Dongmei Li - Academia.edu (original) (raw)

Papers by Dongmei Li

Scientific reports, Jan 5, 2017

The lack of new antifungal compounds with unique mechanisms of action is a concern for therapeuti... more The lack of new antifungal compounds with unique mechanisms of action is a concern for therapeutic management of patients. To identify inhibitors against human pathogenic fungi, we screened ~3000 compounds provided by the Developmental Therapeutics Program of NIH/NCI against a panel of pathogenic fungi including Candida species, Aspergillus fumigatus, and Cryptococcus neoformans. NSC319726 (a thiosemicarbazone) had broad antifungal activity in the range of 0.1-2.0 µg/ml and was also inhibitory to fluconazole-resistant isolates of Candida species. Synergy was demonstrated with NSC319726 and azoles, as well as caspofungin. The inhibitory concentration 50% (IC) of NSC319726 was 35-800-fold higher than the Minimum Inhibitory Concentration 50% (MICvalues), which indicates low compound toxicity to human cells in vitro. Transcriptome analysis of treated and untreated C. albicans using Gene Ontology (GO) revealed a large cluster of down regulated genes that encode translational proteins, es...

FEMS yeast research, 2015

The mitochondrion plays wide-ranging roles in eukaryotic cell physiology. In pathogenic fungi, th... more The mitochondrion plays wide-ranging roles in eukaryotic cell physiology. In pathogenic fungi, this central metabolic organelle mediates a range of functions related to disease, from fitness of the pathogen to developmental and morphogenetic transitions to antifungal drug susceptibility. In this review, we present the latest findings in this area. We focus on likely mechanisms of mitochondrial impact on fungal virulence pathways through metabolism and stress responses, but also potentially via control over signaling pathways. We highlight fungal mitochondrial proteins that lack human homologs, and which could be inhibited as a novel approach to antifungal drug strategy.

Journal of proteome research, Jan 21, 2016

Candida albicans is the most common human fungal pathogen for both immunocompetent and immunocomp... more Candida albicans is the most common human fungal pathogen for both immunocompetent and immunocompromised individuals. Lysine succinylation is a frequently occurring post-translational modification that is found in many organisms; however, the role of succinylation is still under investigation. Here, we initiated a first screening of lysine succinylation in C. albicans. We identified 1550 succinylation sites from 389 proteins in C. albicans, demonstrating that succinylation is conservative in this organism. However, the lysine succinylation sites showed some difference in C. albicans, with the overlapping rates between C. albicans and other species ranging from 55% for Saccharomyces cerevisiae, 40% for human, 35% for mouse, and to only 16% for Escherichia coli. The further bioinformatics analysis indicated that the succinylated proteins were involved in a wide range of cellular functions with diverse subcellular localizations. Furthermore, we discovered that lysine succinylation coul...

Virulence, Feb 18, 2016

Mitochondria are essential for cell growth and survival of most fungal pathogens. Energy (ATP) pr... more Mitochondria are essential for cell growth and survival of most fungal pathogens. Energy (ATP) produced during oxidation/reduction reactions of the electron transport chain (ETC) Complexes I, III and IV (CI, CIII, CIV) fuel cell synthesis. The mitochondria of fungal pathogens are understudied even though more recent published data suggest critical functional assignments to fungal-specific proteins. Proteins of mammalian mitochondria are grouped into 16 functional categories. In this review, we focus upon 11 proteins from 5 of these categories in fungal pathogens, OXPHOS, protein import, stress response, carbon source metabolism, and fission/fusion morphology. As these proteins also are fungal-specific, we hypothesize that they may be exploited as targets in antifungal drug discovery. We also discuss published transcriptional profiling data of mitochondrial CI subunit protein mutants, in which we advance a novel concept those CI subunit proteins have both shared as well as specific r...

