Method Validation and Measurement Uncertainty (MU) Evaluation on Enrofloxacin and Ciprofloxacin in the Aquatic Products.

This study aimed to investigate a detection method of enrofloxacin and ciprofloxacin to be avail for strictly supervising the quality and safety of aquatic products. The results displayed that the optimal extraction conditions for enrofloxacin and ciprofloxacin were the following five aspects: 15 g dosages of Na2SO4 to dehydrate, 8‰ of acetonitrile and 50% hydrochloric acid to deproteinization, 2 mL dosages of n-hexane to degrease, 10 min of ultrasonic time, and 20 min of extraction (stand) time. Meanwhile, it was also obtained for the optimal detection performance indexes of the recovery, precision, and accuracy from the tests of shrimp, grass carp, and tilapia. In particular, the expanded uncertainties were 2.8601 and 0.8613, and the factors of both the calibration curves (Urel(C)) and the analysis of the experiment (Urel(E)) were the two MU main contributors for enrofloxacin and ciprofloxacin together with the results above 40%. Consequently, the developed novel method was suited for the determination of the enrofloxacin and ciprofloxacin residues in aquatic products and would contribute to reinforce in supervision and inspection of the quality and safety of aquatic products.

A GATA-type transcription factor SreA affects manganese susceptibility by regulating the expression of iron uptake-related genes.

SreA has been identified as a GATA-type transcription factor that represses iron uptake to avoid iron excess during iron sufficiency. However, knowledge about whether SreA also affects the homeostasis of other divalent metal ions is limited. In this study, by screening Aspergillus fumigatus transcription factor deletion mutant libraries, we demonstrate that the sreA deletion mutant shows the greatest tolerance to MnCl2 among the tested divalent metal ions. Fe and Mn stimuli are able to enhance the expression of SreA with the different time-dependent manner, while the expression of SreA contributes to Mn2+ tolerance. Lack of SreA results in abnormally increased expression of a series of siderophore biosynthesis genes and iron transport-related genes, especially under MnCl2 treatment. Further mechanistic exploration indicated that lack of SreA exacerbates abnormal iron uptake, and iron excess inhibits cellular Mn content; thus, deletion of sreA results in Mn tolerance. Thus, findings in this study have demonstrated a new unexplored function for the transcription factor SreA in regulation of the Mn2+ tolerance.

The C-22 sterol desaturase Erg5 is responsible for ergosterol biosynthesis and conidiation in Aspergillus fumigatus.

Aspergillus fumigatus is the most prevalent saprophytic fungi and can cause severe invasive aspergillosis in immunocompromised individuals. For infection of A. fumigatus, the small hydrophobic conidia have been shown to play a dominant role. In this study, we found that deletion of erg5, a C-22 sterol desaturase gene which function in the last two steps of ergosterol biosynthesis, was sufficient to block ergosterol biosynthesis and conidiation. The deletion phenotype was further verified by a conditional expression strain of erg5 using the inducible tet-on system. Strikingly, erg5 mutant displays increased susceptibility to antifungal azoles itraconazole. RNA sequencing analysis showed that erg5 deficiency resulted in changes in transcription mainly related to lipid, carbohydrate, and amino acid metabolism. Genes encoding ergosterol biosynthesis-related enzymes were found to be up-regulated in erg5 null mutants. However, genes involved in asexual development, including upstream regulators, melanin biosynthesis enzymes, heterotrimeric G proteins, and MAPK signaling, were down-regulated to various degrees. Furthermore, metabolomic study revealed that erg5 deficiency also resulted in altered lipid and amino acid metabolism, which was consistent with our transcriptomics analysis. Collectively, our study established a link between ergosterol biosynthesis and asexual development at the transcriptomics and metabolomics level in A. fumigatus.

In vitro and in vivo Efficacy of a Synergistic Combination of Itraconazole and Verapamil Against Aspergillus fumigatus.

