Toshiyuki Mio

Isolation of CaSLN1 and CaNIK1, the genes for osmosensing histidine kinase homologues, from the pathogenic fungus Candida albicans

Recent studies have revealed that fungi possess a mechanism similar to bacterial two-component systems to respond to extracellular changes in osmolarity. In Saccharomyces cerevisiae, Sln1p contains both histidine kinase and receiver (response regulator) domains and acts as an osmosensor protein that regulates the downstream HOG1 MAP kinase cascade. SLN1 of Candida albicans was functionally cloned using an S. cerevisiae strain in which SLN1 expression was conditionally suppressed. Deletion analysis of the cloned gene demonstrated that the receiver domain of C. albicans Sln1p was not necessary to rescue SLN1-deficient S. cerevisiae strains. Unlike S. cerevisiae, a null mutation of C. albicans SLN1 was viable under regular and high osmotic conditions, but it caused a slight growth retardation at high osmolarity. Southern blotting with C. albicans SLN1 revealed the presence of related genes, one of which is highly homologous to the NIK1 gene of Neurospora crassa. Thus, C. albicans harbours both SLN1- and NIK1-type histidine kinases.

Tetracycline-regulatable system to tightly control gene expression in the pathogenic fungus Candida albicans

ABSTRACT Conventional tools for elucidating gene function are relatively scarce in Candida albicans, the most prevalent human fungal pathogen. To this end, we developed a convenient system to control gene expression in C. albicans by the tetracycline-regulatable (TR) promoters. When the sea pansy Renilla reniformisluciferase gene (RLUC1) was placed under the control of this system, doxycycline (DOX) inhibited the luciferase activity almost completely. In the absence of DOX, the RLUC1 gene was induced to express luciferase at a level 400- to 1,000-fold higher than that in the presence of DOX. The same results were obtained in hypha-forming cells. The replacement ofN-myristoyltransferase or translation elongation factor 3 promoters with TR promoters conferred a DOX-dependent growth defect in culture media. Furthermore, all the mice infected with these mutants, which are still virulent, survived following DOX administration. Consistently, we observed that the number of these mutant cells recovered from the mouse kidneys was significantly reduced following DOX administration. Thus, this system is useful for investigating gene functions, since this system is able to function in both in vitro and in vivo settings.

The Eukaryotic UDP-N-Acetylglucosamine Pyrophosphorylases GENE CLONING, PROTEIN EXPRESSION, AND CATALYTIC MECHANISM

A search of the yeast data base for a protein homologous to Escherichia coliUDP-N-acetylglucosamine pyrophosphorylase yieldedUAP1 (UDP-N-acetylglucosaminepyrophosphorylase), the Saccharomyces cerevisiae gene for UDP-N-acetylglucosamine pyrophosphorylase. The Candida albicans and human homologs were also cloned by screening a C. albicans genomic library and a human testis cDNA library, respectively. Sequence analysis revealed that the human UAP1 cDNA was identical to previously reported AGX1. A null mutation of the S. cerevisiae UAP1 (ScUAP1) gene was lethal, and when expressed under the control of ScUAP1 promoter, bothC. albicans and Homo sapiens UAP1(CaUAP1 and HsUAP1) rescued theScUAP1-deficient S. cerevisiae cells. All the recombinant ScUap1p, CaUap1p, and HsUap1p possessed UDP-N-acetylglucosamine pyrophosphorylase activitiesin vitro. The yeast Uap1p utilizedN-acetylglucosamine-1-phosphate as the substrate, and together with Agm1p, it produced UDP-N-acetylglucosamine from N-acetylglucosamine-6-phosphate. These results demonstrate that the UAP1 genes indeed specify eukaryotic UDP-GlcNAc pyrophosphorylase and that phosphomutase reaction precedes uridyltransfer. Sequence comparison with other UDP-sugar pyrophosphorylases revealed that amino acid residues, Gly112, Gly114, Thr115, Arg116, Pro122, and Lys123 of ScUap1p are highly conserved in UDP-sugar pyrophosphorylases reported to date. Among these amino acids, alanine substitution for Gly112, Arg116, or Lys123 severely diminished the activity, suggesting that Gly112, Arg116, or Lys123 are possible catalytic residues of the enzyme.

Involvement of cell wall β‐glucan in the action of HM‐1 killer toxin

HM‐1 killer toxin secreted from Hansenula mrakii inhibits the growth of Saccharomyces cerevisiae cells by interfering with β‐1,3‐glucan synthesis. We found that HM‐1 killer toxin killed intact cells but not protoplasts. In addition, cells lacking the functional KRE 6 allele (kre6Δ) became resistant to higher concentration of HM‐1 killer toxin. As reported by Roemer and Bussey [(1991) Proc. Natl. Acad. Sci. 88 11295–11299], cells lacking functional KPE6 had a reduced level of the cell wall β‐1,6‐glucan compared to that in cells harboring the normal KRE6. These results suggest that the cell wall ⨿‐glucan is involved in the action of HM‐1 killer toxin. Addition of HM‐1 killer toxin with several kinds of oligosaccharides revealed that either ⨿‐1,3‐ or β‐1,6‐glucan blocked the cytocidal action of HM‐1 killer toxin whereas α‐1,4‐glucan and chitin did not. Mannan also interfered with HM‐1 killer toxin action, but this inhibitory effect was much weaker than that observed with β‐1,3‐ or β‐1,6‐glucans. Thus, it appears that the cell wall β‐glucan interacts with HM‐1 killer toxin, and that this toxin‐β‐glucan commitment is required for the action of HM‐1 killer toxin.