FEMS Yeast Research, 2009

Mitogen activated protein kinase (MAPK) cascades are signal transduction mechanisms present in eu... more Mitogen activated protein kinase (MAPK) cascades are signal transduction mechanisms present in eukaryotic cells that allow adaptation to environmental changes. MAPK activity is mainly regulated by dual phosphorylation in a TXY motif present in the kinase subdomain VIII as well as dephosphorylation by specific phosphatases. The Cek1 MAPK is involved in filamentous growth in Candida albicans and is an important determinant of virulence in this microorganism; its activation is controlled by the Sho1 adaptor protein. Here we show that Cek1 phosphorylation is regulated by quorum sensing (QS). Cek1 phosphorylation is prevented by farnesol, a compound that also regulates the dimorphic transition in this fungus. Farnesol also induced the activation of Mkc1, the MAPK of the cell integrity pathway. The role of farnesol in Cek1 phosphorylation is independent of the Chk1 histidine kinase, a putative QS sensor, as revealed by genetic analysis. In addition, Cek1, not Hog1, is degraded by proteasome, as revealed by the use of a conditional lethal protein degradation mutant. Our data therefore describe two different mechanisms (QS and protein degradation) that control a MAPK pathway that regulates virulence in a fungal pathogen.

Fungal Genetics and Biology, 2009

Several published functions associated with the CHK1 histidine kinase of Candida albicans resembl... more Several published functions associated with the CHK1 histidine kinase of Candida albicans resemble those of the MAPK Cek1p and its cognate receptor Sho1p (SSU81). To explore this further, we have compared mutants lacking the proteins mentioned above and have constructed a double sho1/ chk1Δ null mutant to determine relationships among these proteins. We observed that the sensitivity to Congo red (CR), calcofluor white (CW), as well as clumping of cells, was slightly increased in the double mutant compared to the single chk1Δ or sho1Δ mutants. However, Cek1p phosphorylation via Sho1p, which occurs during log phase growth in the presence or absence of CR in Wt cells, does not require Chk1p. These data suggest that Chk1p and Sho1p are components of parallel but independent signal pathways. In addition, bulk mannan of strains was analyzed by GPC/MS and NMR. Compared to Wt and a CHK1 gene-reconstituted strain (CHK23) that contained, high, intermediate and low Mw mannan species, we found that the mannan of strains CHK21 (chk1Δ null), the cek1Δ null, and the double mutant consisted only of low Mw mannan. The sho1Δ null mutant only demonstrated a reduced intermediate type of mannan. Alcian blue binding was lower in cek1Δ, chk1Δ, and the double sho1/chk1Δ null mutant lacking high and intermediate Mw mannan than in the sho1Δ null which had a partial loss of intermediate Mw mannan only. We conclude that the Chk1p HK is part of a functionally similar but parallel pathway to the Sho1p-Cek1p pathway that confers resistance to the cell wall inhibitors CR and CW. However, a functional relationship in mannan biosynthesis of Chk1p and Cek1p exists that only partially requires Sho1p.

Medical Mycology Case Reports, 2013

Curvularia is a dematiaceous mold that infects plant species and is found in the soil. In humans,... more Curvularia is a dematiaceous mold that infects plant species and is found in the soil. In humans, it is known to cause keratitis after trauma to the eye. We report the findings of persistent fungal endophthalmitis in a 74-year-old female patient who had undergone prior cataract surgery. Mold identification and antifungal susceptibilities were done on 2 separate samples of vitreous fluid and they were found to be consistent with Curvularia lunata by the use of PCR amplification methods.

PloS one, 2016

The Goa1p of Candida albicans regulates mitochondrial Complex I (CI) activities in its role as a ... more The Goa1p of Candida albicans regulates mitochondrial Complex I (CI) activities in its role as a putative CI accessory protein. Transcriptional profiling of goa1∆ revealed a down regulation of genes encoding β-oligomannosyl transferases. Herein, we present data on cell wall phenotypes of goa1∆ (strain GOA31). We used transmission electron microscopy (TEM), GPC/MALLS, and NMR to compare GOA31 to a gene-reconstituted strain (GOA32) and parental cells. We note by TEM a reduction in outer wall fibrils, increased inner wall transparency, and the loss of a defined wall layer close to the plasma membrane. GPC-MALLS revealed a reduction in high and intermediate Mw mannan by 85% in GOA31. A reduction of β-mannosyl but not α-mannosyl linkages was noted in GOA31 cells. β-(1,6)-linked glucan side chains were branched about twice as often but were shorter in length for GOA31. We conclude that mitochondrial CI energy production is highly integrated with cell wall formation. Our data also suggest ...

