RESUMO
Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their active center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.
Assuntos
Ácido Aspártico/genética , DNA Bacteriano/metabolismo , Endonucleases/metabolismo , Inteínas/fisiologia , Mycobacterium leprae/enzimologia , Recombinases Rec A/genética , Sequência de Aminoácidos , Ácido Aspártico/metabolismo , Domínio Catalítico , Análise Mutacional de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Endonucleases/química , Endonucleases/genética , Dados de Sequência Molecular , Mycobacterium leprae/genética , Estabilidade Proteica , Recombinases Rec A/metabolismo , Alinhamento de Sequência , Cloreto de SódioRESUMO
Leprosy is the major cause of non-traumatic neuropathy. Herein, we investigated the role of ninjurin 1, an adhesion molecule involved in nerve regeneration in leprosy. Our results demonstrated that M. leprae stimulates in vitro up-regulation of ninjurin mRNA in cultured Schwann and blood cells as well as in vivo mRNA and protein expression in leprosy nerve biopsies. A polymorphism (asp110ala) was investigated in a case-control study (1123 individuals) and no association was found with leprosy per se or with disseminated forms. Nevertheless, ala110 was associated with functional nerve impairment (OR=2.42; p=0.02 for ala/ala) and with lower mRNA levels. Our data suggests that asp110ala could be a valuable genetic marker of nerve damage in leprosy.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Hanseníase/complicações , Hanseníase/genética , Fatores de Crescimento Neural/genética , Nervos Periféricos/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Alanina/genética , Substituição de Aminoácidos/genética , Ácido Aspártico/genética , Moléculas de Adesão Celular Neuronais/química , Análise Mutacional de DNA , Feminino , Marcadores Genéticos/genética , Predisposição Genética para Doença/genética , Testes Genéticos , Genótipo , Humanos , Imunidade Inata/genética , Hanseníase/fisiopatologia , Masculino , Pessoa de Meia-Idade , Fatores de Crescimento Neural/química , Nervos Periféricos/patologia , Nervos Periféricos/fisiopatologia , Doenças do Sistema Nervoso Periférico/diagnóstico , Doenças do Sistema Nervoso Periférico/fisiopatologia , RNA Mensageiro/metabolismo , Regulação para Cima/genéticaRESUMO
Similar to other mycobacterial diseases, susceptibility to Buruli ulcer (Mycobacterium ulcerans infection) may be determined by host genetic factors. We investigated the role of SLC11A1 (NRAMP1) in Buruli ulcer because of its associations with both tuberculosis and leprosy. We enrolled 182 Buruli ulcer patients (102 with positive laboratory confirmation) and 191 healthy neighbourhood-matched controls in Ghana, and studied three polymorphisms in the SLC11A1 gene: 3' UTR TGTG ins/del, D543N G/A, and INT4 G/C. Finger prick blood samples from study subjects were dried on filter papers (FTA) and processed. D543N was significantly associated with Buruli ulcer: the odds ratio (adjusted for gender, age, and region of the participant) of the GA genotype versus the GG genotype was 2.89 (95% confidence intervals (CI): 1.41-5.91). We conclude that a genetic polymorphism in the SLC11A1 gene plays a role in susceptibility to develop Buruli ulcer, with an estimated 13% population attributable risk.