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Master's Dissertation
DOI
https://doi.org/10.11606/D.17.2020.tde-04012021-111327
Document
Author
Full name
Fernanda Carvalho Leal
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2020
Supervisor
Committee
Silva Neto, José Freire da (President)
Carvalho, Cristiane Rodrigues Guzzo
Pupo, Monica Tallarico
Title in Portuguese
Papel de proteínas VgrG parálogas no sistema de secreção do tipo VI de Chromobacterium violaceum
Keywords in Portuguese
Chromobacterium violaceum
Competição bacteriana
Proteínas efetoras
Proteínas VgrGs parálogas
Sistema de secreção do tipo VI
Sistemas de secreção bacterianos
Abstract in Portuguese
O sistema de secreção do tipo VI (T6SS) injeta efetores tóxicos dentro das células alvo expelindo um aparato de punção composto por proteínas Hcp, VgrG e PAAR. Ainda não se sabe exatamente como as proteínas efetoras se ligam à VgrG ou o motivo da existência de várias cópias de VgrG em algumas bactérias. Recentemente, nosso grupo mostrou que a bactéria Gram-negativa Chromobacterium violaceum possui um T6SS funcional com atividade antibacteriana. O objetivo do presente trabalho é investigar os genes vgrGs do T6SS de C. violaceum. As análises in silico revelaram que C. violaceum possui seis genes vgrGs (vgrG1 a vgrG6), com os genes vgrG1 e vgrG2 localizados no cluster principal do T6SS e os demais quatro genes vgrGs localizados em clusters órfãos. As seis proteínas VgrGs apresentaram alta identidade de sequência (de 70% a 93%) e a mesma organização de domínios, com todas possuindo um potencial domínio adaptador no C-terminal. Linhagens mutantes foram obtidas com deleção individual de cada gene vgrG ou deleção sequencial até obter uma linhagem mutante sêxtupla (ΔvgrG1-6). Cada gene vgrG foi introduzido no mutante sêxtuplo. Os ensaios de competição contra bactérias como Pseudomonas aeruginosa utilizando todas estas linhagens mutantes revelaram que a VgrG3 é a mais importante para a atividade antibacteriana do T6SS, pois apenas o mutante individual ΔvgrG3 teve forte redução na capacidade de matar a P. aeruginosa; a partir do mutante triplo ΔvgrG1-3 houve perda total da competição mediada pelo T6SS; e apenas vgrG3 resgatou a capacidade de competição do mutante sêxtuplo. Ensaios de Western blot foram utilizados para avaliar a secreção de Hcp nos mutantes vgrGs de C. violaceum como um indicativo do funcionamento do T6SS. Os dados indicaram que, dos seis mutantes individuais, apenas ΔvgrG1 e ΔvgrG3 tiveram uma diminuição da secreção de Hcp. A partir da deleção tripla ΔvgrG1-3 não houve mais secreção de Hcp. A ausência de secreção no mutante sêxtuplo foi totalmente revertida na presença de vgrG3 e fracamente recuperada com os outros genes vgrGs, indicando maior relevância de VgrG3. Ensaios de β-galactosidase revelaram baixa atividade promotora de vgrG2 e vgrG6 e uma expressão dependente da fase de crescimento nos promotores de vgrG3 e vgrG4. O vgrG3 foi o gene mais expresso em qualquer condição. Portanto, os dados obtidos neste trabalho indicam pouca redundância entre as seis VgrGs de C. violaceum, uma vez que a VgrG3 possui papel preponderante em relação às outras cinco VgrGs na montagem de um T6SS funcional com atividade antibacteriana.
Title in English
Role of paralogous VgrG proteins in the Chromobacterium violaceum type VI secretion system
Keywords in English
Bacterial competition
Bacterial secretion systems
Chromobacterium violaceum
Effector proteins
Type VI secretion system
VgrG paralogs
Abstract in English
The type VI secretion system (T6SS) deliveries toxic effectors inside target cells by expelling a puncturing apparatus composed of Hcp, VgrG, and PAAR proteins. It is not yet known precisely how the effector proteins bind to VgrG or the reason for the existence of multiple copies of VgrG in some bacteria. Recently, our group has shown that the Gram-negative bacterium Chromobacterium violaceum has a functional T6SS with antibacterial activity. In this work, we aim to investigate the vgrG genes of the C. violaceum T6SS. In silico analysis revealed that C. violaceum has six vgrG genes (vgrG1 to vgrG6), with the vgrG1 and vgrG2 genes located in the main T6SS cluster and the other four vgrG genes found in orphan clusters. The six VgrG proteins showed high sequence identity (from 70% to 93%) and the same organization of domains, with all of them having a potential adapter domain at the C-terminal. Mutant strains were obtained with individual deletion of each vgrG gene or sequential deletion until a sextuple mutant strain was obtained (ΔvgrG1-6). Each vgrG gene was introduced into the sextuple mutant. Competition tests against bacteria such as Pseudomonas aeruginosa, using all these mutant strains, revealed that VgrG3 is the most important for the T6SS antibacterial activity, since only the individual mutant ΔvgrG3 had a strong reduction in the ability to kill P. aeruginosa; from the triple mutant ΔvgrG1-3, there was total loss of competition mediated by T6SS; and only the vgrG3 rescued the competition ability of the sextuple mutant. Western blot assays were used to evaluate Hcp secretion in the C. violaceum vgrG mutants as an indicator of T6SS activity. The data indicated that, of the six individual mutants, only ΔvgrG1 and ΔvgrG3 had a decrease in Hcp secretion. From the ΔvgrG1-3 triple deletion, there was no more Hcp secretion. The absence of secretion in the sextuple mutant was completely reversed in the presence of vgrG3 and poorly recovered with the other vgrGs genes, indicating greater relevance of VgrG3. β-galactosidase assays revealed low promoter activity of vgrG2 and vgrG6 and an expression dependent on the growth phase in the promoters of vgrG3 and vgrG4. vgrG3 was the most expressed gene in any condition. Therefore, the data obtained in this work indicate little redundancy among the six VgrGs of C. violaceum, since VgrG3 has a preponderant role in relation to the other five VgrGs in the assembly of a functional T6SS with antibacterial activity.
 
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Publishing Date
2021-01-27
 
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