Masakazu Niimi

University of Otago, Taieri, Otago Region, New Zealand

Are you Masakazu Niimi?

Claim your profile

Publications (63)192.76 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to confirm an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-beta mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonised for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonised full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123 (R123), daunorubicin, tetramethylrhodamine (TMR), FK506 or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance.
    Molecular Pharmaceutics 08/2014; · 4.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Resistance of Candida albicans to widely used azoles poses serious clinical problems, as C. albicans is the predominant cause of systemic fungal infections in immunocompromised individuals. The major mechanism of azole resistance is the overexpression of ATP-binding cassette (ABC) transporters such as C. albicans Cdr1p. A detailed analysis of 244 fungal pleiotropic drug resistance (PDR) type ABC transporters revealed highly conserved amino acid motifs in the two large fungal PDR transporter-specific extracellular loops (EL3 and EL6) that are essential for its function and localisation in the plasma membrane. Previous studies identified E704 in EL3 as a critically important amino acid for proper folding and localisation of Cdr1p. Objective: This project investigated the hypothesis that the conserved amino acid R546 interacts with E704 in Cdr1p and compensates for the loss of function caused by the E704K mutation. Methods: The R546E mutation was introduced into wild-type Cdr1p to make a single mutant, and into previously constructed EL3.1 mutant (E704K) to make a double mutant using PCR-based site-directed mutagenesis and transformation of Saccharomyces cerevisiae ADD. Phenotypes of the mutants were analysed using drug susceptibility and pump chemosensitisation assays. Results: Changing R546 to E546 had no effect on the transport function of Cdr1p. Cdr1p-R546E behaved similarly to the wild-type enzyme and responded equally well to known Cdr1p efflux pump inhibitors, although the double mutant R546E/E704K became slightly more susceptible to test drugs than the parent EL3.1. Conclusions: R546 does not appear to interact with E704. Also, it was clear that R546 is not in close contact with E704, disproving the hypothesis that R546 creates a salt-bridge with E704 that is important for the proper folding and function of the enzyme. Nevertheless, the single ADD/CDR1A-R546E and double ADD/CDR1A-R546E.E704K mutants are useful for further analysis of Cdr1p function.
    IADR General Session and Exhibition 2014; 06/2014
  • Source
    Erwin Lamping, Masakazu Niimi, Richard D Cannon
    [Show abstract] [Hide abstract]
    ABSTRACT: A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5[prime] UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5[prime] UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs ([increment]G = -15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs ([increment]G = -4.4 kcal/mol) inhibited Cdr1p expression by ~50%. We have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression.
    Microbial Cell Factories 07/2013; 12(1):74. · 3.31 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mannans are mannose polymers attached to cell wall proteins in all Candida species, including the pathogenic fungus Candida albicans. Mannans are sensed by pattern recognition receptors expressed on innate immune cells. However, the detailed structural patterns affecting immune sensing are not fully understood because mannans have a complex structure that includes α- and β-mannosyl linkages. In this study, we focused on the β-1,2-mannosides of N-linked mannan in C. albicans because this moiety is not present in the non-pathogenic yeast Saccharomyces cerevisiae. To investigate the impact of β-1,2-mannosides on immune sensing, we constructed a C. albicans ∆mnn4/∆bmt1 double deletant. Thin-layer chromatography and nuclear magnetic resonance analyses revealed that the deletant lacked β-1,2-mannosides in N-linked mannan. Mannans lacking the β-1,2-mannosides induced the production of higher levels of inflammatory cytokines, including IL-6, IL-12p40 and TNF-α, in mice dendritic cells compared to wild-type mannan. Our data show that β-1,2-mannosides in N-linked mannan reduce the production of inflammatory cytokines by dendritic cells.
