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Scott G Filler

Publications and source records attributed to Scott G Filler.

35 records · Page 2Linked to original sources
PubMed2004

Functional and structural diversity in the Als protein family of Candida albicans.

The human fungal pathogen Candida albicans colonizes and invades a wide range of host tissues. Adherence to host constituents plays an important role in this process. Two members of the C. albicans Als protein family (Als1p and Als5p) have been found to mediate adherence; however, the functions of other members of this family are unknown. In this study, members of the ALS gene family were cloned and expressed in Saccharomyces cerevisiae to characterize their individual functions. Distinct Als proteins conferred distinct adherence profiles to diverse host substrates. Using chimeric Als5p-Als6p constructs, the regions mediating substrate-specific adherence were localized to the N-terminal domains in Als proteins. Interestingly, a subset of Als proteins also mediated endothelial cell invasion, a previously unknown function of this family. Consistent with these results, homology modeling revealed that Als members contain anti-parallel beta-sheet motifs interposed by extended regions, homologous to adhesins or invasins of the immunoglobulin superfamily. This finding was confirmed using circular dichroism and Fourier transform infrared spectrometric analysis of the N-terminal domain of Als1p. Specific regions of amino acid hypervariability were found among the N-terminal domains of Als proteins, and energy-based models predicted similarities and differences in the N-terminal domains that probably govern the diverse function of Als family members. Collectively, these results indicate that the structural and functional diversity within the Als family provides C. albicans with an array of cell wall proteins capable of recognizing and interacting with a wide range of host constituents during infection.

Amino Acid Motifs↗
PubMed2004

Functional analysis of the Candida albicans ALS1 gene product.

ALS1 encodes a cell surface protein that mediates adherence of Candida albicans to endothelial cells. The predicted Als1p has an N-terminal region, which contains a signal peptide; a middle region, which contains 20 36-amino acid tandem repeats; and a C-terminal region, which contains a glycosylphosphotidylinositol-anchorage sequence. We used site-directed mutagenesis to delineate the regions in Als1p required for endothelial cell adherence and cell surface expression of the protein. Mutant alleles of ALS1 containing either deletions or insertions were expressed in the normally non-adherent Saccharomyces cerevisiae. These transformants were analysed for endothelial cell adherence and cell surface expression of Als1p. We found that mutations centred around amino acid 285 in the N-terminus completely abolished adherence, but had no effect on cell surface expression of Als1p. Deletion of 15 of the tandem repeats reduced adherence by 50%, whereas deletion of all abolished adherence completely, even though cell surface expression of the N-terminus of Als1p was maintained. Insertions into the C-terminus at amino acids 413 and 254 upstream of the stop codon resulted in a modest loss of adherence, while cell surface expression of Als1p was maintained. An insertion at amino acid 249 in the C-terminus caused complete loss of both adherence and cell surface expression, even though the glycosylphosphotidylinositol-anchorage sequence remained intact. These data suggest a model of Als1p in which the endothelial cell binding region is localized within its N-terminus, the tandem repeats are essential for the proper presentation of the binding site, and the C-terminus is required for localizing Als1p to the cell surface.

Antibodies, Blocking↗
PubMed2004

Enantioselectivity of inhibition of cytochrome P450 3A4 (CYP3A4) by ketoconazole: Testosterone and methadone as substrates.

Racemic ketoconazole (KTZ) was the first orally active azole antifungal agent used in clinical practice and has become widely used in the treatment of mucosal fungal infections associated with AIDS immunosuppression and cancer chemotherapy. However, the use of KTZ has been limited because of adverse drug-drug interactions. KTZ blocks ergosterol biosynthesis by inhibiting the fungal cytochrome P450 (CYP51). KTZ is also a potent inhibitor of human cytochrome P450 3A4 (CYP3A4) enzyme, the major drug-metabolizing CYP isozyme in the human liver. We examined the enantioselective differences of KTZ in the inhibition of human CYP3A4 and in antifungal action. Dextro- and levo-KTZ exhibited modest enantioselective differences with respect to CYP3A4 inhibition of testosterone and methadone metabolism. For both substrates levo-KTZ was approximately a 2-fold more potent inhibitor. We examined the enantioselective differences in the in vitro activity of KTZ against medically relevant species of Candida and Aspergillus, as well as Cryptococcus neoformans. Overall, levo-KTZ was 2-4-fold more active than dextro-KTZ. Therefore, levo-KTZ is a more potent inhibitor of CYP3A4 and has stronger in vitro antifungal activity. Chirality 16:79-85, 2004.

Antifungal Agents↗
PubMed2004

Novel inhalational murine model of invasive pulmonary aspergillosis.

