RESUMEN
Traditional approaches to drug discovery are frustratingly inefficient and have several key limitations that severely constrain our capacity to rapidly identify and develop novel experimental therapeutics. To address this, we have devised a second-generation target-based whole-cell screening assay based on the principles of competitive fitness, which can rapidly identify target-specific and physiologically active compounds. Briefly, strains expressing high, intermediate, and low levels of a preselected target protein are constructed, tagged with spectrally distinct fluorescent proteins (FPs), and pooled. The pooled strains are then grown in the presence of various small molecules, and the relative growth of each strain within the mixed culture is compared by measuring the intensity of the corresponding FP tags. Chemical-induced population shifts indicate that the bioactivity of a small molecule is dependent upon the target protein's abundance and thus establish a specific functional interaction. Here, we describe the molecular tools required to apply this technique in the prevalent human fungal pathogen Candida albicans and validate the approach using two well-characterized drug targets-lanosterol demethylase and dihydrofolate reductase. However, our approach, which we have termed target abundance-based fitness screening (TAFiS), should be applicable to a wide array of molecular targets and in essentially any genetically tractable microbe. IMPORTANCE Conventional drug screening typically employs either target-based or cell-based approaches. The first group relies on biochemical assays to detect modulators of a purified target. However, hits frequently lack drug-like characteristics such as membrane permeability and target specificity. Cell-based screens identify compounds that induce a desired phenotype, but the target is unknown, which severely restricts further development and optimization. To address these issues, we have developed a second-generation target-based whole-cell screening approach that incorporates the principles of both chemical genetics and competitive fitness, which enables the identification of target-specific and physiologically active compounds from a single screen. We have chosen to validate this approach using the important human fungal pathogen Candida albicans with the intention of pursuing novel antifungal targets. However, this approach is broadly applicable and is expected to dramatically reduce the time and resources required to progress from screening hit to lead compound.
RESUMEN
The fungal vacuole is a large acidified organelle that performs a variety of cellular functions. At least a sub-set of these functions are crucial for pathogenic species of fungi, such as Candida albicans, to survive within and invade mammalian tissue as mutants with severe defects in vacuolar biogenesis are avirulent. We therefore sought to identify chemical probes that disrupt the normal function and/or integrity of the fungal vacuole to provide tools for the functional analysis of this organelle as well as potential experimental therapeutics. A convenient indicator of vacuolar integrity based upon the intracellular accumulation of an endogenously produced pigment was adapted to identify Vacuole Disrupting chemical Agents (VDAs). Several chemical libraries were screened and a set of 29 compounds demonstrated to reproducibly cause loss of pigmentation, including 9 azole antifungals, a statin and 3 NSAIDs. Quantitative analysis of vacuolar morphology revealed that (excluding the azoles) a sub-set of 14 VDAs significantly alter vacuolar number, size and/or shape. Many C. albicans mutants with impaired vacuolar function are deficient in the formation of hyphal elements, a process essential for its pathogenicity. Accordingly, all 14 VDAs negatively impact C. albicans hyphal morphogenesis. Fungal selectivity was observed for approximately half of the VDA compounds identified, since they did not alter the morphology of the equivalent mammalian organelle, the lysosome. Collectively, these compounds comprise of a new collection of chemical probes that directly or indirectly perturb normal vacuolar function in C. albicans.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Vacuolas/efectos de los fármacos , Antifúngicos/química , Candida albicans/genética , Candida albicans/fisiología , Línea Celular , Colorantes , Cumarinas , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Mutación , Pigmentación/genética , Pigmentos Biológicos/genética , Pigmentos Biológicos/metabolismo , Vacuolas/fisiología , Vacuolas/ultraestructuraRESUMEN
Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/patogenicidad , Candidiasis Invasiva/patología , Candidiasis Vulvovaginal/patología , Morfolinas/farmacología , Oxidorreductasas/genética , Esteroide Isomerasas/genética , Vacuolas/fisiología , Animales , Candida albicans/efectos de los fármacos , Candidiasis Invasiva/microbiología , Candidiasis Vulvovaginal/microbiología , Catepsina A/metabolismo , Farmacorresistencia Fúngica/genética , Ergosterol/biosíntesis , Ergosterol/genética , Femenino , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/genética , Hifa/genética , Hifa/crecimiento & desarrollo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Oxidorreductasas/antagonistas & inhibidores , Esteroide Isomerasas/antagonistas & inhibidores , Vacuolas/efectos de los fármacosRESUMEN
The azole antifungals block ergosterol biosynthesis by inhibiting lanosterol demethylase (Erg11p). The resulting depletion of cellular ergosterol and the accumulation of "toxic" sterol intermediates are both thought to compromise plasma membrane function. However, the effects of ergosterol depletion upon the function of intracellular membranes and organelles are not well described. The purpose of this study was to characterize the effects of azole treatment upon the integrity of the Candida albicans vacuole and to determine whether, in turn, vacuolar trafficking influences azole susceptibility. Profound fragmentation of the C. albicans vacuole can be observed as an early consequence of azole treatment, and it precedes significant growth inhibition. In addition, a C. albicans vps21Δ/Δ mutant, blocked in membrane trafficking through the late endosomal prevacuolar compartment (PVC), is able to grow significantly more than the wild type in the presence of several azole antifungals under standard susceptibility testing conditions. Furthermore, the vps21Δ/Δ mutant is able to grow despite the depletion of cellular ergosterol. This phenotype resembles an exaggerated form of "trailing growth" that has been described for some clinical isolates. In contrast, the vps21Δ/Δ mutant is hypersensitive to drugs that block alternate steps in ergosterol biosynthesis. On the basis of these results, we propose that endosomal trafficking defects may lead to the cellular "redistribution" of the sterol intermediates that accumulate following inhibition of ergosterol biosynthesis. Furthermore, the destination of these intermediates, or the precise cellular compartments in which they accumulate, may be an important determinant of their toxicity and thus ultimately antifungal efficacy.
Asunto(s)
Antifúngicos/metabolismo , Antifúngicos/farmacología , Azoles/metabolismo , Azoles/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/metabolismo , Endosomas/metabolismo , Farmacorresistencia Fúngica , Ergosterol/metabolismo , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Vacuolas/efectos de los fármacosRESUMEN
Vulvovaginal candidiasis (VVC), caused by Candida albicans, affects women worldwide. Animal and clinical studies suggest that the immunopathogenic inflammatory condition of VVC is initiated by S100 alarmins in response to C. albicans, which stimulate polymorphonuclear neutrophil (PMN) migration to the vagina. The purpose of this study was to extend previous in vitro data and determine the requirement for the alarmin S100A8 in the PMN response and to evaluate pattern recognition receptors (PRRs) that initiate the response. For the former, PMN migration was evaluated in vitro or in vivo in the presence or absence of S100 alarmins initiated by several approaches. For the latter, vaginal epithelial cells were evaluated for PRR expression and C. albicans-induced S100A8 and S100A9 mRNAs, followed by evaluation of the PMN response in inoculated PRR-deficient mice. Results revealed that, consistent with previously reported in vitro data, eukaryote-derived S100A8, but not prokaryote-derived recombinant S100A8, induced significant PMN chemotaxis in vivo. Conversely, a lack of biologically active S100A8 alarmin, achieved by antibody neutralization or by using S100A9(-/-) mice, had no effect on the PMN response in vivo. In PRR analyses, whereas Toll-like receptor 4 (TLR4)- and SIGNR1-deficient vaginal epithelial cells showed a dramatic reduction in C. albicans-induced S100A8/S100A9 mRNAs in vitro, inoculated mice deficient in these PRRs showed PMN migration similar to that in wild-type controls. These results suggest that S100A8 alarmin is sufficient, but not necessary, to induce PMN migration during VVC and that the vaginal PMN response to C. albicans involves PRRs in addition to SIGNR1 and TLR4, or other induction pathways.
