An important point in the development of an antimicrobial agent is whether its target molecules are essential for growth of the microorganism. From this viewpoint, we focused attention on essential genes as potential tar...An important point in the development of an antimicrobial agent is whether its target molecules are essential for growth of the microorganism. From this viewpoint, we focused attention on essential genes as potential targets of antifungal agents in the pathogenic yeast Candida. Here we introduce recent attempts for screening, identification, and characterization of essential genes from a haploid yeast Candida glabrata, using temperature-sensitive mutants. Our experimental results suggesting the essentiality of C. albicans PHO85, the homologue of which is known as a negative regulator of the PHO system and as a non-essential gene in Saccharomyces cerevisiae are also described.
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-se...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.
In Cryptococcus neoformans the DNA content of cells having tiny buds varied rather widely, depending on growth phases and strains used. Typically, buds of C. neoformans emerged soon after initiation of DNA synthesis in t...In Cryptococcus neoformans the DNA content of cells having tiny buds varied rather widely, depending on growth phases and strains used. Typically, buds of C. neoformans emerged soon after initiation of DNA synthesis in the early exponential phase. However, bud emergence was delayed to G2 during transition to the stationary phase, and in the early stationary phase budding scarcely occurred, although roughly half of the cells completed DNA synthesis. The timing of budding in C. neoformans was shifted to later cell cycle points with progression of the growth phase of the culture. Similarly, a deficit in oxygen was demonstrated to delay the timing of budding, prolong the G2 phase and cause accumulation of cells after DNA synthesis, but before commitment to budding. The C. neoformans homologue of the main cell cycle control gene CDC28/Cdc2 was isolated using degenerate RT-PCR. The full-length coding region was then amplified using primers to target the regions around the start and stop codons. The gene was called CnCdk1 and was found to have high homologies to S. cerevisiae CDC28 and S. pombe cdc2. To determine its function, its ability to rescue S. cerevisiae cdc28-temperature sensitive mutants was tested. S. cerevisiae cdc28-4 and cdc28-1N strains transformed with the pYES2-CnCdk1 construct exhibited growth at the restrictive temperature. Results of the sequence analysis and the ability of CnCdk1 to complement the S. cerevisiae cdc28-ts mutations support its assumed role as the CDC28/cdc2 homologue in C. neoformans.
The article describes various features of aspergillosis and a discussed the role of calatases produced by Aspergillus fumigatus during infection. Since a large body of invasive Aspergillus infection occurs as an opportun...The article describes various features of aspergillosis and a discussed the role of calatases produced by Aspergillus fumigatus during infection. Since a large body of invasive Aspergillus infection occurs as an opportunistic infection in variously impaired defense mechanisms, there is a wide spectrum of histopathological features of lesions demonstrated at the site of infection. Accordingly, histopathology of the lesions can be understood as a phenotypical representation of interaction between differently impaired functions of neutrophils and macrophages and virulence factors of invading Aspergilli. Consideration of previous pathological knowledge regarding infection and inflammation provides much important information to predict the pathophysiology of a patient. Meanwhile, detoxification of hydrogen peroxide by catalases has been proposed as a way to overcome this host response. A. fumigatus produces three active catalases, one from conidia and two from mycelia. CatAp, a spore specific monofunctional catalase, is resistant to heat and metal ions. In spite of their increased sensitivity to H(2)O(2), killing of catA conidia by alveolar macrophages, virulence in animals was similar to wild type conidia. In contrast to mycelial Cat1p, and CatAp catalases, the mycelial Cat2p is a bifunctional catalase-peroxidase enzyme and is also sensitive to heat, metal ions and detergent. Surprisingly, the mycelium of the double cat1 cat2 mutant with no catalase activity has only a slightly increased sensitivity to H(2)O(2) and was as sensitive to the killing of polymorphonuclear neutrophils as the wild type strain. However, it showed a delayed infection in the rat model of aspergillosis compared to the wild type strain. Consequently, it should be emphasized that conidial catalase is not a virulence factor but that mycelial catalases transiently protect the fungus from the host defence reactions.
The conidia of filamentous fungi can be easily blown into the air and tend to be contaminants in the laboratory environment. We developed a new "safety culture tube for fungi" to prevent biohazards and a procedure for co...The conidia of filamentous fungi can be easily blown into the air and tend to be contaminants in the laboratory environment. We developed a new "safety culture tube for fungi" to prevent biohazards and a procedure for collecting conidia for passage or fixing strains was proposed.
