Candida albicans Msi3p, a homolog of the Saccharomyces cerevisiae Sse1p of the Hsp70 family, is involved in cell growth and fluconazole tolerance.

We investigated the cellular function of Msi3p, belonging to the heat shock protein 70 family, in Candida albicans. The mutant strain tetMSI3 was generated, in which MSI3 was controlled by a tetracycline-repressive promoter, because there is evidence to suggest that MSI3 is an essential gene. We controlled the MSI3 expression level by doxycycline (DOX) and compared its phenotype with that of a control strain with the tetracycline-repressive promoter and a wild-type copy MSI3. The results indicated that MSI3 was essential for cell growth. In addition, all the tetMSI3-infected mice survived after DOX administration. Drug susceptibility tests indicated that repression of MSI3 expression resulted in hypersensitivity to fluconazole and conferred fungicidal activity to fluconazole. The expression levels of MSI3 and calcineurin-dependent genes were upregulated in response to fluconazole in the control strain. In tetMSI3, the upregulation of MSI3 was lost, and the expression level of the calcineurin-dependent genes was no longer elevated in response to fluconazole and was not affected by DOX, indicating that the upregulation of MSI3 expression was required for the induction of the calcineurin-dependent gene expression. These data suggest that Msi3p confers fluconazole tolerance by partially influencing the calcineurin signaling pathway and also other tolerance mechanisms.

In vitro efficacy of continuous mild heat stress on the antifungal susceptibility of Candida albicans biofilm formation.

Elevation in the temperature induces heat stress to both host cells and the invading pathogen. This study aimed to determine whether continuous mild heat stress (increased temperature without causing significant damage to host cells) can increase susceptibility of biofilm formation of the opportunistic fungal pathogen Candida albicans to low concentrations of three typical antifungal agents. In this way the side effects associated with higher concentrations of the antifungal agents on host cells would be reduced. Fluconazole and micafungin at concentrations ranging from 0.0625 to 2 µg/mL and amphotericin B at concentrations ranging from 0.0625 to 1 µg/mL inhibited less than 20% of cells in biofilm formation. Biofilm formation at 39 or 41°C compared to 37°C resulted in increased susceptibility to the three agents, but especially micafungin. These data suggest that mild heat stress (39°C) would be valuable for increasing the effectiveness of low concentrations of antifungal agents against C. albicans biofilm formation. Thus, the concept of continuous mild heat stress at the site of insertion of medical devices or catheters combined with antifungal agents could be beneficial.

Transcriptional changes in Candida albicans Genes by both farnesol and high cell density at an early stage of morphogenesis in N-acetyl-D-glucosamine medium.

Quorum sensing through farnesol, a quorum sensing molecule, regulates virulence and morphogenesis in Candida albicans. Farnesol and high cell density of C. albicans repress hyphal formation in a minimal medium containing N-acetyl-D-glucosamine. Global transcription profiling at an early stage of quorum sensing by C. albicans in the N-acetyl-D-glucosamine medium was analyzed. Twenty-two of a total of 53 genes responded to both farnesol and high cell density. From in silico analysis and previous published data, nine of these genes including those encoding amino acid biosynthesis were controlled by the Gcn4p regulator. Nine other genes which included genes encoding central carbon metabolism were controlled by negative regulators including Nrg1p, Tup1p, Ssn6p, and/or Mig1p. Other genes not controlled by these regulators included genes related to oxidative stress, glucose metabolism, and agglutination. Expression of genes related to amino acid biosynthesis and central carbon metabolism in this study is similar to a previous report of transcription profiling in C. albicans following its internalization by phagocyte cells and adaptation to host challenges.

[Quorum-sensing system in Candida albicans].

The bacterial behavior system controlled by the cell density is described as quorum-sensing. The system is triggered via autoinducers. Various kinds of autoinducers have been identified from different bacteria. Quorum-sensing signals via autoinducers are involved in regulation of important virulences such as exotoxin, protease, and pigment production. Therefore, this system in pathogenic bacteria has a critical role in the regulation of bacterial pathogenicity. In the pathogenic fungus Candida albicans, an extracellular quorum-sensing molecule that regulates hyphal formation by this organism has been identified in recent years. Candida albicans has been shown to form biofilm on many medical devices, therefore quorum-sensing in this organism has been especially focused on from the aspect of biofilm formation.

Antibacterial activities and bonding of MMSA/TBB resin containing amphiphilic lipids.