    Medical mycology: official publication of the International Society for Human and Animal Mycology 10/2012; · 2.13 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Overexpression of the Candida albicans ATP-binding cassette transporter CaCdr1p causes clinically significant resistance to azole drugs including fluconazole (FLC). Screening of a ~1.89 × 10(6) member D-octapeptide combinatorial library that concentrates library members at the yeast cell surface identified RC21v3, a 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative of the D-octapeptide D-NH(2) -FFKWQRRR-CONH(2) , as a potent and stereospecific inhibitor of CaCdr1p. RC21v3 chemosensitized Saccharomyces cerevisiae strains overexpressing CaCdr1p but not other fungal ABC transporters, the C. albicans MFS transporter CaMdr1p or the azole target enzyme CaErg11p, to FLC. RC21v3 also chemosensitized clinical C. albicans isolates overexpressing CaCDR1 to FLC, even when CaCDR2 was overexpressed. Specific targeting of CaCdr1p by RC21v3 was confirmed by spontaneous RC21v3 chemosensitization-resistant suppressor mutants of S. cerevisiae expressing CaCdr1p. The suppressor mutations introduced a positive charge beside, or within, extracellular loops 1, 3, 4 and 6 of CaCdr1p or an aromatic residue near the extracytoplasmic end of transmembrane segment 5. The mutations did not affect CaCdr1p localization or CaCdr1p ATPase activity but some increased susceptibility to the CaCdr1p substrates FLC, rhodamine 6G, rhodamine 123 and cycloheximide. The suppressor mutations showed that the drug-like CaCdr1p inhibitors FK506, enniatin, milbemycin α11 and milbemycin β9 have modes of action similar to RC21v3.
    Molecular Microbiology 07/2012; 85(4):747-67. · 5.03 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A mechanism for the acquisition of high-level echinocandin resistance in Candida glabrata was investigated. FKS mutants were constructed to: determine whether clinically significant micafungin resistance requires a hot-spot mutation in FKS1 and a premature stop codon in FKS2, as was observed in a clinical isolate; select for variants with reduced susceptibility and locate mutations in FKS genes; and assess the roles of FKS1 and FKS2. A panel of FKS mutants was constructed using micafungin-susceptible parents by site-directed mutagenesis. Drug susceptibility, gene expression and glucan synthase activities were compared between mutants. Mutations acquired by selection were identified by DNA sequence analysis of FKS genes from selected variants. Single FKS deletants were constructed and their phenotypes examined. Introduction of the hot-spot mutation in FKS1 alone conferred an intermediate reduction in susceptibility, and the premature stop codon in FKS2 alone had no effect on susceptibility, while severely reduced susceptibility equivalent to that of the clinical isolate required both mutations. Exposure of susceptible strains to micafungin yielded variants with an intermediate reduction in susceptibility that possessed a hot-spot mutation in FKS1. Further exposure to micafungin yielded variants with severely reduced susceptibility that acquired various single mutations in FKS2. The phenotypes of Δfks1 and Δfks2 mutants indicate that the two FKS genes are functionally redundant, while deletion of both FKS1 and FKS2 conferred synthetic lethality. In the laboratory mutants of C. glabrata, clinically significant reduced susceptibility to micafungin required single nucleotide changes in both FKS1 and FKS2, and both genes encoded β-1,3-glucan synthase catalytic subunits.
    Journal of Antimicrobial Chemotherapy 04/2012; 67(7):1666-76. · 5.34 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Members of the pleiotropic drug resistance (PDR) family of ATP binding cassette (ABC) transporters consist of two homologous halves, each containing a nucleotide binding domain (NBD) and a transmembrane domain (TMD). The PDR transporters efflux a variety of hydrophobic xenobiotics and despite the frequent association of their overexpression with the multidrug resistance of fungal pathogens, the transport mechanism of these transporters is poorly understood. Twenty-eight chimeric constructs between Candida albicans Cdr1p (CaCdr1p) and Cdr2p (CaCdr2p), two closely related but functionally distinguishable PDR transporters, were expressed in Saccharomyces cerevisiae. All chimeras expressed equally well, localized properly at the plasma membrane, retained their transport ability, but their substrate and inhibitor specificities differed significantly between individual constructs. A detailed characterization of these proteins revealed structural features that contribute to their substrate specificities and their transport mechanism. It appears that most transmembrane spans of CaCdr1p and CaCdr2p provide or affect multiple, probably overlapping, substrate and inhibitor binding site(s) similar to mammalian ABC transporters. The NBDs, in particular NBD1 and/or the ∼150 amino acids N-terminal to NBD1, can also modulate the substrate specificities of CaCdr1p and CaCdr2p.
