Class 2016
Grant: PD/BD/116940/2016
PhD thesis
New insights into the interaction of Burkholderia cenocepacia with host cells: Trimeric autotransporter adhesins as pathogenicity factors
Supervisor
• Arsénio M Fialho, Professor @ IST/ULisboa, PT
Co-Advisor
• Dalila Mil-Homens, Researcher @ IST/ULisboa, PT
Collaborators
• Michelle Kilcoyne, Lecturer in Glycosciences @ National University of Galway, School of Natural Sciences, Ireland
PhD Degree complete: Discussion on December 22, 2020; Pass with Distinction
THESIS ABSTRACT
Burkholderia cenocepacia is an opportunistic pathogen capable of establishing infection and causing disease in Cystic Fibrosis (CF) patients and other immunocompromised individuals. The intrinsic resistance to several classes of antibiotics, the large panel of virulence factors and ability to adapt to various environments facilitate B. cenocepacia colonization and persistence in their hosts. The B. cenocepacia arsenal of virulence factors is wide and diversified and include specialized surface exposed proteins. Among those, Trimeric Autotransporter Adhesins (TAAs) represent a unique class of proteins known to play relevant roles in pathogenicity. The general objective of this thesis is to gain an in-depth knowledge regarding the early steps of B. cenocepacia-host interaction, particularly elucidating the roles played by TAAs in that process. The results allowed the functional characterization of two B. cenocepacia TAAs – BcaC and BCAM2418. Both were found to be multifunctional proteins and important players during the bacterial adhesion to cells and extracellular matrix components. The expression of those TAA genes proved to be under fine-tuned regulation. bcaC was negatively regulated by a neighbor histidine kinase while BCAM2418 expression increased upon initial contact of bacterium with the host cell. Elimination of O-glycans both from mucins and surface of bronchial epithelial cells was found to turned BCAM2418 gene expression off and impaired bacterial adherence. The use of an anti-BCAM2418 antibody caused a marked reduction in the adhesion of B. cenocepacia to bronchial cells and mucins and an inhibition of in vivo virulence. Using glycan microarrays, host glycan epitopes for BCAM2418 were predicted and Burkholderia species were found to interact in different ways with distinct classes of CF-associated carbohydrate moieties. Finally, B. cenocepacia transcriptional modulation and adaptation to the host upon the initial contacts was assessed using giant plasma membrane vesicles (GPMVs) as a cell-like alternative. RNA-sequencing analysis demonstrated that the early physical interactions between bacteria and GPMVs caused a shift in the transcription of genes related to central metabolism, transport systems, and virulence traits, including many associated with the adhesion phenomena. Overall, the work supports the importance of the early contacts between bacteria and host cell as a crucial step for B. cenocepacia infection and opened new insights regarding the bacterial adaptation that take place in the early stages of infection.
RESUMO DA TESE
Burkholderia cenocepacia é um patogénico oportunista capaz de estabelecer infeção e causar doença em pacientes com Fibrose Cística (FC) e outros indivíduos imunocomprometidos. A resistência intrínseca a várias classes de antibióticos, a vasto painel de fatores de virulência e a capacidade de adaptação a diferentes ambientes, facilitam a colonização e persistência de B. cenocepacia no hospedeiro. O arsenal de fatores de virulência de B. cenocepacia é amplo e diversificado e inclui proteínas de superfície altamente especializadas. Entre estas, Adesinas Triméricas Autotransportadas (ATAs) representam uma classe única de proteínas que são conhecidas por desempenhar papéis importantes na patogenicidade. O objetivo geral desta tese é aprofundar o conhecimento relativo aos passos iniciais de interação B. cenocepacia-hospedeiro, elucidando em particular os papéis desempenhados por ATAs nesse processo. Os resultados permitiram uma caracterização funcional de duas ATAs de B. cenocepacia – BcaC e BCAM2418. Ambas demonstraram ser proteínas multifuncionais e importantes intervenientes durante a adesão bacteriana a células e componentes da matriz extracelular. A expressão desses ATAs genes provou estar sobre uma regulação estrita. bcaC demonstrou ser regulado negativamente por uma histidina cinase vizinha enquanto que a expressão de BCAM2418 aumentou durante os contactos inicias da bactéria com a célula hospedeira. A eliminação de O-glicanos tanto de mucinas como da superfície de células do epitélio bronquial provou diminuir a expressão do gene BCAM2418 e inviabilizar a adesão bacteriana. O uso de um anticorpo anti-BCAM2418 causou uma demarcada redução na adesão de B. cenocepacia a células do brônquio e mucinas e a inibição de virulência in vivo. Usando microarrays de glicanos, epítopos glicosilados do hospedeiro foram identificados como alvos de interação com BCAM2418, e espécies do género Burkholderia demonstraram interagir de diferentes formas com variadas classes de carboidratos associados a FC. Por fim, a modulação transcricional e adaptação de B. cenocepacia em consequência dos primeiros contactos com o hospedeiro foi analisada com recurso a vesículas gigantes de membrana plasmática (VGMPs) como alternativa celular. Sequenciação de RNA indicou que os primeiros contactos físicos entre bactéria e VGMPs causam uma mudança na transcrição de genes relacionados com o metabolismo central, sistemas de transporte e características de virulência, incluindo muitas associadas com o fenómeno de adesão. Em suma, o trabalho desenvolvido nesta tese suporta a importância das interações iniciais entre bactéria e a célula hospedeira como passo fundamental para a infeção de B. cenocepacia, e trouxe novos conhecimentos relativos à adaptação bacteriana quer ocorre nos estadios iniciais de infeção.