Future Microbiology, 2014

ABSTRACT: New data suggest that the global incidence of several types of fungal diseases have tr... more ABSTRACT: New data suggest that the global incidence of several types of fungal diseases have traditionally been under-documented. Of these, mortality caused by invasive fungal infections remains disturbingly high, equal to or exceeding deaths caused by drug-resistant tuberculosis and malaria. It is clear that basic research on new antifungal drugs, vaccines and diagnostic tools is needed. In this review, we focus upon antifungal drug discovery including in vitro assays, compound libraries and approaches to target identification. Genome mining has made it possible to identify fungal-specific targets; however, new compounds to these targets are apparently not in the antimicrobial pipeline. We suggest that ‘repurposing’ compounds (off patent) might be a more immediate starting point. Furthermore, we examine the dogma on antifungal discovery and suggest that a major thrust in technologies such as structural biology, homology modeling and virtual imaging is needed to drive discovery.

Current therapeutic research, clinical and experimental, 2014

The Women's Interagency HIV Study was established in 1993 to study the natural history of HIV... more The Women's Interagency HIV Study was established in 1993 to study the natural history of HIV disease among women in the United States. It currently has enrolled 2,895 women testing positive for HIV infection and 972 women without HIV infection recruited from 6 national metropolitan locations. The clinical database information collected for each HIV-positive individual included CD4 cell counts, viral load, and antiviral treatment to evaluate HIV prognosis and related conditions in women. To provide a baseline for fluconazole treatment prospects in women who test positive for HIV infection. As part of the ongoing Women's Interagency HIV Study project, we investigated the fluconazole susceptibility of Candida spp. isolated from women with HIV in comparison to volunteer women without HIV. The implication of antifungal treatment on fluconazole susceptibility was evaluated by reviewing antifungal medication use for the past 2 years in each participant. In addition, genotyping of ...

Mycopathologia, 2013

Background-Candidiasis in HIV/AIDS patients continues to be a public health problem. Antifungal t... more Background-Candidiasis in HIV/AIDS patients continues to be a public health problem. Antifungal therapies are not always effective and may result in complications, such as the development of drug-resistant strains of Candida species. Objectives-This study evaluated the impact of probiotic consumption on Candida colonization of the oral and vaginal mucosa. Patients/Methods-A pilot study was conducted in 24 women (17 HIV-infected, 7 HIVuninfected) from the Women's Interagency HIV Study. The women underwent a 60-day initiation period with no probiotic consumption, followed by two 15-day consumption periods, with a

