Identification of two proteins induced by exposure of the pathogenic fungus Candida glabrata to fluconazole

Candida glabrata is an increasingly important cause of opportunistic fungal infection of humans and appears to be intrinsically resistant to the triazole antifungal fluconazole. However, the mechanisms responsible for reduced susceptibility to azole drugs are not understood. Fluconazole exposure rapidly induced expression of a 169-kDa protein band in plasma membrane fractions of C. glabrata cells. Mass spectrometry of trypsin-digested peptide fragments showed that the induced protein band comprised the ATP binding cassette-type drug efflux transporter CgCdr1p. CgCdr1p was also functionally overexpressed in S. cerevisiae and similarly identified by mass spectrometry. A 61-kDa protein band in the plasma membrane fraction from C. glabrata was also induced by fluconazole exposure. Mass spectrometric peptide fingerprinting identified this band as lanosterol 14alpha-demethylase, the enzyme in the ergosterol biosynthesis pathway targeted by fluconazole. The rapid induction of a multidrug efflux pump and/or overproduction of lanosterol 14alpha-demethylase are mechanisms that could make C. glabrata appear intrinsically resistant to fluconazole. Mass spectrometric fingerprint analysis of SDS-PAGE separated plasma membrane fractions combined with heterologous hyper-expression provides a convenient method for protein identification and functional evaluation of induced proteins, even in an organism where the genome sequence database is incomplete.     

PKC Signaling Regulates Drug Resistance of the Fungal Pathogen Candida albicans via Circuitry Comprised of Mkc1, Calcineurin,and Hsp90

Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.     

深部真菌感染临床特点分析

目的了解深部真菌感染患者的性别、年龄、感染部位、住院科室、菌种分布及真菌耐药情况,为临床防治真菌感染提供研究依据。方法收集荆州市中心医院2009年1月至2009年12月微生物实验室分离的真菌446株,采用科马嘉显色琼脂及API220C Aux鉴定系统鉴定,并使用ATMTMFUNGUS3真菌药敏卡进行体外药敏试验。结果临床真菌感染男性占72%,以老年患者为主,大于60岁者占54.9%;感染的真菌主要分布于呼吸内科和ICU,分别占35.5%、24.9%;主要感染部位为呼吸道,占91.3%;主要菌种为白假丝酵母菌、热带念株菌、近平滑假丝酵母菌、光滑念株菌和克柔念株菌,分别占64.2%、13.2%、9.6%、7.6%和5.4%;合并细菌感染的感染真菌100株,占22.2%,细菌中以革兰阴杆性菌为主,占96%;药敏试验结果显示真菌对各抗真菌药具有较好的敏感性。结论临床真菌感染已日益突出,以呼吸科及ICU患者老年男性为主,儿童真菌感染亦不容忽视,感染菌种以白假丝酵母菌和热带念株菌为主,临床应加强对这些真菌感染的预防和监测,防止真菌感染。     

Calcineurin Is Required for Pseudohyphal Growth, Virulence, and Drug Resistance in Candida lusitaniae

Candida lusitaniae is an emerging fungal pathogen that infects immunocompromised patients including HIV/AIDS, cancer, and neonatal pediatric patients. Though less prevalent than other Candida species, C. lusitaniae is unique in its ability to develop resistance to amphotericin B. We investigated the role of the calcium-activated protein phosphatase calcineurin in several virulence attributes of C. lusitaniae including pseudohyphal growth, serum survival, and growth at 37°C. We found that calcineurin and Crz1, a C. albicans Crz1 homolog acting as a downstream target of calcineurin, are required for C. lusitaniae pseudohyphal growth, a process for which the underlying mechanism remains largely unknown in C. lusitaniae but hyphal growth is fundamental to C. albicans virulence. We demonstrate that calcineurin is required for cell wall integrity, ER stress response, optimal growth in serum, virulence in a murine systemic infection model, and antifungal drug tolerance in C. lusitaniae. To further examine the potential of targeting the calcineurin signaling cascade for antifungal drug development, we examined the activity of a calcineurin inhibitor FK506 in combination with caspofungin against echinocandin resistant C. lusitaniae clinical isolates. Broth microdilution and drug disk diffusion assays demonstrate that FK506 has synergistic fungicidal activity with caspofungin against echinocandin resistant isolates. Our findings reveal that pseudohyphal growth is controlled by the calcineurin signaling cascade, and highlight the potential use of calcineurin inhibitors and caspofungin for emerging drug-resistant C. lusitaniae infections.     