    Molecular Microbiology 09/2011; 82(2):416 - 433. · 5.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Zn[2]-Cys[6] binuclear transcription factors Upc2p and Ecm22p regulate the expression of genes involved in ergosterol biosynthesis and exogenous sterol uptake in Saccharomyces cerevisiae. We identified two UPC2/ECM22 homologues in the pathogenic fungus Candida glabrata which we designated CgUPC2A and CgUPC2B. The contribution of these two genes to sterol homeostasis was investigated. Cells that lack CgUPC2A (upc2AΔ) exhibited enhanced susceptibility to the sterol biosynthesis inhibitors, fluconazole and lovastatin, whereas upc2BΔ-mutant cells were as susceptible to the drugs as wild-type cells. The growth of upc2AΔ cells was also severely attenuated under anaerobic conditions. Lovastatin treatment enhanced the expression of ergosterol biosynthetic genes, ERG2 and ERG3 in wild-type and upc2BΔ but not in upc2AΔ cells. Similarly, serum-induced expression of ERG2 and ERG3 was completely impaired in upc2AΔ cells but was unaffected in upc2BΔ cells, whereas serum-induced expression of the sterol transporter gene CgAUS1 was impaired in both upc2AΔ and upc2BΔ cells. These results suggest that in C. glabrata CgUPC2A but not in CgUPC2B is the main transcriptional regulator of the genes responsible for maintaining sterol homeostasis as well as susceptibility to sterol inhibitors.
    Genes to Cells 01/2011; 16(1):80-9. · 2.73 Impact Factor
  • Article: Chimeras of
    [Show abstract] [Hide abstract]
    ABSTRACT: Members of the pleiotropic drug resistance (PDR) family of ATP binding cassette (ABC) transporters consist of two homologous halves, each containing a nucleotide binding domain (NBD) and a transmembrane domain (TMD). The PDR transporters efflux a variety of hydrophobic xenobiotics and despite the frequent association of their overexpression with the multidrug resistance of fungal pathogens, the transport mechanism of these transporters is poorly understood. Twenty‐eight chimeric constructs between Candida albicans Cdr1p (CaCdr1p) and Cdr2p (CaCdr2p), two closely related but functionally distinguishable PDR transporters, were expressed in Saccharomyces cerevisiae. All chimeras expressed equally well, localized properly at the plasma membrane, retained their transport ability, but their substrate and inhibitor specificities differed significantly between individual constructs. A detailed characterization of these proteins revealed structural features that contribute to their substrate specificities and their transport mechanism. It appears that most transmembrane spans of CaCdr1p and CaCdr2p provide or affect multiple, probably overlapping, substrate and inhibitor binding site(s) similar to mammalian ABC transporters. The NBDs, in particular NBD1 and/or the ∼150 amino acids N‐terminal to NBD1, can also modulate the substrate specificities of CaCdr1p and CaCdr2p.
    Molecular Microbiology 01/2011; 82(2). · 5.03 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: To determine the mechanism of intermediate- and high-level echinocandin resistance, resulting from heterozygous and homozygous mutations in GSC1 (FKS1), in both laboratory-generated and clinical isolates of Candida albicans. The DNA sequences of the entire open reading frames of GSC1, GSL1 (FKS3) and RHO1, which may contribute to the beta-1,3-glucan synthase of a micafungin-susceptible strain and a resistant clinical isolate, were compared. A spontaneous heterozygous mutant isolated by selection for micafungin resistance, and a panel of laboratory-generated homozygous and heterozygous mutants that possessed combinations of the echinocandin-susceptible and -resistant alleles, or mutants with individual GSC1 alleles deleted, were used to compare levels of echinocandin resistance and inhibition of glucan synthase activity. DNA sequence analysis identified a mutation, S645P, in both alleles of GSC1 from the clinical isolate. GSL1 had two homozygous amino acid changes and five non-synonymous nucleotide polymorphisms due to allelic variation. The predicted amino acid sequence of Rho1p was conserved between strains. Reconstruction of the heterozygous (S645/S645F) and homozygous (S645F/S645F) mutation showed that the homozygous mutation conferred a higher level of micafungin resistance (4 mg/L) than the heterozygous mutation (1 mg/L). Exposure of the heterozygous mutant to micafungin resulted in a loss of heterozygosity. Kinetic analysis of beta-1,3-glucan synthase activity showed that the homozygous and heterozygous mutations gave echinocandin susceptibility profiles that correlated with their MIC values. A homozygous hot-spot mutation in GSC1, caused by mutation in one allele and then loss of heterozygosity, is required for high-level echinocandin resistance in C. albicans. Both alleles of GSC1 contribute equally and independently to beta-1,3-glucan synthase activity.
