Katsuhiko Kamei

NikA/TcsC Histidine Kinase Is Involved in Conidiation, Hyphal Morphology, and Responses to Osmotic Stress and Antifungal Chemicals in Aspergillus fumigatus

The fungal high osmolarity glycerol (HOG) pathway is composed of a two-component system (TCS) and Hog1-type mitogen-activated protein kinase (MAPK) cascade. A group III (Nik1-type) histidine kinase plays a major role in the HOG pathway of several filamentous fungi. In this study, we characterized a group III histidine kinase, NikA/TcsC, in the life-threatening pathogenic fungus, Aspergillus fumigatus. A deletion mutant of nikA showed low conidia production, abnormal hyphae, marked sensitivity to high osmolarity stresses, and resistance to cell wall perturbing reagents such as congo red and calcofluor white, as well as to fungicides such as fludioxonil, iprodione, and pyrrolnitrin. None of these phenotypes were observed in mutants of the SskA response regulator and SakA MAPK, which were thought to be downstream components of NikA. In contrast, in response to fludioxonil treatment, NikA was implicated in the phosphorylation of SakA MAPK and the transcriptional upregulation of catA, dprA, and dprB, which are regulated under the control of SakA. We then tested the idea that not only NikA, but also the other 13 histidine kinases play certain roles in the regulation of the HOG pathway. Interestingly, the expression of fos1, phkA, phkB, fhk5, and fhk6 increased by osmotic shock or fludioxonil treatment in a SakA-dependent manner. However, deletion mutants of the histidine kinases showed no significant defects in growth under the tested conditions. Collectively, although the signal transduction network related to NikA seems complicated, NikA plays a crucial role in several aspects of A. fumigatus physiology and, to a certain extent, modulates the HOG pathway.

NBRP databases: databases of biological resources in Japan.

The National BioResource Project (NBRP) is a Japanese project that aims to establish a system for collecting, preserving and providing bioresources for use as experimental materials for life science research. It is promoted by 27 core resource facilities, each concerned with a particular group of organisms, and by one information center. The NBRP database is a product of this project. Thirty databases and an integrated database-retrieval system (BioResource World: BRW) have been created and made available through the NBRP home page (http://www.nbrp.jp). The 30 independent databases have individual features which directly reflect the data maintained by each resource facility. The BRW is designed for users who need to search across several resources without moving from one database to another. BRW provides access to a collection of 4.5-million records on bioresources including wild species, inbred lines, mutants, genetically engineered lines, DNA clones and so on. BRW supports summary browsing, keyword searching, and searching by DNA sequences or gene ontology. The results of searches provide links to online requests for distribution of research materials. A circulation system allows users to submit details of papers published on research conducted using NBRP resources.

The role of AtfA and HOG MAPK pathway in stress tolerance in conidia of Aspergillus fumigatus

Aspergillus fumigatus is a life-threatening pathogenic fungus, whose conidium is the infectious agent of aspergillosis. To better understand the mechanism underlying the long-term viability of conidia, we characterized a bZip transcription factor, AtfA, with special reference to stress-tolerance in conidia. The atfA deletion mutant conidia showed significant sensitivity to high temperature and oxidative stress. The trehalose content that accumulated in conidia was reduced in the mutant conidia. Transcriptome analysis revealed that AtfA regulated several stress-protection-related genes such as catA, dprA, scf1, and conJ at the conidiation stage. The upstream high-osmolarity glycerol pathway was also involved in conferring stress tolerance in conidia because ΔpbsB showed stress sensitivity and reduced trehalose in conidia. However, a mutant lacking the SakA mitogen-activated protein kinase (MAPK) produced normal conidia. We investigated another MAPK, MpkC, in relation with SakA, and the double deletion mutant, ΔsakA,mpkC, was defective in conidia stress tolerance. We concluded that MpkC is able to bypass SakA, and the two MAPKs redundantly regulate the conidia-related function of AtfA in A. fumigatus.