Reduced virulence of Candida albicans mutants lacking the GNA1 gene encoding glucosamine-6-phosphate acetyltransferase.

The yeast GNA1 gene encodes glucosamine-6-phosphate acetyltransferase which catalyses the reaction of glucosamine 6-phosphate with acetyl-CoA to form N-acetylglucosamine 6-phosphate, a fundamental precursor in UDP-N-acetylglucosamine biosynthesis. Candida albicans mutants lacking GNA1 were viable in the presence of N-acetylglucosamine. To confirm the physiological importance of C. albicans GNA1, the virulence of a C. albicans gna1Delta null mutant was examined in a mouse model of candidiasis. When injected intravenously into mice, the virulence of the C. albicans gna1Delta null mutant was significantly attenuated. The reduced virulence appeared to be the result of rapid clearance from host tissue. These data suggest that C. albicans GNA1 is required for survival of the fungus in host animals, probably because an insufficient level of N-acetylglucosamine is available from the host tissues.

Isolation and characterization of the Candida albicans gene for mRNA 5′-triphosphatase: association of mRNA 5′-triphosphatase and mRNA 5′-guanylyltransferase activities is essential for the function of mRNA 5′-capping enzyme in vivo1

The amino acid sequence of the Saccharomyces cerevisiae mRNA 5′‐triphosphatase (TPase) diverges from those of higher eukaryotes. In order to confirm the sequence divergence of TPases in lower and higher eukaryotes, the Candida albicans gene for TPase was identified and characterized. This gene designated CaCET1 ( . lbicans mRNA 5′‐ apping nzyme triphosphatase ) has an open reading frame of 1.5 kb, which can encode a 59‐kDa protein. Although the N‐terminal one‐fifth of S. cerevisiae TPase (ScCet1p) is missing in CaCet1p, CaCet1p shares significant sequence similarity with ScCet1p over the entire region of the protein; the recombinant CaCet1p, which was expressed as a fusion protein with glutathione S‐transferase (GST), displayed TPase activity in vitro. CaCET1 rescued CET1‐deficient S. cerevisiae cells when expressed under the control of the ADH1 promoter, whereas the human capping enzyme derivatives that are active for TPase activity but defective in mRNA 5′‐guanylyltransferase (GTase) activity did not. Yeast two‐hybrid analysis revealed that C. albicans Cet1p can bind to the S. cerevisiae GTase in addition to its own partner, the C. albicans GTase. In contrast, neither the full‐length human capping enzyme nor its TPase domain interacted with the yeast GTase. These results indicate that the failure of the human TPase activity to complement an S. cerevisiae cet1Δ null mutation is attributable, at least in part, to the inability of the human capping enzyme to associate with the yeast GTase, and that the physical association of GTase and TPase is essential for the function of the capping enzyme in vivo.

Crystal Structures of N-Acetylglucosamine-phosphate Mutase, a Member of the α-d-Phosphohexomutase Superfamily, and Its Substrate and Product Complexes

N-Acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthetic process of UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a UDP sugar that serves as a biosynthetic precursor of glycoproteins, mucopolysaccharides, and the cell wall of bacteria. Thus, a specific inhibitor of AGM1 from pathogenetic fungi could be a new candidate for an antifungal reagent that inhibits cell wall synthesis. AGM1 catalyzes the conversion of N-acetylglucosamine 6-phosphate (GlcNAc-6-P) into N-acetylglucosamine 1-phosphate (GlcNAc-1-P). This enzyme is a member of the α-d-phosphohexomutase superfamily, which catalyzes the intramolecular phosphoryl transfer of sugar substrates. Here we report the crystal structures of AGM1 from Candida albicans for the first time, both in the apoform and in the complex forms with the substrate and the product, and discuss its catalytic mechanism. The structure of AGM1 consists of four domains, of which three domains have essentially the same fold. The overall structure is similar to those of phosphohexomutases; however, there are two additional β-strands in domain 4, and a circular permutation occurs in domain 1. The catalytic cleft is formed by four loops from each domain. The N-acetyl group of the substrate is recognized by Val-370 and Asn-389 in domain 3, from which the substrate specificity arises. By comparing the substrate and product complexes, it is suggested that the substrate rotates about 180° on the axis linking C-4 and the midpoint of the C-5—O-5 bond in the reaction.

