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Vasco Ariston de Carvalho Azevedohttp://lattes.cnpq.br/1020477751003832Anne Cybelle Pinto Gomidehttp://lattes.cnpq.br/4175954849617381Izabela Coimbra Ibraim2021-03-19T10:03:50Z2021-03-19T10:03:50Z2018-11-14http://hdl.handle.net/1843/35277O gene ciuA codifica uma proteína de superfície associada a um sistema de captação de ferro dependente de sideróforos, porém seu papel na aquisição de ferro nessa bactéria ainda é pouco conhecido. O ferro é crucial no crescimento e desenvolvimento de bactérias, porém no hospedeiro as frações livres disponíveis de ferro são inferiores às necessárias para o crescimento bacteriano. Em bactérias intracelulares como C. pseudotuberculosis, a homeostase de ferro no interior do hospedeiro é controlada transcricionalmente pela concentração de ferro no meio e envolve a regulação de genes voltados para a subversão dos sistemas de restrição nutricional imposto pelo hospedeiro. Trabalhos anteriores realizados por nosso grupo, levaram a construção de uma linhagem mutante (Cp13) para o gene ciuA, através de mutagênese aleatória utilizando o transposon TnFuZ na linhagem T1 selvagem de Corynebacterium pseudotuberculosis. A interrupção deste gene, contribuiu para a redução da virulência e viabilidade intracelular da linhagem, além de induzir níveis significativos de anticorpos e alta taxa de sobrevivência em camundongos imunizados. Diante do exposto, este trabalho teve como objetivo principal avaliar o crescimento e a resposta transcricional de C. pseudotuberculosis em resposta à restrição de ferro, bem como a caracterização funcional de genes associados a virulência, entre linhagem T1 e mutante Cp13. Assim, a análise de expressão diferencial de ambas as linhagens, foi realizada a partir de culturas incubadas em meio com baixa disponibilidade de ferro (low iron – LI), obtido pelo agente quelante 2,2-bipiridina (BIP), e meio com alta disponibilidade de ferro (high iron – HI). Neste contexto, os dados aqui apresentados demonstraram uma redução significativa na densidade celular e na taxa de crescimento da linhagem mutante em comparação a linhagem parental em culturas LI. No total, 120 e 86 genes diferencialmente expressos (GDEs) foram identificados nas cepas T1 e Cp13 em resposta à restrição de ferro, respectivamente. Dentre esses genes, 36 GDEs foram compartilhados entre as duas linhagens, sendo que 26 apresentaram um perfil de expressão semelhante entre as linhagens. Dois fatores transcricionais ripA e hrrA, regulados por DtxR, foram identificados entre os GDEs compartilhados, demonstrando uma complexa rede regulatória controlada pela disponibilidade de ferro. Sete dos 36 GDEs codificam proteínas de superfície e transportadores de hemina, sendo quatro genes localizados em ilhas genômicas associados a fatores de virulência. Nossos resultados demonstraram que a restrição intracelular da utilização de ferro é fundamental para a resposta de C. pseudotuberculosis à disponibilidade do metal. Alterações significativas também foram observadas em relação aos GDEs identificados exclusivamente entre as linhagens, indicando divergência entre a resposta adaptativa da linhagem mutante em relação a linhagem parental. Em conjunto, os resultados do presente trabalho fornecem uma análise abrangente da rede regulatória e da resposta transcricional da linhagem T1 selvagem e Cp13 mutante de C. pseudotuberculosis, acrescentando informações importantes envolvendo o processo de adaptação desse importante patógeno à restrição de ferro.The ciuA gene codes for a surface protein which is associated to a siderophore-base iron acquisition system; however, its role in the acquisition of iron in C. pseudotuberculosis is still largely unknown. Iron is crucial for the growth and development of many bacterial pathogens; however, within the host, the concentration that is freely accessible is much lower than the concentration required for bacterial growth. In intracellular bacteria, like C. pseudotuberculosis, iron homeostasis is transcriptionally controlled by the availability of iron in the environment, which allows the bacteria to counteract the limitation of the metal imposed by the host. In a previous work conducted by our group, we described the construction of a ciuA gene mutant strain (Cp13), generated by using the in vivo insertional mutagenesis of the reporter transposon-based system TnFuZ in the T1 strain of C. pseudotuberculosis. The disruption of the ciuA gene led to a decrease in virulence