The production of chlamydospores is a diagnostic tool used to identify Candida albicans; these structures also represent a model for morphogenetic research. The time required to produce them with standard methods is 48-7...The production of chlamydospores is a diagnostic tool used to identify Candida albicans; these structures also represent a model for morphogenetic research. The time required to produce them with standard methods is 48-72 hours in rice meal agar and tensoactive agents. This time can be shorted using liquid media such as cornmeal broth (CMB) and dairy supplements. Five media were tested: CMB plus 1% Tween-80, CMB plus 5% milk, CMB plus 5% milk serum, milk serum, and milk serum plus 1% Tween-80, under different incubation conditions: at 28 degrees C and 37 degrees C in a metabolic bath stirring at 150 rpm, and at 28 degrees C in a culture stove. The reading time points were established at 8 and 16 hours. The best results were obtained at 16 hours with CMB plus 5% milk under incubation at 28 degrees C and stirring at 150 rpm. The next most efficient methods were CMB plus 5% milk serum and CMB plus 1% Tween-80, under the same incubation conditions. The other media were ineffective in producing chlamydospores. The absence of stirring at 28 degrees C prevented the formation of chlamydospores within the set time points, and incubation at 37 degrees C decreased their production. This paper reports that the time to form C. albicans chlamydospores can be reduced.
Early identification of Candida isolates to the species level is necessary for effective antifungal therapy, and can also facilitate control of hospital infections. Phenotype-based methods for identifying Candida species...Early identification of Candida isolates to the species level is necessary for effective antifungal therapy, and can also facilitate control of hospital infections. Phenotype-based methods for identifying Candida species are often difficult and time-consuming. Molecular biological techniques provide a useful alternative approach. In the present study, the ITS1-5.8S-ITS2 regions of fungal rRNA genes were amplified with universal primers in 20 standard strains. Digestion of the PCR products with one restriction enzyme, MspI, allowed discrimination of medically important Candida species, including C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, and C. guilliermondii. Using this method, we successfully identified 137 clinical isolates of Candida. Among them, C. albicans was identified as the most common species, followed by C. parapsilosis, C. tropicalis, C. glabrata, C. krusei, and C. guilliermondii. This method is a simple, rapid, and cost-effective method for differentiation between species that is applicable in clinical laboratories.
A novel elastase inhibitor from Aspergillus flavus (AFLEI) was isolated, and biochemical properties of AFLEI were examined. Column chromatography using diethylaminoethyl (DE) 52-Cellulose and Sephadex G-75 was used to pu...A novel elastase inhibitor from Aspergillus flavus (AFLEI) was isolated, and biochemical properties of AFLEI were examined. Column chromatography using diethylaminoethyl (DE) 52-Cellulose and Sephadex G-75 was used to purify the inhibitor. The final preparation was found to be homogeneous as indicated by a single band after disc polyacrylamide gel (PAGE) and isoelectric focusing electrophoreses. AFLEI had a molecular weight of 7,525.8 as determined by TOF-MS (time of flight mass spectrometry). The elastolytic activity of elastases from A. flavus, A. fumigatus and human leukocytes were inhibited by AFLEI. However, this activity from porcine pancreas elastase, trypsin, chymotrypsin, thrombin, and Ac1-Proteinase from snake venom was not affected by AFLEI. The fibrinogenase activity of the elastase from A. flavus was inhibited by AFLEI. AFLEI was inhibited by alpha2-macroglobulin. However, ethylenediaminetetraacetic acid (EDTA-2Na), benzamidine, chymostatin, tosyl phenylalanine chloromethyl ketone (TPCK) and dithiothreitol (DTT) did not show any inhibitory effect on the elastase inhibitory activity of AFLEI.
