Using extended Bigelow meta-regressions for modelling the effects of temperature, pH, °Brix on the inactivation of heat resistant moulds.

The management of Heat Resistant Moulds (HRMs) is considered a great challenge for the juice fruit industry. Neosartorya, Byssochlamys and Talaromyces are three out of the main genera isolated from fruit juices that show great resistance to heat treatments. Several inactivation parameters can be found in the literature, however all of them were carried out in specific food matrices and using diverse inactivation methods. Thus, this meta-analysis study synthesizes the thermal resistance parameters of the three HRMs by adjusting extended Bigelow-based meta-regression models to data on inactivation experiments conducted in different liquid media. The meta-analytical data, extracted from publications between 1969 and 2017, was composed of decimal reduction time (D), inactivation method, temperature of inactivation, pH, °Brix, age of spores, and type of medium (model, juice, concentrates). Pooled D* values (D at 90 °C, pH 3.5 and 12° Brix) were estimated for B. fulva (1.95 min; 95% CI: 1.21-3.11 min), Talaromyces (4.03 min; 95% CI: 3.43-4.74 min), Neosartorya (0.5.35 min; 95% CI: 4.10-7.08 min), and B. nivea (10.32 min; 95% CI: 5.81-18.4 min). It was found that increasing the soluble solids in concentrates tends to cause a lower decrease in the heat resistance of Neosartorya and Talaromyces than increasing the soluble solids in model liquid or juices (p = 0.001; 0.012). In general, the screw-capped tubes and three neck round inactivation methods render higher D* values (p < 0.05) than the thermal death tubes, the polyethylene bag and the capillary methods. Spores of Talaromyces (overall zpH = 7.56; 95% CI: 5.13-13.5) and Neosartorya (overall zpH = 7.07; 95% CI: 5.04-10.8) appear to be more thermal sensitive to a decrease in medium pH than spores of Byssochlamys (overall zpH = 4.34; 95% CI: 3.20-6.73). The meta-regression models presented in this study can be valuable for estimating pooled inactivation kinetic parameters to be used by the fruit juice industry in the management of thermal processes and in the determination of shelf-life.

Ergot alkaloids contribute to virulence in an insect model of invasive aspergillosis.

Neosartorya fumigata (Aspergillus fumigatus) is the most common cause of invasive aspergillosis, a frequently fatal lung disease primarily affecting immunocompromised individuals. This opportunistic fungal pathogen produces several classes of specialised metabolites including products of a branch of the ergot alkaloid pathway called fumigaclavines. The biosynthesis of the N. fumigata ergot alkaloids and their relation to those produced by alternate pathway branches in fungi from the plant-inhabiting Clavicipitaceae have been well-characterised, but the potential role of these alkaloids in animal pathogenesis has not been studied extensively. We investigated the contribution of ergot alkaloids to virulence of N. fumigata by measuring mortality in the model insect Galleria mellonella. Larvae were injected with conidia (asexual spores) of two different wild-type strains of N. fumigata and three different ergot alkaloid mutants derived by previous gene knockouts and differing in ergot alkaloid profiles. Elimination of all ergot alkaloids significantly reduced virulence of N. fumigata in G. mellonella (P < 0.0001). Mutants accumulating intermediates but not the pathway end product fumigaclavine C also were less virulent than the wild type (P < 0.0003). The data indicate that ergot alkaloids contribute to virulence of N. fumigata in this insect model and that fumigaclavine C is important for full virulence.

Hydrophilins in the filamentous fungus Neosartorya fischeri (Aspergillus fischeri) have protective activity against several types of microbial water stress.