We developed a novel model of invasive aspergillosis (IA) that recapitulates human disease. Mice were immunosuppressed with cyclophosphamide and cortisone acetate and then infected in an aerosol chamber. This procedure reproducibly delivered 1 x 10(3) to 3 x 10(3) conidia to the lungs. Lethal pulmonary IA developed over 2 weeks and was prevented by amphotericin B.

Amphotericin B↗
PubMed2004

Relationship between Candida albicans virulence during experimental hematogenously disseminated infection and endothelial cell damage in vitro.

Candida albicans must penetrate the endothelial cell lining of the vasculature to invade the deep tissues during a hematogenously disseminated infection. We compared 27 C. albicans mutants with their wild-type parent for their capacity to damage endothelial cells in vitro and cause a lethal infection in mice following tail vein inoculation. Of 10 mutants with significantly impaired capacity to damage endothelial cells, all had attenuated virulence. Therefore, the endothelial cell damage assay can be used as a screen to identify some virulence factors relevant to hematogenously disseminated candidiasis.

Animals↗
PubMed2003

Interactions of Aspergillus fumigatus with endothelial cells: internalization, injury, and stimulation of tissue factor activity.

Invasive aspergillosis causes significant mortality among patients with hematologic malignancies. This infection is characterized by vascular invasion and thrombosis. To study the pathogenesis of invasive aspergillosis, we investigated the interactions of Aspergillus fumigatus conidia and hyphae with endothelial cells in vitro. We found that both forms of the organism induced endothelial cell microfilament rearrangement and subsequent endocytosis. Conidia were endocytosed 2-fold more avidly than hyphae, and endocytosis was independent of fungal viability. Endocytosed conidia and hyphae caused progressive endothelial cell injury after 4 hours of infection. Live conidia induced more endothelial cell injury than did live hyphae. However, endothelial cell injury caused by conidia was dependent on fungal viability, whereas injury caused by hyphae was not, indicating that conidia and hyphae injure endothelial cells by different mechanisms. Neither live nor killed conidia increased tissue factor activity of endothelial cells. In contrast, both live and killed hyphae stimulated significant endothelial cell tissue factor activity, as well as the expression of tissue factor antigen on the endothelial cell surface. These results suggest that angioinvasion and thrombosis caused by A fumigatus hyphae in vivo may be due in part to endothelial cell invasion, induction of injury, and stimulation of tissue factor activity.

Actin Cytoskeleton↗
PubMed2003

Genetic basis for differential activities of fluconazole and voriconazole against Candida krusei.

Invasive infections caused by Candida krusei are a significant concern because this organism is intrinsically resistant to fluconazole. Voriconazole is more active than fluconazole against C. krusei in vitro. One mechanism of fluconazole resistance in C. krusei is diminished sensitivity of the target enzyme, cytochrome P450 sterol 14alpha-demethylase (CYP51), to inhibition by this drug. We investigated the interactions of fluconazole and voriconazole with the CYP51s of C. krusei (ckCYP51) and fluconazole-susceptible Candida albicans (caCYP51). We found that voriconazole was a more potent inhibitor of both ckCYP51 and caCYP51 in cell extracts than was fluconazole. Also, the ckCYP51 was less sensitive to inhibition by both drugs than was caCYP51. These results were confirmed by expressing the CYP51 genes from C. krusei and C. albicans in Saccharomyces cerevisiae and determining the susceptibility of the transformants to voriconazole and fluconazole. We constructed homology models of the CYP51s of C. albicans and C. krusei based on the crystal structure of CYP51 from Mycobacterium tuberculosis. These models predicted that voriconazole is a more potent inhibitor of both caCYP51 and ckCYP51 than is fluconazole, because the extra methyl group of voriconazole results in a stronger hydrophobic interaction with the aromatic amino acids in the substrate binding site and more extensive filling of this site. Although there are multiple differences in the predicted amino acid sequence of caCYP51 and ckCYP51, the models of the two enzymes were quite similar and the mechanism for the relative resistance of ckCYP51 to the azoles was not apparent.

Amino Acid Sequence↗
PubMed2003

Calcineurin is essential for Candida albicans survival in serum and virulence.

Calcineurin is a calcium-activated protein phosphatase that is the target of the immunosuppressants cyclosporin A and FK506. In T cells, calcineurin controls nuclear import of the NF-AT transcription factor and gene activation. In plants and fungi, calcineurin functions in stress responses (e.g., temperature, cations, and pH) and is necessary for the virulence of the fungal pathogen Cryptococcus neoformans. Here we show that calcineurin is also required for the virulence of another major fungus that is pathogenic to humans, Candida albicans. C. albicans calcineurin mutants had significantly reduced virulence in a murine model of systemic infection. In contrast to its role in C. neoformans, calcineurin was not required for C. albicans survival at 37 degrees C. Moreover, C. albicans calcineurin mutant strains exhibited no defects in known Candida virulence traits associated with host invasion, including filamentous growth, germ tube formation, and adherence to and injury of mammalian cells. C. albicans calcineurin mutant strains failed to colonize and grow in the kidneys of infected animals and were unable to survive when exposed to serum in vitro. Our studies illustrate that calcineurin has evolved to control aspects of the virulence of two divergent fungal pathogens via distinct mechanisms that can be targeted to achieve broad-spectrum antifungal action.