Asunto(s)
Calgranulina A/metabolismo , Candida albicans/inmunología , Candidiasis Vulvovaginal/inmunología , Movimiento Celular/efectos de los fármacos , Células Epiteliales/inmunología , Neutrófilos/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Animales , Calgranulina A/genética , Calgranulina B/genética , Calgranulina B/metabolismo , Candidiasis Vulvovaginal/microbiología , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Ratones , Ratones Noqueados , Neutrófilos/efectos de los fármacosRESUMEN
Gene knockout and transgenic mice are important tools that are widely used to dissect the mammalian hosts' responses to microbial invasion. A novel alternative is to engineer the pathogen itself to secrete host factors that stimulate or suppress specific immune defense mechanisms. Herein, we have described and validated an approach to facilitate the production and export of ectopic host proteins, from the most prevalent human fungal pathogen, Candida albicans. Our strategy utilized a prepropeptide from the C. albicans secreted aspartic proteinase, Sap2p. The prepeptide facilitates entry of Sap2p into the secretory pathway, while the propeptide maintains the protease as an inactive precursor, until proteolytic cleavage in the Golgi apparatus releases the mature protein. The Sap2p prepropeptide coding sequence was linked to that of two mammalian calcium-binding proteins, S100A8 and S100A9, which are associated with symptomatic vaginal candidiasis. The resulting expression constructs were then introduced into C. albicans. While the S100A8 protein is secreted into the growth medium intact, the S100A9 protein is apparently degraded during transit. Nonetheless, culture supernatants from both S100A8 and S100A9 expressing C. albicans strains acted as potent chemoattractants for a macrophage-like cell line and polymorphonuclear leukocytes. Thus, the pathogen-derived mammalian proteins possessed the expected biological activity.
Asunto(s)
Candida albicans/genética , Factores Inmunológicos/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/metabolismo , Animales , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Secuencia de Bases , Calgranulina A/genética , Calgranulina A/metabolismo , Calgranulina B/genética , Calgranulina B/metabolismo , Candida albicans/metabolismo , Candida albicans/fisiología , Línea Celular , Quimiotaxis/efectos de los fármacos , Femenino , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Factores Inmunológicos/genética , Factores Inmunológicos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Disruption of vacuolar biogenesis in the pathogenic yeast Candida albicans causes profound defects in polarized hyphal growth. However, the precise vacuolar pathways involved in yeast-hypha differentiation have not been determined. Previously we focused on Vps21p, a Rab GTPase involved in directing vacuolar trafficking through the late endosomal prevacuolar compartment (PVC). Herein, we identify two additional Vps21p-related GTPases, Ypt52p and Ypt53p, that colocalize with Vps21p and can suppress the hyphal defects of the vps21Δ/Δ mutant. Phenotypic analysis of gene deletion strains revealed that loss of both VPS21 and YPT52 causes synthetic defects in endocytic trafficking to the vacuole, as well as delivery of the virulence-associated vacuolar membrane protein Mlt1p from the Golgi compartment. Transcription of all three GTPase-encoding genes is increased under hyphal growth conditions, and overexpression of the transcription factor Ume6p is sufficient to increase the transcription of these genes. While only the vps21Δ/Δ single mutant has hyphal growth defects, these were greatly exacerbated in a vps21Δ/Δ ypt52Δ/Δ double mutant. On the basis of relative expression levels and phenotypic analysis of gene deletion strains, Vps21p is the most important of the three GTPases, followed by Ypt52p, while Ypt53p has an only marginal impact on C. albicans physiology. Finally, disruption of a nonendosomal AP-3-dependent vacuolar trafficking pathway in the vps21Δ/Δ ypt52Δ/Δ mutant, further exacerbated the stress and hyphal growth defects. These findings underscore the importance of membrane trafficking through the PVC in sustaining the invasive hyphal growth form of C. albicans.