A double-blind comparative study of the safety and efficacy of caspofungin versus micafungin in the treatment of candidiasis and aspergillosis

The safety and efficacy profile of caspofungin and micafungin in Japanese patients with fungal infections were directly compared in this prospective, randomized, double-blind study. The proportion of patients who developed significant drug-related adverse event(s) (defined as a serious drug-related adverse event or a drug-related adverse event leading to study therapy discontinuation) was compared in 120 patients [caspofungin 50xa0mg, or 50xa0mg following a 70-mg loading dose on Day 1 (hereinafter, 70/50xa0mg) group: 60 patients; micafungin 150xa0mg: 60 patients]. The overall response rate was primarily evaluated in the per-protocol set (PPS) population. The proportion of patients who developed significant drug-related adverse events was 5.0xa0% (3/60) in the caspofungin group and 10.0xa0% (6/60) in the micafungin group [95xa0% confidence interval (CI) for the difference: −15.9xa0%, 5.2xa0%]. The favorable overall response in the PPS population for patients with esophageal candidiasis, invasive candidiasis, and chronic pulmonary aspergillosis including aspergilloma was 100.0xa0% (6/6), 100.0xa0% (3/3), and 46.7xa0% (14/30) in the caspofungin group, and 83.3xa0% (5/6), 100.0xa0% (1/1), and 42.4xa0% (14/33) in the micafungin group, respectively. In Japanese patients with Candida or Aspergillus infections, there was no statistical difference in the safety between caspofungin and micafungin. Consistent with other data on these two agents, the efficacy of caspofungin and micafungin was similar.

Epidemiological and Genomic Landscape of Azole Resistance Mechanisms in Aspergillus Fungi

Invasive aspergillosis is a life-threatening mycosis caused by the pathogenic fungus Aspergillus. The predominant causal species is Aspergillus fumigatus, and azole drugs are the treatment of choice. Azole drugs approved for clinical use include itraconazole, voriconazole, posaconazole, and the recently added isavuconazole. However, epidemiological research has indicated that the prevalence of azole-resistant A. fumigatus isolates has increased significantly over the last decade. What is worse is that azole-resistant strains are likely to have emerged not only in response to long-term drug treatment but also because of exposure to azole fungicides in the environment. Resistance mechanisms include amino acid substitutions in the target Cyp51A protein, tandem repeat sequence insertions at the cyp51A promoter, and overexpression of the ABC transporter Cdr1B. Environmental azole-resistant strains harboring the association of a tandem repeat sequence and punctual mutation of the Cyp51A gene (TR34/L98H and TR46/Y121F/T289A) have become widely disseminated across the world within a short time period. The epidemiological data also suggests that the number of Aspergillus spp. other than A. fumigatus isolated has risen. Some non-fumigatus species intrinsically show low susceptibility to azole drugs, imposing the need for accurate identification, and drug susceptibility testing in most clinical cases. Currently, our knowledge of azole resistance mechanisms in non-fumigatus Aspergillus species such as A. flavus, A. niger, A. tubingensis, A. terreus, A. fischeri, A. lentulus, A. udagawae, and A. calidoustus is limited. In this review, we present recent advances in our understanding of azole resistance mechanisms particularly in A. fumigatus. We then provide an overview of the genome sequences of non-fumigatus species, focusing on the proteins related to azole resistance mechanisms.

Multi-azole resistant Aspergillus fumigatus harboring Cyp51A TR46/Y121F/T289A isolated in Japan.

Multi-azole resistant Aspergillus fumigatus carrying TR46/Y121F/T289A was isolated from a patient in Japan in Dec 2013. This strain grouped into the same clade of the ones which were clinically isolated in France and Germany. A.xa0fumigatus harboring this mutation could be rapidly diffused outside the Eurasian continent.

Identification of Fungal Pathogens by Visible Microarray System in Combination with Isothermal Gene Amplification

The increasing incidence of infectious diseases caused by fungi in immunocompromised patients has encouraged researchers to develop rapid and accurate diagnosis methods. Identification of the causative fungal species is critical in deciding the appropriate treatment, but it is not easy to get satisfactory results due to the difficulty of fungal cultivation and morphological identification from clinical samples. In this study, we established a microarray system that can identify 42 species from 24 genera of clinically important fungal pathogens by using a chemical color reaction in the detection process. The array uses the internal transcribed spacer region of the rRNA gene for identification of fungal DNA at the species level. The specificity of this array was tested against a total of 355 target and nontarget fungal species. The fungal detection was succeeded directly from 103xa0CFU/ml for whole blood samples, and 50xa0fg DNA per 1xa0ml of serum samples indicating that the array system we established is sensitive to identify infecting fungi from clinical sample. Furthermore, we conducted isothermal amplification in place of PCR amplification and labeling. The successful identification with PCR-amplified as well as isothermally amplified target genes demonstrated that our microarray system is an efficient and robust method for identifying a variety of fungal species in a sample.

Comparative transcriptome analysis revealing dormant conidia and germination associated genes in Aspergillus species: an essential role for AtfA in conidial dormancy.

