Construction of <i>Mycobacterium tuberculosis cdd</i> knockout and evaluation of invasion and growth in macrophages

Cytidine deaminase (MtCDA), encoded by cdd gene (Rv3315c), is the only enzyme identified in nucleotide biosynthesis pathway of Mycobacterium tuberculosis that is able to recycle cytidine and deoxycytidine. An M. tuberculosis knockout strain for cdd gene was obtained by allelic replacement. Evaluation of mRNA expression validated cdd deletion and showed the absence of polar effect. MudPIT LC-MS/MS data indicated thymidine phosphorylase expression was decreased in knockout and complemented strains. The cdd disruption does not affect M. tuberculosis growth both in Mid- dlebrook 7H9 and in RAW 264.7 cells, which indicates that cdd is not important for macrophage invasion and virulence.

Key words:
Mycobacterium tuberculosis; cdd gene; cytidine deaminase

Authorship

Anne Drumond Villela ⁺+ Corresponding author: anne.villela@pucrs.br

Pontifícia Universidade Católica do Rio Grande do Sul, Centro de Pesquisas em Biologia Molecular e Funcional, Instituto Nacional de Ciência e Tecnologia em Tuberculose, Porto Alegre, RS, BrasilPontifícia Universidade Católica do Rio Grande do SulBrasilPorto Alegre, RS, BrasilPontifícia Universidade Católica do Rio Grande do Sul, Centro de Pesquisas em Biologia Molecular e Funcional, Instituto Nacional de Ciência e Tecnologia em Tuberculose, Porto Alegre, RS, Brasil

Valnês S Rodrigues-Junior

Antônio Frederico Michel Pinto

Virgínia Carla de Almeida Falcão

Pontifícia Universidade Católica do Rio Grande do Sul, Programa de Pós-Graduação em Biologia Celular e Molecular, Porto Alegre, RS, BrasilPontifícia Universidade Católica do Rio Grande do SulBrasilPorto Alegre, RS, BrasilPontifícia Universidade Católica do Rio Grande do Sul, Programa de Pós-Graduação em Biologia Celular e Molecular, Porto Alegre, RS, Brasil

Zilpa Adriana Sánchez-Quitian

Paula Eichler

Cristiano Valim Bizarro

Luiz Augusto Basso

Pontifícia Universidade Católica do Rio Grande do Sul, Programa de Pós-Graduação em Medicina e Ciências da Saúde, Porto Alegre, RS, BrasilPontifícia Universidade Católica do Rio Grande do SulBrasilPorto Alegre, RS, BrasilPontifícia Universidade Católica do Rio Grande do Sul, Programa de Pós-Graduação em Medicina e Ciências da Saúde, Porto Alegre, RS, Brasil

Diógenes Santiago Santos

+ Corresponding author: anne.villela@pucrs.br

AUTHORS’ CONTRIBUTION

ADV constructed and validated the Mycobacterium tuberculosis knockout and complemented strains, performed the growth curve, and wrote the manuscript; ADV and VSRJ performed macrophage infection experiments; AFMP, PE and CVB carried out MudPIT LC-MS/MS experiments and performed data analysis; VCAF participated in all biosafety level 3 experiments; ZASQ constructed the plasmids; LAB contributed in the analysis of results and manuscript revision; and DSS conceived the study and participated in its design and coordination.

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Figures

Figures (3)

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Fig. 1
genomic environment of cdd gene in Mycobacterium tuberculosis (A), regions cloned into pPR27xylE vector (B), agarose gel electrophoresis of polymerase chain reaction (PCR) products from knockout clones (C), and mRNA expression of cdd, deoA, and add genes (D). (A) Genomic region of cdd gene (402 bp) containing unique internal NotI site and flanking genes; the operon cdd-add is indicated. (B) The cdd gene and flanking regions were amplified by PCR from M. tuberculosis H37Rv genomic DNA, and the cdd gene was disrupted by the insertion of a kanamycin cassette (kan^R) into NotI site (cdd::kan^R). The cdd::kan^R fragment was cloned into pPR27xylE vector using XbaI restriction site. Annealing regions of gene-specific screening primers forward (Primer F) and reverse (Primer R) for the possible knockout strains of cdd gene are indicated. (C) Agarose gel electrophoresis of PCR products from possible knockout clones. M - molecular marker 1 kb plus DNA Ladder (Invitrogen), PCRs were carried out with: Lane 1 -M. tuberculosis H37Rv genomic DNA, Lanes 2-10 - possible knockout clones genomic DNA. (D) mRNA expression of cdd, deoA, and add genes. M - molecular marker 1 kb plus DNA Ladder (Invitrogen). Lanes 1-6: cdd cDNA amplification (686 bp). Lanes 7-12: deoA cDNA amplification (1,284 bp). Lanes 13-18: add cDNA amplification (1,098 bp). cDNA synthesis from M. tuberculosis H37Rv (wild-type) was performed with reverse transcriptase enzyme (RT+) (lanes 1, 7, 13) or without (RT-) (lanes 2, 8, 14). cDNA synthesis from cdd knockout strain (KO) RT+ (lanes 3, 9, 15) and RT- (lanes 4, 10, 16). cDNA synthesis from complemented strain (CP) RT+ (lanes 5, 11, 17) and RT- (lanes 6, 12, 18).