    Journal of Antimicrobial Chemotherapy 03/2010; 65(5):842-52. · 5.34 Impact Factor
  • Masakazu Niimi, Norman A Firth, Richard D Cannon
    [Show abstract] [Hide abstract]
    ABSTRACT: Fungi comprise a minor component of the oral microbiota but give rise to oral disease in a significant proportion of the population. The most common form of oral fungal disease is oral candidiasis, which has a number of presentations. The mainstay for the treatment of oral candidiasis is the use of polyenes, such as nystatin and amphotericin B, and azoles including miconazole, fluconazole, and itraconazole. Resistance of fungi to polyenes is rare, but some Candida species, such as Candida glabrata and C. krusei, are innately less susceptible to azoles, and C. albicans can acquire azole resistance. The main mechanism of high-level fungal azole resistance, measured in vitro, is energy-dependent drug efflux. Most fungi in the oral cavity, however, are present in multispecies biofilms that typically demonstrate an antifungal resistance phenotype. This resistance is the result of multiple factors including the expression of efflux pumps in the fungal cell membrane, biofilm matrix permeability, and a stress response in the fungal cell. Removal of dental biofilms, or treatments to prevent biofilm development in combination with antifungal drugs, may enable better treatment and prevention of oral fungal disease.
    Odontology 02/2010; 98(1):15-25. · 1.58 Impact Factor
  • Masakazu Niimi
    [Show abstract] [Hide abstract]
    ABSTRACT: Systemic fungal infections, caused by a wide variety of fungi, contribute to high mortality in humans with immunocompromised conditions. However, there are few classes of antifungal drugs available, limiting therapeutic options. Azoles are the most commonly used class of antifungals to treat many fungal infections, but resistance to azoles can be induced or, for some fungi, is an inherent property. One of major mechanisms of azole resistance is overexpression of drug efflux pumps in fungal cell membranes, such as the energy-dependent ATP-binding cassette (ABC) transporters. We have developed a protein hyperexpression system to facilitate functional analysis of efflux pumps using a Saccharomyces cerevisiae strain as the host for heterologous expression. The system is well suited for the hyperexpression of individual fungal ABC transporters for structural and functional studies. Furthermore, the recombinant yeast strains expressing heterologous membrane proteins can be used to screen for compounds that overcome fungal drug resistance. Also in this review, the mechanisms of azole resistance in Candida glabrata and C. krusei will be considered in relation to the recent increase in the incidence of Candida infections caused by non-albicans Candida. The development of possible novel antifungal agents will also be discussed.
    Nippon Ishinkin Gakkai Zasshi 01/2010; 51(2):79-86.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Drug susceptibility tests were performed with a series of Candida spp. in media supplemented with serum or bile. The azole susceptibilities of several medically important Candida spp., including C. albicans but not C. parapsilosis, were significantly reduced in supplemented media. These findings have important implications for the mechanisms of acquired azole resistance in pathogenic fungi.
    Japanese journal of infectious diseases 08/2009; 62(4):306-8. · 1.51 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Fungi cause serious infections in the immunocompromised and debilitated, and the incidence of invasive mycoses has increased significantly over the last 3 decades. Slow diagnosis and the relatively few classes of antifungal drugs result in high attributable mortality for systemic fungal infections. Azole antifungals are commonly used for fungal infections, but azole resistance can be a problem for some patient groups. High-level, clinically significant azole resistance usually involves overexpression of plasma membrane efflux pumps belonging to the ATP-binding cassette (ABC) or the major facilitator superfamily class of transporters. The heterologous expression of efflux pumps in model systems, such Saccharomyces cerevisiae, has enabled the functional analysis of efflux pumps from a variety of fungi. Phylogenetic analysis of the ABC pleiotropic drug resistance family has provided a new view of the evolution of this important class of efflux pumps. There are several ways in which the clinical significance of efflux-mediated antifungal drug resistance can be mitigated. Alternative antifungal drugs, such as the echinocandins, that are not efflux pump substrates provide one option. Potential therapeutic approaches that could overcome azole resistance include targeting efflux pump transcriptional regulators and fungal stress response pathways, blockade of energy supply, and direct inhibition of efflux pumps.