and intracellular viability of the mutant strain. In addition, immunization of mice with the Cp13 strain elicited significantly IgG titers and higher survival rate (80%). The ciuA gene codes for a surface protein which is associated to a siderophore-base iron acquisition system; however, its role in the acquisition of iron in C. pseudotuberculosis is still largely unknown. Iron is crucial for the growth and development of many bacterial pathogens; however, within the host, the concentration that is freely accessible is much lower than the concentration required for bacterial growth. In intracellular bacteria, like C. pseudotuberculosis, iron homeostasis is transcriptionally controlled by the availability of iron in the environment, which allows the bacteria to counteract the limitation of the metal imposed by the host. Herein, our primary goal was to comprehensively characterize the transcriptional response of C. pseudotuberculosis under iron restriction. Differential gene expression analyses of the wild-type strain (T1 strain) and the (ciuA) Cp13 mutant strain (Cp13 strain) were conducted in low iron (LI) cultures, supplemented with the iron-chelator 2,2’-dipyridyl-DIP, in relation to high iron (HI) cultures in both strains. In this context, the data presented here shows a significant reduction in cellular density and the growth rate of the Cp13 mutant strain in relation to the parental strain under iron restriction. In total, 120 and 86 differentially expressed genes (DEGs) were identified in the T1 and Cp13 strains in response to iron restriction, respectively. 36 of these genes were identified in both strains and 26 of these genes had a similar expression pattern in both strains. Two transcription factors, known to be regulated by DtxR, were also identified between the overlapped DEGs of both strains, indicating a complex regulatory network controlled by the availability of iron in C. pseudotuberculosis. Out of the 26 genes, 7 DEGs encode high-affinity hemin-binding proteins. Interestingly, 4 hemin-binding genes were identified located within genomic islands harboring known virulence factors, corroborating with the association of iron acquisition and virulence in pathogenic bacteria. We have also observed that the intracellular restriction of iron usage is key to the iron starvation response of C. pseudotuberculosis. Significant differences between mutant and parental strains were also observed in relation to the strain-specific DEGs, indicating divergence in the adaptive responses of the mutant and parental strains in response to iron restriction. Altogether, these findings provide a comprehensive analysis of the regulatory network and transcriptional response of the Cp13 mutant and T1 parental strain of C. pseudotuberculosis, adding relevant insights into the transcriptional adaptation of this important pathogen within an iron-restricted environment.CNPq - Conselho Nacional de Desenvolvimento Científico e TecnológicoporUniversidade Federal de Minas GeraisPrograma de Pós-Graduação em BioinformaticaUFMGBrasilICB - INSTITUTO DE CIÊNCIAS BIOLOGICAShttp://creativecommons.org/licenses/by-nc-nd/3.0/pt/info:eu-repo/semantics/openAccessCorynebacterium pseudotuberculosisFerroHomeostasePerfilação da expressão gênicaCorynebacterium pseudotuberculosisHomeostase de ferroFatores transcricionais regulados por ferroExpressão gênica diferencialAnálise do transcriptoma de Corynebacterium pseudotuberculosis em resposta à limitação por ferroinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisreponame:Repositório Institucional da UFMGinstname:Universidade Federal de Minas Gerais (UFMG)instacron:UFMGORIGINALTese_Izabela Ibraim_revisada.pdfTese_Izabela Ibraim_revisada.pdfapplication/pdf5652391https://repositorio.ufmg.br/bitstream/1843/35277/1/Tese_Izabela%20Ibraim_revisada.pdf60245a11bc789094ff8f46b96e4e3eebMD51CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8811https://repositorio.ufmg.br/bitstream/1843/35277/2/license_rdfcfd6801dba008cb6adbd9838b81582abMD52LICENSElicense.txtlicense.txttext/plain; charset=utf-82119https://repositorio.ufmg.br/bitstream/1843/35277/3/license.txt34badce4be7e31e3adb4575ae96af679MD531843/352772021-03-19 07:03:50.519oai:repositorio.ufmg.br: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Repositório InstitucionalPUBhttps://repositorio.ufmg.br/oaiopendoar:2021-03-19T10:03:50Repositório Institucional da UFMG - Universidade Federal de Minas Gerais (UFMG)false
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