Identification and susceptibility against fluconazole and albaconazole of 100 yeasts' strains isolated from vaginal discharge

Vulvovaginal candidiasis is a condition that affects a great number of fertile women. It is considered the second cause of genital infection after vaginosis due to GAM complex. Candida albicans is the most frequent isolated species from vaginal discharge. However, sometimes more than one yeast species could be found in the same clinical sample that are more resistant to antifungal drugs. Nowadays, it is necessary to identify properly up to species level the isolated microorganism and to determine the antifungal susceptibility profile. One hundred strains obtained from vaginal discharge of 94 patients suffering acute vulvovaginal candidiasis were studied. The identification of the isolates showed: C. albicans 86%, Candida glabrata 6%, Candida inconspicua 3%, Candida krusei 2% and Candida intermedia, Candida holmii and Trichosporon asahii one case each. Minimal inhibitory concentrations (MIC) of all the yeasts against fluconazole and albaconazole were performed. C. glabrata, C. krusei and C. inconspicua were the most resistant against fluconazole, on the other hand albicans was susceptible to this drug. All the isolates presented MIC against albaconazole much lower than fluconazole.     

Conventional dendritic cells mount a type I IFN response against Candida spp. requiring novel phagosomal TLR7-mediated IFN-β signaling

Human fungal pathogens such as the dimorphic Candida albicans or the yeast-like Candida glabrata can cause systemic candidiasis of high mortality in immunocompromised individuals. Innate immune cells such as dendritic cells and macrophages establish the first line of defense against microbial pathogens and largely determine the outcome of infections. Among other cytokines, they produce type I IFNs (IFNs-I), which are important modulators of the host immune response. Whereas an IFN-I response is a hallmark immune response to bacteria and viruses, a function in fungal pathogenesis has remained unknown. In this study, we demonstrate a novel mechanism mediating a strong IFN-β response in mouse conventional dendritic cells challenged by Candida spp., subsequently orchestrating IFN-α/β receptor 1-dependent intracellular STAT1 activation and IFN regulatory factor (IRF) 7 expression. Interestingly, the initial IFN-β release bypasses the TLR 4 and TLR2, the TLR adaptor Toll/IL-1R domain-containing adapter-inducing IFN-β and the β-glucan/phagocytic receptors dectin-1 and CD11b. Notably, Candida-induced IFN-β release is strongly impaired by Src and Syk family kinase inhibitors and strictly requires completion of phagocytosis as well as phagosomal maturation. Strikingly, TLR7, MyD88, and IRF1 are essential for IFN-β signaling. Furthermore, in a mouse model of disseminated candidiasis we show that IFN-I signaling promotes persistence of C. glabrata in the host. Our data uncover for the first time a pivotal role for endosomal TLR7 signaling in fungal pathogen recognition and highlight the importance of IFNs-I in modulating the host immune response to C. glabrata.     

Susceptibility to fluconazole and amphotericin B in clinical Candida isolates.

Susceptibility to fluconazole and amphotericin B in 84 clinical isolates of Candida was determined by a macrodilution method (NCCLS). Amphotericin B was very active (CMI < 1.25 microg/ml) against C. tropicalis and C. parapsilosis. Less than 5% of C. albicans and/or C. glabrata isolates presented low susceptibility to the drug (CMI 80 > 2.50 microg/ml). Fluconazole was less active against C. glabrata and C. krusei (CMI 80 > 100 microg/ml). The susceptibility profile for fluconazole indicated the importance to the treatment of identification to species level.     

Biofilm formation by Candida albicans on various prosthetic materials and its fluconazole sensitivity: a kinetic study

Candida albicans has the ability to colonize various materials used in prostheses. In this report, we have studied the kinetics of biofilm formation on prosthetic materials and their susceptibility to fluconazole at various stages of development. Results indicated that C. albicans efficiently adheres to and colonizes polystyrene, polyvinylchloride, silicon, and polycarbonate surfaces. Candida albicans biofilm formation was observed to be both strain- and substrate dependent. Adhesion of cells to solid substrates was found sufficient to induce fluconazole resistance. Drug susceptibility at different stages of biofilm growth showed that Candida biofilms on these substrates are highly resistant to fluconazole. The study focuses on the limitations of fluconazole to combat biofilm-related infections and emphasizes the need for better therapeutic strategies against prosthesis-associated C. albicans infections.     

