Objective To study the correlation between expression level of als3 gene and the in vivo biofilm formation of Candida albicans in mice.Methods The real-time polymerase chain reaction (PCR) assay was used to detect als3 gene expressions of the clinical Candida albicans isolates from February 2016 to August 2016 in Tianjing No.1 Central Hospital.According to the expression levels of als3 gene, Candida albicans isolates were divided into high and low-expression groups.Thirty C57 mice were randomly assigned to high-expression group (n=15), low-expression group (n=5) and blank group (n=5).Animal model of Candida albicans biofilm was established based on venous catheter and intraperitoneal injection of Candida albicans.Catheters were removed after two weeks;inverted microscope was used for the observation of Candida albicans biofilm formation and transmission electron microscope was used for the observation of its ultrastructure.After irrigating the catheter, the growth of Candida albicans was observed;real-time PCR was used to detect the expression levels of als3 gene 12, 24, and 48 h after the catheter being removed.In this study, t test was used for measurement data and chi-square test was used for rate comparisons.Results In high-expression group, 11 strains (11/15) formed biofilms.In als3 low-expression group, only one strain (1/10) formed biofilm.The difference between these two group was statistically significant (x2=9.64,P<0.05).In als3 high-expression group, two mice died and 8 strains (8/13) formed biofilms, while in low-expression group, there were only 2 strains (2/10) formed biofilms.The difference between these two group was statistically significant (x2=4.02,P<0.05).Thickened Candida albicans membranes and increased mitochondria in high-expression group were observed under transmission electron microscope.In als3 high-expression group, 9 of 13 catheter cultures were positive.However, in als3 low-expression group, 5 of 10 catheter cultures were positive.The difference between these two group was not statistically significant (x2=0.99, P>0.05).In the als3 high-expression group, the expression of als3 gene declined gradually during the biofilm formation.In the als3 low-expression group, the change of als3 gene expression was not obvious.The expressions of als3 gene over time between two groups were significantly different (t=8.7, 10.3 and 9.2, respectively, all P<0.05).Conclusion The high expression of als3 gene in Candida albicans facilitates the formation of biofilm in vivo.

The experiment was conducted by directed evolution strategy (error-prone PCR) to improve the activity of aflatoxin detoxifzyme with the high-throughput horse radish peroxidas and recessive brilliant green (HRP-RBG) screening system. We built up a mutant library to the order of 10(4). Two rounds of EP-PCR and HRP-RBG screening were used to obtain three optimum mutant strains A1773, A1476 and A2863. We found that mutant A1773 had upper temperature tolerance of 70 degrees C and that its enzyme activity was 6.5 times higher than that of the parent strain. Mutant strains A1476 worked well at pH 4.0 and its enzyme activity was 21 times higher than that of the parent strain. Mutant A2863 worked well at pH 4.0 and pH 7.5, and its enzyme activity was 12.6 times higher than that of the parent strain. With DNA sequencing we found that mutant A1773 revealed two amino acid substitutions, Glu127Lys and Gln613Arg. Mutant A1476 revealed four amino acid substitutions: Ser46Pro, Lys221Gln, Ile307Leu and Asn471lle. Mutant A2863 revealed four amino acid substitutions: Gly73Ser, Ile307Leu, Va1596Ala and Gln613Arg. The results provided a useful illustration for the deep understanding of the relationship between the function and structure of aflatoxin detoxifzyme.
Aflatoxin B1 ; antagonists & inhibitors ; chemistry ; Amino Acid Substitution ; Directed Molecular Evolution ; Enzyme Activation ; Enzyme Stability ; Multienzyme Complexes ; genetics ; metabolism ; Mutant Proteins ; genetics ; metabolism ; Point Mutation ; Polymerase Chain Reaction ; methods ; Protein Engineering