Asunto(s)
Candida albicans/enzimología , Candida albicans/crecimiento & desarrollo , Proteínas Fúngicas/metabolismo , Hifa/enzimología , Hifa/crecimiento & desarrollo , Vacuolas/enzimología , Proteínas de Unión al GTP rab/metabolismo , Candida albicans/citología , Compartimento Celular , Endocitosis , Endosomas/metabolismo , Proteínas Fúngicas/química , Mutación/genética , Fenotipo , Conformación Proteica , Transporte de Proteínas , Estrés Fisiológico , Proteínas de Unión al GTP rab/químicaRESUMEN
Extracellular matrix factors within the tumor microenvironment that control resistance to chemotherapeutics are poorly understood. This study focused on understanding matrix adhesion pathways that control the oral carcinoma response to cisplatin. Our studies revealed that adhesion of HN12 and JHU012 oral carcinomas to carcinoma matrix supported tumor cell proliferation in response to treatment with cisplatin. Proliferation in response to 30 µM cisplatin was not observed in HN12 cells adherent to other purified extracellular matrices such as Matrigel, collagen I, fibronectin or laminin I. Integrin ß1 was important for adhesion to carcinoma matrix to trigger proliferation after treatment with cisplatin. Disruption of talin expression in HN12 cells adherent to carcinoma matrix increased cisplatin induced proliferation. Pharmacological inhibitors were used to determine signaling events required for talin deficiency to regulate cisplatin induced proliferation. Pharmacological inhibition of NF-kB reduced proliferation of talin-deficient HN12 cells treated with 30 µM cisplatin. Nuclear NF-kB activity was assayed in HN12 cells using a luciferase reporter of NF-kB transcriptional activity. Nuclear NF-kB activity was similar in HN12 cells adherent to carcinoma matrix and collagen I when treated with vehicle DMSO. Following treatment with 30 µM cisplatin, NF-kB activity is maintained in cells adherent to carcinoma matrix whereas NF-kB activity is reduced in collagen I adherent cells. Expression of talin was sufficient to trigger proliferation of HN12 cells adherent to collagen I following treatment with 1 and 30 µM cisplatin. Talin overexpression was sufficient to trigger NF-kB activity following treatment with cisplatin in carcinoma matrix adherent HN12 cells in a process disrupted by FAK siRNA. Thus, adhesions within the carcinoma matrix create a matrix environment in which exposure to cisplatin induces proliferation through the function of integrin ß1, talin and FAK pathways that regulate NF-kB nuclear activity.
Asunto(s)
Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Matriz Extracelular/metabolismo , Neoplasias de la Boca/metabolismo , FN-kappa B/metabolismo , Talina/metabolismo , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Uniones Célula-Matriz/efectos de los fármacos , Uniones Célula-Matriz/metabolismo , Proteína Sustrato Asociada a CrK/metabolismo , Matriz Extracelular/efectos de los fármacos , Humanos , Integrina beta1/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Modelos Biológicos , Neoplasias de la Boca/patología , Transducción de Señal/efectos de los fármacosRESUMEN
The vacuole has crucial roles in stress resistance and adaptation of the fungal cell. Furthermore, in Candida albicans it has been observed to undergo dramatic expansion during the initiation of hyphal growth, to produce highly "vacuolated" subapical compartments. We hypothesized that these functions may be crucial for survival within the host and tissue-invasive hyphal growth. We also considered the role of the late endosome or prevacuole compartment (PVC), a distinct organelle involved in vacuolar and endocytic trafficking. We identified two Rab GTPases, encoded by VPS21 and YPT72, required for trafficking through the PVC and vacuole biogenesis, respectively. Deletion of VPS21 or YPT72 led to mild sensitivities to some cellular stresses. However, deletion of both genes resulted in a synthetic phenotype with severe sensitivity to cellular stress and impaired growth. Both the vps21Delta and ypt72Delta mutants had defects in filamentous growth, while the double mutant was completely deficient in polarized growth. The defects in hyphal growth were not suppressed by an "active" RIM101 allele or loss of the hyphal repressor encoded by TUP1. In addition, both single mutants had significant attenuation in a mouse model of hematogenously disseminated candidiasis, while the double mutant was rapidly cleared. Histological examination confirmed that the vps21Delta and ypt72Delta mutants are deficient in hyphal growth in vivo. We suggest that the PVC and vacuole are required on two levels during C. albicans infection: (i) stress resistance functions required for survival within tissue and (ii) a role in filamentous growth which may aid host tissue invasion.