重症监护病房真菌谱及耐药性6年变化趋势

目的 调查我院重症监护室（ICU）近6 a临床分离真菌株的菌种结构及耐药性变化.方法 2007 ～2012年从我院1435例ICU患者中分离出的1 435株真菌均为首次分离的菌株.采用Cox-Stuart趋势检验回顾性分析了分离真菌株的菌种结构和主要真菌对常用抗真菌药物耐药率的变化趋势.结果 2007 ～2012年间,我院tCU真菌检出阳性标本总数从159株/a逐年增加至396株/a.其中,白念珠菌、近平滑念珠菌、曲霉的构成比波动范围分别为34.6％ ～ 50.3％、6.3％～9.6％、1.3％～3.2％,2010 ～2012年较2007 ～2009年构成比总体均有上升趋势,P=0.02.热带念珠菌、光滑念珠菌、克柔念珠菌构成比波动范围分别为13.6％ ～ 17.3％、9.4％ ～ 22.6％、1.4％ ～5.2％,P=0.18,构成比均无明显上升或下降趋势.2010 ～2012年较2007 ～2009年,白念珠菌对氟康唑和伊曲康唑耐药率的变化均呈降低趋势,P=0.02.热带念珠菌对氟康唑和伊曲康唑耐药率的变化均呈降低趋势,P =0.02;光滑念珠菌对伊曲康唑耐药率的变化总体呈上升趋势,P =0.02.结论 我院ICU近6 a来白念珠菌仍然为临床最常见的真菌分离株,白念珠菌、近平滑念珠菌及曲霉占总真菌分离株的百分比有上升趋势.白念珠菌及热带念珠菌对氟康唑和伊曲康唑耐药率均呈降低趋势,光滑念珠菌对伊曲康唑耐药率呈上升趋势.     

Study of acute vulvovaginitis in sexually active adult women, with special reference to candidosis, in patients of the Francisco J. Muñiz Infectious Diseases Hospital

The results of microbiological vaginal secretions samples obtained from 749 women (from July 2001 to July 2002) were studied in the Bacteriology Unit of the Francisco Javier Mu?iz Hospital from Buenos Aires. All patients suffered acute vulvovaginitis were child bearing and sexually active women, 334 of them were HIV-positive. The following are the results of the microbiological studies: Lactobacillus spp 50.6%, Gardnerella vaginalis 25.6%, Candida spp 17.4%, Trichomonas vaginalis 5.3%, Neisseria gonorrhoeae 0.3% and B group Streptococcus 0.8%. Candida vaginitis was significantly more frequent in HIV-positive patients, (21.6% vs 14%; p = 0.0086); meanwhile, trichomoniasis was less common although the difference was not statistically significant (3.6 vs 6.7%, p = 0.0810). The following Candida species were isolated in this study: Candida albicans 76.8%, Candida glabrata 15.6%, Candida parapsilosis 2.9%, Candida tropicalis 1.5% and Candida krusei 0.7%. Eight cases (6.2%) of vaginitis were produced by two Candida species (C. albicans and C. glabrata), and in three cases (2.17%) Saccharomyces cerevisiae were isolated. Five women suffering acute vaginitis with Candida spp presented another etiologic agent of vaginal infection, three cases T. vaginalis and two cases G. vaginalis. The following are some of the most important findings of this study: 1) Half of the patients presented a normal microbial biota; 2) Candida spp vaginitis was significantly more frequent among HIV-positive women; 3) we observed a high incidence of Candida glabrata infections (15.9%), 4) 6.2% of vaginal candidiasis were caused by more than one Candida species and, 5) the susceptibility pattern of C. albicans and C. glabrata isolates against fluconazole was similar to the one observed in other studies. The majority of C. albicans isolates were susceptible to fluconazole (MIC90 = 0.5 microg/ml) meanwhile C. glabrata strains were much less susceptible to this drug (MIC50 and MIC90 = 32 microg/ml).     

Characterization of Candida Species from Different Populations in Taiwan

The opportunistic Candida species existing as part of commensal microbiota in humans are usually the etiological agents causing infections. We investigated whether isolates collected from different age groups, hospital units, and sources have distinct characteristics. A total of 913 isolates comprising 395 Candida albicans, 230 Candida tropicalis, 202 Candida glabrata, 62 Candida parapsilosis, 13 Candida krusei, and 11 of other six species were analyzed. Urine was the most common source (41.2%), followed by sputum (16.3%), blood (15.2%), and others (27.3%). Candida albicans and C. parapsilosis were more prevalent in the working group [from 19 to 65 years], whereas C. tropicalis and C. glabrata were more prevalent in the elder one (≥ 66 years). We found that the age of patients and the source of isolates affect the distribution of species. On the other hand, the drug susceptibility of isolates was associated with fungal species and whether patients were hospitalized.     