The incidence of aspergillosis continues to rise sharply, while the progress made in expanding the antifungal drug arsenal remains extremely slow, indicating an urgent need for new strategies. Previous studies have shown that the calcium signaling pathway, which is evolutionarily conserved in mammals and fungi, is involved in regulating the tolerance of azoles in fungi. In this study, we performed a preliminary screening among various combinations of different clinical calcium channel blockers and different antifungal drugs. We found that the combination of itraconazole and verapamil showed the best synergistic effect against Aspergillus fumigatus. Thereafter, using the checkboard assays we observed synergistic effects of the combination treatment against most of the A. fumigatus strains tested, including itraconazole-sensitive and itraconazole-resistant strains, with a fractional inhibitory concentration index (FICI) < 0.5. Furthermore, we showed that verapamil strongly decreased the cytosolic calcium transients following itraconazole stimulation by an aequorin-mediated method. Moreover, verapamil influenced the efflux of rhodamine 6G, an azole mimic substance. An ergosterol assay revealed that verapamil alone had no effect on ergosterol biosynthesis, but the combination of itraconazole and verapamil treatment decreased the ergosterol level. Further murine assays were performed using a luciferase-probed bioluminescence imaging method. Drug combination therapy reduced lung burden and improved survival rate. In conclusion, verapamil is a promising candidate to enhance the antifungal activity of itraconazole against A. fumigatus. In addition, our study suggests the effectiveness of an emerging approach based on bioluminescence imaging in monitoring the efficacy of drug combination therapy for invasive aspergillosis.

The Zn2Cys6-type transcription factor LeuB cross-links regulation of leucine biosynthesis and iron acquisition in Aspergillus fumigatus.

Both branched-chain amino acids (BCAA) and iron are essential nutrients for eukaryotic cells. Previously, the Zn2Cys6-type transcription factor Leu3/LeuB was shown to play a crucial role in regulation of BCAA biosynthesis and nitrogen metabolism in Saccharomyces cerevisiae and Aspergillus nidulans. In this study, we found that the A. fumigatus homolog LeuB is involved in regulation of not only BCAA biosynthesis and nitrogen metabolism but also iron acquisition including siderophore metabolism. Lack of LeuB caused a growth defect, which was cured by supplementation with leucine or iron. Moreover, simultaneous inactivation of LeuB and HapX, a bZIP transcription factor required for adaptation to iron starvation, significantly aggravated the growth defect caused by inactivation of one of these regulators during iron starvation. In agreement with a direct role in regulation of both BCAA and iron metabolism, LeuB was found to bind to phylogenetically conserved motifs in promoters of genes involved in BCAA biosynthesis, nitrogen metabolism, and iron acquisition in vitro and in vivo, and was required for full activation of their expression. Lack of LeuB also caused activation of protease activity and autophagy via leucine depletion. Moreover, LeuB inactivation resulted in virulence attenuation of A. fumigatus in Galleria mellonella. Taken together, this study identified a previously uncharacterized direct cross-regulation of BCCA biosynthesis, nitrogen metabolism and iron homeostasis as well as proteolysis.

Aspergillus fumigatus Afssn3-Afssn8 Pair Reverse Regulates Azole Resistance by Conferring Extracellular Polysaccharide, Sphingolipid Pathway Intermediates, and Efflux Pumps to Biofilm.

Antifungal treatment is often ineffectual, partly because of biofilm formation. In this study, by using a combined forward and reverse genetic strategy, we identified that nucleus-localized AfSsn3 and its partner AfSsn8, which constitute a Cdk8-cyclin pair, are required for azole resistance in Aspergillus fumigatus Deletion of Afssn3 led to increased absorption and utilization of glucose and amino acids. Interestingly, absorption and utilization of glucose accelerated the extracellular polysaccharide formation, while utilization of the amino acids serine, threonine, and glycine increased sphingolipid pathway intermediate accumulation. In addition, the absence of Afssn3 induced the activity of the efflux pump proteins. These factors indicate the mature biofilm is responsible for the major mechanisms of A. fumigatus resistance to azoles in the ΔAfssn3 mutant. Collectively, the loss of Afssn3 led to two "barrier" layers between the intracellular and extracellular spaces, which consequently decreased drug penetration into the cell.

Innate and adaptive immune response to chronic pulmonary infection of hyphae of Aspergillus fumigatus in a new murine model.

