Class 2017
PhD Grant: PD/BD/135137/2017
PhD thesis
Unveiling the non-coding transcriptome and the role of ProQ on Burkholderia cenocepacia virulence
Supervisor
• Jorge Leitão, Professor @ IST/ULisboa, PT
Collaborators
• Joana Rita Rodrigues Feliciano
• Sílvia Andreia Bento da Silva Sousa Barbosa
THESIS ABSTRACT
Post-transcriptional regulation of gene expression in bacteria impacts several processes like energy metabolism, stress response, as well as processes related to bacterial virulence. Small non-coding RNAs (sRNAs) and RNA chaperones are major players in gene expression post-transcriptional regulation. Despite the identification of hundreds of bacterial sRNAs and a few RNA chaperones, their roles on bacteria physiology and virulence remain largely unknown in several pathogens. This is the case of Burkholderia cepacia complex (Bcc), a group of opportunistic pathogens with relatively large genomes and multiple resistances to antibiotics. On this thesis we describe the development of a methodology to identify sRNAs expressed by B. cenocepacia during infection ofCaenorhabditis elegans. 108 new and 31 previously described sRNAs with a predicted Rho independent terminator were identified. The RIT11b, a sRNA found downregulated under C. elegans infection conditions, was shown to be directly involved in B. cenocepacia virulence and to affect biofilm formation and motility. In addition, RIT11b overexpression reduced the expression of the direct targets dusA and pyrC, found to be upregulated on C. elegansinfection by B. cenocepacia, and involved in biofilm formation, epithelial cell adherence and chronic infections in other organisms. Furthermore, we functionally characterized BCAS0010 encoding a ProQ-like protein with a conserved ProQ/FinO central domain, and a fist-like predicted structure. As other ProQ-like chaperones, this protein influences the B. cenocepacia mobility, biofilm formation and virulence. Several mRNAs and sRNAs altered during C. elegansinfection were identified as potential targets of ProQ, which apparently prefers to bind to their structured GC-rich ends. Results on ProQ direct regulatory networks also shows its ability to regulate the expression of the flagellar-related protein FliC and three biofilm-related sRNAs. Altogether, these results contribute to better understand the role played by sRNAs and RNA chaperones on Bcc virulence and in their relation with the host.
RESUMO DA TESE
Os pequenos RNAs não codificantes (sRNAs) e chaperonas de RNA desempenham papéis preponderantes na regulação pós-transcricional da expressão génica em bactérias, afetando vários processos, como o metabolismo energético, resposta ao stress e virulência. Apesar da identificação de centenas de sRNAs e algumas chaperonas de RNA, os seus papéis na fisiologia e virulência das bactérias permanecem amplamente desconhecidos. É o caso do complexo Burkholderia cepacia (Bcc), um grupo de patogénios oportunistas com genomas relativamente grandes e resistência a múltiplos antibióticos. Neste trabalho foi desenvolvida uma metodologia para identificar sRNAs expressos por B. cenocepacia durante a infeção de Caenorhabditis elegans. Foram identificados 108 sRNAs novos e 31 previamente descritos. O sRNA RIT11b, subexpresso em condições de infeção, mostrou estar diretamente envolvido na virulência de B. cenocepacia e afetar a formação de biofilme e a motilidade. Além disso, a sobreexpressão de RIT11b reduziu a expressão dos alvos diretos dusA e pyrC, sobreexpressos por B. cenocepaciadurante a infeção de C. elegans e envolvidos na formação de biofilme, adesão a células epiteliais e infeções crónicas noutros organismos. Foi ainda caracterizado funcionalmente o gene BCAS0010, que codifica uma proteína do tipo ProQ, com um domínio central ProQ/FinO conservado. Tal como outras chaperonas do tipo ProQ, esta proteína influencia a motilidade, a formação de biofilme e a virulência de B. cenocepacia. Vários mRNAs e sRNAs diferencialmente expressos durante a infeção em C. elegans foram identificados como potenciais alvos de ProQ, que aparentemente prefere ligar às suas extremidades estruturadas e ricas em GC. Foi ainda demonstrada a capacidade de ProQ regular a expressão da proteína flagelar FliC e três sRNAs envolvidos na formação de biofilme. Estes resultados contribuem no seu conjunto para uma melhor compreensão do papel desempenhado pelos sRNAs e chaperonas de RNA na virulência das bactérias do Bcc e na sua relação com o hospedeiro.