To evaluate the currently used Japanese Society for Medical Mycology (JSMM) method for testing the azole susceptibility of yeasts, the activities of fluconazole and itraconazole were tested against recently collected cli...To evaluate the currently used Japanese Society for Medical Mycology (JSMM) method for testing the azole susceptibility of yeasts, the activities of fluconazole and itraconazole were tested against recently collected clinical isolates of Candida spp. (n=946) and compared with the National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 microdilution reference method. Favorable correlation with the M27-A2 method was not seen for isolates of C. albicans, C. tropicalis or other Candida spp., particularly their trailing-growth isolates. However, the degree of correlation and agreement of MIC values were markedly improved when testing was performed by the modified JSMM method in which the end-point to be read was changed from IC80 (for the current JSMM method) to IC50. These results suggest that there is an urgent need to revise the current JSMM method.
Cryptococcus neoformans is an opportunistic fungal infectious pathogen in immunocompromised patients with acquired immunodeficiency syndrome and hematological malignancies. Recently, innate immune cells, such as NK, NKT...Cryptococcus neoformans is an opportunistic fungal infectious pathogen in immunocompromised patients with acquired immunodeficiency syndrome and hematological malignancies. Recently, innate immune cells, such as NK, NKT and ganmma delta T cells, have been found critical for determining the quality of acquired immunity by affecting the direction of Th1-Th2 balance. Th1-type immune response is important for the host defense against C. neoformans, and innate immunity may involve this process. In the present review, the accumulated knowledge including our own data on the role of innate immune lymphocytes in the host defense to this fungal pathogen are summarized, focusing on NKT and ganmma delta T cells.
Neutrophils are believed to be the first line of defense against invading microorganisms, but in vivo roles of reactive oxygens produced by neutrophils are not well known. Myeloperoxidase (MPO) catalyzes reaction of hydr...Neutrophils are believed to be the first line of defense against invading microorganisms, but in vivo roles of reactive oxygens produced by neutrophils are not well known. Myeloperoxidase (MPO) catalyzes reaction of hydrogen peroxide with chloride ion to produce hypochlorous acid that is used for microbial killing by phagocytic cells. To define the in vivo role of MPO, we generated mice having no peroxidase activity in their neutrophils or monocytes. MPO-deficient (MPO-KO) mice showed severely reduced cytotoxicity to Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, and other microorganisms, demonstrating that an MPO-dependent oxidative system is important for host defense against fungi. However, the significance of MPO compared to the NADPH-oxidase is still unclear because individuals with MPO deficiency are usually healthy in contrast to patients with chronic granulomatous disease (CGD) who present clinical symptoms early in life. To better understand the contributions of MPO and NADPH-oxidase to antifungal defense mechanisms, we compared the susceptibility of MPO-KO mice and CGD mice to infections by C. albicans. Interestingly, at the highest dose, the mortality of MPO-KO mice was comparable to CGD mice, but was the same as normal mice at the lowest dose. These results suggest that MPO and NADPH-oxidase are equally important for early host defense against a large inocula of Candida. Our present results suggest that MPO-deficient individuals could exhibit similar problems as CGD patients if exposed to a large number of microorganisms.
1,3-Beta-glucans is a major cell wall component in fungi. Receptor molecules relating to innate immunity may recognize such cell wall products, and affect host defense systems. A beta-glucan receptor, dectin-1, is a C-ty...1,3-Beta-glucans is a major cell wall component in fungi. Receptor molecules relating to innate immunity may recognize such cell wall products, and affect host defense systems. A beta-glucan receptor, dectin-1, is a C-type lectin and may contribute to the innate immune responses. To examine the role of dectin-1 in recognition of 1,3-beta-glucans and subsequent activation of intracellular signaling, the molecular characteristics of a carbohydrate recognition domain (CRD) of dectin-1 were investigated. The binding ability to beta-glucans was abolished by mutating two amino acid residues, Trp221 and His223, on the CRD. Dectin-1 increased TLR2-mediated NF-kappaB activation in response to zymosan. However, dectin-1 alone could not affect the activation pathway for NF-kappaB, nor did co-expression of dectin-1 mutant and TLR2 increase the NF-kappaB activation. These results suggest that dectin-1 may have a co-stimulatory effect on leukocyte activation in response to fungal infection.