    Clinical microbiology reviews 05/2009; 22(2):291-321, Table of Contents. · 14.69 Impact Factor
  • Kyoko Niimi, Masakazu Niimi
    [Show abstract] [Hide abstract]
    ABSTRACT: The echinocandin (candin) class of antifungal drugs inhibit beta-1,3-glucan synthase and block synthesis of beta-1,3-glucan , an important polysaccharide in fungal cell walls. Candins are used widely for treatment of systemic infections caused by Candida and Aspergillus because of their high potency and low toxicity to humans. The incidence of candin resistance has been rare compared to that of azole resistance, although candin-resistant clinical isolates of C. albicans, C. glabrata, C. krusei and C. tropicalis have been reported in the USA and Europe in recent years. These isolates possess hundred-fold higher MIC values for candins than sensitive strains, as well as candin-resistant beta-1,3-glucan synthase activities. Their candin resistance is associated with amino acid substitutions in the echinocandin resistant region (Ech) of the FKS gene that encodes a catalytic subunit of the beta-1,3-glucan synthase. However, the effect of these amino acid substitutions on the drug-protein interaction and the molecular basis for the resistance is unknown. The exposure of fungi to candin drugs induces stress responses that activate networks involving transcriptional regulators and components controlling signal transduction of the pathways responsible for maintenance of fungal cell wall integrity. The fungal cell wall is still an attractive drug target and further investigation into the mechanisms of candin resistance and structural analysis of the beta-1,3-glucan synthase protein complex will facilitate the development of broad spectrum inhibitors of fungal cell wall synthesis.
    Nippon Ishinkin Gakkai Zasshi 02/2009; 50(2):57-66.
  • Koichi Tanabe, Kyoko Niimi, Masakazu Niimi
    [Show abstract] [Hide abstract]
    ABSTRACT: Drug resistance in various organisms including cancer cells, bacteria and fungi is a serious issue for human disease therapy, including use of anticancer drugs, antibiotics and antifungals, respectively. Candida strains resistant to the azole class of antifungal drugs, have been isolated frequently from HIV patients following prophylaxis with azole drugs such as fluconazole. Therefore, it is important to be aware of the emergence of drug-resistant clinical isolates, despite the recent introduction of new, effective classes of antifungal drugs such as the azole voriconazole, and the candin micafungin. In this review we discuss the molecular mechanisms underlying resistance to the azole and candin antifungal agents.
    Nippon rinsho. Japanese journal of clinical medicine 01/2009; 66(12):2273-8.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Most Candida krusei strains are innately resistant to fluconazole (FLC) and can cause breakthrough candidemia in immunocompromised individuals receiving long-term prophylactic FLC treatment. Although the azole drug target, Erg11p, of C. krusei has a relatively low affinity for FLC, drug efflux pumps are also believed to be involved in its innate FLC resistance. We describe here the isolation and characterization of Abc1p, a constitutively expressed multidrug efflux pump, and investigate ERG11 and ABC1 expression in C. krusei. Examination of the ERG11 promoter revealed a conserved azole responsive element that has been shown to be necessary for the transcription factor Upc2p mediated upregulation by azoles in related yeast. Extensive cloning and sequencing identified three distinct ERG11 alleles in one of two C. krusei strains. Functional overexpression of ERG11 and ABC1 in Saccharomyces cerevisiae conferred high levels of resistance to azoles and a range of unrelated Abc1p pump substrates, while small molecule inhibitors of Abc1p chemosensitized C. krusei to azole antifungals. Our data show that despite the presence of multiple alleles of ERG11 in some, likely aneuploid, C. krusei strains, it is mainly the low affinity of Erg11p for FLC, together with the constitutive but low level of expression of the multidrug efflux pump Abc1p, that are responsible for the innate FLC resistance of C. krusei.
