Objectives: The amino sugar N-acetylglucosamine (GlcNAc) is well known to induce the mycelial growth of Candida albicans. We had reported that GlcNAc aggravated murine oral and gastrointestinal candidiasis1, 2), and C. albicans adheres to chitin, polymer of GlcNAc3). Here we investigated the effect of chitin oligosaccharides, the oligomer of GlcNAc, on mycelial growth of C. albicans and murine oral candidiasis.
Methods: C. albicans strain TIMM1768, TIMM2640 and TIMM3136 recovered from patients with candidiasis were used. Chitin oligosaccharides (GlcNAc) n (n = 1–6) were produced by partial hydrolysis of crab chitin by the method of Rupley4). One hundred microliters of C. albicans cell suspension were aliquoted into 96-well microtiter plates (1 x 104 cfu/well for germ-tube formation, 1 x 103 cfu/well for mycelial growth analysis), 100-μl serial dilutions of chitin oligosaccharides (GlcNAc, (GlcNAc)3 or (GlcNAc)4) were then added the wells which made final concentration of 50 mM/ml to 3.13 mM/ml and incubated at 37℃ in 5% CO2 in air for 2h or 4h for germ-tube formation, for 20h for mycelial growth analysis. Animal experiments were performed according to the guidelines for the care and use of animals approved by Teikyo University (approval number 15-026). The experimental procedures for oral candidiasis model in mice were according to Takakura N 5).
Results: Chitin oligosaccharides inhibited mycelial growth of C. albicans in vitro. Inhibition of mycelial growth detected by the length of hyphae from 4h-culture of C. albicans; hyphae length elongation of C. albicans cells in the control culture, 25mM GlcNAc-, 25mM (GlcNAc)3- and 25mM (GlcNAc)4-supplemented culture were 89.77±20.38, 86.1±22.7, 67.18±27.96, and 42.25±22.4μm, respectively. Average length of the elongated hyphae in the case of (GlcNAc)3 or (GlcNAc)4 were significantly less than control or monomer (P<0.01). After 20h culture, each volumes of hyphae attached to the bottom of the culture plates were measured by crystal violet staining method. The hyphal volume of C. albicans after 20h-culture were decreased by the presence of the oligomer of GlcNAc (degree of polymerization = 2 to 6). The inhibitory effect of mycelial growth were observed in the case of three strains of C. albicans, TIMM1768, TIMM2640 and TIMM3163 (having fluconazole-resistant phenotype). To clarify the effect of chitin oligosaccharides in vivo, their effects on experimental murine oral candidiasis were examined. The mice infected with C. albicans were administrated 50μL of 12.5mM (GlcNAc)1-4 four times (5min, 3h, 24h, 27h). Although mice given GlcNAc showed significant aggravation of oral symptoms of candidiasis after two days (P<0.01), mice given (GlcNAc)2-4 showed significant improvement when compared with those of GlcNAc in oral symptoms (P<0.01).
Conclusion: application of chitin oligosaccharide as a nutritional supplement may have a better effect on oral health in people susceptible to oral or gastrointestinal candidiasis.
Acknowledgements; this work was supported in part by a Grant-in-Aid for scientific research (C) (JP16K08790) to SAI.
Reference
1. Ishijima SA et al. Med Mycol, 50; 252-258 (2012)
2. Ishijima SA et al. Med Mycol, 56E; E31-E39 (2015)
3. Ishijima SA et al. Med Mycol J, 58E; E15-E21 (2017)
4. Rupley JA. Biochem Biophys Acta, 83; 245-255 (1964)
5. Takakura N et al. Microbiol Immunol 47: 321-326 (2003)

Chitin oligosaccharides (oligomer of GlcNAc) protect mycelial growth of Candida albicans in vitro and in vivo.