Experimental Study On The Correlation Between Phospholipase Of Candida Albicans And The Resistance To Fluconazole

Candida albicans is the most common human opportunistic pathogenic fungus and the most important organism of nosocomial infection.Recently the infection caused by Candida has dramatically increased,.The mortality rate of bloodstream infections was always at the high level which systemic infections caused by C.albicans had been estimated to be up to 67.9%.C.albicans had become a major lethal cause of hospital-acquired infectious patients especially in immunocompromised patients or immunologically debilitated individuals.With the wide use of antifungal agents,the condition of resistance in fungus became more and more severe,especially the resistance to azole antifungal agents.It was reported that 15.5%or more C.albicans was resistance to fluconazole.It was a big challenge to control the increased candidal infections effectively.There was an important correlation between antidrug of candida and virulent factors.Study indicated that gene disruption of a multidrug resistance MDR₁ leaded to dramatic attenuation of virulence in C.albicans.Resistant candidal strains with high-virulence exhibited over-expression of MDR and CDR,which encoded an ABC drug efflux pump.The resistant isolates of C.albicans were easier to translate from yeast cells to hyphae.The extracellular secreted proteinase and phospholipase activities were found to be higher with the emergence of fungal resistance.The virulence of resistant strains was usually more active than that of susceptible strains.It was an important and severe scientific task in the 21 century to study the relationship between resistance to antifungal drug and virulent factors of Candida,to reduce the virulence and pathogeny,and to prevent the emergency of resistance and cure the candidosis at lastA close correlation existed between pathogenicity of candida and virulent factors. Candida discriminated and invaded the host by adhesion,secreted enzymes which could enhance the virulence,phenotypic switching and the change between hyphal forms and spore forms which was good to adapt fungus to various environment and physiological conditions.Therefore,attributes that putatively contribute to candida virulence included adhesion,hyphal formation,phenotypic switching and extracellular hydrolytic enzyme production.Extracellular phospholipase played an important role in the pathogenesis of candida.Phospholipase targeted membrane phospholipids and digested these components,leading to cell lysis and direct host cell damage that lysis had been proposed as a major mechanism contributing to fungal virulence.Four types of phospholipase had been reported in C.albicans including phospholipase A,phospholipase B,phospholipase C and phospholipase D. Phospholipase A played a role in accelerating inflammation during candidal infection. Phospholipase C and phospholipase D were associated with the phenotypic switching and signal transduction.Phospholipase B could damage the integrality of host cell membrane and facilitate the penetration of the infecting agent by digesting membrane phospholipids components,which was beneficial to invade host in early stages. Phospholipase B was divided into phospholipase B1(PLB1) and phospholipase B2(PLB2).PLB1 was a secreted protein,which displayed a molecular weight of 16KD and consisted of 605 amino acids.It had function of hydrolyst and lbysophospholipase-transacylase(LPTA).A few studies had shown PLB1-deficient strains attenuated candidal virulence and pathogenicity,therefore,PLB1 was necessary during candidal infection as one of virulent factors.The 14a-lanosterol demethylase of fungus was involved in an important step in the biosynthesis of ergosterol which was essential for fungus to build their plasma membrane and was the target enzyme of azole drugs.Azole drugs acted efficiently by inhibiting the enzyme to damage the integrity of cell membrane.There were many related reasons why the fungus was resistance to antifungal drugs including immune dysfunction,taking the same medicine repeatly,infectious severity of the patient, resistance heredity to a antifungal agent and so on.Some molecular mechanisms of resistance to the azole drugs had been elucidated:overexpression of efflux pumps had been implicated in azole,of which encoding gene were CDR₁,CDR₂ and MDR₁; Point mutations in the gene(ERG₁₁) encoding the target of the azoles increased expression of ERG₁₁ which had been identified to be associated with azole resistance; Other mechanism included formation of biofilm and azole sequestration caused by vesicular vacuoles of the resistant isolates.However,it was suggested that decreased virulence of C.albicans isolates was associated with increasing the value of MICs to fluconazole(FCZ) in some cases but not in others and showed that the more virulent isolates caused infections which could be successfully treated,whereas the less virulent isolates caused infections which were refractory to fluconazole therapy. Clearly,the relationship was complex between virulence and resistance to azole.There were a limited number of reports on the correlation between phospholipase and resistance to antifungal agents.Only one in our country which determined the relationship between virulence production of secreted aspartyl proteinase and phospholipase of C.albicans and the developments of the resistance to FCZ by experimentally inducing resistant strains in liquid medium where FCZ concentration was increased gradually.We sought to determine if difference in phospholipase activity,pathogenicity and expression of PLB1 mRNA and PLB1 protein existed between fluconazole-susceptible and fluconazole-resistant C.albieans strains.We hoped the study was good to elucidate the role of phospholipase on emergence of candidal resistance to azole,so as to effectively control candidal infection and antifungal drug resistanceObjectives:1.Study the difference of phospholipase activity between fluconazole-resistant and fluconazole-susceptible C.albicans isolates. 2.Comparative study pathogenicity difference of C.albicans between fluconazole -resistant and fluconazole-susceptible strains by mice models.3.Comparative study expression difference of PLB1 mRNA between fluconazole-resistant and fluconazole-susceptible C.albicans strains by semi-quantitative RT-PCR4.Comparative study expression difference of PLB1 protein secreted by C.albicans between fluconazole-resistant and fluconazole-susceptible strains by Western blot analysisMethods:1.Screening the fluconazole-resistant and fluconazole-susceptible C.albicans strainsAll testing isolates were obtained from outpatients in QiLu Hospital,ShanDong University.C.albicans was confirmed by germ tube test,chlamydospore formation test and YBC identification test.Susceptibility testing to FCZ was performed by using the broth microdilution assay as described in the NCCLS document M27-A (National Committee for Clinical Laboratory Standards).The inoculum suspension was prepared with a final inoculum of 0.5-2.5×10³CFU/mL.The final concentration of the antifungal agent was 0.125-64μg/mL The microplates were incubated at 35℃and MIC endpoints were read after 48h of incubation.Drug free and yeast control were included.C.albicans ATCC22029 was tested as a quantity control.2.Determination of phospholipase activityThe yeast suspension was adjusted to 5×10⁷CFU/mL.About 10μL of droplets of suspension of each C.albicans isolate was plated on the surface of the egg yolk medium.The plates sealed were incubated at 37℃for 48h.The phospholipase activity of the isolates was interpreted positive when a precipitation zone was visible around the growth colony formed on the plate.The value of phospholipase production(Pz) was determined by the ratio of the diameter of the colony to the total diameter of the colony plus the precipitation zone.Each candidal isolate was tested in duplicate.A Pz value of one indicated negative for phospholipase,and less than one(Pz<1) indicated the degree of phospholipase positivity.3.Determination of pathogenicity by mice models30 mice used in the study were divided into fluconazole-resistant group and fluconazole-susceptible group by randomization,and each group included 15 mice. each isolate was prepared inoculum suspension(1-1.5×10⁷CFU/mL).Those mice were infected with fluconazole-resistant and fluconazole-susceptible strains respectively at the same time.Each mouse was injected with 0.2mL of cell suspension into the tail vein.The survival time of the mice were monitored twice a day for 30 days.4.RT-PCR analyses:Total RNA was extracted from C.albicans using E.Z.N.A Yeast RNA kit reagent kit according to the manufacturers instmctions.Spectrophoto-metry detected the value of OD₂₆₀/OD₂₈₀ to determine the purity and concentration of total RNA.In the semiquantitative analysis of PLB1 mRNA expression,the oligonucleotide PLB1 primers used were:5'-TCACCAACGAA GTCCCATCT-3'(forward)and 