| In recent years,the clinical treatment of cancer,the usage of immunosuppressive agents in organ transplantation,the increasing number of AIDS patients and other diseases caused by immunosuppression are all main causes of the increasing clinical fungal infection.Since the limited clinical fungal detection,poor diagnostic tools and specificity,fungal infections have already risen to the fourth place in hospital blood-borne infections.The mortality rate of systemic fungal infection is still as high as 40%-70%.Due to the limited clinical antifungal agents and the severe azole resistance caused by long-term clinical use of azoles,the drug resistance rate of Candida albicans clinical isolates are approximately 40%.Therefore,it is urgent to elucidate the mechanism of azole resistance and discover new antifungal treatment for curing fungal infections.A lot of progress about drug-resistant mechanism has been made in recent years,mainly concerning azole synthesis pathway and the mutation and high expression of drug efflux pump-related genes.Since,the ergosterol synthesis pathway is highly related to azole resistance,there is a need to make a thorough elucidation of its regulatory network.Upc2p,for example,has been demonstrated to play a role in maintaining sterol synthesis pathway through regulating the expression of genes related to sterol synthase.However,in Saccharomyces cerevisiae,the regulation of the synthesis of sterols is not only limited to the level of transcription but also to the post-translational regulation.Since the post-translational regulation is rarely reported in C.albicans,which still needs further studies.Our research is focused on gene ORF19.273 in C.albicans which is homologous to NSG2,an encoding gene of hydroxymethylglutaryl acetyl-CoA reductase(HMG-CoA)reductase in S.cerevisiae.This research is aim at studying the effect of ORF19.273 on sterol synthesis pathway and azole resistance.First of all,C.albicans ORF19.273 knockout and reintroduction strains were constructed by HIS 1-ARG4-LEU2 nutrition screening strategy and fusion PCR method.ORF19.273 was demonstrated to have no effect on strain proliferation rate or the formation of hyphae and biofilm in C.albicans.As for drug susceptibility,the deletion of ORF19.273 significantly enhanced the susceptibility to azoles but reduced susceptibility to amphotericin B.However,ORF19.273 disruption did not affect the susceptibility to lovastatin and terbinafine which target on upstream action site of azoles.Also,the deletion of ORF 19.273 had no effect on fenpropimorph which target on downstream action site of azoles.The fact that no difference existed in the susceptibility to lovastatin suggested that the function of ORF19.273 in C.albicans and NSG2 in S.cerevisiae is not completely consistent.Transmission electron microscopy analysis showed that the cell membrane of ORF19.273 deleted strains was slightly damaged.Under the treatment of fluconazole,the damage of ORF19.273 deficient cell membrane was more serious.Moreover,the deletion of ORF 19.273 resulted in the changes of cell membrane permeability in C.albicans.The deletion of ORF19.273 increased the permeability of propidium iodide(PI)and resulted in the damage of cell membrane structure and function.In C.albicans,ergosterol synthesis is an important substance that affects azole drug sensitivity and cell membrane integrity.By gas chromatography-mass spectrometry(GC-MS)analysis of the percentage of sterols in the parental strain and ORF19.273 null mutants and sterols in the recovery mutants,we found that the sterols Obtusifoliol and 14a-methylfecosterol containing 14a-methyl were accumulated intracellularly.With fluconazole treatment,the main sterols accumulated in the parental strains was lanosterol.However,the accumulated sterols of the ORF19.273 null muntants mainly contained 14a-methylfecosterol,obtusifoliol,and eburicol,indicating that ORF19.273 regulating the sterol balance in C.albicans.C.albicans sterols distribution is an important mechanism in maintaining its tolerance to azole drugs.Therefore,we illustrated that ORF19.273 regulated the proportion of 14a-methyl sterols and improved C.albicans tolerance to azole drugs.In murine systemic C.albicans infection,the renal fungal burden of mice infected with ORF19.273 null mutants was significantly lower than those with the parental strains after fluconazole treatment.Treated with 2mg/kg fluconazole,mice infected with ORF19.273 null mutants showed a higher survival of mice infected with ORF 19.273 null mutants.This study confirmed that ORF19.273 plays an importance role in maintaining the integrity of C.albicans cell membrane and the resistance of C.albicans to azoles by regulating the levels of obtusifoliol and 14a-methylfecosterol.In addition to chemical antifungal treatment,immunotherapies of fungal infections are also widely studied in recent years.As an opportunistic pathogen,the pathogenicity of C.albicans is closely related with the state of host immune system.In recent years,many effective immunotherapies including antifungal monoclonal antibodies,live attenuated vaccines,and recombinant protein vaccines have been reported in the literature,but studies on the mechanism of action of vaccines are relatively rare.By studying the mechanism of different vaccines and the key process in immune system clearing fungi,we could provide new ideas for developing antifungal therapies.In this study,we found that the transcription factor FL08 disrupted C.albicans,as a genetically modified attenuated strain,could activate immune protection in wild-type mice.After primary infeciton with flo8? viable cells,mice could elicit protective responses to secondary invasive C.albicans infection,S.aureus infection or A.fumigatus infection and P.aeruginosa infection in the lungs.Through the long-term vaccination strategy,we found that flo8? live mutants could stimulate mice producing high levels of antibodies to C.albicans and the vaccinated serum could enhance the ability of macrophages in killing C.albicans in vitro.The short-term vaccination strategy showed that the protective effect caused by flo8? live mutants not only depends on the antibodies,but also depends on the stimulation of anti-inflammatory cytokines IL-10.IL-10 could reduce the acute inflammatory injury caused by the secondary infection with virulent strains SC5314 and reduce the recruitment of monocytes and neutrophils in vivo.At the same time,IL-10 could protect the thymus against atrophy through up-regulating the expression of anti-apoptotic genes BCL-2 and BCL-XL,which defends against the apoptosis of CD4+ CK8+ T cells caused by virulent C.albicans infection.By analyzing the changes of the cell wall components in flo8 null mutants,we found that the mannans on the surface of cell wall,especially the serum-induced flo8 mutants mannans,stimulated the production of IL-10 in vitro and in vivo.After intraperitoneal vaccinated with mannans extracted from flo8 null mutants,the renal fungal burden of mice inoculated with SC5314 was decreased and the survival rate was increased,which indicated that mannan was the basis of immunoprotection induced by flo8 null mutants.In addition,stimulating macrophages in vitro indicated that serum-induced flo8 null muants could stimulate macrophages to produce high levels of NO.However,standard strains SC5314 requires co-stimulation with Pam3CSK4 to stimulate macrophages producing NO,which indicated more antigens exposured on the surface of flo8 null mutants.After extraction and isolation,we determined that the n-butanol moiety in the chloroform-methanol extracts of C.albicans stimulated macrophages to produce NO.Unexpectedly,in vivo studies confirmed that NO is not involved in the immune protective effect induced by the flo8 null mutants.Taken together,the flo8 null mutants vaccinnation protects the thymus of mice increases the survival rate of mice after secondary virulent C.albicans infection by increasing CARD9-mediated IL-10 production.These immune protective mechanisms could provide new theoretical guidance for the development of vaccines and antifungal therapies. |
PDF Full Text Request