BackgroundFungal conidia are usually dormant unless the extracellular conditions are right for germination. Despite the importance of dormancy, little is known about the molecular mechanism underlying entry to, maintenance of, and exit from dormancy. To gain comprehensive and inter-species insights, transcriptome analyses were conducted across Aspergillus fumigatus, Aspergillus niger, and Aspergillus oryzae.ResultsWe found transcripts of 687, 694, and 812 genes were enriched in the resting conidia compared with hyphae in A. fumigatus, A. niger, and A. oryzae, respectively (conidia-associated genes). Similarly, transcripts of 766, 1,241, and 749 genes were increased in the 1xa0h-cultured conidia compared with the resting conidia (germination-associated genes). Among the three Aspergillus species, we identified orthologous 6,172 genes, 91 and 391 of which are common conidia- and germination-associated genes, respectively. A variety of stress-related genes, including the catalase genes, were found in the common conidia-associated gene set, and ribosome-related genes were significantly enriched among the germination-associated genes. Among the germination-associated genes, we found that calA-family genes encoding a thaumatin-like protein were extraordinary expressed in early germination stage in all Aspergillus species tested here. In A. fumigatus 63xa0% of the common conidia-associated genes were expressed in a bZIP-type transcriptional regulator AtfA-dependent manner, indicating that AtfA plays a pivotal role in the maintenance of resting conidial physiology. Unexpectedly, the precocious expression of the germination-associated calA and an abnormal metabolic activity were detected in the resting conidia of the atfA mutant, suggesting that AtfA was involved in the retention of conidial dormancy.ConclusionsA comparison among transcriptomes of hyphae, resting conidia, and 1xa0h-grown conidia in the three Aspergillus species revealed likely common factors involved in conidial dormancy. AtfA positively regulates conidial stress-related genes and negatively mediates the gene expressions related to germination, suggesting a major role for AtfA in Aspergillus conidial dormancy.

Multiple Scedosporium apiospermum abscesses in a woman survivor of a tsunami in northeastern Japan: a case report

IntroductionScedosporium apiospermum is increasingly recognized as a cause of localized and disseminated mycotic infections in near-drowning victims.Case presentationWe report the case of a 59-year-old Japanese woman who was a survivor of a tsunami in northeastern Japan and who had lung and brain abscesses caused by S. apiospermum. Initially, an aspergillus infection was suspected, so she was treated with micafungin. However, computed tomography scans of her chest revealed lung abscesses, and magnetic resonance images demonstrated multiple abscesses in her brain. S. apiospermum was cultured from her bronchoalveolar lavage fluid, and antimycotic therapy with voriconazole was initiated. Since she developed an increase in the frequency of premature ventricular contractions, an adverse drug reaction to the voriconazole was suspected. She was started on a treatment of a combination of low-dose voriconazole and liposomal amphotericin B. After combination therapy, further computed tomography scans of the chest and magnetic resonance images of her brain showed a demarcation of abscesses.ConclusionsVoriconazole appeared to have a successful record in treating scedosporiosis after a near drowning but, owing to several adverse effects, may possibly not be recommended. Thus, a combination treatment of low-dose voriconazole and liposomal amphotericin B may be a safe and effective treatment for an S. apiospermum infection. Even though a diagnosis of scedosporiosis may be difficult, a fast and correct etiological diagnosis could improve the patients chance of recovery in any case.

An atypical Paracoccidioides brasiliensis clinical isolate based on multiple gene analysis.

An atypical isolate of Paracoccidioides brasiliensis (IFM54648), recovered from the sputum of a Brazilian man, was not detected in immunodiffusion tests for paracoccidioidomycosis and in species-specific PCR for the major antigen 43-kDa glycoprotein coding gene (gp43). The mycological characteristics of the isolate were similar to those of a typical P. brasiliensis. A total of 8 genes were sequenced from IFM54648, and the sequences were compared between the new isolate and other reference isolates and database sequences. We analyzed fragments of the gene sequences that code for gp43, the internal transcribed spacer regions of ribosomal RNA, the D1/D2 domains of the large subunit ribosomal RNA, glucan synthase, chitin synthase, glyoxalase I mRNA, 70-kDa heat-shock protein mRNA and urease. The gene sequences were 98.9-100% identical between IFM54648 and Pb01 (another atypical isolate). When compared to the other typical isolates, the identities were generally lower than 98%. A phylogenetic tree constructed using gp43 sequences showed that IFM54648 clustered with Pb01 at a considerable distance from other isolates. Therefore, this isolate is likely related to Pb01, which has recently been shown to be genetically distinct from other isolates of this species.