Candida albicans mannan consists of the alpha-1,6-linked backbone moiety and the alpha-1,2- and alpha-1,3-linked side chains. It also contains alpha-1,6-branched mannose units, beta-1,2-linked mannose units, and phosphat...Candida albicans mannan consists of the alpha-1,6-linked backbone moiety and the alpha-1,2- and alpha-1,3-linked side chains. It also contains alpha-1,6-branched mannose units, beta-1,2-linked mannose units, and phosphate groups. The cell wall mannans of the genus Candida possess three types of beta-1,2 linked mannose units. One is linked via the phosphodiester linkage, the second type is connected to an alpha-1,2-linked mannose unit, and the third type is attached to an alpha-1,3-linked mannose unit. These beta-1,2-linked mannose units showed a strong antigenicity and produce the characteristic NMR chemical shifts. Using two-dimensional NMR techniques, we will practically determine the structure of these polysaccharides in a nondestructive manner.
We studied the clinical features of 59 chronic pulmonary aspergillosis cases (aspergilloma, chronic necrotizing pulmonary aspergillosis) which we experienced in our hospital. To diagnose this disease, X-rays, sputum cult...We studied the clinical features of 59 chronic pulmonary aspergillosis cases (aspergilloma, chronic necrotizing pulmonary aspergillosis) which we experienced in our hospital. To diagnose this disease, X-rays, sputum culture and serologic tests were mainly examined, X-ray findings were a fungus ball type in 47% of cases and thickened wall of a cavity type in 32%. Positive sputum culture found was A. fumigatus 78%, A. niger 13% and A. flavus 2%. Positive rates of serologic tests showed precipitating antibody 81% and antigen 11%; 39% of beta-D glucan exceeded the reference value. As clinical symptoms, bloody sputum and hemoptysis were found at high frequency. Antifungal agents were administered intravenously or topically for treatment, primarily AMPH-B, ITCZ and MCFG. As adjuvant therapy, we administered Ulinastatin which is an elastase inhibitor for use against hemoptysis, and we performed steroid combination for cases considered to be associated with allergy. In all of 6 cases of chronic necrotizing pulmonary aspergillosis which were administered MCFG, X-ray findings improved. A pathogenic factor, elastase was isolated from Aspergillus spp., and we also found the elastase inhibitor from this series. Five of 12 strains of A. fumigatus, and one of 2 strains of A. flavus expressed elastase inhibitory activity when we screened for the culture supernatant of various Aspergillus spp. of a clinical isolate. Elastase inhibitory activity from A. niger was very weak. Culture supernatants from 5 strains of A. fumigatus and one strain of A. flavus were stable for a fever, and human leucocyte elastase was inhibited, but these did not inhibit porcine pancreas elastase. We are aiming at clinical application and plan to continue further study.
Solid organ transplantation is becoming increasingly common in Japan. Despite invasive fungal infections being less common than bacterial and viral infections, fungal infections still result in a higher mortality rate. E...Solid organ transplantation is becoming increasingly common in Japan. Despite invasive fungal infections being less common than bacterial and viral infections, fungal infections still result in a higher mortality rate. Empiric and pre-emptive therapy plays an important role in management of invasive fungal infections, because successful treatment is difficult after a definite diagnosis of an invasive disease, especially invasive aspergillosis. Given this situation, to improve outcome, high risk patients need to be identified and antifungal prophylaxis is mandatory in preventing the development of the disease. However, antifungal prophylaxis for solid organ transplant recipients remains controversial. New antifungal agents might change the choice of fungal prevention and treatment.
Recent situation of HIV-related mycosis was discussed in this paper, with the analysis of 1) annual report of HIV trends in Japan by the AIDS epidemiology committee, 2) report of HIV-related opportunistic infections (OIs...Recent situation of HIV-related mycosis was discussed in this paper, with the analysis of 1) annual report of HIV trends in Japan by the AIDS epidemiology committee, 2) report of HIV-related opportunistic infections (OIs) collected by the AIDS-OIs research group funded by the Ministry of Health, Labour and Welfare, and 3) 17 cases of HIV-related aspergillosis collected by the author. Annual AIDS cases were increasing, and their major diseases were included with the following mycosis: pneumosystis pneumonia 35.7%, candidiasis 19.1%, and cryptococcosis 2.4%. There were two foreigner's cases of histoplasmosis and no coccidioidosis. Candidiasis was likely to be shown in Japanese patients and cryptococcosis was in foreigners. Outcome of cryptococcosis was very poor as 32.7% of patients died. There were 17 HIV-related aspergillosis, which consisted of 13 cases of lung diseases, 2 of brain lesions, and one each of sinus and stomach disease. Remarkable risk factor of HIV-related aspergillosis was decrease of CD4 cell count less than 10/microl, in addition to the usual risk factors of aspergillosis. Outcome of aspergillosis was very poor, as all treated cases died except one recent case treated with voriconazole.
