Molecular Characterization of the N-Acetylglucosamine Catabolic Genes in Candida africana, a Natural N-Acetylglucosamine Kinase (HXK1) Mutant.

BackgroundIn this study we report the genetic characterization, including expression analysis, of the genes involved in the uptake (NGT1) and catabolism (HXK1/NAG5, DAC1/NAG2, NAG1) of the aminosugar N-acetylglucosamine (GlcNAc) in Candida africana, a pathogenic biovariant of Candida albicans that is naturally unable to assimilate the GlcNAc.ResultsDNA sequence analysis of these genes revealed a number of characteristic nucleotide substitutions including a unique and distinctive guanine insertion that shifts the reading frame and generates a premature stop codon (TGA) 154 bp downstream of the ATG start codon of the HXK1 gene encoding the GlcNAc-kinase, a key enzyme of the GlcNAc catabolic pathway. However, all examined genes produced transcripts even though different levels of expression were observed among the Candida isolates examined. In particular, we found an HXK1-idependent relationship of the NGT1 gene and a considerable influence of the GlcNAc-kinase functionality on the transcription of the DAC1 and NAG1 genes. Additional phenotypic analysis revealed that C. africana isolates are hyperfilamentous in the first 24-48h of growth on filament-inducing media and revert to the yeast morphological form after 72h of incubation on these media.ConclusionsOur results show that C. africana is a natural HXK1 mutant, displaying a number of phenotypic characteristics distinct from typical C. albicans isolates

Molecular Characterization of the N-Acetylglucosamine Catabolic Genes in Candida africana, a Natural N-Acetylglucosamine Kinase (HXK1) Mutant.

BackgroundIn this study we report the genetic characterization, including expression analysis, of the genes involved in the uptake (NGT1) and catabolism (HXK1/NAG5, DAC1/NAG2, NAG1) of the aminosugar N-acetylglucosamine (GlcNAc) in Candida africana, a pathogenic biovariant of Candida albicans that is naturally unable to assimilate the GlcNAc.ResultsDNA sequence analysis of these genes revealed a number of characteristic nucleotide substitutions including a unique and distinctive guanine insertion that shifts the reading frame and generates a premature stop codon (TGA) 154 bp downstream of the ATG start codon of the HXK1 gene encoding the GlcNAc-kinase, a key enzyme of the GlcNAc catabolic pathway. However, all examined genes produced transcripts even though different levels of expression were observed among the Candida isolates examined. In particular, we found an HXK1-idependent relationship of the NGT1 gene and a considerable influence of the GlcNAc-kinase functionality on the transcription of the DAC1 and NAG1 genes. Additional phenotypic analysis revealed that C. africana isolates are hyperfilamentous in the first 24-48h of growth on filament-inducing media and revert to the yeast morphological form after 72h of incubation on these media.ConclusionsOur results show that C. africana is a natural HXK1 mutant, displaying a number of phenotypic characteristics distinct from typical C. albicans isolates

Morphologies of <i>C</i>. <i>albicans</i> and <i>C</i>. <i>africana</i> in YNB medium plus GlcNAc (5 mM final concentration).

<p>Images were taken at different intervals of time (T_0h, T_8h and T_24h). Results from different concentrations of GlcNAc (ranging from 0.195–100 mM) showed similar levels of filamentation (not shown).</p

Effect of GlcNAc on mRNA expression levels of the <i>HXK1</i>, <i>NGT1</i>, <i>DAC1</i> and <i>NAG1</i> genes in <i>C</i>. <i>albicans</i> ATCC10231, <i>C</i>. <i>africana</i> CBS1016, <i>C</i>. <i>africana</i> UPV/EHU 97135 (A-D) and the MGC1 strain (E-H).

<p>Values are normalized to <i>ACT1</i> as an internal control. Data are expressed as fold changes with respect to untreated cells. The experiments were performed in triplicate and results are expressed as means ± SEM. Statistical analysis were performed using a One-Way-ANOVA with Bonferroni’s Multiple comparison post-test. A <i>P value</i> < 0.05 was considered significant.</p

Morphologies of <i>C</i>. <i>albicans</i> and <i>C</i>. <i>africana</i> colonies obtained in this study after 7 days of incubation at 30°C and 37°C on YEPD plus 10% serum and Spider medium.

<p>Morphologies of <i>C</i>. <i>albicans</i> and <i>C</i>. <i>africana</i> colonies obtained in this study after 7 days of incubation at 30°C and 37°C on YEPD plus 10% serum and Spider medium.</p

Primers used for amplification, sequencing and expression analysis.

<p>Primers used for amplification, sequencing and expression analysis.</p

Partial <i>HXK1-</i>gene sequencing of <i>C</i>. <i>albicans</i> ATCC10231, <i>C</i>. <i>africana</i> CBS11016 and MGC1 strain showing the region containing the guanine insertion.

<p>In the MGC1 strain, a heterozygous site containing both <i>C</i>. <i>albicans</i> and <i>C</i>. <i>africana</i> specific nucleotides is also shown (* indicates the <i>C</i>. <i>albicans</i> and <i>C</i>. <i>africana</i> peaks that overlap in the heterozygous site). The respective growth of the strains in YNB medium plus GlcNAc is also shown.</p

Microscopic characteristics of <i>C</i>. <i>albicans</i> and <i>C</i>. <i>africana</i> isolates grown on YEPD plus 10% serum and Spider medium at 37°C for 24 and 72h.

<p>YEPD control is also shown at 24h (at 72h the YEPD control was nearly identical to the 24h time point and thus is not shown). Images were taken from the first dilution (inoculum size ~10⁴ cells/μl). Magnification 20x.</p