The lipophilic fungal pathogen Malassezia spp. must acquire long-chain fatty acids (LCFAs) from outside the cell. To clarify the mechanism of LCFA acquisition, we investigated fatty acid uptake by this fungus and identified the long-chain acyl-CoA synthetase (ACS) gene FAA1 in three Malassezia spp.: M. globosa, M. pachydermatis, and M. sympodialis. These FAA1 genes could compensate for the double mutation of FAA1 and FAA4 in Saccharomyces cerevisiae, suggesting that Malassezia Faa1 protein recognizes exogenous LCFAs. MgFaa1p and MpFaa1p utilized a medium-chain fatty acid, lauric acid (C12:0). Interestingly, the ACS inhibitor, triacsin C, affected the activity of the Malassezia Faa1 proteins but not that of S. cerevisiae. Triacsin C also reduced the growth of M. globosa, M. pachydermatis, and M. sympodialis. These results suggest that triacsin C and its derivatives are potential compounds for the development of new anti-Malassezia drugs.

Fatty acyl-CoA synthetase (Faa) activates fatty acid (FA) by converting the FA into the CoA ester in the cell. In the present study, we characterized a FAA homologue (CaFAA4) from the opportunistic pathogen Candida albicans. Most organisms can not only synthesize long-chain fatty acyl-CoAs (LCFA-CoAs) endogenously using a fatty acid synthase (Fas) activity but also can uptake long-chain fatty acids (LCFAs) from the extracellular environment and convert them into LCFA-CoAs via a vectorial acylation system. The budding yeast Saccharomyces cerevisiae possesses two LCFA-CoA synthetases, ScFaa1p and ScFaa4p. The disruption of ScFAA1 and ScFAA4 leads to synthetic lethality in the presence of a fatty acid synthesis inhibitor-cerulenin. The homologue-CaFAA4-rescued the lethality of an S. cerevisiae Scfaa1-Scfaa4 double mutant in the presence of cerulenin. On the other hand, a C. albicans faa4 mutant was unable to grow in the presence of cerulenin even if LCFAs were provided exogenously. Moreover, a biofilm analysis showed that the metabolic activity of the Cafaa4 mutant was approximately 40% lower than that of the wild-type parent, even though there was no significant difference in cell number or cell morphology between these strains. Notably, the Cafaa4 mutant showed increased susceptibility to micafungin during biofilm formation, a phenotype that presumably can be attributed to the impaired metabolism of the mutant strain. These results indicated that CaFaa4p is the unique C. albicans Faa protein responsible for activating LCFAs and is involved in the metabolism of biofilms.

The uptake of cholesterol from the host is closely linked to the proliferation of pathogenic fungi and protozoa during infection. For some pathogenic fungi, cholesterol uptake is an important strategy for decreasing susceptibility to antifungals that inhibit ergosterol biosynthesis. In this study, we show that Candida glabrata ERG25, which encodes an enzyme that demethylates 4,4-dimethylzymosterol, is required for cholesterol uptake from host serum. Based on the screening of C. glabrata conditional knockdown mutants for each gene involved in ergosterol biosynthesis, ERG25 knockdown was found to decrease lethality of infected mice. ERG25 knockdown impairs the plasma membrane localization of the sterol importer Aus1p, suggesting that the accumulated 4,4-dimethylzymosterol destabilizes the lipid domain with which Aus1p functionally associates. ERG25 knockdown further influences the structure of the membrane compartment of Can1p (MCC)/eisosomes (ergosterol-rich lipid domains), but not the localization of the membrane proteins Pma1p and Hxt1p, which localize to sterol-poor domains. In the sterol-rich lipid domain, Aus1p-contining domain was mostly independent of MCC/eisosomes, and the nature of these domains was also different: Ausp1-contining domain was a dynamic network-like domain, whereas the MCC/eisosomes was a static dot-like domain. However, deletion of MCC/eisosomes was observed to influence the localization of Aus1p after Aus1p was transported from the endoplasmic reticulum (ER) through the Golgi apparatus to the plasma membrane. These findings suggest that ERG25 plays a key role in stabilizing sterol-rich lipid domains, constituting a promising candidate target for antifungal therapy.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease characterized by complex lung pathogenesis affecting approximately three million people worldwide. While the molecular and cellular details of the IPF mechanism is emerging, our current understanding is centered around the lung itself. On the other hand, many human diseases are the products of complex multi-organ interactions. Hence, we postulate that a dysfunctional crosstalk of the lung with other organs plays a causative role in the onset, progression and/or complications of IPF. In this study, we employed a generative computational approach to identify such inter-organ mechanism of IPF. The approach works as follows: 1) To find unexpected relatedness of IPF to other diseases of non-lung organs and to identify molecular features that define such relatedness, 2) To identify differentially expressed genes between the lung tissues of IPF vs. those of non-IPF pulmonary disease patients, 3) To detect ligand-receptor relationships across multiple organs and their upstream and downstream signaling pathways in 1) and 2), 4) To generate a map of the inter-organ IPF mechanism with the molecular and cellular resolution. This approach found unexpected molecular relatedness of IPF to neoplasm, diabetes, Alzheimer′s disease, obesity, atherosclerosis, and arteriosclerosis. Furthermore, as a potential mechanism underlying this relatedness, we uncovered a putative molecular crosstalk system across the lung and the liver. In this inter-organ system, a secreted protein, kininogen 1, from hepatocytes in the liver interacts with its receptor, bradykinin receptor B1 in the lung. This ligand-receptor interaction across the liver and the lung leads to the activation of calmodulin pathways in the lung, leading to the activation of interleukin 6 and phosphoenolpyruvate carboxykinase 1 pathway across these organs. Furthermore, we retrospectively identified several pre-clinical and clinical evidence supporting this inter-organ mechanism of IPF. In conclusion, such feedforward and feedback system across the lung and the liver provides a unique opportunity for the development of the treatment and/or diagnosis of IPF. Furthermore, the result illustrates a generative computational framework for machine-mediated synthesis of mechanisms that facilitates and complements the traditional experimental approaches in biomedical sciences.

AbstractComputational deconvolution of transcriptome data of organs/tissues uncovers their structural and functional complexities at cellular resolution without performing single-cell RNA-sequencing experiments. However, the deconvolution of highly heterogenous diverse organs/tissues remains a challenge. Herein, we report “cell type-specific weighting-factors” that are essential for accurate deconvolution, but critically lacking in the existing methods. We computed such weighting-factors for 97 cell-types across 10 mouse organs and demonstrate their effective usage in the Bayesian framework to generate their virtual single-cell RNA-sequencing data, hence accurately estimating both cell-type ratios and the complete transcriptome of each cell-type in these organs. The method also efficiently detects the temporal changes of such cell type-profiles during organ pathogenesis in disease models. Furthermore, we present its potential utility for human organ/bulk-tissue deconvolution. Taken together, the weighting-factors reported herein and their computation for new cell-types and/or new species such as human are essential tools/resources for studying high-resolution biology and disease.