Kenjirou Nakamura

Extracellular proteolytic activity of Cryptococcus neoformans.

Eight strains ofCryptococcus neoformans var.neoformans isolated from AIDS patients in the Infectious Disease Institute, University of Turin, Italy, were examined for growth and extracellular proteolytic activity in culture with solid and liquid media. All of the strains grew well on Yeast Carbon Base (YCB) agar medium supplemented with both 0.1% (w/v) bovine serum albumin (BSA) and 0.01% (w/v) polypeptone (Pp), and produced a clear proteolytic zone around their colonies, whereas they exhibited less growth and proteolytic activity on YCB medium supplemented with BSA alone. Strain #8 with a strong proteolytic activity was cultured in three different liquid media. Its growth was limited in YCB medium supplemented with 0.1% BSA, but was moderate in that with 0.01% Pp. Enhanced growth was supported by the addition of both BSA and Pp to the YCB medium. The relative value of the final cellular yields obtained with the above YCB-0.1% BSA, YCB-0.01% Pp and YCB-0.1% BSA-0.01% Pp media was approximately 1:10:20. In the culture with YCB medium containing both BSA and Pp, a rapid decrease in the amount of BSA was demonstrated by a spectrophotometric assay and gel electrophoresis of the culture supernatant after the log-to-stationary phase. The proteolytic activity in the culture supernatant became detectable after the log phase when tested with skim milk agarose plates. These results allowed us to conclude thatCr. neoformans var.neoformans is able to secrete protease and to utilize protein as a source of nitrogen.

Oxygen as a possible tropic factor in hyphal growth of Candida albicans

Hyphae ofCandida albicans elongated towards the oxygen-rich direction when exposed to gradients of oxygen concentration in thin-layer and capillary-tube cultures with corn meal (CM) agar. The thin-layer culture was prepared by covering a drop of molten CM agar containingC. albicans cells with a cover slip in Petri dishes. Cells located in the central region of the thin-layered medium neither grew nor produced hyphae. Cells in the marginal regions at first directed their hyphae in arbitrary directions after forming a small colony. Hyphae then gradually changed their direction of elongation and eventually oriented towards the nearest margin. Under anaerobiosis, cells seeded in the thin-layered medium did not grow even in the marginal regions. When exposed to air, the cells in the marginal regions rapidly began to form hyphae which elongated towards the nearest margin. To prepare an oxygen gradient in capillary-tube cultures, CM agar, and dilute and dense cell suspensions in CM agar were introduced sequentially into the capillary tubes, and the end closest to the dense cell suspension was sealed with paraffin. Among cells in the dilute layer, only that located closest to the meniscus grew well and extended hyphae towards the meniscus, where oxygen concentrations were highest. These studies suggest a positive aerotropic response in the hyphal growth ofC. albicans.

Diversity of laccase among Cryptococcus neoformans serotypes

The pathogenic yeast C. neoformans is classified into three varieties with five serotypes; var. grubii (serotype A), var. neoformans (serotype D), var. gattii (serotypes B and C), and serotype AD. Melanin is a virulence factor in the species, and its biosynthesis is catalyzed by laccase, encoded by the LAC1 gene. In order to estimate the natural variability of the LAC1 gene among Cryptococcus serotypes, the laccase protein sequence from 55 strains was determined and the phylogenetic relationships between cryptococcal and related fungal laccases revealed. The deduced laccase proteins consisted of 624 amino acid residues in serotypes A, D and AD, and 613 to 615 residues in serotypes B and C. Intra‐serotype amino acid variation was marginal within serotypes A and D, and none was found within serotypes AD and C. Maximum amino acid replacement occurred in two serotype B strains. The similarity in the deduced sequence ranged from 80 to 96% between serotypes. The sequence in the copper‐binding regions was strongly conserved in the five serotypes. The laccases of the five serotypes were grouped together in the same clade of the phylogenetic tree reconstructed from different fungal laccases, suggesting a monophyletic clade.

Comparisons of the laccase gene among serotypes and melanin-deficient variants of Cryptococcus neoformans.

Cryptococcus neoformans is a fungus causing life‐threatening infections in immunocompromised hosts. Melanin production is a major virulence factor of this fungus and the initial steps of dihydroxyphenylalanine (DOPA)‐melanin biosynthesis pathways are catalyzed by laccase. To understand phylogenetic relationships among serotypes of three varieties, partial sequences (about 600 bases) of the laccase gene (CNLAC1) were determined in a total of 64 strains, including 10 melanin‐deficient variants. The phylogenetic tree constructed from the nucleotide sequence grouped the 64 strains into the clusters corresponding to the three varieties. The diversity of the fragment sequences was very minor among strains of each of var. grubii and var. neoformans. Strains in var. gattii, however, were subdivided into two groups, although differences between serotypes B and C were not large. The sequences of the melanin‐deficient variants were almost completely homologous to those of the melanin‐producing strains in the same serotype. Results of laccase assay and northern blot analysis suggested that the lower melanin production in the variants was associated with lower transcription of the laccase gene.

