Análise funcional de genes de "Candidatus" Liberibacter asiaticus, bactéria associada ao huanglongbing dos citros
Carregando...
Data
2018-08-30
Autores
Dutra, Michele Fernanda Souza
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
O Huanglongbing (HLB) é a doença mais devastadora dos citros. No Brasil, o HLB está associado a presença de "Candidatus" Liberibacter asiaticus (Las) e Ca. L. americanus (Lam), ambas transmitidas pelo psilídeo Diaphorina citri. O sequenciamento e anotação dos profagos SC1 e SC2 no genoma de Las, identificou uma possível bacteriocina do tipo colicina Ia em SC1, e uma possível proteína de imunidade a colicina em SC2. Lam apresenta profagos similares no seu genoma, porém SP2 é degenerado, sem proteína de imunidade. Como houve uma inversão notável na ocorrência de Las em relação à Lam no Brasil, com o predomínio atual de Las, a presença de ambos os profagos pode conferir uma vantagem adaptativa a Las pela atividade da bacteriocina. Adicionalmente, Las tem lisozimas cromossômicas e de profago, que também podem estar envolvidas em competição bacteriana. A incapacidade de cultivo axênico de Las e Lam requer o uso de sistemas heterólogos para estudos funcionais. Genes com potencial envolvimento na biologia do profago e na competição bacteriana, como as colicinas e lisozimas foram avaliados. As lisozimas CLIBASIA_04790 e ED07_RS0204515, obtidas dos isolados de Las 3PA e Las 4PA, e as colicinas SC1_gp060 e lam_678 obtidas de isolados de Las 2PA, Las Poona e Lam São Paulo foram clonadas em vetor de expressão pET28a e superexpressas em E. coli BL21 (DE3). O crescimento destas bactérias, mensurado pelas médias dos valores de absorbância (OD600), mostraram efeito tóxico das construções com lisozimas, sendo que clones de E. coli BL21 (DE3) expressando CLIBASIA_04790 (pMD15) e ED07_RS0204515 (pMD17) cresceram cerca de 60 e 70% menos, respectivamente, quando comparados aos controles. Ensaios de SDS-PAGE e Western Blot com anticorpo anti-His6X, evidenciaram a presença de ambas as proteínas na fração insolúvel, de 20 kDa (pMD15) e 12 kDa (pMD17), confirmando a expressão das lisozimas. Para as colicinas, somente o clone expressando SC1_gp060 proveniente do isolado Poona (pMD16) mostrou efeito em E. coli BL21 (DE3), com redução de 70% no crescimento quando comparado aos controles. Estes resultados auxiliam a desvendar a relação entre genes com potencial vantagem adaptativa e seu envolvimento na biologia das Liberibactérias e podem justificar seu uso biotecnológico em plantas geneticamente modificadas contra o HLB.
Huanglongbing (HLB) is the most devastating disease of citrus. In Brazil, HLB is associated with the presence of "Candidatus" Liberibacter asiaticus (Las) and Ca. L. americanus (Lam), both transmitted by psyllid Diaphorina citri. Sequencing and annotation of prophages SC1 and SC2 in the genome of Las, reveals the presence of a putative bacteriocin, colicin Ia in SC1, while in SC2 a possible colicin immunity-protein was found. Lam has similar prophages in the genome, though SP2 is degenerated, without immunity gene. As there was a striking inversion in the prevalence of Las in detriment of Lam, the presence of both prophages may confer an adaptative advantage to Las, due to the activity of the bacteriocin. Additionally, Las encodes both a chromosomal and phage lysozyme that may also be involved in bacterial competition. The inability of axenic cultivation of Las and Lam requires the use of heterologous systems for functional studies. Genes with potential involvement in prophage biology and bacterial competition, such as colicins and lysozymes, were evaluated. The lysozymes ORFs CLIBASIA_04790 and ED07_RS0204515, from Las isolates 3PA and Las 4PA, and the colicins SC1_gp060 and lam_678 obtained from isolates Las 2PA, Las Poona and Lam São Paulo were cloned into expression vector pET28a and superexpressed in E. coli BL21 (DE3) cells. The growth rate of these bacteria, measured by means of absorbance values (OD600), showed toxic effect of transformants expressing lysozymes, whereas E. coli BL21 (DE3) clones expressing CLIBASIA_04790 (pMD15) and ED07_RS0204515 (pMD17) grew about 60 and 70% when compared to controls. SDS-PAGE and Western Blot assays were performed with anti-His6X antibody, showing the presence of both proteins in the insoluble fraction, with 20 kDa (pMD15) and 12 kDa (pMD17), confirming lysozyme expression. In the case of the colicin, only the clone expressing SC1_gp060 from Las Poona strain (pMD16) showed toxic effect, showing reduction of 70% in growth rates when compared to the controls. These results help to unravel the relationship between genes with potential adaptive advantage and their involvement in the biology of Liberibacteria and may justify their biotechnological use in genetically modified plants against HLB.
Huanglongbing (HLB) is the most devastating disease of citrus. In Brazil, HLB is associated with the presence of "Candidatus" Liberibacter asiaticus (Las) and Ca. L. americanus (Lam), both transmitted by psyllid Diaphorina citri. Sequencing and annotation of prophages SC1 and SC2 in the genome of Las, reveals the presence of a putative bacteriocin, colicin Ia in SC1, while in SC2 a possible colicin immunity-protein was found. Lam has similar prophages in the genome, though SP2 is degenerated, without immunity gene. As there was a striking inversion in the prevalence of Las in detriment of Lam, the presence of both prophages may confer an adaptative advantage to Las, due to the activity of the bacteriocin. Additionally, Las encodes both a chromosomal and phage lysozyme that may also be involved in bacterial competition. The inability of axenic cultivation of Las and Lam requires the use of heterologous systems for functional studies. Genes with potential involvement in prophage biology and bacterial competition, such as colicins and lysozymes, were evaluated. The lysozymes ORFs CLIBASIA_04790 and ED07_RS0204515, from Las isolates 3PA and Las 4PA, and the colicins SC1_gp060 and lam_678 obtained from isolates Las 2PA, Las Poona and Lam São Paulo were cloned into expression vector pET28a and superexpressed in E. coli BL21 (DE3) cells. The growth rate of these bacteria, measured by means of absorbance values (OD600), showed toxic effect of transformants expressing lysozymes, whereas E. coli BL21 (DE3) clones expressing CLIBASIA_04790 (pMD15) and ED07_RS0204515 (pMD17) grew about 60 and 70% when compared to controls. SDS-PAGE and Western Blot assays were performed with anti-His6X antibody, showing the presence of both proteins in the insoluble fraction, with 20 kDa (pMD15) and 12 kDa (pMD17), confirming lysozyme expression. In the case of the colicin, only the clone expressing SC1_gp060 from Las Poona strain (pMD16) showed toxic effect, showing reduction of 70% in growth rates when compared to the controls. These results help to unravel the relationship between genes with potential adaptive advantage and their involvement in the biology of Liberibacteria and may justify their biotechnological use in genetically modified plants against HLB.
Descrição
Palavras-chave
"Candidatus" Liberibacter spp., Competição bacteriana, Colicinas, Lisozimas, Expressão heteróloga, Profagos