Animals↗
PubMed2003

Parenchymal organ, and not splenic, immunity correlates with host survival during disseminated candidiasis.

We examined the relationship between host survival and renal and splenic immune responses in a murine model of hematogenously disseminated candidiasis. Male BALB/c mice were infected via tail vein injection with wild-type C. albicans or with an isogenic, Deltaefg1/Deltaefg1 hypha-deficient mutant. Host survival, organ fungal burden, intracellular cytokine content of splenic and kidney lymphocytes, and whole-organ cytokine profiles were determined. Wild-type C. albicans induced type 2 splenocyte responses with both nonfatal and fatal inocula. In the kidney, conversely, wild-type inocula causing no or low mortality induced type 1 responses and 100% fatal inocula induced type 2 or interleukin-10 (IL-10)-dominant responses. Hypha-deficient mutant C. albicans caused no or low mortality while inducing type 1 responses in both the spleen and kidney. To our knowledge, this is the first demonstration that host survival during systemic infection correlates with the type of immune response engendered in a nonlymphoid, parenchymal organ and not with the response in the spleen. Furthermore, the results provide in vivo confirmation that hyphal formation by C. albicans induces type 2 or IL-10-dominant host responses in tissues.

Animals↗
PubMed2002

Candida albicans stimulates local expression of leukocyte adhesion molecules and cytokines in vivo.

The host inflammatory response to the opportunistic pathogen Candida albicans determines susceptibility to disseminated infection. This study used immunohistochemical analysis to correlate microbiologic findings and pathologic changes with local expression of cytokines and leukocyte adhesion molecules in mice with disseminated candidiasis. After being inoculated intravenously as blastospores, the organisms filamented extensively in the kidneys while remaining predominantly as blastospores or short germ tubes in the lung, liver, and spleen. Very few leukocytes accumulated around the invading organisms until 24 h after inoculation. The leukocytes at the infection site appeared to amplify the inflammatory response. They expressed interleukin-1beta, tumor necrosis factor-alpha, intercellular adhesion molecule 1, and platelet-endothelial cell adhesion molecule 1. Therefore, the morphology of the organism varies with the infection site. Furthermore, leukocyte recruitment occurs relatively late in the infection, and this recruitment is likely amplified by proinflammatory mediators produced by the leukocytes themselves.

Animals↗
PubMed2002

Predictive value of HIV-1 viral load on risk for opportunistic infection.

OBJECTIVE: The relationship between HIV-1 viral load and the risk for opportunistic infection (OI) was examined in Adult AIDS Clinical Trial Group (AACTG) 722, a virology substudy of AACTG 323: a phase 4 randomized study designed to examine the use of chronic suppressive versus episodic fluconazole therapy. METHODS: The primary analysis used a case-control sampling scheme with two controls per "case" (subjects that developed an OI) matched by gender, age, and time on study. Forty-five cases and matched controls were identified and used in the analysis. RESULTS: Study 722 accrued 518 subjects between 5/97 and 11/99. Forty-five subjects developed serious OIs or refractory candidiasis. Median baseline CD4 count was 24 cell/mm3 for cases and 46 for controls (p =.003). Median viral load (VL) was 5.02 log10 copies/mL for cases and 4.08 for controls (p =.002). Multivariate analysis found four independent variables associated with time to OI: baseline VL and CD4 (RR = 2.2 per log increment and 6.0 per 50-cell increment, respectively), a one log increase in VL at any time (RR = 15), and history of an OI (RR = 5.2). CONCLUSIONS: VL and changes in VL were independently associated with risk of development of OIs in a prospective study and should be considered by clinicians when assessing patients for risk of OI.

AIDS-Related Opportunistic Infections↗
PubMed2002

Candida albicans Als1p: an adhesin that is a downstream effector of the EFG1 filamentation pathway.