Microbiology, 2004

The two-component histidine kinase Chk1p of Candida albicans has been implicated in the regulatio... more The two-component histidine kinase Chk1p of Candida albicans has been implicated in the regulation of cell wall biosynthesis. Deletion of CHK1 results in avirulence that in part may be due to the increased sensitivity of mutant strains to polymorphonuclear leukocytes. The mutant also does not adhere to human oesophageal tissue in vitro, probably as a consequence of its altered cell wall. In the current study, a CHK1 promoter-lacZ reporter (CHK1prlacZ) construct was expressed in wild-type C. albicans strain CAI4 and in two-component signal transduction mutants to determine the effect of environmental stress conditions on the regulation of CHK1 and the co-regulatory activities among these proteins. It is shown that lacZ expression varied according to the type of growth conditions and incubation time; expression was also influenced by the strain background. lacZ expression in CAI4 was greater at 37 6C and at a pH of 3?5 and in the presence of 4 mM H 2 O 2 , 0?1 mM menadione, 10 % serum or 1?5 M NaCl compared to cells grown at 30 or 42 6C. The increases in expression were time-dependent and not observed until cells were incubated for 120 min in these conditions (P<0?05). As a correlate of the increase in transcription of CHK1-lacZ in the presence of H 2 O 2 , the chk1 mutant was more sensitive than wild-type and revertant cells to H 2 O 2 in vitro. In addition to strain CAI4, we also measured CHK1p-lacZ reporter activity of mutants deleted in genes encoding other two-component proteins such as the response regulator gene SSK1, the histidine kinases, SLN1 and NIK1, and the HOG1 MAP kinase. Of these proteins, Ssk1p and Sln1p are presumed to mediate phosphotransfer to the HOG1 [hyperosmotic glycerol] MAP kinase pathway during oxidative and perhaps osmotic stress in C. albicans. Compared to strain CAI4, lacZ reporter activity increased significantly in the ssk1 mutant under all growth conditions after a 10 and 120 min incubation (P<0?0001). lacZ expression in the ssk1 mutant was less at 42 6C compared to all other growth conditions (P<0?05). Furthermore, lacZ reporter activity also increased in the hog1 mutant of C. albicans. These data suggest that SSK1 and HOG1 indirectly or directly negatively regulate CHK1 under most growth conditions tested. In the sln1 mutant, downregulation of CHK1 was observed in all growth conditions compared to strain CAI4 (P<0?05), while regulation of lacZ in the nik1 mutant was similar to strain CAI4 except when cells were incubated in the presence of 4 mM H 2 O 2 for 120 min (P<0?05). Western blot analysis was used to determine the role of Chk1p in phosphorylation of Hog1p under oxidative or osmotic stress. It was found that Hog1p was phosphorylated in the chk1 mutant similar to wild-type CAF2-1 cells, although the temporal events of phosphorylation differed slightly in mutant cells. These results show that transcription of CHK1, as measured by the lacZ reporter assay, is statistically increased when cells are exposed to several types of stress or when incubated in 10 % serum in a mutant-specific background and at a specific time point. Of importance, our data also suggest that lacZ expression is indirectly or directly regulated by the HOG1 MAP kinase pathway, although a determination of its position in this pathway or in a cross-talking pathway awaits additional studies.

Medical Mycology, 2008

Aspergillus fumigatus, an important human fungal pathogen, encounters high levels of reactive oxy... more Aspergillus fumigatus, an important human fungal pathogen, encounters high levels of reactive oxygen species following its ingestion by phagocytes. Reactive oxygen species are important mediators of the fungicidal activities of phagocytes. In yeasts, YAP1 encodes for transcriptional factors that contribute to their oxidative stress response and given the importance of the stress response, we hypothesized that the YAP1 homologue in A. fumigatus plays a similar role in this fungus. In this study, we found that Afyap1, the Yap1 homologue of A. fumigatus, confers protection against oxidative stress. Replacement of Afyap1 with the marker gene pyrG (DAfyap1) resulted in hypersensitivity of A. fumigatus to oxidants such as H 2 O 2 and menadione. In contrast, an A. fumigatus strain harboring multiple-copy Afyap1 was resistant to these two oxidants as well as the oxidant diamide. However, DAfyap1 and strain harboring multiple-copy Afyap1 were comparable in their virulence to a wild-type A. fumigatus strain in a murine model of invasive pulmonary aspergillosis. Taken together, these results demonstrate that Afyap1 is involved in oxidative stress response but is not an essential virulence factor for A. fumigatus.

Infection and Immunity, 2005

The isolation and partial functional characterization of the two-component response regulator SSK... more The isolation and partial functional characterization of the two-component response regulator SSK1 gene of Candida albicans was previously reported. Compared to wild-type (CAF2-1) and gene-reconstituted (SSK23) strains, the ssk1 null strain (SSK21) was avirulent in a murine model of hematogenously disseminated candidiasis and less able to adhere to human esophageal cells. More recent data indicate that SSK21 is sensitive to 4 to 8 mM H2O2 in vitro than CAF2-1 and SSK23. Furthermore, microarray studies indicate that the regulation of two classes of genes, those encoding cell wall functions and stress adaptation, are altered in the ssk1 mutant. In the present study, the susceptibility of strains CAF2-1, SSK21, and SSK23 to killing by human polymorphonuclear neutrophils (PMNs) was assessed. Results are also described for a newly constructed ssk1 mutant (SSK24) in which the URA3 gene is integrated into its native locus. Our results indicate that killing of SSK21 and SSK24 was significan...