5'-CAACGAAGCGGTGTTGTCTA-3'(reverse).EFB1 gene was amplified in the same PCR reaction condition as the internal control.The EFB1 primers were: 5'-ATTGAACGAATTCTTGGCTGAC-3'(forward)and 5'-CATCTTCTTCAACAGCAGCTTG-3'(reverse),and expected PCR product,526 bp.The cycling conditions were as follows:preparation denaturation at 95℃for 5 min.denaturation at 95℃for 45sec,annealing at 58℃for 45see,extension at 72℃for 45see and 35 cycling was amplified for the target regions of PLB1,311 bp. final extension at 72℃for 7 min.The PCR products were examined on a 1.5% agarose gel with ethidium bromide staining.The gel image was captured with a GeI Dos 2000 Camera system.IOD value of PLB1 and EFB1 were determined by Molecular Analyst Quantification Software.5.Western blot analyses:C.albicans isolates(6×10⁶CFU/mL) were grown in YPD broth medium containing 4%glucose at 35℃for 12h on a rotary shaker(200rpm).The cell-free supematant was obtained by centrifuging the medium at 3000gfor 10 min and was then filtered.Solid ammonium sulfate was then added to 65%saturation.The salted-out proteins were redissolved and dialyzed and then applied to a DEAE-cellulose column.The cells were lyses using P0013B RIPA Lysis Buffer to obtain the intracellular protein.Protein concentration was determined using BCA Protein Assay Kit.Equal amount of protein were separated on the polyacrylamide gel by SDS-PAGE and transferred to a nitrocellulose membrane.The transferred blots were incubated with 5%non-fat dry milk and probed with an primary antibody at 4℃ovemight.Anti-PLB1 antibody and anti-β-actin antibody were used at 1:3000 and 1:500 dilution respectively.After several washes,the membranes were incubated at room temperature for 2h with the secondary antibody conjugated to HRP, and then washed again.The blots were then visualized with enhanced chemiluminescence(ECL).The IOD values of PLB1 andβ-actin protein expression were determined by the TotalLab1.0 Analyst Quantification software.Results:1.Determination of the MIC values:The MIC of FCZ was read as the lowest concentration at which 80%decrease in turbidity relative to the growth control was observed,and 15 fluconazole-resistant strains(MIC>64μg/mL) and 15 fluconazole-susceptible strains(MIC<8μg/mL) were obtained respectively.2.Comparison of phospholipase activity:12 of 15 fluconazole-resistant strains and 7 of 15 fluconazole-susceptible strains produced distinct precipitation zone implicated the phospholipase activity.The positive rate of phospholipase was 80.0% in fluconazole-resistant strains and 46.7%in fluconazole-susceptible strains.The mean values of Pz were(0.786±0.055) and(0.913±0.078) respectively.Significant difference was observed in the phospholipase activity between fluconazole-resistant strains and fluconazole-susceptible strains statistically(P<0.01).The phospholipase activity of fluconazole-resistant strains was proved to be more superior than that of fluconazole-susceptible strains.3.Comparison of pathogenicity:12 of 15 infected mice in resistant group died within 30 days,40%of mice died in susceptible group.Significant differences were proved in the mortality and average survive time between two groups(P<0.05).The virulence and pathogenicity of fluconazole-resistant group were stronger than that of fluconazole-susceptible group.4.Comparison of PLB1 mRNA expression:The value of OD₂₆₀/OD₂₈₀ detected by spectrophotometry comply with the standards.The target regions of PLB1 contained 311bp examined on agarose gel.There was significant difference in PLB1 mRNA expression between resistant and susceptible strains(P<0.05).The level of PLB1 mRNA expression was higher in fluconazole-resistant C.albicans strains.5.Comparison of PLB1 protein expression:β-actin was selected as an internal control.IOD value of the band intensity of extracellular and intracellular PLB1 protein showed a significant increase in fluconazole-resistant C.albicans strains.The level of extracellular and intracellular PLB1 protein in fluconazole-resistant strains were higher than those in fluconazole-susceptible strains(p<0.05).Conclusions:Phospholipase activity,pathogenicity to mice,expression of PLB1 mRNA and expression of PLB1 protein in fluconazole-resistant isolates of C.albieans were higher than those in fluconazole-susceptible isolates.There was relationship between the phospholipase and resistance to antifungal drugs in C.albicans.Phospholipase maybe play a role in the emergence of resistance to antifungal drug.