Jae-Hyung Mah

Upstream and Downstream Regulation of Asexual Development in Aspergillus fumigatus

ABSTRACT The opportunistic human pathogen Aspergillus fumigatus produces a large quantity of asexual spores (conidia), which are the primary agent causing invasive aspergillosis in immunocompromised patients. We investigated the mechanisms controlling asexual sporulation (conidiation) in A. fumigatus via examining functions of four key regulators, GpaA (Gα), AfFlbA (RGS), AfFluG, and AfBrlA, previously studied in Aspergillus nidulans. Expression analyses of gpaA, AfflbA, AffluG, AfbrlA, and AfwetA throughout the life cycle of A. fumigatus revealed that, while transcripts of AfflbA and AffluG accumulate constantly, the latter two downstream developmental regulators are specifically expressed during conidiation. Both loss-of-function AfflbA and dominant activating GpaAQ204L mutations resulted in reduced conidiation with increased hyphal proliferation, indicating that GpaA signaling activates vegetative growth while inhibiting conidiation. As GpaA is the primary target of AfFlbA, the dominant interfering GpaAG203R mutation suppressed reduced conidiation caused by loss of AfflbA function. These results corroborate the hypothesis that functions of G proteins and RGSs are conserved in aspergilli. We then examined functions of the two major developmental activators AfFluG and AfBrlA. While deletion of AfbrlA eliminated conidiation completely, null mutation of AffluG did not cause severe alterations in A. fumigatus sporulation in air-exposed culture, implying that, whereas the two aspergilli may have a common key downstream developmental activator, upstream mechanisms activating brlA may be distinct. Finally, both AffluG and AfflbA mutants showed reduced conidiation and delayed expression of AfbrlA in synchronized developmental induction, indicating that these upstream regulators contribute to the proper progression of conidiation.

Growth and Developmental Control in the Model and Pathogenic Aspergilli

The filamentous fungi comprise a ubiquitous group of heterotrophic organisms living as saprophytes, parasites, or symbionts. The basis for fungal vegetative growth is the continued and coordinated expansion of a series of fungal cell tips into a linear or complex structure. Fungi differentiate into a variety of structures including spores, which are the effective means of genome protection, survival, and propagation. Spores are also the primary agent for infecting host organisms for many human- and plant-pathogenic fungi. Asexual sporulation is a prevalent mode of reproduction for a diverse group of fungi, which results in the production of vast numbers of mitotically derived spores (reviewed in reference 2). The genus Aspergillus represents the most widespread fungi in our environment, which all reproduce asexually by forming long chains of conidiospores (or conidia) radiating from a central structure known as a conidiophore (Fig. 1) (reviewed in reference 3). The impact of various Aspergillus species on humans runs the range from “good” to “bad.” For instance, several species such as Aspergillus oryzae and Aspergillus niger are used in industry for enzyme production and food processing. In contrast, Aspergillus flavus and Aspergillus parasiticus can produce the most potent naturally present carcinogen, aflatoxin, which can contaminate various foods and feeds (reviewed in reference 43). Moreover, the opportunistic human pathogen Aspergillus fumigatus has become the most prevalent airborne fungal pathogen, causing severe and usually fatal invasive aspergillosis in immunocompromised patients (reviewed in reference 22). Aspergillus nidulans has served as an excellent model system for studying various biological questions, primarily due to the ease of genetic analysis through meiotic (sexual) recombination and the development of sophisticated molecular tools (32). These properties have provided a better understanding of the mechanisms controlling growth, development, secondary metabolism, and other aspects of cell biology in filamentous fungi (3, 35, 50, 57). The availability of the genome sequences of several aspergilli facilitates comparative genomic, genetic, and functional studies. In particular, knowledge and information obtained from a model fungus can be effectively tested in less genetically tractable aspergilli. Recent studies of a number of Aspergillus species have proven that a model fungus can provide a useful framework for understanding the biology of agriculturally and/or medically relevant aspergilli (6, 7, 38, 41, 49, 57). For instance, the novel nuclear protein LaeA has been shown to function as a global regulator of secondary metabolism as well as a regulator of morphogenetic virulence factors in the genus Aspergillus (6, 7). This review summarizes our current understanding of the genetic mechanisms controlling asexual development (conidiation) and vegetative growth in the model (A. nidulans) and pathogenic (A. fumigatus) aspergilli.