The purpose of this study was to investigate the antibacterial activity of MMA/TBB resin containing newly developed amphiphilic lipids. The amphiphilic lipids, C10-L-Ala/pts and C12-L-Ala/pts, synthesized from the reaction of n-alkyl alcohol and L-alanine were dissolved in MMA at concentrations of 0.5, 1.0, 1.5, and 2.0 mol%. Resin mixtures of PMMA powder and each MMA liquid containing lipid and TBB were prepared for all tests. Both lipids gave antibacterial effect to MMA/ TBB resin. The addition of C12-L-Ala/pts to MMA resulted in a significantly higher antibacterial activity than the addition of C10-L-Ala/pts. In terms of bond strength, the bond strength of MMA/TBB resin to bovine dentin was significantly decreased by the addition of amphiphilic lipids. But for enamel, the bond strength of MMA/TBB resin with amphiphilic lipids was clinically acceptable for orthodontic brackets. In conclusion, amphiphilic lipids will be useful as a component of adhesive resin to give the latter an antibacterial effect.

A poly(gamma-glutamic acid)-amphiphile complex as a novel nanovehicle for drug delivery system.

Recently, many studies have focused on biomedical and pharmaceutical applications of self-assembled nanoparticles. In addition, several biodegradable nanoparticles have been reported to possess poor dispersion stability and poor size-controllability. However, these nanoparticles require complicated fabrication procedures using synthesis techniques. We developed an efficient method for producing nanoparticles derived from a biological origin of molecule poly(gamma-glutamic acid) (gamma-PGA), a cationic lipid, and doxorubicin (Dox). The complex had a size of 510 nm and was able to encapsulate over 90% of the added Dox. An in vivo assay of antitumor activity demonstrated that the complex had significant antitumor activity in sarcoma 180-bearing mice, and was effectively accumulated in solid tumors based on the EPR effect. The data suggested that this complex is a promising formulation of gamma-PGA for targeted delivery to solid tumors. gamma-PGA-12GP2 complexes may possess several unique advantages, including simplicity of nanoparticle preparation, high drug-carrying capacity, appropriate size to allow deeper penetration based on EPR effect into solid tumors, and lack of necessity to modify the chemical structure of the drugs. These data indicate that the gamma-PGA-12GP2 complexes are potentially useful in cancer chemotherapy.

[Farnesol as a quorum-sensing molecule in Candida albicans].

Farnesol is one of the quorum sensing molecules of Candida albicans. In this report, we discuss the effects of farnesol on: 1. growth of Candida albicans in vitro and in vivo; 2. the incorporation of biomolecules into the cell wall of Candida albicans; and 3. cytokine expression by the immune system. Our results indicate genes of Candida albicans expressed at an early stage of quorum-sensing. Half of these genes are known and two-thirds of known genes are up-regulated by two types of transcription factors.

Transcriptional profiling of the early stages of germination in Candida albicans by real-time RT-PCR.

By using real-time RT-PCR, we profiled the expression of CGR1, CaMSI3, EFG1, NRG1, and TUP1 in Candida albicans strains JCM9061 and CAI4 under several conditions, including induction of morphological transition, heat shock, and treatment with calcium inhibitors. Expression of CaMSI3 changed under these growth conditions except during heat shock. CGR1 expression increased during the early stages of hyphal growth in JCM9061, while expression was strain-dependent during heat shock. Both EFG1 and NRG1 were similarly expressed under hypha-inducing conditions and heat shock. Expression of TUP1 was slightly different from the expression of EFG1 or NRG1.

Isolation and sequencing of the Candida albicans MSI3, a putative novel member of the HSP70 family.

We have reported previously that the expression of CGR1 increased at an early stage of the yeast-mycelial transition (morphogenesis) in Candida albicans. We now show that Cgr1p interacts in a yeast two-hybrid system with the C. albicans Msi3p (CaMsi3p), a putative novel member of the heat shock protein 70 (HSP70) family. The DNA sequence of CaMSI3 encodes a predicted protein of 702 amino acids with a molecular mass of 78.6 kDa. The amino acid sequence of CaMsi3p is 63% identical to Msi3p/Sse1p of the HSP70 family of Saccharomyces cerevisiae. Further, CaMSI3 complemented the temperature-sensitive phenotype of the msi3(-) mutant of S. cerevisiae. Other heat shock proteins of C. albicans are required for morphogenesis and are highly antigenic. These observations suggest that CaMSI3 may well provide functions for this organism unrelated to a heat shock function. The DDBJ Accession No. for the sequence reported in this paper is AB061274.