RESUMO
A BLAST search of the Candida Genome Database with the Saccharomyces cerevisiae LYS4 sequence known to encode homoaconitase (HA) revealed ORFs 19.3846 and 19.11327. Both alleles of the LYS4 gene were sequentially disrupted in Candida albicans BWP17 cells using PCR-based methodology. The null lys4Δ mutant exhibited lysine auxotrophy in minimal medium but was able to grow in the presence of l-Lys and α-aminoadipate, an intermediate of the α-aminoadipate pathway, at millimolar concentrations. The presence of d-Lys and pipecolic acid did not trigger lys4Δ growth. The C. albicans lys4Δ mutant cells demonstrated diminished germination ability. However, their virulence in vivo in a murine model of disseminated neonatal candidiasis appeared identical to that of the wild-type strain. Moreover, there was no statistically significant difference in fungal burden of infected tissues between the strains.
Assuntos
Candida albicans/fisiologia , Proteínas Fúngicas/genética , Técnicas de Inativação de Genes , Ácido 2-Aminoadípico/metabolismo , Estruturas Animais/microbiologia , Animais , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Candidíase/microbiologia , Candidíase/patologia , Contagem de Colônia Microbiana , Meios de Cultura/química , Modelos Animais de Doenças , Lisina/metabolismo , Camundongos , VirulênciaRESUMO
Two genes, LYS21 and LYS22, encoding isoforms of homocitrate synthase, an enzyme catalysing the first committed step in the lysine biosynthetic pathway, were disrupted in Candida albicans using the SAT1 flipper strategy. The double null lys21Δ/lys22Δ mutant lacked homocitrate synthase activity and exhibited lysine auxotrophy in minimal media that could be fully rescued by the addition of 0.5-0.6 mM L: -lysine. On the other hand, its virulence in vivo in the model of disseminated murine candidiasis appeared identical to that of the mother, wild-type strain. These findings strongly question a possibility of exploitation of homocitrate synthase and possibly also other enzymes of the lysine biosynthetic pathway as targets in chemotherapy of disseminated fungal infections.
Assuntos
Candida albicans/enzimologia , Candida albicans/crescimento & desenvolvimento , Técnicas de Inativação de Genes , Oxo-Ácido-Liases/genética , Oxo-Ácido-Liases/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Animais , Candida albicans/patogenicidade , Candidíase/microbiologia , Candidíase/mortalidade , Meios de Cultura/química , Modelos Animais de Doenças , Feminino , Genes Fúngicos , Lisina/metabolismo , Camundongos , Análise de Sobrevida , VirulênciaRESUMO
Expression plasmids containing recombinant genes encoding three His(6)-tagged versions of the enzyme, glucosamine-6-phosphate synthase from Candida albicans, were constructed and overexpressed in Escherichia coli. The gene products were purified by metal-affinity chromatography to near homogeneity with 77-80% yield and characterized in terms of size and enzymatic properties. Presence of oligohistidyl tags at either of two ends did not affect enzyme quarternary structure but strongly influenced its catalytic activity. The His6-N-tagged enzyme completely lost an ability of glucosamine-6-phosphate formation and amidohydrolase activity but retained the hexosephosphate-isomerising activity. On the other hand, two His6-C-tagged versions of glucosamine-6-phosphate synthase exhibited amidohydrolase activity almost equal to that of the wild-type enzyme but only 18% of its hexosephosphate-isomerising activity and about 1.5% of the synthetic activity.