Resistance of Enterobacter sakazakii (Cronobacter spp.) to environmental stresses.

Aim:  To gain a better understanding of the survival and persistence of Enterobacter sakazakii in severe environments.

Biodegradable Poly(butylene adipate‐co‐terephthalate) Films Incorporated with Nisin: Characterization and Effectiveness against Listeria innocua

Biodegradable poly(butylene adipate-co-terephthalate) (PBAT) films incorporated with nisin were prepared with concentrations of 0, 1000, 3000, and 5000 international units per cm(2) (IU/cm(2)). All the films with nisin inhibited Listeria innocua, and generated inhibition zones with diameters ranging from 14 to 17 mm. The water vapor permeability and oxygen permeability after the addition of nisin ranged from 3.05 to 3.61 x 10(11) g m m(-2) s(-1) Pa(-1) and from 4.80 x 10(7) to 11.26 x 10(7) mL.m.m(-2).d(-1).Pa(-1), respectively. The elongation at break (epsilon(b)) was not altered by the incorporation of nisin (P > 0.05). Significant effect was found for the elastic modulus (E) and the tensile strength (sigma(s)) (P < 0.05). The glass transition and melting temperatures with the presence of nisin ranged from -36.3 to -36.6 degrees C and from 122.5 to 124.2 degrees C, respectively. The thermal transition parameters such as the crystallization and melting enthalpies and crystallization temperature were influenced significantly (P < 0.05) by incorporation of nisin into films. The X-ray diffraction patterns exhibited decreasing levels of intensity (counts) as the concentration of nisin increased in a range of 2theta from 8 degrees to 35 degrees . Formation of holes and pores was observed from the environmental scanning electron microscopy images in the films containing nisin, suggesting interaction between PBAT and nisin.

Biogenic amine formation and bacterial contribution in Natto products

Twenty-one Natto products currently distributed in Korea were analysed for biogenic amine contents and tested to determine physicochemical and bacterial contributions to biogenic amine formation. Among them, nine products (about 43%) had β-phenylethylamine or tyramine contents greater than the toxic dose (30mg/kg and 100mg/kg, respectively) of each amine, although no products showed total amounts of biogenic amines above the harmful level (1000mg/kg), which indicates that the amounts of biogenic amines in some Natto products are not within the safe level for human health. From four different Natto products, that contained noticeable levels of β-phenylethylamine and tyramine, 80 bacterial strains were isolated. All the strains were identified to be Bacillus subtilis and highly capable of producing β-phenylethylamine and tyramine. Therefore, it seems likely that the remarkable contents of β-phenylethylamine and tyramine in Natto predominantly resulted from the strains highly capable of producing those amines present in the food.

Paenibacillus tyraminigenes sp. nov. isolated from Myeolchi-jeotgal, a traditional Korean salted and fermented anchovy

A bacterial strain H3029, a gram-positive, rod-shaped, oxidase-negative, endospore-forming bacterium that characteristically produces tyramine from tyrosine, was isolated from a Myeolchi-jeotgal, a traditional Korean salted and fermented anchovy (Engraulis japonicus). The H3029 strain showed a high ability to produce 4140 microg/ml of tyramine from the culture broth containing 5000 microg/ml tyrosine. On the other hand, the strain produced a relatively low level of other putrefactive amines, at 973 microg/ml of putrescine and 147 microg/ml of cadaverine from the media, with each 5000 microg/ml of ornithine hydrochloride and lysine hydrochloride. The H3029 strain produced no detectable level of histamine (detection limit of 4 microg/ml) from the media containing 5000 microg/ml of histidine hydrochloride. Meanwhile, tyramine, the main product of the strain, showed the antimicrobial activity at the level of over 1 mg/disk against Staphylococcus aureus by agar diffusion test, and the mutagenicity in Ames test at 0.1 mg/plate using Salmonella typhimurium TA98 and TA1535. On the basis of the polyphasic taxonomic study, the H3029(T) strain was assigned a novel species of the genus Paenibacillus as Paenibacillus tyraminigenes sp. nov. The type strain of which is strain H3029(T) (=KCTC 10694BP(T)).