Calcineurin is essential for survival during membrane stress in Candida albicans

The immunosuppressants cyclosporin A (CsA) and FK506 inhibit the protein phosphatase calcineurin and block T-cell activation and transplant rejection. Calcineurin is conserved in microorganisms and plays a general role in stress survival. CsA and FK506 are toxic to several fungi, but the common human fungal pathogen Candida albicans is resistant. However, combination of either CsA or FK506 with the antifungal drug fluconazole that perturbs synthesis of the membrane lipid ergosterol results in potent, synergistic fungicidal activity. Here we show that the C.albicans FK506 binding protein FKBP12 homolog is required for FK506 synergistic action with fluconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance (CNB1-1/CNB1) abolished FK506-fluconazole synergism. Candida albicans mutants lacking calcineurin B (cnb1/cnb1) were found to be viable and markedly hypersensitive to fluconazole or membrane perturbation with SDS. FK506 was synergistic with fluconazole against azole-resistant C.albicans mutants, against other Candida species, or when combined with different azoles. We propose that calcineurin is part of a membrane stress survival pathway that could be targeted for therapy.     

Multicenter survey of in vitro antifungal resistance in yeasts of medical importance isolated from Spanish patients

Twelve Spanish laboratories collected 325 yeast clinical isolates during a 30 day's period, among them 224 Candida albicans, 30 Candida glabrata, and 27 Candida parapsilosis. In vitro antifungal susceptibility to amphotericin B, ketoconazole, fluconazole and itraconazole was determined by an agar diffusion test (Neo-Sensitabs, Rosco, Denmark). All the isolates tested were susceptible in vitroto amphotericin B and nearly all (97.2%) to itraconazole. In vitrosusceptibility to fluconazole and ketoconazole was high (90.2% and 91.4% of isolates, respectively) but showed variations depending on the species tested. Resistance to fluconazole and ketoconazole was low in C. albicans (4% and 3%, respectively), but 30% of Candida guilliermondii and 36% of C. glabrata isolates were resistant to fluconazole. Ketoconazole resistance was observed in 40% of C. glabrata, and 17% of Candida tropicalis. Resistance to antifungal drugs is very low in Spain and it is related to non-C. albicans isolates.     

The Hsp90 Co-Chaperone Sgt1 Governs Candida albicans Morphogenesis and Drug Resistance

The molecular chaperone Hsp90 orchestrates regulatory circuitry governing fungal morphogenesis, biofilm development, drug resistance, and virulence. Hsp90 functions in concert with co-chaperones to regulate stability and activation of client proteins, many of which are signal transducers. Here, we characterize the first Hsp90 co-chaperone in the leading human fungal pathogen, Candida albicans. We demonstrate that Sgt1 physically interacts with Hsp90, and that it governs C. albicans morphogenesis and drug resistance. Genetic depletion of Sgt1 phenocopies depletion of Hsp90, inducing yeast to filament morphogenesis and invasive growth. Sgt1 governs these traits by bridging two morphogenetic regulators: Hsp90 and the adenylyl cyclase of the cAMP-PKA signaling cascade, Cyr1. Sgt1 physically interacts with Cyr1, and depletion of either Sgt1 or Hsp90 activates cAMP-PKA signaling, revealing the elusive link between Hsp90 and the PKA signaling cascade. Sgt1 also mediates tolerance and resistance to the two most widely deployed classes of antifungal drugs, azoles and echinocandins. Depletion of Sgt1 abrogates basal tolerance and acquired resistance to azoles, which target the cell membrane. Depletion of Sgt1 also abrogates tolerance and resistance to echinocandins, which target the cell wall, and renders echinocandins fungicidal. Though Sgt1 and Hsp90 have a conserved impact on drug resistance, the underlying mechanisms are distinct. Depletion of Hsp90 destabilizes the client protein calcineurin, thereby blocking crucial responses to drug-induced stress; in contrast, depletion of Sgt1 does not destabilize calcineurin, but blocks calcineurin activation in response to drug-induced stress. Sgt1 influences not only morphogenesis and drug resistance, but also virulence, as genetic depletion of C. albicans Sgt1 leads to reduced kidney fungal burden in a murine model of systemic infection. Thus, our characterization of the first Hsp90 co-chaperone in a fungal pathogen establishes C. albicans Sgt1 as a global regulator of morphogenesis and drug resistance, providing a new target for treatment of life-threatening fungal infections.     

