Effect Of Glucosamine On The Virulence Of Crz1 In Cryptococcal Infection In Mice

Objective: To study the effect of glucosamine on cryptococcal virulence,and confirm whether glucosamine can reduce the ability of infecting animals with cryptococcus neoformans,and characterized the role of Crz1 in cryptococcus neoformans when strains wwre injected into animals with or without glucosamine.Method: Mice aged 6-8 weeks with weight 15-22 g were selected and fed in standard animal houses.The experiment was divided into three parts:(1)verify that glucosamine(Glc N)effects on the virulence of cryptococcus neoformans;(2)test whether Crz1 plays a role in the virulence of cryptococcus in animals.(3)test the effect of Crz1 on animals under the influence of Glc N.The mice were numbered,their weight before inoculation was recorded,and their weights were observed and measured every day.Changes in activity were recorded.Before inoculation,cryptococcus H99 was cultured overnight in YPD medium.After PBS washing,the suspension concentration of H99 cells was 2x106cells/ml.Before inoculation,the cell suspension was diluted with PBS for 10 x,and the cell counter was used to reach the concentration of 2x105cells/ml.After anaesthesia,mice containing 1x104 CFU 50μL of this fungus would be dripped into the nasal cavity and be inhaled,thus the mice model of cryptococcus infection established.For Glc N feeding mice group,we fed 7% Glc N a week before the infection,and then inject relative cryptococcus strain,after 7 days,anesthesia was applied,the mice were put to death by cervical vertebra dislocation and the lungs were collected and homogenized in PBS suspension,with final 10-105 times dilution,and each dilution were spread evenly over the YNB plates,The plates were cultured in 30 incubator.Result: The results of experiment A showed that: compared with the mice drinking water,the lung fungal burden of the experimental mice drinking glucosamine water was reduced(P<0.05).However,the lung fungal burden of the mice fed with Glc N and inoculated WT+5% Glc N showed no significant statistical difference compared with the A2 group fed Glc N but inoculated with WT strain alone.The results of experiment B showed that there was no significant difference in the pulmonary fungalburden in the three groups of mice,WT group,Crz1 overexpression group and Crz1 knockout group.Compared with WT group,the lung fungal burden of Crz1 overexpression group was decreased(P<0.05),while the lung fungal burden of Crz1 knockout group was increased(P<0.05),showing statistically significant differences.Conclusion: In the experiment,the infection model was established by inhalation of cryptococcus in mice.Through the analysis of fungal load count data,it can be concluded that drinking glucosamine can significantly reduce the virulence/infection of animals infected with cryptococcus.In this process,Crz1 plays a role in regulating and responding to glucosamine.In the absence of glucosamine,Crz1 has no significant effect on cryptococcal virulence.However,under the effect of glucosamine,the virulence of Crz1 overexpressed strain is significantly reduced,and cryptococcal infection of Crz1 knockout strain in mice is likely to spread.