Antifungal Effects And Potential Mechanisms Of Lonidamine Combined With Fluconazole Against Candida Albicans

BackgroundIn recent years,the morbidity and mortality of deep fungal infections had increased sharply due to the increase of chemotherapy and radiotherapy among cancer patients,the widespread use of antibiotics and immunosuppressants,the increase number of patients with AIDS.Candida albicans(C.albicans,CA)is an opportunistic pathogen which could cause deep fungal infections.The clinically used antifungal agents are limited,and azoles are widely used to cope with fungal infections since they are low-toxic and highly effective,which has resulted in an increasing number of drug-resistant C.albicans.In order to cope with fungal resistance,the use of drug combination has attracted a lot of attention.Lonidamine(Lonidamine,LND)is an anti-tumor drug with a broad anti-cancer spectrum and low side effects.At present,it often combines with other anti-tumor drugs to treat a variety of tumor diseases clinically.The mechanism of LND is mainly to affect the energy metabolism of cancer cells,including inhibiting the oxygen consumption of cancer cells and the activity of hexokinase in the glycolytic pathway.In addition,studies have shown that the glucose metabolism pathway is closely related to the infecting process of C.albicans,and the glycolytic pathway could provide energy for C albicans invading the host’s plasma and tissue,and hexokinase Ⅱ(HXK2)is vital for C.albicans virulence.At present,there is no research report investigating the antifungal activity of the LND-fluconazole(FLC)combination against C.albicans.ObjectivesTo study and evaluate in vitro and in vivo antifungal activities of LND-FLC combination against C.albicans,and to explore the further potential synergistic antifungal mechanisms.MethodA broth microdilution method was used to evaluate the minimum inhibitory concentration(MIC)of LND alone or in combination with FLC against planktonic C.albicans cells,the XTT colorimetric experiment was used to determine the anti-biofilm activities of the LND-FLC combination against C.albicans biofilms at different time periods.The in vivo antifungal effect of LND combined with FLC was evaluated by constructing insect fungal infection models,including recording the survival numbers of Galleria mellonella in different drug groups,the amount of Candida albicans cells,and the degree of histopathological damage in larvae tissues.Besides,in order to further explore the potential antifungal mechanism of the combination of LND and FLC,the following experiments were carried out:observing and photographing the effects of different groups of drugs on Candida albicans hyphae formation in Spider medium through a microscope;the fluorescent dye DCFH-DA and FITC-VAD-FMK was used to explore the accumulation of ROS levels and the activation of metacaspase in resistant C.albicans,respectively;rhodamine 6G was used to determine whether LND could influence the efflux pump activity on drug-resistant C.albicans cell membrane.Results(1)LND-FLC combination exhibited synergistic antifungal activity on drug-resistant planktonic C.albicansLND alone had no obvious antifungal effect on the twelve C.albicans strains used in the experiment,but when LND combined with FLC,it had a synergistic antifungal effect on six FLC-resistant strains,and the MIC values of FLC could be significantly reduced:from≥ 512μg/mL to 0.5μg/mL,the FICI value was between 0.06-0.13.At the same time,although LND plus FLC had no obvious synergistic activities for the other 6 FLC-sensitive strains,FICI>0.5,the minimum inhibitory concentration of LND could also be reduced from≥ 512μg/mL to 32-64μg/mL.(2)LND-FLC combination showed synergistic effect on preformed biofilmThe results showed that LND+FLC had a synergistic effect against C.albicans biofilms with a short pre-formation time(≤8 h),and the minimum inhibitory concentration of FLC could be decreased from≥ 1024μg/mL to 2-8μg/mL.FICIs<0.5.We also observed that with the prolongation of the biofilm formation time,although LND+FLC showed irrelevant effects on the biofilms pre-formed at 12 h and 24h,the MIC value of FLC can be reduced by 128-512 times at 12 h.(3)LND-FLC combination could prolong survival rate,reduce fungal burden and tissue damage of Galleria mellonella larvaeLND plus FLC exhibited a synergistic antifungal effect in Galleria mellonella larvae.Compared with the control group and drug alone groups,the numbers of surviving larvae in LND-FLC combination group were increased and the mortality rate was greatly reduced;the fungal load of C.albicans in larvae was reduced.At the same time,LND-FLC could significantly reduce the size and number of black clumps in the tissues,and only a few tiny clumps were scattered in the tissues,which could reduce the damage to the larval tissue.(4)LND-FLC combination could inhibit the hyphal growth and morphological transition of C.albicansComparing to the control group,FLC alone could slightly reduce the number of hyphae,but had no significant effect on hyphae length.LND alone could shorten the hyphae length of C.albicans,but couldn’t reduce the numbers of hyphae,and most of them were in an aggregated state.In LND and FLC combination group,there was some round yeast cells could be observed,the hyphae length of C.albicans was significantly shortened,and the number was reduced,indicating that the LND-FLC combination could inhibit the morphological transition of C.albicans hyphae from yeast state to hyphae state.(5)LND-FLC combination could activate the metacaspase in C.albicans,but was unrelated to accumulate ROS levelsThe results showed that the ROS levels in the LND+FLC group were similar to other three groups(P>0.05).In addition,we didn’t observe fluorescence in the control group and the drug-only group under the microscope.However,we observed that not only fungal cells numbers were decreased,but also bright green fluorescence was emitted,indicating that the metacaspase in C.albicans was activated.The results demonstrating that the synergistic antifungal mechanisms of LND-FLC combination may be associated with the activation of metacaspase.(6)LND was independent of inhibiting the activity of drug transportersOverexpression of drug efflux genes is one of the most common mechanisms of C.albicans resistance.In this part,we used Rh 6G instead of FLC to carry out the experiment.The results showed that there was almost no difference in the uptake and efflux curves of Rh 6G between the LND group and the control group(P>0.05),indicating that the synergistic antifungal effects of LND and FLC were independent of inhibiting drug transporters.ConclusionThis study exhibited a new pharmacological effect of LND,that is,LND in combination with FLC had a synergistic antifungal activity against planktonic cells and preformed biofilms(≤8 h)of C.albicans.In addition,mechanistic studies have clarified that the synergistic antifungal effect may be related to the inhibition of biofilm formation,the hyphae morphological transformation,and the metacaspase activation of fungi,but it was independent of accumulating ROS levels or inhibiting the drug transporter activity,other mechanisms still needed to be further studied.This thesis hopes to provide a theoretical basis for overcoming the resistance of fungi and finding new antifungal drugs based on the glucose metabolism pathway.