Comparison of disk diffusion test and Etest for voriconazole and fluconazole susceptibility testing

The distribution for voriconazole and fluconazole susceptibility was determined by Etest and disk diffusion test in 143 clinical isolates. The majority of the strains of Aspergillus spp., Candida krusei, C. inconspicua, C norvegensis and Saccharomyces cerevisiae displayed resistance or decreased susceptibility to fluconazole in contrast to voriconazole. The absolute categorical agreement for voriconazole and fluconazole susceptibility results by the disk method and Etest was 90.5 and 74.8 % respectively. The error rate bounding analysis showed only 0.7 % of false susceptible results ( very major error) with voriconazole, but 2.8 % with fluconazole. Fluconazole can be used as a surrogate factor to predict voriconazole susceptibility but with lower reliability for susceptible-dose dependent and resistance category, especially in Candida glabrata isolates. The results of the disk method were not substantially influenced by the composition of media (Mueller-Hinton agar vs antimycotic Sensitivity Test agar), even if with the latter the results had fewer tendencies to produce false susceptibility of C.glabrata isolates to both of the triazole drugs. Disk test as well as Etest were shown to represent suitable methods for routine evaluation of susceptibility of clinical isolates of pathogenic fungi, including aspergilli, to fluconazole and voriconazole.     

临床分离161株念珠菌菌种鉴定及氟康唑药敏试验分析

目的调查临床分离的念珠菌种类及其对氟康唑的敏感性。方法采用科玛嘉念珠菌显色培养基和YBC平板对山东大学齐鲁医院细菌室分离到的161株念珠菌进行鉴定,并对分离出的白念珠菌分别采用NCCLS推荐的微量稀释法和ROSCO药敏纸片法对氟康唑进行药物敏感性试验。结果在161株念珠菌中白念珠菌为69．57％,热带念珠菌为19．88％,近平滑念珠菌为4．97％,克柔念珠菌为2．48％,光滑念珠菌为1．86％,其他念珠菌为1．24％。两种方法药敏结果显示：112株白念珠菌对氟康唑的敏感率分别为96．43％和97．32％,仅有2株耐药。结论白念珠菌仍然是我院分离率最高的念珠菌,其次是热带念珠菌和近平滑念珠菌。白念珠菌对氟康唑仍敏感,耐药菌株极少。     

Extracellular ATP activates the PLC/PKC/ERK signaling pathway through the P2Y2 purinergic receptor leading to the induction of early growth response 1 expression and the inhibition of viability in human endometrial stromal cells

ATP is an extracellular signaling molecule that activates specific G protein-coupled P2Y receptors in most cell types to mediate diverse biological effects. ATP has been shown to activate the phospholipase C (PLC)/diacylglycerol/protein kinase C (PKC) pathway in various systems. However, little is known about the signaling events in human endometrial stromal cells (hESCs). The objective of this study was to examine the presence of the P2Y2 receptor and the effects of exogenous ATP on the intracellular mitogen-activated protein kinases (MAPKs) signaling pathway, immediate early genes expression, and cell viability in hESCs. Western blot analysis, gene array analysis, and MTT assay for cell viability were performed. The current study demonstrated the existence of the P2Y2 purinergic receptor in hESCs. UTP and ATP activated MAPK in a dose- and time-dependent manner. Suramin (a P2-purinoceptor antagonist), neomycin (a PLC inhibitor), staurosporin (a PKC inhibitor), and PD98059 (a MEK inhibitor) significantly attenuated the ATP-induced activation of MAPK. ATP activated ERK1/2 and induced translocation of activated ERK1/2 to the nucleus. The gene array for 23 genes associated with members of the mitogenic pathway cascade and immediate early genes revealed that the expression of early growth response 1 was increased. In addition, MTT assay revealed an inhibition effect of ATP on cell viability. ATP activated MAPKs through the P2Y2 purinoceptor/PLC/PKC/ERK signaling pathway and induced translocation of ERK1/2 into the nucleus. Further, ATP induced the expression of early growth response 1 and inhibited cell viability in hESCs.