Infection and Immunity, 2002

We previously demonstrated that genes encoding a putative two-component histidine kinase (CHK1) o... more We previously demonstrated that genes encoding a putative two-component histidine kinase (CHK1) or a response regulator (CSSK1) are each required for virulence in a murine model of hematogenously disseminated candidiasis and that strains with each gene deleted are also defective in morphogenesis under certain growth conditions. In the present study, the role of these two genes in the adherence to and colonization of reconstituted human esophageal tissue (RHE) is described. We compared strains of Candida albicans with deletions of chk1 (strain CHK21) and cssk1 (strain CSSK21) to wild-type cells (CAF2), as well as strains with CHK1 and CSSK1 reconstituted (strains CHK23 and CSSK23, respectively). Adherence and colonization of RHE were evaluated in periodic acid-Schiff-stained sections, as well as by SEM. We observed that both deletion-containing strains colonized the RHE to a lesser extent than did CAF2 and that the percent germination by both strains was reduced in comparison to that...

[ Research paper thumbnail of The role of the Candida albicans histidine kinase [ CHK1 ) gene in the regulation of cell wall mannan and glucan biosynthesis](https://attachments.academia-assets.com/85894054/thumbnails/1.jpg)

FEMS Yeast Research, 2003

The human pathogen Candida albicans encodes at least three putative two-component histidine kinas... more The human pathogen Candida albicans encodes at least three putative two-component histidine kinase signal transduction proteins, including Chk1p and a response regulator protein (Cssk1p). Strains deleted in CHK1 are avirulent in a murine model of hematogenously disseminated disease. The specific function of Chk1p has not been established, but hyphae of the chk1 mutant exhibit extensive flocculation while yeast forms are less adherent to reconstituted human esophageal tissue, indicating that this protein may regulate cell surface properties. Herein, we analyze glucan, mannan and chitin profiles in strains deleted in chk1 (CHK21) compared to a genereconstituted strain (CHK23) and a parental strain CAF2. Total alkali-soluble hexose from the cell wall of the chk1 mutant (strain CHK21) was significantly reduced. Western blots of cell wall extracts from CHK21, CHK23 and CAF2 reacted with a Mab to the acidstable mannan fraction revealed extensive staining of lower molecular mass species in strain CHK21 only. FACE (fluorophore assisted carbohydrate electrophoresis) was used to characterize the oligosaccharide side chains of L-eliminated (O-linked), acid-hydrolyzed (acidlabile phosphomannan) and acetolysis (acid-stable mannan) extracted fractions of total mannan. The profiles of O-linked as well as the acid-labile oligosaccharides were similar in both CAF2 and CHK21, but the acid-stable oligosaccharide side chains were significantly truncated. We also characterized the L-glucan from each strain using NMR, and found that both the degree of polymerization and the ratio of (1-3)/(1-6) linkages was lower in CHK21 relative to wild-type cells. The sensitivity of CHK21 to antifungal drugs and inhibitors was unaffected. In summary, our data have identified a new function for a histidine kinase two-component signal protein in a human pathogenic fungus.

Eukaryotic Cell, 2009

Using a Tn7transposon library ofCandida albicans, we have identified a mutant that exhibited sens... more Using a Tn7transposon library ofCandida albicans, we have identified a mutant that exhibited sensitivity in drop plate assays to oxidants such as menadione and hydrogen peroxide. To verify the role of the mutated gene in stress adaptation, null mutants were constructed and phenotypically characterized. Because of its apparent functions in growth and oxidant adaptation, we have named the geneGOA1. Goa1p appears to be unique to the CTG subclade of theSaccharomycotina, includingC. albicans. Mutants ofC. albicanslackinggoa1(strain GOA31) were more sensitive to 6 mM H2O2and 0.125 mM menadione than the wild type (wt) or a gene-reconstituted (GOA32) strain. The sensitivity to oxidants correlated with reduced survival of the GOA31 mutant in human neutrophils and avirulence compared to control strains. Other phenotypes of GOA31 include reduced growth and filamentation in 10% serum, Spider, and SLAD agar media and an inability to form chlamydospores. Since Goa1p has an N-terminal mitochondrio...