PUBLICATIONS
Papers
Pais P, Vagueiro S, Mil-Homens D, Pimenta AI, Viana R, Okamoto M, Chibana H, Fialho AM, Teixeira MC. A new regulator in the crossroads of oxidative stress resistance and virulence in Candida glabrata: the transcription factor CgTog1. Virulence. 2020 Dec;11(1):1522-1538. doi: 10.1080/21505594.2020
Pimenta AI, Mil-Homens D, Pinto SN, Fialho AM. Phenotypic characterization of trimeric autotransporter adhesin-defective bcaC mutant of Burkholderia cenocepacia: cross-talk towards the histidine kinase BCAM0218. Microbes Infect. 2020; S1286-4579(20)30096-4. doi:10.1016/j.micinf.2020.05.018
Pimenta AI, Mil-Homens D, Fialho AM. Burkholderia cenocepacia-host cell contact controls the transcription activity of the trimeric autotransporter adhesin BCAM2418 gene. MicrobiologyOpen. 2020;9(4):e998. doi:10.1002/mbo3.998
Oliveira H, Mendes A, Fraga AG, Ferreira A, Pimenta AI, Mil-Homens D, Fialho AM, Pedrosa J, Azeredo, J. K2 Capsule Depolymerase Is Highly Stable, Is Refractory to Resistance, and Protects Larvae and Mice from Acinetobacter baumannii Sepsis [published correction appears in Appl Environ Microbiol. 2020 Mar 18;86(7):]. Appl Environ Microbiol. 2019;85(17):e00934-19. doi:10.1128/AEM.00934-19
Pimenta AI, Kilcoyne M, Bernardes N, Mil-Homens D, Joshi L, Fialho AM. Burkholderia cenocepacia BCAM2418-induced antibody inhibits bacterial adhesion, confers protection to infection and enables identification of host glycans as adhesin targets. Cell Microbiol. 2021 Aug;23(8):e13340. doi: 10.1111/cmi.13340.
Pimenta AI, Bernardes N, Alves, MM, Mil-Homens D, Fialho AM. Burkholderia cenocepacia transcriptome analysis during the early contacts with bronchial epithelial cell-derived giant plasma-membrane vesicles. Sci Rep. 2021 Mar 11;11(1):5624. doi: 10.1038/s41598-021-85222-5.
Cavalheiro M, Pereira D, Leitão C, Pais P, Ndlovu E, Pimenta AI, Santos R, Takahashi-Nakaguchi A, Okamoto M, Ola M, Chibana H, Fialho AM, Butler G, Dague E, Teixeira MC, From the first touch to biofilm establishment by the human pathogen Candida glabrata: a genome-wide to nanoscale view. Commun Biol. 2021 Jul 20;4(1):886. doi: 10.1038/s42003-021-02412-7.
Oral Communications
Pimenta AI, Mil-Homens D, Fialho AM. “Defining the role of Burkholderia cenocepacia Trimeric Autotransporter Adhesin BCAM2418 in the early stages of bacteria-host- cell interactions”, MICROBIOTEC’17, December 2017, Porto, Portugal.
Pimenta, AI., Mil-Homens, D., Fialho, AM. “Differential Gene Expression of Burkholderia cenocepacia Trimeric Autotransporter Adhesin BCAM2418 in the Early Stages of Bacteria-Host-Cell Interactions”, 21st IBCWG, 2-5 May, 2018, Dublin, Ireland
Poster Communications
Pais P, Galocha M, Califórnia R, Pimenta AI, Mil-Homens D, Fialho AM, Butler G, Teixeira MC. Functional characterization of the transcription factor CgMrr1 suggests new players governing azole drug resistance in Candida glabrata. 14th ASM Conference on Candida and Candidiasis. April 2018, Providence, RI, USA.
Pedro NA, Lima C, Pimenta AI, Pinto SC, Alves M, Mira, NP. Study of the interaction between vaginal lactobacilli, Candida albicans and Candida glabrata: from physiological aspects to transcriptomic analyses. Advanced Lecture Course on Human Fingal Pathogens. May 2019, Nice, France.
Salazar SB, Silva S, Mil-Homens D, Pimenta AI, Chibana H, Azeredo J, Fialho AM, Mira NP. The Candida glabrata CgHaa1-dependent system is required for biofilm formation, adhesion to epithelial cells and maximal virulence against Galleria mellonella. Advanced Lecture Course on Human Fingal Pathogens. May 2019, Nice, France.
Pimenta AI, Mil-Homens D, Fialho AM. Differential Gene Expression of Burkholderia cenocepacia BCAM2418 Trimeric Autotransporter Adhesin in the initial steps of bacteria-host interaction. 8th Congress of European Microbiologists (FEMS). July 2019, Glasgow, Scotland.
Pimenta AI, Mil-Homens D, Fialho AM. A novel sensor histidine kinase controls Burkholderia cenocepacia trimeric autotransporter adhesin BCAM0219 expression – role in host cell adhesion, hemagglutination and biofilm formation. Gordon Conference on Microbial Adhesion and Signal Transduction. July 2019, Newport, RI, USA.
Congress Attendance Grant given by the Federation of European Microbiological Societies (FEMS), at 2019 8th Congress of European Microbiologists, Glasgow, Scotland.
DOCTORAL PROGRAM (36 ECTS)
• General Doctoral Training (6 ECTS)
• Advanced Experimental Techniques and methodologies (6 ECTS)
• Bioentrepreneurship (6 ECTS)
• Outreach and Teaching Skills (6 ECTS)
• Functional and Comparative Genomics (6 ECTS)
• Molecular and Cellular Microbiology (6 ECTS)
Current position/address
Researcher @Bacterial Energy Metabolism Lab, Instituto de Tecnologia Química e Biológica António Xavier (ITQB-NOVA), Nova University of Lisbon, Av. da República, 2780-157 Oeiras, Portugal