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Previous issue date: 2017-03-30 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Funda??o de Amparo ? Pesquisa do Estado do Rio Grande do Sul (FAPERGS) / Tuberculosis (TB) has become the leading global cause of death from infectious
diseases. In 2015, according to WHO, 10.4 million new cases of tuberculosis worldwide have
emerged. Currently the commonly used treatments are not effective against the forms of disease
resistant to the most effective anti-TB drugs, and drugs with new mechanisms of action are
needed. Mycobacterium tuberculosis dihydroneopterin aldolase (MtDHNA /FolB) is a folate
enzyme encoded by the folB gene, which has important properties that make it a potential target
for the synthesis of new antimicrobial agents. As a first step for target validation in the
antimicrobial drug development pipeline, it is important to prove that the gene encoding a
putative target is essential for pathogen?s viability. In this study, using site directed
mutagenesis, biochemical analyzes and gene knockout experiments, we demonstrated that the
folB gene is essential for the survival of Mtb, and furthermore we prove that this essentiality
depends on the aldolase/epimerase activities of the MtFolB protein. The wild-type gene (wt)
and the point mutants K99A and Y54F were cloned and expressed, and the corresponding
recombinant proteins were purified and monitored for the activities of aldolase, epimerase and
oxygenase using HPLC. In contrast to the wild-type MtFolB (wt) enzyme, both mutants had
neither aldolase nor epimerase activities under the conditions tested. The Y54F mutant
maintained oxygenase activity, whereas for the K99A mutant it was possible to detect
oxygenase activity only in the presence of HP and GA as substrates. Knockout experiments
showed that the folB gene is essential for the survival of Mtb under the conditions tested.
However, unlike the wild-type copy, when the sequences encoding the K99A or Y54F mutants
were used for complementation, no viable colonies were obtained, indicating that these point
mutants could not rescue the cells after the folB knockout. These results indicate that aldolase
and/or epimerase activities are crucial for the survival of Mtb. The construction of
Mycobacterium tuberculosis folB-GFP fusion (Mtb) strains containing wild-type folB gene
sequence or a deleted C-terminal mutant (folB?C), devoid of the sequence presumably
necessary for anchoring the enzyme within nanocage compartments, were performed and
together with other cell biology methods described in this work will be used for a better
understanding of MtDHNA/FolB cellular functions and for the validation of this enzyme as a
therapeutic target. / A tuberculose (TB) tornou-se a principal causa mundial de morte por doen?as infecciosas. Em
2015, de acordo com a OMS, surgiram 10,4 milh?es de novos casos de tuberculose no mundo.
Atualmente os tratamentos comumente utilizados n?o s?o eficientes contra as formas da doen?a
resistentes aos f?rmacos anti-TB mais eficazes, sendo necess?rios f?rmacos com novos
mecanismos de a??o. A di-hidroneopterina aldolase de Mycobacterium tuberculosis
(MtDHNA/FolB) ? uma enzima da via do folato, codificada pelo gene folB, que apresenta
caracter?sticas importantes que a tornam um potencial alvo para s?ntese de novos agentes
antimicrobianos. Neste estudo, por meio de mutag?nese s?tio-direcionada, an?lises bioqu?micas
e experimentos de nocaute g?nico, demostramos que o gene folB ? essencial para a
sobreviv?ncia de Mtb, e al?m disso provamos que essa essencialidade depende das atividades
de aldolase/epimerase da prote?na MtFolB. O gene do tipo selvagem (wt) e os mutantes pontuais
K99A e Y54F foram clonados e expressos, e as prote?nas recombinantes correspondentes foram
purificadas e monitoradas para as atividades de aldolase, epimerase e oxigenase utilizando
HPLC. Em contraste com a enzima MtFolB selvagem (wt), ambas as mutantes n?o
apresentaram atividade de aldolase nem de epimerase nas condi??es testadas. A mutante Y54F
manteve a atividade da oxigenase, enquanto que para a mutante K99A foi poss?vel detectar a
atividade de oxigenase apenas na presen?a de HP e GA como substratos. Os experimentos de
nocaute mostraram que o gene folB ? essencial para a sobreviv?ncia de Mtb sob as condi??es
testadas. Entretanto, diferentemente da c?pia selvagem, quando as sequ?ncias que codificam os
mutantes K99A ou Y54F foram utilizadas para complementa??o, n?o foram obtidas col?nias
vi?veis, indicando que estes mutantes pontuais n?o poderiam resgatar as c?lulas ap?s o nocaute
do gene folB. Esses resultados indicam que as atividades de aldolase e/ou epimerase s?o cruciais
para a sobreviv?ncia de Mtb. A constru??o de cepas com fus?o folB-GFP de Mycobacterium
tuberculosis (Mtb) que cont?m a sequ?ncia do tipo selvagem do gene folB ou um mutante com
o C-terminal deletado (folB?C), desprovida da sequ?ncia supostamente necess?ria para a
ancoragem da enzima dentro dos compartimentos de nanocargas, foram realizadas e juntamente
com outros m?todos de biologia celular descritos neste trabalho tamb?m poder?o ser utilizados
para um melhor entendimento das fun??es celulares apresentadas por MtDHNA/FolB e para
valida??o dessa enzima como potencial alvo terap?utico.
| Identifer | oai:union.ndltd.org:IBICT/oai:tede2.pucrs.br:tede/7420 |
| Date | 30 March 2017 |
| Creators | Falc?o, Virg?nia Carla de Almeida |
| Contributors | Bizarro, Cristiano Valim |
| Publisher | Pontif?cia Universidade Cat?lica do Rio Grande do Sul, Programa de P?s-Gradua??o em Biologia Celular e Molecular, PUCRS, Brasil, Faculdade de Bioci?ncias |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da PUC_RS, instname:Pontifícia Universidade Católica do Rio Grande do Sul, instacron:PUC_RS |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 8198246930096637360, 600, 600, 600, 600, 600, 36528317262667714, -1634559385931244697, 2075167498588264571, -4379409248623720768 |
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