyIN/full

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Anexo 2.1. O artigo publicado foi tema de um editorial escrito pela Dr. Angela Nobbs,

University of Bristol, Bristol, UK.

Por questões de direitos autoriais da revista, o editorial não será inserido nesta tese, mas

será disponibilizado aos componentes da banca, e pode ser acessado no link: Virulence

8(1), 2017: http://www.tandfonline.com/doi/full/10.1080/21505594.2016.1212157.

http://www.tandfonline.com/doi/full/10.1080/21505594.2016.1212157

76

Anexo 3. Certificado de aprovação do Comitê de Ética no Uso de Animais

CEUA/UNICAMP.

77

Anexo 4. Certificado de aprovação para atividades em contenção com organismos

geneticamente modificados e seus derivados (CIBio – FOP/UNICAMP).

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