Micafungin, a new class of the antifungal agent "echinocandin" released in 2002, and voriconazole, a new triazole antifungal agent released in 2005 in Japan have in vitro activities against Aspergillus spp. Results of la...Micafungin, a new class of the antifungal agent "echinocandin" released in 2002, and voriconazole, a new triazole antifungal agent released in 2005 in Japan have in vitro activities against Aspergillus spp. Results of large-scale clinical trials in Europe and the United States showed voriconazole to have superior efficacy against invasive pulmonary aspergillosis in comparison with conventional amphotericin B, and caspofungin, a member of the echinocandins, was effective as an empirical antifungal therapy in patients with persistent fever and neutropenia. In this way, choices of therapeutic medicine for aspergillosis are increasing more and more, and it is expected that the method of treatment will change greatly in future. On the other hand, we need to establish a new standard therapy for aspergillosis to avoid the clinical disruption caused by the variety of pharmaceutical choice caused. In this report, we describe the role of new antifungal agents for non-fumigatus Aspergillus infections, and the breakthrough in counteracting fungal infection using these new drugs.
Hematopoietic stem cell transplantation has been established as a curative treatment for advanced hematologic malignancies. Transplantation with a reduced-intensity conditioning regimen has been developed, and the minima...Hematopoietic stem cell transplantation has been established as a curative treatment for advanced hematologic malignancies. Transplantation with a reduced-intensity conditioning regimen has been developed, and the minimal toxicity of reduced-intensity stem cell transplantation (RIST) has made this procedure available for patients of advanced age or with organ dysfunction. The response of malignant lymphoma and some solid tumors to RIST has been observed. RIST with unrelated donors and umbilical cord blood has been studied. Fungal infection is an important complication of RIST. Since the prognosis of fungal infection is poor, the management has been focused on its prophylaxis. Given recent progression in RIST management, the strategy of infectious prophylaxis has also changed. Equipment in the hospital is important for fungal infection; however, the median day of the development of fungal infection is day 100, when most patients are followed as outpatients. The focus of fungal management after RIST is oral antifungal agents rather than in-hospital equipment. Various antifungal agents have recently been developed and applied for clinical use, and many of these have been developed simultaneously for the first time. A major change in antifungal management will probably occur in the next several years.
Serological surrogate tests for invasive fungal infection have been used practically in Japan. The Aspergillus galactomannan antigen detection kit by enzyme linked immunosorbent assay is the most reliable test for making...Serological surrogate tests for invasive fungal infection have been used practically in Japan. The Aspergillus galactomannan antigen detection kit by enzyme linked immunosorbent assay is the most reliable test for making a diagnosis of invasive aspergillosis. But in recent years, occurrences of false positive and negative results have also been reported by several investigators. Therefore, evaluation of the results of this assay should be done carefully in the clinical stage. Moreover, some other methods to detect the Aspergillus antigen or anti Aspergillus antibody in serum have also been reported. The problem of false positive results due to the frequent occurrence of non-specific reaction in the alkaline treatment, chromogenic automated kinetic assay to measure (1-->3)-beta-D-glucan had been noticed in Japan. But this important problem was resolved by improvement of the pretreatment reagent in this kit in July 2005. In this manuscript, I describe recent trends of serological surrogate tests for Aspergillus infection and the process of improvement of the (1-->3)-beta-D-glucan measurement kit.
There are several different types of repeated sequences in the genome of Candida albicans, including the MRS (Major repeated sequence). In 2004, the whole genome sequence of C. albicans was published. Assembly of the seq...There are several different types of repeated sequences in the genome of Candida albicans, including the MRS (Major repeated sequence). In 2004, the whole genome sequence of C. albicans was published. Assembly of the sequences to chromosomal length contigs was not achieved, mainly due to interruption of the sequences by MRS. However, MRS including Ca3, 27A and RPS have been playing important roles in a number of epidemiological studies and basic biological investigations into C. albicans chromosome loss events and associated phenotypic changes. Here we summarize structural analyses from subrepeat sequences to the chromosome level, and functional analyses of MRS.