Effect of heterologous auxotrophy markers on adaptation to various stresses in Candida albicans / 第二军医大学学报

ABSTRACT

Objective To investigate the effect of heterologous auxotrophy markers from non-Candida albicans (C. albicans) yeasts (C.dubliniensis HIS1\[C.d.HIS1\], C. maltosa LEU2\[C.m.LEU2\] and C.dubliniensis ARG4\[C.d.ARG4\]) on adaptation to various stresses of C. albicans knockout mutants (by His1-Leu2-Arg4 strategy). Methods Fusion PCR strategy was used to construct recombination fragment, which was then used to transform SN152 by lithium acetate method. Genomic PCR was used to confirm the integration of the three selection markers. Spot assays were employed to assess the sensitivities of different reintegrated strains to different stresses. Results The results of PCR showed that seven reintegrated strains containing C.m. LEU2, C.d. HIS1 and C.d. ARG4 were successfully constructed. The results of spot assay showed that there were no differences between the seven reintegration strains and SN152 in responses to the tested stresses, including pH stress, oxidative stress, antifungal agent stress, metal ion stress, cell wall stress, osmotic stress and DNA damage agent stress. We also found that only strains which integrated C.d. HIS1 marker could survive from the 0.02% SDS stress. Conclusion The three heterologous selection marker C.m. LEU2, C.d. HIS1 and C.d. ARG4 do not influence the sensitivity of C. albicans to most stresses; comparison can be made between C. albicans knockout mutants with different markers, and SN152 can be used as parental control. While assessing the sensitivity to SDS, the results vary with different selection markers, and SN152 can not be used as a unified control; therefore comparison should be made with C.d. HIS1 kept consistent.