Asunto(s)
Candida albicans/crecimiento & desarrollo , Candida albicans/patogenicidad , Endosomas/enzimología , GTP Fosfohidrolasas/metabolismo , Vacuolas/enzimología , Animales , Candidiasis/microbiología , GTP Fosfohidrolasas/genética , Eliminación de Gen , Hifa/crecimiento & desarrollo , Ratones , Ratones Endogámicos BALB C , VirulenciaRESUMEN
BACKGROUND: Putative high mutation rates of RNA viruses are believed to mediate undesirable phenomena, such as emergence of drug resistance. However, very little is known about biochemical fidelity rates for viral RNA-dependent RNA polymerases. Using a recently developed in vitro polymerase assay for poliovirus polymerase 3Dpol [Arnold and Cameron (2000) JBC 275:5329], we measured fidelity for each possible mismatch. Polymerase fidelity, in contrast to sequence error rate, is biochemically defined as kpol/Kd of {(correct plus incorrect) divided by incorrect} incorporations, such that a larger value connotes higher fidelity. RESULTS: To derive kpol/Kd for correct base incorporation, we performed conventional pre-steady state single turnover measurements, yielding values that range from 0.62 to 9.4 microM-1 sec-1. Pre-steady state measurements for incorrect base incorporation were less straightforward: several anomalous phenomena interfered with data collection. To obtain pre-steady state kinetic data for incorrect base incorporation, three strategies were employed. (1) For some incorrect bases, a conventional approach was feasible, although care was taken to ensure that only single turnovers were being assessed. (2) Heparin or unlabeled RNA traps were used to simulate single turnover conditions. (3) Finally, for some incorrect bases, incorporation was so poor that single datapoints were used to provide kinetic estimates. Overall, we found that fidelity for poliovirus polymerase 3Dpol ranges from 1.2 x 10(4) to 1.0 x 10(6) for transition mutations and 3.2 x 10(5) to 4.3 x 10(7) for transversion mutations. CONCLUSION: These values are unexpectedly high showing that high RNA virus sequence variation is not due to intrinsically low polymerase fidelity. Based on unusual enzyme behavior that we observed, we speculate that RNA mismatches either directly or indirectly cause enzyme RNA dissociation. If so, high sequence variation of RNA viruses may be due to template-switch RNA recombination and/or unknown fitness/selection phenomena. These findings may lead to a mechanistic understanding of RNA virus error catastrophe and improved anti-viral strategies.
Asunto(s)
Poliovirus/enzimología , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Secuencia de Bases , Simulación por Computador , ADN Viral/genética , Cinética , Mutación , Poliovirus/genética , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/química , Proteínas Virales/metabolismoRESUMEN
Nucleic acid polymerases have similar structures and motifs. The function of an aspartic acid (conserved in all classes of nucleic acid polymerases) in motif A remains poorly understood in RNA-dependent RNA polymerases. We mutated this residue to alanine in a poliovirus replicon. The resulting mutant could still replicate, although at a reduced level. In addition, mutation A231C (also in motif A) yielded high levels of replication. Taken together these results show that poliovirus polymerase conserved residues D233 and A231 are not essential to poliovirus replicon function.