PUBLICATIONS
Papers
Pita, T., Feliciano, J.R., Leitão, J.H., “Extracellular RNAs in Bacterial Infections: From Emerging Key Players on Host-Pathogen Interactions to Exploitable Biomarkers and Therapeutic Targets”. International Journal of Molecular Sciences, 21(24):9634, 2020:
Feliciano, J.R.; Seixas, A.M.M.; Pita, T.; Leitão, J.H.. “Comparative Genomics and Evolutionary Analysis of RNA-Binding Proteins of Burkholderia cenocepacia J2315 and Other Members of the B. cepacia Complex”. Genes, 11 2 (2020): 231. http://dx.doi.org/10.3390/genes11020231.
Feliciano, J.R. , Seixas, A.M.M., Pita, T., Leitão, J.H., “Comparative Genomics and Evolutionary Analysis of RNA-binding Proteins of Burkholderia cenocepacia J2315 and other members of the B. cepacia Complex”, Genes 11, 231, 2020
Pita, T., Feliciano, J.R., Leitão, J.H., “Small Non-coding Regulatory RNAs from Pseudomonas aeruginosa and Burkholderia cepacia complex”, International Journal of Molecular Sciences, 19(12): 3759, 2018.
Sousa, S.A., Feliciano, J.R. , PitaT., Guerreiro, S.I., Leitão, J.H., “Burkholderia cepacia complex virulence gene expression regulation: a review”, Genes 8, 43, 2017.
Sousa, S.A., Morad, M., Feliciano, J.R., Pita, T., Nady, S., El-Hennamy, R.E., Abdel- Rahman, M., Cavaco, J., Pereira, L., Barreto, C., Leitão, J.H., “The Burkholderia cenocepacia OmpA-like protein BCAL2958: Identification, characterization, and detection of anti-BCAL2958 antibodies in serum from B. cepacia complex-infected Cystic Fibrosis patients”, AMB Express 6:41, 2016.
Oral Communications
Pita, T., Feliciano, J.R., Leitão, J.H., “Characterization of small noncoding RNAs expressed by Burkholderia cenocepacia when infecting Caenorhabditis elegans”, Microbiotec21 – Webconference, 2021, Nova University Lisbon (Lisbon,Portugal)
Feliciano, J.R., Pita, T., Sousa, S.A., Leitão, J.H., “The RNA chaperones Hfq and Hfq2 of Burkholderia cepacia complex: Functional analysis and biological roles”, 21st International Burkholderia cepacia Working Group meeting, 2-5 May 2019, Dublin, Ireland
Poster Communications
Pita, T., Feliciano, J.R., Sousa, S.A., Leitão, J.H., “Characterization of small non coding RNAs differentially expressed by Burkholderia cenocepacia when infecting Caenorhabditis elegans”, MicroBiotec2019 December 5-7, 2019, Coimbra, Portugal
Feliciano, J.R., Michaux, C., Bischler,T., Pita, T., Barquist, L., Sousa, S.A., Seixas, A.A., Vogel, J., Leitão, J.H., The distinct in vivo RNA global profiles of the Hfq, Hfq2 and ProQ from Burkholderia cenocepacia K56-2”, 8th Congress of European Microbiologists (FEMS), 2019, Glasgow, Scotland
Pita T, Feliciano JR, Balugas B, Sousa, SA, Leitão JH, “Towards the identification of the virulence-associated sRNAs from the Burkholderia cepacia complex”, MicroBiotec2017 December 7-8, 2017, Porto, Portugal
DOCTORAL PROGRAM (36 ECTS)
Curricular units:
• 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)