Effects of minerals on sporulation and heat resistance of Clostridium sporogenes

In this study, various mineral supplements, such as chloride salts (CaCl2, MgCl2, MnCl2, FeCl2 and KCl) supplying cations and calcium salts (CaCl2, CaCO3, CaSO4, Ca(OH)2 and CaHPO4) supplying anions, were tested if they could stimulate the sporulation of Clostridium sporogenes, a surrogate microorganism for C. botulinum. Of the cations tested, the addition of CaCl2 showed a slightly, but not significantly, greater increase in spore levels within 3 weeks of incubation, compared to that of the other cations. The optimum concentration of CaCl2 was 0.5%, which yielded nearly 10(4) CFU/ml of spores. Of the anions tested, CaCO3 promoted sporulation within one week, which was the most effective compound for promoting rapid sporulation among the minerals tested. CaSO4 produced a pattern of sporulation similar to that of CaCl2. While CaHPO4 resulted in the maximum production of spores after 4 weeks, Ca(OH)2 failed to induce sporulation. With an optimized concentration of 0.5% CaCO3, the spore yield was approximately 10(5) CFU/ml. The spores prepared in sporulation medium with CaCO3 (pH 5.0) had slightly, but not significantly, higher D values than those produced with CaCl2 (pH 5.0) at temperatures ranging from 113 to 121 degrees C. However, no significant differences were observed in Z values (both 10.76 degrees C). In a large scale spore production, D(121 degrees C) values of the spore crops prepared with CaCl2 and CaCO3 and resuspended in phosphate buffer (pH 7.0) were found to be both 0.92 min. In conclusion, our data suggest that CaCO3 is highly effective in reducing sporulation time as well as enhancing heat resistance.

Influences of milk components on biofilm formation of Cronobacter spp. (Enterobacter sakazakii)

Aim:  To determine the critical component(s) of skim milk for biofilm formation of Cronobacter species.

Comparison of viability and heat resistance of Clostridium sporogenes stored at different temperatures.

The objective of this study was to determine the influence of storage temperature on the viability and heat resistance of Clostridium sporogenes spores. Spore suspension containing both spores and vegetative cells was divided into 3 groups to be stored at different temperatures of -20 (freezing), 4 (refrigerating), and 25 degrees C (ambient temperature). Samples stored for different times within the 2 mo were tested for viability by comparison of colony counts on plates and for heat resistance by determining D values at 121 degrees C. No significant differences were found in the viability of vegetative cells during the storage period, regardless of storage temperatures tested, while the viability of the spores stored for more than 4 wk was significantly higher at 4 degrees C than at -20 degrees C. The heat resistance of spores stored at 4 degrees C for more than 4 wk was remarkably higher than that at 25 degrees C, but similar to that at -20 degrees C throughout the storage period. Consequently, it turned out that a refrigerating temperature of 4 degrees C is satisfactory for storage of C. sporogenes spores in maintaining viability and heat resistance. This study suggests that storage temperature influences the viability and heat resistance of C. sporogenes spores.

Formation and destruction of biogenic amines in Chunjang (a black soybean paste) and Jajang (a black soybean sauce)

Chunjang and Jajang samples were analysed for biogenic amine contents by using HPLC equipped with a UV-Vis detector. Chunjang samples contained relatively large amounts of histamine (up to 273mg/kg) and tyramine (up to 131mg/kg), whereas Jajang samples had relatively small amounts of biogenic amines (mostly less than 40mg/kg). There appeared to be a strong relationship between biogenic amine contents in Chunjang and Jajang, and the biogenic amines in Chunjang were found to be pyrolysed during frying thereof to prepare Jajang. Meanwhile, the total plate counts of Chunjang samples ranged from 5 to 8logcfu/g, and most strains that were isolated from Chunjang samples were identified to be Bacillus subtilis (91.0%). The strains isolated from a sample in which relatively small amounts of biogenic amines were detected showed significantly weak abilities to produce biogenic amines. This indicates that biogenic amine contents in Chunjang are primarily attributed to bacterial abilities to produce biogenic amines.