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Fig. 2
evaluation of MtCDA, MtTP and MtAD expression. MtCDA, MtTP and MtAD normalised spectral abundance factor (NSAF) in wild-type (WT), knockout strain (KO) and complemented strain (CP) cytoplasmic fractions identified by Multidimentional Protein Identification Technology (MudPIT). Bars represent average identification in technical triplicates. Asterisks represent significant differences between WT and KO or CP strains of each protein by one-way ANOVA analysis followed by Bonferroni post-test, *p < 0.05, ***p < 0.001.

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Fig. 3
growth curve (A) and macrophage infection (B) with wild-type H37Rv (WT), knockout for cdd gene (KO), and complemented (CP) Mycobacterium tuberculosis strains. (A) Growth curves were started at an OD₆₀₀ of 0.01 in Middlebrook 7H9 medium, in triplicate. (B) The macrophage infection results are expressed as mean numbers of the logarithms of colony-forming unit (CFU) per well of each strain of three independent measurements.

Fig. 1 genomic environment of cdd gene in Mycobacterium tuberculosis (A), regions cloned into pPR27xylE vector (B), agarose gel electrophoresis of polymerase chain reaction (PCR) products from knockout clones (C), and mRNA expression of cdd, deoA, and add genes (D). (A) Genomic region of cdd gene (402 bp) containing unique internal NotI site and flanking genes; the operon cdd-add is indicated. (B) The cdd gene and flanking regions were amplified by PCR from M. tuberculosis H37Rv genomic DNA, and the cdd gene was disrupted by the insertion of a kanamycin cassette (kan^R) into NotI site (cdd::kan^R). The cdd::kan^R fragment was cloned into pPR27xylE vector using XbaI restriction site. Annealing regions of gene-specific screening primers forward (Primer F) and reverse (Primer R) for the possible knockout strains of cdd gene are indicated. (C) Agarose gel electrophoresis of PCR products from possible knockout clones. M - molecular marker 1 kb plus DNA Ladder (Invitrogen), PCRs were carried out with: Lane 1 -M. tuberculosis H37Rv genomic DNA, Lanes 2-10 - possible knockout clones genomic DNA. (D) mRNA expression of cdd, deoA, and add genes. M - molecular marker 1 kb plus DNA Ladder (Invitrogen). Lanes 1-6: cdd cDNA amplification (686 bp). Lanes 7-12: deoA cDNA amplification (1,284 bp). Lanes 13-18: add cDNA amplification (1,098 bp). cDNA synthesis from M. tuberculosis H37Rv (wild-type) was performed with reverse transcriptase enzyme (RT+) (lanes 1, 7, 13) or without (RT-) (lanes 2, 8, 14). cDNA synthesis from cdd knockout strain (KO) RT+ (lanes 3, 9, 15) and RT- (lanes 4, 10, 16). cDNA synthesis from complemented strain (CP) RT+ (lanes 5, 11, 17) and RT- (lanes 6, 12, 18).

Fig. 2 evaluation of MtCDA, MtTP and MtAD expression. MtCDA, MtTP and MtAD normalised spectral abundance factor (NSAF) in wild-type (WT), knockout strain (KO) and complemented strain (CP) cytoplasmic fractions identified by Multidimentional Protein Identification Technology (MudPIT). Bars represent average identification in technical triplicates. Asterisks represent significant differences between WT and KO or CP strains of each protein by one-way ANOVA analysis followed by Bonferroni post-test, *p < 0.05, ***p < 0.001.

Fig. 3 growth curve (A) and macrophage infection (B) with wild-type H37Rv (WT), knockout for cdd gene (KO), and complemented (CP) Mycobacterium tuberculosis strains. (A) Growth curves were started at an OD₆₀₀ of 0.01 in Middlebrook 7H9 medium, in triplicate. (B) The macrophage infection results are expressed as mean numbers of the logarithms of colony-forming unit (CFU) per well of each strain of three independent measurements.

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Construction of <i>Mycobacterium tuberculosis cdd</i> knockout and evaluation of invasion and growth in macrophages

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[1] + Corresponding author: anne.villela@pucrs.br