Radiographic features of experimentalCandida arthritis in rats

Sprague-Dawley rats were inoculated intravenously (i.v.) withCandida albicans, and limb joints showing signs ofCandida-induced arthritis were subjected to radiographic and histologic examination. New bone formation and bone resorption were morbidly enhanced in bones sampled from the arthritic joints. Sparsely distributed needle-shaped calcified deposits began to be formed on bony surfaces in parallel with the onset of joint swelling. The calcified deposits gradually became denser and then covered the bony surfaces almost entirely, giving rise to an exostosis-like profile. In addition to the new bone formation, bone resorption was also observed in regions adjacent to the sites of new bone formation, and punched-out bone lesions were produced. Eventually, severe deformation of joint bones due to new bone formation and bone resorption was evident. Reflecting these unusual radiographic changes, abundant osteoblasts and osteoclasts were demonstrated histologically in the bones. On the basis of these results, possible mechanisms for the induction of arthritis byCandida infection are discussed.

Study of the oral carriage of Candida sp. in dental students and staff—Identification of Candida sp. and background survey

Abstract Purpose The purpose of this study is to investigate the relationship between oral candidal carriage in able-bodied persons (dental students and staff) and health condition using a epidemiological method with questionnaire, and use the results for an educational campaign for the promotion of health. Methods The candidal carriage was examined by culture method using swabs from tongue surfaces. The identification of Candida spp. of the culture positive specimens was performed by a multiplex polymerase chain reaction method. Questionnaire items included age, gender, body mass index, pedometer use, oral conditions, regularity of hospital visits, medication, dental visits, oral care, exercise habits, alcohol habits, and smoking habits. Results A total 482 participants were surveyed over a period of two years, males: 269 (mean, 36 years); females: 213 (33 years). Oral candidal carriage was 18.3%. Candida albicans accounted for 80.7% of isolated Candida spp. After analysis using a stepwise method, three items (age, smoking habits, and exercise habits) were selected as the variables in the model. The adjusted odds ratios (95% confidence intervals) for the three model variables were 1.84 (1.10–3.08) for exercise habits, 1.93 (1.00–3.69) for smoking, and 0.96 (0.94–0.98) for age. Logistic regression analysis suggested an association between candidal carriage, exercise habits, and smoking. Conclusions Because lack of exercise, as well as smoking are well-known to be detrimental factors to the maintenance of good health, candidal carriers do not appear to be in a condition of good health. In other words, candidal carriage may be a health warning.

Systemic hematogenous dissemination of mouse oral candidiasis is induced by oral mucositis

The causes of fungemia include immunosuppression and neutropenia stemming from diverse factors as well as the placement of central venous catheters. However, the relationship between fungemia and the oral cavity has not been substantiated. In this study, we explored the pathological conditions of Candida albicans—derived oral candidiasis in a mouse model, which always develops oral mucositis as a complication. In oral candidiasis, the hyphae of C. albicans are believed to primarily invade the stratum granulosum, but not the subepithelium, of the mucous membrane. We provide histological evidence that in concomitant oral mucositis, the hyphae infiltrate the subepithelium and blood vessels. Blood cultures and tissue samples revealed the onset of fungemia only in the mucositis-induced groups. Positive numbers of colony-forming units were found in groups A (chemotherapy), B (chemotherapy + mucositis) and C (mucositis), but were highest in group B. Some organs revealed positive CFU in groups B and C. The presence of fungal DNA in blood plasma and tissue was confirmed by PCR. The fungal DNA frequency was significantly higher in the mucositis group when compared with the non-mucositis group. The results suggest that fungi first invade the subepithelium and then the blood vessels, from which they disseminate throughout the body, and that oral mucositis is an important risk factor for fungemia. This study clearly demonstrates the relationship between oral mucositis, fungemia, and the potential systemic fungal dissemination, which has not been previously proven. Our findings highlight the importance of oral care for patients at risk of fungemia.

White-to-opaque switching is involved in the phospholipase B production of Candida dubliniensis on Price’s egg yolk agar

Measuring the production of Candida dubliniensis (C. dubliniensis) phospholipase B (PLase B) by the Price’s method has long been considered to be unattainable because the levels of PLase produced are undetectable. In this study, C. dubliniensis, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis and C. tropicalis were shown to produce PLase B and form clear white zones around their colonies when peptone, a component of the original Price’s egg yolk (OP) agar, is replaced with a yeast nitrogen base (YNB). This new medium is named modified Price’s (MP) agar. Based on this finding, we propose a new modified Price’s (NMP) agar containing 0.75% peptone and 0.25% YNB, which enabled measurement of PLase B production by C. dubliniensis and C. albicans with results consistent with those obtained for C. albicans grown on OP agar. We strongly believe that the MP and NMP agars are very useful for screening PLase B production by C. dubliniensis and non-albicans Candida spp. Moreover, the addition of several bioactive agents (the proteinase inhibitors pepstatin A and saquinavir, the calcineurin inhibitors cyclosporine A and tacrolimus, the cell-permeable cAMP analog dBcAMP, and the quorum-sensing molecule farnesol) to the OP agar enhanced PLase B production by C. dubliniensis. During the course of our study to clarify the reason why PLase B was not produced, we found that C. dubliniensis cells grown on OP agar undergo a white-to-opaque transition, which may explain why they showed minimal production of PLase B on this medium.