Eukaryotic Cell, 2003

Ssk1p ofCandida albicansis a putative response regulator protein of the Hog1 two-component signal... more Ssk1p ofCandida albicansis a putative response regulator protein of the Hog1 two-component signal transduction system. InSaccharomyces cerevisiae, the phosphorylation state of Ssk1p determines whether genes that promote the adaptation of cells to osmotic stress are activated. We have previously shown thatC. albicans SSK1does not complement thessk1mutant ofS. cerevisiaeand that thessk1mutant ofC. albicansis not sensitive to sorbitol. In this study, we show that theC. albicans ssk1mutant is sensitive to several oxidants, including hydrogen peroxide,t-butyl hydroperoxide, menadione, and potassium superoxide when each is incorporated in yeast extract-peptone-dextrose (YPD) agar medium. We used DNA microarrays to identify genes whose regulation is affected by thessk1mutation. RNA from mutant cells (strain CSSK21) grown in YPD medium for 3 h at 30°C was reverse transcribed and then compared with similarly prepared RNA from wild-type cells (CAF2). We observed seven genes from mutant cells ...

Cellular Microbiology, 2013

We have previously characterized several fungal-specific proteins from the human pathogen Candida... more We have previously characterized several fungal-specific proteins from the human pathogen Candida albicans that either encode subunits of mitochondria Complex I (CI) of the electron transport chain (ETC) or regulate CI activity (Goa1p). Herein, the role of energy production and cell wall gene expression is investigated in the mitochondria mutant goa1Δ. We show that down regulation of cell wall-encoding genes in the goa1Δ results in sensitivity to cell wall inhibitors such as congo red and calcofluor white, reduced phagocytosis by a macrophage cell line, reduced recognition by macrophage receptors, and decreased expression of cytokines such as IL-6, IL-10, and IFN-γ. In spite of the reduced recognition by macrophages, the goa1Δ is still killed to the same extent as control strains. We also demonstrate that expression of the epithelial cell receptors E-cadherin and EGFR is also reduced in the presence of goa1Δ. Together, our data demonstrate the importance of mitochondria in the expression of cell wall biomolecules and the interaction of C. albicans with innate immune and epithelial cells. Our underlying premise is that mitochondrial proteins such as Goa1p and other fungal-specific mitochondrial proteins regulate critical functions in cell growth and in virulence. As such, they remain as valid drug targets for antifungal drug discovery.

Biochemistry, 2001

Manganese peroxidase (MnP) is a heme-containing enzyme produced by white-rot fungi and is part of... more Manganese peroxidase (MnP) is a heme-containing enzyme produced by white-rot fungi and is part of the extracellular lignin degrading system in these organisms. MnP is unique among Mn binding enzymes in its ability to bind and oxidize Mn II and efficiently release Mn III. Initial site-directed mutagenesis studies identified the residues E35, E39, and D179 as the Mn binding ligands. However, an E39D variant was recently reported to display wild-type Mn binding and rate of oxidation, calling into question the role of E39 as an Mn ligand. To investigate this hypothesis, we performed computer modeling studies which indicated metal-ligand bond distances in the E39D variant and in an E35D-E39D-D179E triple variant which might allow Mn binding and oxidation. To test the model, we reconstructed the E35D and E39D variants used in the previous study, as well as an E39A single variant and the E35D-E39D-D179E triple variant of MnP isozyme 1 from Phanerochaete chrysosporium. We find that all of the variant proteins are impaired for Mn II binding (K m increases 20-30-fold) and Mn II oxidation (k cat decreases 50-400-fold) in both the steady state and the transient state. In particular, mutation of the E39 residue in MnP decreases both Mn binding and oxidation. The catalytic efficiency of the E39A variants decreased ∼10 4-fold, while that of the E39D variant decreased ∼10 3-fold. Contrary to initial modeling results, the triple variant performed only as well as any of the single Mn ligand variants. Interestingly, the catalytic efficiency of the triple variant decreased only 10 4-fold, which is ∼10 2-fold better than that reported for the E35Q-D179N double variant. These combined studies indicate that precise geometry of the Mn ligands within the Mn binding site of MnP is essential for the efficient binding, oxidation, and release of Mn by this enzyme. The results clearly indicate that E39 is a Mn ligand and that mutation of this ligand decreases both Mn binding and the rate of Mn oxidation.