    Antimicrobial Agents and Chemotherapy 12/2008; 53(2):354-69. · 4.57 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Histoplasmosis is an infectious disease caused by inhaling spores of the fungal pathogen H. capsulatum and in Japan is considered an imported mycosis. However, some patients in Japan with histoplasmosis have no history of traveling overseas nor of risk of occupational exposure to Histoplasma. To investigate the possibility of native distribution of Histoplasma in Japan, 187 bat guano samples from 67 bat-inhabited caves in 17 prefectures were collected. These were examined for H. capsulatum by culture and Histoplasma-specific PCR in three independent laboratories. No H. capsulatum was detected by either method, therefore H. capsulatum is unlikely to be present in bat guano in Japanese caves.
    Microbiology and Immunology 10/2008; 52(9):455-9. · 1.55 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein phosphatases are critical for the regulation of many cellular processes. Null mutants of 21 putative protein phosphatases of Candida albicans were constructed by consecutive allele replacement using the URA3 and ARG4 marker genes. A simple silkworm model of C. albicans infection was used to screen the panel of mutants. Four null mutant (cmp1Delta, yvh1Delta, sit4Delta, and ptc1Delta) strains showed attenuated virulence in the silkworm model relative to that of control and parental strains. Three of the mutants, the cmp1Delta, yvh1Delta, and sit4Delta mutants, had previously been identified as affecting virulence in a conventional mouse model, indicating the validity of the silkworm model screen. Disruption of the putative protein phosphatase gene PTC1 of C. albicans, which has 52% identity to the Saccharomyces cerevisiae type 2C protein phosphatase PTC1, significantly reduced virulence in the silkworm model. The mutant was also avirulent in a mouse model of disseminated candidiasis. Reintroducing either of the C. albicans PTC1 alleles into the disruptant strain, using a cassette containing either allele under the control of a constitutive ACT1 promoter, restored virulence in both infection models. Characterization of ptc1Delta revealed other phenotypic traits, including reduced hyphal growth in vitro and in vivo, and reduced extracellular proteolytic activity. We conclude that PTC1 may contribute to pathogenicity in C. albicans.
    Eukaryotic Cell 09/2008; 7(10):1640-8. · 3.59 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Fluconazole (FLC) remains the antifungal drug of choice for non-life-threatening Candida infections, but drug-resistant strains have been isolated during long-term therapy with azoles. Drug efflux, mediated by plasma membrane transporters, is a major resistance mechanism, and clinically significant resistance in Candida albicans is accompanied by increased transcription of the genes CDR1 and CDR2, encoding plasma membrane ABC-type transporters Cdr1p and Cdr2p. The relative importance of each transporter protein for efflux-mediated resistance in C. albicans, however, is unknown; neither the relative amounts of each polypeptide in resistant isolates nor their contributions to efflux function have been determined. We have exploited the pump-specific properties of two antibody preparations, and specific pump inhibitors, to determine the relative expression and functions of Cdr1p and Cdr2p in 18 clinical C. albicans isolates. The antibodies and inhibitors were standardized using recombinant Saccharomyces cerevisiae strains that hyper-express either protein in a host strain with a reduced endogenous pump background. In all 18 C. albicans strains, including 13 strains with reduced FLC susceptibilities, Cdr1p was present in greater amounts (2- to 20-fold) than Cdr2p. Compounds that inhibited Cdr1p-mediated function, but had no effect on Cdr2p efflux activity, significantly decreased the resistance to FLC of seven representative C. albicans isolates, whereas three other compounds that inhibited both pumps did not cause increased chemosensitization of these strains to FLC. We conclude that Cdr1p expression makes a greater functional contribution than does Cdr2p to FLC resistance in C. albicans.
    Antimicrobial Agents and Chemotherapy 09/2008; 52(11):3851-62. · 4.57 Impact Factor

Publication Stats

1k Citations
192.76 Total Impact Points

Institutions

  • 1996–2014
    • University of Otago
      • • Department of Oral Sciences
      • • Faculty of Dentistry
      Taieri, Otago Region, New Zealand
  • 2002–2009
    • National Institute of Infectious Diseases, Tokyo
      Edo, Tōkyō, Japan
  • 2008
    • Juntendo University
      • Department of Infection Control Science
      Edo, Tōkyō, Japan
  • 2005
    • Meiji Pharmaceutical University
      Edo, Tōkyō, Japan
    • Tokyo Women's Medical University
      • School of Medicine
      Edo, Tōkyō, Japan