In Candida albicans, adaptation to environmental pH is relevant to its pathogenicity. Calcium signaling pathway involves in many stress responses and often accompany with Ca2+ fluctuation. We constructed CCH1 and MID1 mutant strains and studied their effect on calcium influx and further investigated the regulation by Crz1p transcription factor. We used PCR-directed gene disruption to construct cch1delta/delta and mid1delta/delta null mutant. By using a flow cytometry-based method we monitored the free cytosolic Ca2+ levels under alkaline stress. Moreover, we constructed pPHO89-LacZ plasmids and by beta-Galactosidase assays, we analyzed the changes of LacZ activities after gene disruption. The results showed that alkaline stress induced calcium burst reduced obviously in cch1delta/delta and mid1delta/delta mutant strains, also for LacZ activities, and fully abolished in crz1delta/delta mutant strain. Finally, by realtime PCR, we confirmed the regulation role of Crz1p in CCH1 and MID1 genes but in a calcineurin independent way. Studies on the effect of calcium pathway on response to alkaline stress will provide an important theoretical basis for Candida albicans infection-oriented treatment and new drug targets.
Calcium Channels ; metabolism ; Candida albicans ; genetics ; metabolism ; physiology ; Fungal Proteins ; genetics ; physiology ; Gene Expression Regulation, Fungal ; Hydrogen-Ion Concentration ; Signal Transduction ; Stress, Physiological ; Transcription Factors ; metabolism ; physiology

By constructing the genomic DNA library of Meiothermus ruber CBS-01, the genes of trehalose phosphate synthase (TPS) and trehalose phosphate phosphatase (TPP) involved in trehalose synthesis were cloned. The genes were cloned into the plasmid pET21a, and expressed in Escherichia coli Rosetta gami (DE3). The activities of these two purified enzymes were confirmed by thin layer chromatography (TLC). Meanwhile, we tested the cellular compatible solutes of M. ruber CBS-01 under different environmental pressure, and found that under hyperosmotic pressure, this strain can accumulate trhalose-6-phosphate, but not trehalose. These results can give more insight to future research in the roles of TPS/TPP and TreS pathway.
Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Glucosyltransferases ; genetics ; metabolism ; Phosphoric Monoester Hydrolases ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism ; Thermus ; enzymology ; genetics ; Trehalose ; biosynthesis

The calcium gate encoded by CCH1 and MID1 genes is the main channel for external calcium absorption. As one of the important secondary messengers, the elevation of calcium concentration could activate some pathways to take part in various cell processes. In this study, we used CCH1 and MID1 mutant strains and also constructed their complementary strains to study the effect of drug tolerance and virulence of Candida albicans after CCH1 or MID1 deletion. By drug plate sensitivity assay and the broth microdilution method, we compared the changes between different strains. Moreover, we added calcium channel blocker and inhibitors to analyze the effect of calcium concentration on drug action. After the deletion of CCH1 or MID1 gene, the strain exhibited an obvious sensitivity to FLUC and ITRA, and the drug action was regulated by the calcium concentration. In a mouse model of intravenous infection, we found that attenuated virulence of cch1delta/delta or mid1delta/delta strain is specifically due to a loss of CCH1 or MID1 gene.
Animals ; Calcineurin ; genetics ; metabolism ; Calcium ; metabolism ; Calcium Channels ; metabolism ; Candida albicans ; drug effects ; genetics ; pathogenicity ; Candidiasis ; microbiology ; Drug Resistance, Fungal ; genetics ; Female ; Fungal Proteins ; genetics ; metabolism ; Gene Deletion ; Mice ; Mice, Inbred ICR ; Virulence

Enterococcus faecalis has been confirmed to be closely related to dental pulp and periapical disease in recent years. Enterococcus faecalis is one of the important bacteria causing persistent or secondary root canal infection and root canal treatment failure. Traditional root canal disinfection drugs such as sodium hypochlorite, chlorhexidine and calcium hydroxide can not completely remove Enterococcus faecalis in the root canal because of the concentration limitation of the drug and the complexity of the root canal system. Therefore, how to effectively resist the Enterococcus faecalis infection in the root canal has become one of the important research directions in the treatment of periodontal pulp periapical disease. In recent years, some new antimicrobial agents and disinfection methods have emerged due to the drug resistance and pathogenicity of Enterococcus faecalis, such as laser, photodynamic, ultrasonic irrigation and ozone therapy. Their combination with traditional root canal irrigation drugs can significantly enhance the ability of traditional drugs to remove Enterococcus faecalis in the root canal. In addition, the emergence of new disinfection methods such as chlorine dioxide, nano-magnesia, superoxidized water and N-acetylcysteine have been shown to have a unique killing effect on Enterococcus faecalis in root canals. At present, most of the new disinfection methods described above are in the in vitro experimental stage, and their stimulation and damage to normal tissue still lack relevant clinical data support; thus, these outcomes need to be further studied.