The Study On Pathogenicity Of CHK Mutants In Candida Albicans

ObjectiveTo understand the differences of pathogenicity between Candida albicans mutant strains CHK26(Ser/Thr MAPK kinase and GAF domains deletion mutant)and CHK21( CHK null strain ) were compared to the parental C.albicans strain CAF-2 in murine model. Thus to identify CHK gene and domain functions in the pathogenicity of C.albicans.Methods1. The murine models of hematogenously disseminated candidiasis were established:18 healthy male Kunming mice were randomized into three groups and six mice each. Animals were injected intravenously via the lateral tail vein with 0.5 ml (106 cells) of a cell suspension of either CAF-2, CHK26 and CHK21.One kidney was removed for histological examination and the other kidney prepared for incubated on YPD.2. Mortality and survival results: 63 murine model of hematogenously disseminated candidiasis were established as previously shown and each group of 21. Groups of mice were observed twice daily and morbidity was recorded.3. Histological examination and TEM of murine kidneys:12 male Kunming mice each groups were established animal model as previously shown and each group of 4 . Two mice each group were sacrificed by CO2 inhalation at 3 and 5 day post infection. One kidney from each mouse was removed and prepared for histological examination, then samples were stained with periodic acid-Schiff stain. The other kidney prepared for TEM samples,examined by TEM.4. Candida colony count in kidneys and spleen of animal models: Established 45 murine infection models as previously shown, and each group of 15. Removed the kidneys and spleen from 5 mice each group in the 1, 3 and 5 day. Weighted and homogenized and quantitated after incubated. Then to compared the numbers of cfu in each group. 5. Wide type and mutant C.albicans strains were examined with transmission electron microscope (TEM) and scanning electron microscope (SEM).Results1. The murine model of hematogenously disseminated candidiasis were successfully established: Each group of mice showed mycelium growth and spores in infected kidney tissue. yeast-like colony to grow in incubated pathological tissue .2. The results of mortality and survival studies: The mortality of group mice infected with wide type CAF-2, CHK26 and CHK21 was 100 percent, 23.8 percent and zero respectively. The live time of mice infected with CHK21 and CHK26 longer than CAF-2, have statistical significance.3. Renal histopathology and transmission electron microscopy results:Detected renal histopathology that purple-red mycelium and spores can be observed in renal tissue pathological section stained with PAS of each group mice. Observed under transmission electron microscopy, we found that renal epithelial cells were visibled fungal spores in murine renal organization which infected with three C.albicans. The mice infected with CAF-2 significantly changed in renal ultrastructure shown the surface of tubular epithelial cells lost microvillis, even shown focal necrosis. Mitochondrial swelling found in renal tubular epithelial cells and found infiltration of neutrophils. The ultrastructure of renal tissue in mice infected with CHK26 were less changes than CAF-2, shown edema of individual renal tubular epithelial cells, increased lysosomes, and infiltrated neutrophils in renal interstitial. CHK21 infected mice displayed normal ultrastructure in general; only observed individual renal tubular epithelial cell swelling and few neutrophil infiltration in renal interstitial.4. Colony-forming units (cfu) of spleen and kidneys organization: Quantitative determinations of the level of each C. albicans strain associated with host tissues suggest that CHK21 was slowly cleared from the kidneys and spleen, CAF-2 was cleared fast and CHK26 between CAF-2 and CHK21.5. Results of transmission electron microscopy and scanning electron microscopy in C.albicans:we observed CHK26 strain of C.albicans cell wall thinner than wide type CAF-2 and showed incomplete cell wall structure which somewhere disappeared. And CHK21 strain's cell wall thickness between CAF-2 and CHK26 strain, but cell wall thickness of thin mixed. Scanning electron microscopy of C.albicans were seen three strains were round or oval in shape, smooth surface.CHK26 and wide type strains can be seen Yeast forms with smooth surfaces showing the formation of polar buds, but surface were randomly distributed budding scars observed in CHK21 strain's cells and the cells adhere to one another.Conclusion1. CHK pays an important role in the pathogenicity of C.albicans. Avirulence of C.albicans CHK21mutants in murine model of hematogenously disse- minated candidiasis, and virulence reduced in CHK26 mutants infected mice.2. The pathological changes of renal tissue in murine hematogenously disseminated candidiasis models , mainly in the renal tubular. After infected by C.albicans, the main ultrastructural changes observed in the murine were swallowed C.albicans in the renal tubular epithelial cells, swelled mitochondrial and infiltrated neutrophil in renal interstitial.4. CHK impacted the construction of the yeast cells wall of C.albicans, and the mainly reason to reduce toxicity.5. Ser/Thr MAPK and GAF domain of CHK have paid an important role in the pathogenicity and to regulate biosynthesis of yeast cells wall in C.albicans.6. Changes in the structure of C.albicans cells wall affected the formation of budding.