Azusa Takahashi-Nakaguchi

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.

Whole-Genome Comparison of Aspergillus fumigatus Strains Serially Isolated from Patients with Aspergillosis

ABSTRACT The emergence of azole-resistant strains of Aspergillus fumigatus during treatment for aspergillosis occurs by a mutation selection process. Understanding how antifungal resistance mechanisms evolve in the host environment during infection is of great clinical importance and biological interest. Here, we used next-generation sequencing (NGS) to identify mutations that arose during infection by A. fumigatus strains sequentially isolated from two patients, one with invasive pulmonary aspergillosis (IPA) (five isolations) and the other with aspergilloma (three isolations). The serial isolates had identical microsatellite types, but their growth rates and conidia production levels were dissimilar. A whole-genome comparison showed that three of the five isolates from the IPA patient carried a mutation, while 22 mutations, including six nonsynonymous ones, were found among three isolates from the aspergilloma patient. One aspergilloma isolate carried the cyp51A mutation P216L, which is reported to confer azole resistance, and it displayed an MIC indicating resistance to itraconazole. This isolate harbored five other nonsynonymous mutations, some of which were found in the afyap1 and aldA genes. We further identified a large deletion in the aspergilloma isolate in a region containing 11 genes. This finding suggested the possibility that genomic deletions can occur during chronic infection with A. fumigatus. Overall, our results revealed dynamic alterations that occur in the A. fumigatus genome within its host during infection and treatment.

Prevalence of Hepatitis C Virus Subgenotypes 1a and 1b in Japanese Patients: Ultra-Deep Sequencing Analysis of HCV NS5B Genotype-Specific Region

Background Hepatitis C virus (HCV) subgenotypes 1a and 1b have different impacts on the treatment response to peginterferon plus ribavirin with direct-acting antivirals (DAAs) against patients infected with HCV genotype 1, as the emergence rates of resistance mutations are different between these two subgenotypes. In Japan, almost all of HCV genotype 1 belongs to subgenotype 1b. Methods and Findings To determine HCV subgenotype 1a or 1b in Japanese patients infected with HCV genotype 1, real-time PCR-based method and Sanger method were used for the HCV NS5B region. HCV subgenotypes were determined in 90% by real-time PCR-based method. We also analyzed the specific probe regions for HCV subgenotypes 1a and 1b using ultra-deep sequencing, and uncovered mutations that could not be revealed using direct-sequencing by Sanger method. We estimated the prevalence of HCV subgenotype 1a as 1.2-2.5% of HCV genotype 1 patients in Japan. Conclusions Although real-time PCR-based HCV subgenotyping method seems fair for differentiating HCV subgenotypes 1a and 1b, it may not be sufficient for clinical practice. Ultra-deep sequencing is useful for revealing the resistant strain(s) of HCV before DAA treatment as well as mixed infection with different genotypes or subgenotypes of HCV.

Nagelamides X–Z, Dimeric Bromopyrrole Alkaloids from a Marine Sponge Agelas sp.

Three new dimeric bromopyrrole alkaloids, nagelamides X-Z (1-3), were isolated from a marine sponge Agelas sp. Nagelamides X (1) and Y (2) possess a novel tricyclic skeleton consisting of spiro-bonded tetrahydrobenzaminoimidazole and aminoimidazolidine moieties. Nagelamide Z (3) is the first dimeric bromopyrrole alkaloid involving the C-8 position in dimerization. The structures of 1-3 were elucidated on the basis of spectroscopic data. Nagelamides X-Z (1-3) exhibited antimicrobial activity.

Zamamiphidin A, a New Manzamine Related Alkaloid from an Okinawan Marine Sponge Amphimedon sp.

A manzamine related alkaloid, zamamiphidin A (1), consisting of a new heptacyclic ring system has been isolated from an Okinawan marine sponge Amphimedon sp. The structure of 1 including the relative stereochemistry was elucidated on the basis of the spectroscopic data. Compound 1 showed antibacterial activity against Staphylococcus aureus (MIC, 32 μg/mL).

Identification of genes involved in the phosphate metabolism in Cryptococcus neoformans

Cryptococcus neoformans is a pathogenic basidiomycetous yeast that can cause life-threatening meningoencephalitis in immuno-compromized patients. To propagate in the human body, this organism has to acquire phosphate that functions in cellular signaling pathways and is also an essential component of nucleic acids and phospholipids. Thus it is reasonable to assume that C. neoformans (Cn) possesses a phosphate regulatory system (PHO system) analogous to that of other fungi. By BLAST searches using the amino acid sequences of the components of the PHO system of Saccharomyces cerevisiae (Sc), we found potential counterparts to ScPHO genes in C. neoformans, namely, acid phosphatase (CnPHO2), the cyclin-dependent protein kinase (CDK) inhibitor (CnPHO81), Pho85-cyclin (CnPHO80), and CDK (CnPHO85). Disruption of each candidate gene, except CnPHO85, followed by phenotypic analysis, identified most of the basic components of the CnPHO system. We found that CnPHO85 was essential for the growth of C. neoformans, having regulatory function in the CnPHO system. Genetic screening and ChIP analysis, showed that CnPHO4 encodes a transcription factor that binds to the CnPHO genes in a Pi-dependent manner. By RNA-seq analysis of the wild-type and the regulatory mutants of the CnPHO system, we found C. neoformans genes whose expression is controlled by the regulators of the CnPHO system. Thus the CnPHO system shares many properties with the ScPHO system, but expression of those CnPHO genes that encode regulators is controlled by phosphate starvation, which is not the case in the ScPHO system (except ScPHO81). We also could identify some genes involved in the stress response of the pathogenic yeast, but CnPho4 appeared to be responsible only for phosphate starvation.