Filamentation and adherence to host cells are critical virulence factors of Candida albicans. Multiple filamentation regulatory pathways have been discovered in C. albicans using Saccharomyces cerevisiae as a model. In S. cerevisiae, these pathways converge on Flo11p, which functions as a downstream effector of filamentation and also mediates cell-cell adherence (flocculation). In C. albicans, such effector(s) have not yet been identified. Here, we demonstrate that the cell surface protein Als1p is an effector of filamentation in C. albicans. We show that Als1p expression is controlled by the transcription factor Efg1p, which is known to be a key regulator of filamentation in C. albicans. Further, disruption of ALS1 inhibited filamentation, and autonomous expression of Als1p restored filamentation in an efg1 homozygous null mutant. Thus, Als1p functions as a downstream effector of the EFG1 filamentation pathway. In addition, we found that Als1p mediates both flocculation and adherence of C. albicans to endothelial cells in vitro. As a cell surface glycoprotein that mediates filamentation and adherence, Als1p has both structural and functional similarity to S. cerevisiae Flo11p. Consistent with our in vitro results, Als1p was required for both normal filamentation and virulence in the mouse model of haematogenously disseminated candidiasis.

Candida albicans↗
PubMed2002

Endocytosis of Candida albicans by vascular endothelial cells is associated with tyrosine phosphorylation of specific host cell proteins.

Candida albicans escapes from the bloodstream by invading the endothelial cell lining of the vasculature. In vitro, C. albicans invades endothelial cells by inducing its own endocytosis. We examined whether this process is regulated by the tyrosine phosphorylation of endothelial cell proteins. We found that endocytosis of wild-type C. albicans was accompanied by the tyrosine phosphorylation of two endothelial cell proteins with molecular masses of 80 and 82 kDa. The phosphorylation of these proteins was closely associated with the endocytosis of C. albicans because these proteins were phosphorylated in response to the endocytosis of both live and killed organisms, but they were not phosphorylated in endothelial cells infected with a poorly endocytosed strain of C. albicans. The tyrosine kinase inhibitors genistein and tyrphostin 47 blocked the phosphorylation of the two endothelial cell proteins and significantly reduced endocytosis of C. albicans. Therefore, C. albicans probably induces its own endocytosis by stimulating the tyrosine phosphorylation of two endothelial cell proteins.

Bacterial Adhesion↗
PubMed2002

Candida albicans Mds3p, a conserved regulator of pH responses and virulence identified through insertional mutagenesis.

Candida albicans is a commensal fungus that causes diverse infections after antibiotic use or immune debilitation. Gene discovery has been limited because the organism is an asexual diploid. We have developed a strategy that yields random homozygous insertion mutants. The strategy has permitted identification of several prospective essential genes. Many of these genes are homologous to nonessential Saccharomyces cerevisiae genes, and some have no S. cerevisiae homolog. These findings may expand the range of antifungal drug targets. We have also identified new genes required for pH-dependent filamentation, a trait previously associated with virulence. One newly identified gene, MDS3, is required for expression in alkaline media of two filamentation-associated genes, HWP1 and ECE1, but is not required for expression of other pH-response genes. In S. cerevisiae, the two MDS3 homologs are required for growth in alkaline media, thus arguing that Mds3p function in adaptation to external pH changes is conserved. Epistasis tests show that Mds3p contributes to virulence and alkaline pH responses independently of the well-characterized Rim101p pH-response pathway.

Animals↗
PubMed2002

A forkhead transcription factor is important for true hyphal as well as yeast morphogenesis in Candida albicans.

Candida albicans is an important pathogen of immunocompromised patients which grows with true hyphal, pseudohyphal, and yeast morphologies. The dynamics of cell cycle progression are markedly different in true hyphal relative to pseudohyphal and yeast cells, including nuclear movement and septin ring positioning. In Saccharomyces cerevisiae, two forkhead transcription factors (ScFKH1 and ScFKH2) regulate the expression of B-cyclin genes. In both S. cerevisiae and Schizosaccharomyces pombe, forkhead transcription factors also influence morphogenesis. To explore the molecular mechanisms that connect C. albicans morphogenesis with cell cycle progression, we analyzed CaFKH2, the single homolog of S. cerevisiae FKH1/FKH2. C. albicans cells lacking CaFkh2p formed constitutive pseudohyphae under all yeast and hyphal growth conditions tested. Under hyphal growth conditions levels of hyphae-specific mRNAs were reduced, and under yeast growth conditions levels of several genes encoding proteins likely to be important for cell wall separation were reduced. Together these results imply that Fkh2p is required for the morphogenesis of true hyphal as well as yeast cells. Efglp and Cphlp, two transcription factors that contribute to C. albicans hyphal growth, were not required for the pseudohyphal morphology of fkh2 mutants, implying that Fkh2p acts in pathways downstream of and/or parallel to Efglp and Cphlp. In addition, cells lacking Fkh2p were unable to damage human epithelial or endothelial cells in vitro, suggesting that Fkh2p contributes to C. albicans virulence.

Candida albicans↗