PURPOSE: The pathogenesis of chronic pulmonary aspergillosis (CPA) has seldom been studied due partly to a lack of animal models. Since hypha is the main morphology colonizing the airway in CPA, it's critical to study the immune reaction to chronic pulmonary infection of hyphae of Aspergillus fumigatus, which also has seldom been studied in vivo before. METHODOLOGY: We established a novel murine model of chronic pulmonary infection of hyphae by challenging immunocompetent mice with tightly-structured hyphae balls intratracheally, and described the ensuing immunoreaction to hyphae and conidia, and the pathogenesis of CPA. RESULTS: Our experiment proved that the hyphae balls could induce a chronic pulmonary infection for 28 days with a considerable recrudescence at day 28 post-infection. Lungs infected with hyphae balls were remarkable for the many neutrophils and macrophages that flooded into airway lumens, with peribronchiolar infiltration of leukocytes. There was a transient increase of Th2 cells and Th17 cells at day 7 post-infection in the lung tissue. In contrast, lungs infected with conidia showed no peribronchiolar infiltration of leukocytes, but an influx of a great number of macrophages, and a much less number of neutrophils in the lumen. Besides, conidia activated the co-response of Th1, Th2 and Th17 cells with an increase of Treg cells in the lung tissue (quite different from most previous studies). CONCLUSION: We established a new murine model of chronic infection of hyphae to mimic the formation of CPA, and provide a new marker for different immune responses to hyphae and conidia.

Cu-sensing transcription factor Mac1 coordinates with the Ctr transporter family to regulate Cu acquisition and virulence in Aspergillus fumigatus.

Copper (Cu) is an essential trace element and is regarded as an important virulence factor in fungal pathogens. Previous studies suggest that a putative Cu-sensing transcription factor Mac1 and the Cu transporter Ctr family play important roles during fungal development and virulence. However, how Cu importers of the Ctr family are involved in the Cu acquisition and what is the functional relationship between them have not been fully investigated yet. Here, we demonstrate that the yeast Mac1 homolog in the opportunistic human pathogen Aspergillus fumigatus is required during colony development under low Cu conditions. Transcriptional profiling combined with LacZ reporter analyses indicate that Cu transporters ctrA2 and ctrC are expressed in an Afmac1-dependent manner upon Cu starvation, and over-expression of ctrA2 or ctrC transporters almost completely rescue the Afmac1-deletion defects, suggesting a redundancy of both transporters in Afmac1-mediated Cu uptake. Genetic analysis showed that ctrC may play a dominant role against Cu starvation relative to ctrA2 and elevated expression of ctrA2 can compensate for ctrC deletion under Cu starvation. Interestingly, both ctrA2 and ctrC deletions can suppress ctrB deletion colony defects. Our findings suggest that Ctr family proteins might coordinately regulate their functions to adapt to different Cu environments. Compared to yeast homologs, Cu family proteins in A. fumigatus may have their own working styles. Most importantly, the Afmac1 deletion strain shows a significantly attenuated pathogenicity in the neutropenic immunocompromised (a combination of cyclophosphamide and hydrocortisone) mice model, demonstrating that Afmac1 is required for pathogenesis in vivo.

Erg4A and Erg4B Are Required for Conidiation and Azole Resistance via Regulation of Ergosterol Biosynthesis in Aspergillus fumigatus.