Genome sequence comparison of Aspergillus fumigatus strains isolated from patients with pulmonary aspergilloma and chronic necrotizing pulmonary aspergillosis

Aspergillus fumigatus is the Aspergillus species most commonly associated with aspergillosis. Of the various presentations of aspergillosis, one of the most frequently observed in cases involving A. fumigatus pulmonary infections is aspergilloma (PA). In such infections one finds a fungus ball composed of fungal hyphae, inflammatory cells, fibrin, mucus, and tissue debris. Chronic necrotizing pulmonary aspergillosis (CNPA), also known as semi-invasive or invasive aspergillosis, is locally invasive and predominantly seen in patients with mild immunodeficiency or with a chronic lung disease. In the present study, with the aid of a next-generation sequencer, we conducted whole genome sequence (WGS) analyses of 17 strains isolated from patients in Japan with PA and CNPA. A total of 99,088 SNPs were identified by mapping the reads to A. fumigatus genome reference strain Af293, and according to genome-wide phylogenetic analysis, there were no correlations between the whole genome sequence typing results and pathologic conditions of patients. Here, we conducted the first multi-genome WGS study to focus on the A. fumigatus strains isolated from patients with PA and CNPA, and comprehensively characterized genetic variations of strains. WGS approach will help in better understanding of molecular mechanisms of aspergillosis cases caused by A. fumigatus.

The CgHaa1-Regulon Mediates Response and Tolerance to Acetic Acid Stress in the Human Pathogen Candida glabrata

To thrive in the acidic vaginal tract, Candida glabrata has to cope with high concentrations of acetic acid. The mechanisms underlying C. glabrata tolerance to acetic acid at low pH remain largely uncharacterized. In this work, the essential role of the CgHaa1 transcription factor (encoded by ORF CAGL0L09339g) in the response and tolerance of C. glabrata to acetic acid is demonstrated. Transcriptomic analysis showed that CgHaa1 regulates, directly or indirectly, the expression of about 75% of the genes activated under acetic acid stress. CgHaa1-activated targets are involved in multiple physiological functions including membrane transport, metabolism of carbohydrates and amino acids, regulation of the activity of the plasma membrane H+-ATPase, and adhesion. Under acetic acid stress, CgHaa1 increased the activity and the expression of the CgPma1 proton pump and contributed to increased colonization of vaginal epithelial cells by C. glabrata. CgHAA1, and two identified CgHaa1-activated targets, CgTPO3 and CgHSP30, are herein demonstrated to be determinants of C. glabrata tolerance to acetic acid. The protective effect of CgTpo3 and of CgHaa1 was linked to a role of these proteins in reducing the accumulation of acetic acid inside C. glabrata cells. In response to acetic acid stress, marked differences were found in the regulons controlled by CgHaa1 and by its S. cerevisiae ScHaa1 ortholog, demonstrating a clear divergent evolution of the two regulatory networks. The results gathered in this study significantly advance the understanding of the molecular mechanisms underlying the success of C. glabrata as a vaginal colonizer.

Manzamenone O, new trimeric fatty acid derivative from a marine sponge Plakortis sp.

A new structurally unique trimeric fatty acid derivative, manzamenone O (1), was isolated from a marine sponge Plakortis sp. Manzamenone O (1) has a novel skeleton consisting of C-C bonded octahydroindenone and dioxabicyclo[3.3.0]octane moieties and three long aliphatic chains. The structure of 1 was elucidated on the basis of spectroscopic data and conformational analysis. Manzamenone O (1) exhibited antimicrobial activity against Micrococcus luteus, Aspergillis niger, and Trichophyton mentagrophytes.

The Ubiquitin Ligase SCFUcc1 Acts as a Metabolic Switch for the Glyoxylate Cycle

Despite the crucial role played by the glyoxylate cycle in the virulence of pathogens, seed germination in plants, and sexual development in fungi, we still have much to learn about its regulation. Here, we show that a previously uncharacterized SCF(Ucc1) ubiquitin ligase mediates proteasomal degradation of citrate synthase in the glyoxylate cycle to maintain metabolic homeostasis in glucose-grown cells. Conversely, transcription of the F box subunit Ucc1 is downregulated in C2-compound-grown cells, which require increased metabolic flux for gluconeogenesis. Moreover, in vitro analysis demonstrates that oxaloacetate regenerated through the glyoxylate cycle induces a conformational change in citrate synthase and inhibits its recognition and ubiquitination by SCF(Ucc1), suggesting the existence of an oxaloacetate-dependent positive feedback loop that stabilizes citrate synthase. We propose that SCF(Ucc1)-mediated regulation of citrate synthase acts as a metabolic switch for the glyoxylate cycle in response to changes in carbon source, thereby ensuring metabolic versatility and flexibility.