Functional cloning and mutational analysis of the human cDNA for phosphoacetylglucosamine mutase: identification of the amino acid residues essential for the catalysis.

In Saccharomyces cerevisiae, phosphoacetylglucosamine mutase is encoded by an essential gene called AGM1. The human AGM1 cDNA (HsAGM1) and the Candida albicans AGM1 gene (CaAGM1) were functionally cloned and characterized by using an S. cerevisiae strain in which the endogenous phosphoacetylglucosamine mutase was depleted. When expressed in Escherichia coli as fusion proteins with glutathione S-transferase, both HsAgm1 and CaAgm1 proteins displayed phosphoacetylglucosamine mutase activities, demonstrating that they indeed specify phosphoacetylglucosamine mutase. Sequence comparison of HsAgm1p with several hexose-phosphate mutases yielded three domains that are highly conserved among phosphoacetylglucosamine mutases and phosphoglucomutases of divergent organisms. Mutations of the conserved amino acids found in these domains, which were designated region I, II, and III, respectively, demonstrated that alanine substitutions for Ser(64) and His(65) in region I, and for Asp(276), Asp(278), and Arg(281) in region II of HsAgm1p severely diminished the enzyme activity and the ability to rescue the S. cerevisiae agm1Delta null mutant. Conservative mutations of His(65) and Asp(276) restored detectable activities, whereas those of Ser(64), Asp(278), and Arg(281) did not. These results indicate that Ser(64), Asp(278), and Arg(281) of HsAgm1p are residues essential for the catalysis. Because Ser(64) corresponds to the phosphorylating serine in the E. coli phosphoglucosamine mutase, it is likely that the activation of HsAgm1p also requires phosphorylation on Ser(64). Furthermore, alanine substitution for Arg(496) in region III significantly increased the K(m) value for N-acetylglucosamine-6-phosphate, demonstrating that Arg(496) serves as a binding site for N-acetylglucosamine-6-phosphate.

Crystal Structure of Uridine-diphospho-N-acetylglucosamine Pyrophosphorylase from Candida albicans and Catalytic Reaction Mechanism

Uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc) is a precursor of the bacterial and fungal cell wall. It is also used in a component of N-linked glycosylation and the glycosylphosphoinositol anchor of eukaryotic proteins. It is synthesized from N-acetylglucosamine-1-phosphate (GlcNAc-1-P) and uridine-5′-triphosphate (UTP) by UDP-GlcNAc pyrophosphorylase (UAP). This is an SN2 reaction; the non-esterified oxygen atom of the GlcNAc-1-P phosphate group attacks the α-phosphate group of UTP. We determined crystal structures of UAP from Candida albicans (CaUAP1) without any ligands and also complexed with its substrate or with its product. The series of structures in different forms shows the induced fit movements of CaUAP1. Three loops approaching the ligand molecule close the active site when ligand is bound. In addition, Lys-421, instead of the metal ion in prokaryotic UAPs, is coordinated by both phosphate groups of UDP-Glc-NAc and acts as a cofactor. However, a magnesium ion enhances the enzymatic activity of CaUAP1, and thus we propose that the magnesium ion increases the affinity between UTP and the enzyme by coordinating to the α- and γ-phosphate group of UTP.

Preclinical antitumor activity of the novel heat shock protein 90 inhibitor CH5164840 against human epidermal growth factor receptor 2 (HER2)‐overexpressing cancers

Heat shock protein 90 (Hsp90), a molecular chaperone that plays a significant role in the stability and maturation of client proteins, including oncogenic targets for cell transformation, proliferation, and survival, is an attractive target for cancer therapy. We identified the novel Hsp90 inhibitor, CH5164840, and investigated its induction of oncogenic client protein degradation, antiproliferative activity, and apoptosis against an NCI‐N87 gastric cancer cell line and a BT‐474 breast cancer cell line. Interestingly, CH5164840 demonstrated tumor selectivity both in vitro and in vivo, binding to tumor Hsp90 (which forms active multiple chaperone complexes) in vitro, and being distributed effectively to tumors in a mouse model, which, taken together, supports the decreased levels of phosphorylated Akt by CH5164840 that we observed in tumor tissues, but not in normal tissues. As well as being well tolerated, the oral administration of CH5164840 exhibited potent antitumor efficacy with regression in NCI‐N87 and BT‐474 tumor xenograft models. In addition, CH5164840 significantly enhanced antitumor efficacy against gastric and breast cancer models when combined with the human epidermal growth factor receptor 2 (HER2)‐targeted agents, trastuzumab and lapatinib. These data demonstrate the potent antitumor efficacy of CH5164840 when administered alone, and its significant combination efficacy when combined with trastuzumab or lapatinib, supporting the clinical development of CH5164840 as an Hsp90 inhibitor for combination therapy with HER2‐targeted agents against HER2‐overexpressing tumors. (Cancer Sci 2012; 103: 342–349)