Ergosterol, a fungus-specific sterol enriched in cell plasma membranes, is an effective antifungal drug target. However, current knowledge of the ergosterol biosynthesis process in the saprophytic human fungal pathogen Aspergillus fumigatus remains limited. In this study, we found that two endoplasmic reticulum-localized sterol C-24 reductases encoded by both erg4A and erg4B homologs are required to catalyze the reaction during the final step of ergosterol biosynthesis. Loss of one homolog of Erg4 induces the overexpression of the other one, accompanied by almost normal ergosterol synthesis and wild-type colony growth. However, double deletions of erg4A and erg4B completely block the last step of ergosterol synthesis, resulting in the accumulation of ergosta-5,7,22,24(28)-tetraenol, a precursor compound of ergosterol. Further studies indicate that erg4A and erg4B are required for conidiation but not for hyphal growth. Importantly, the Δerg4A Δerg4B mutant still demonstrates wild-type virulence in a compromised mouse model but displays remarkable increased susceptibility to antifungal azoles. Our data suggest that inhibitors of Erg4A and Erg4B may serve as effective candidates for adjunct antifungal agents with azoles. IMPORTANCE: Knowledge of the ergosterol biosynthesis pathway in the human opportunistic pathogen A. fumigatus is useful for designing and finding new antifungal drugs. In this study, we demonstrated that the endoplasmic reticulum-localized sterol C-24 reductases Erg4A and Erg4B are required for conidiation via regulation of ergosterol biosynthesis. Moreover, inactivation of both Erg4A and Erg4B results in hypersensitivity to the clinical guideline-recommended antifungal drugs itraconazole and voriconazole. Therefore, our finding indicates that inhibition of Erg4A and Erg4B might be an effective approach for alleviating A. fumigatus infection.

A Putative Mitochondrial Iron Transporter MrsA in Aspergillus fumigatus Plays Important Roles in Azole-, Oxidative Stress Responses and Virulence.

Iron is an essential nutrient and enzyme co-factor required for a wide range of cellular processes, especially for the function of mitochondria. For the opportunistic fungal pathogen Aspergillus fumigatus, the ability to obtain iron is required for growth and virulence during the infection process. However, knowledge of how mitochondria are involved in iron regulation is still limited. Here, we show that a mitochondrial iron transporter, MrsA, a homolog of yeast Mrs4p, is critical for adaptation to iron-limited or iron-excess conditions in A. fumigatus. Deletion of mrsA leads to disruption of iron homeostasis with a decreased sreA expression, resulted in activated reductive iron assimilation (RIA) and siderophore-mediated iron acquisition (SIA). Furthermore, deletion of mrsA induces hypersusceptibility to azole and oxidative stresses. An assay for cellular ROS content in ΔmrsA combined with rescue from the mrsA-defective phenotype by the antioxidant reagent L-ascorbic acid indicates that the increased sensitivity of ΔmrsA to the azole itraconazole and to oxidative stress is mainly the result of abnormal ROS accumulation. Moreover, site-directed mutation experiments verified that three conserved histidine residues related to iron transport in MrsA are required for responses to oxidative and azole stresses. Importantly, ΔmrsA causes significant attenuation of virulence in an immunocompromised murine model of aspergillosis. Collectively, our results show that the putative mitochondrial iron transporter MrsA plays important roles in azole- and oxidative-stress responses and virulence by regulating the balance of cellular iron in A. fumigatus.

FigA, a putative homolog of low-affinity calcium system member Fig1 in Saccharomyces cerevisiae, is involved in growth and asexual and sexual development in Aspergillus nidulans.

Calcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. In Saccharomyces cerevisiae, at least two different calcium uptake systems have been identified: the high-affinity calcium influx system (HACS) and the low-affinity calcium influx system (LACS). Compared to the HACS, the LACS in fungi is not well known. In this study, FigA, a homolog of the LACS member Fig1 from S. cerevisiae, was functionally characterized in the filamentous fungus Aspergillus nidulans. Loss of figA resulted in retardant hyphal growth and a sharp reduction of conidial production. Most importantly, FigA is essential for the homothallic mating (self-fertilization) process; further, FigA is required for heterothallic mating (outcrossing) in the absence of HACS midA. Interestingly, in a figA deletion mutant, adding extracellular Ca(2+) rescued the hyphal growth defects but could not restore asexual and sexual reproduction. Furthermore, quantitative PCR results revealed that figA deletion sharply decreased the expression of brlA and nsdD, which are known as key regulators during asexual and sexual development, respectively. In addition, green fluorescent protein (GFP) tagging at the C terminus of FigA (FigA::GFP) showed that FigA localized to the center of the septum in mature hyphal cells, to the location between vesicles and metulae, and between the junctions of metulae and phialides in conidiophores. Thus, our findings suggest that FigA, apart from being a member of a calcium uptake system in A. nidulans, may play multiple unexplored roles during hyphal growth and asexual and sexual development.