Hydrophilins are proteins that occur in all domains of life and protect cells and organisms against drought and other stresses. They include most of the late embryogenesis abundant (LEA) proteins and the heat shock protein (HSP) Hsp12. Here, the role of a predicted LEA-like protein (LeamA) and two Hsp12 proteins (Hsp12A and Hsp12B) of Neosartorya fischeri was studied. This filamentous fungus forms ascospores that belong to the most stress-resistant eukaryotic cells described to date. Heterologous expression of LeamA, Hsp12A and Hsp12B resulted in increased tolerance against salt and osmotic stress in Escherichia coli. These proteins were also shown to protect lactate dehydrogenase against dry heat and freeze-thaw cycles in vitro. Deletion of leamA caused diminished viability of sexual ascospores after drought and heat. This is the first report on functionality of Hsp12 and putative LeamA proteins derived from filamentous fungi, and their possible role in N. fischeri ascospore resistance against desiccation, high temperature and osmotic stress is discussed.

Mannitol is essential for the development of stress-resistant ascospores in Neosartorya fischeri (Aspergillus fischeri).

The polyol mannitol is one of the main compatible solutes in Neosartorya fischeri and accumulates in conidia and ascospores. Here, it is shown that biosynthesis of mannitol in N. fischeri mainly depends on mannitol 1-phosphate dehydrogenase (MpdA). Reporter studies and qPCR analysis demonstrated that mpdA is moderately expressed in vegetative hyphae and conidiophores, while it is highly expressed during development of ascospores. Deletion of mpdA reduced mannitol in whole cultures as much as 85% of the wild type, while trehalose levels had increased more than 4-fold. Decreased mannitol accumulation had no effect on mycelial growth irrespective of heat- or oxidative stress. Notably, conidia of the ΔmpdA strain had higher mannitol and lower trehalose levels. They were more sensitive to heat stress. The most distinct phenotype of mpdA deletion was the absence of full development of ascospores. Formation of cleistothecia, and asci was not affected. The ascus cell wall, however, did not dissolve and asci contained incompletely formed or aborted ascospores. Addition of the Mpd inhibitor nitrophenide to the wild type strain also resulted in disturbed ascospore formation. Taken together, these results show that mannitol has a role in sexual development of N. fischeri and in stress resistance of conidia.

Effect of different fruit components, aeration and cold storage at 5°C on the germination of ascospores of Neosartorya fischeri.

UNLABELLED: Germination is the process by which a spore is transformed from a dormant state of metabolic activity to one of high activity. Effects of different fruit components, aeration and cold storage at 5°C on the germination of ascospores of Neosartorya fischeri after heat treatment were investigated by measuring rate and percentage germination and outgrowth of fungal biomass. Some activated spores reverted back to dormancy after 12 and 24 h of storage at 5°C. Ascospores germinated easily in acetate buffers. Germination of ascospores was retarded in medium containing citric acid and sugars. Ascospores cultured under low aeration showed no signs of germination. Different food components and growth conditions have varying effects on germination of ascospores. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this study can be used in the fruit industry to predict the effect of some fruit components on the germination of heat-resistant ascospores. The impact of cold storage on ascospores, which may be activated by heat schedules used to manufacture some types of fruit products, will help in determining the best storage conditions for enhanced shelf life to be adopted immediately after heat treatment. These findings also help to determine for how long a fruit juice is safe at room temperature after production before mycotoxin synthesis by heat-resistant fungi sets in.

[Sexuality and pathogenicity in Aspergillus fumigatus: is there any relationship?]. / Sexualidad y patogenicidad en Aspergillus fumigatus: ¿existe alguna relación?

BACKGROUND: Aspergillus fumigatus, like many other fungal species of clinical relevance, has been traditionally regarded as an asexual organism. However, in last few years several pieces of evidence question this such assumption, suggesting that the sexual state of A. fumigatus may still be undiscovered. These investigations have finally led to the recent discovery of a teleomorph stage of A. fumigatus, which has been named Neosartorya fumigata. AIMS: To review the most important findings on A. fumigatus sexuality and discuss the possible implications of such findings on its pathogenicity. METHODS: A bibliographic search was performed to find the main works that study the sexuality of fungal pathogens and, especially, of A. fumigatus. Moreover, data from our recent investigations in this field were also introduced to the discussion. RESULTS: The existence of a teleomorph for A. fumigatus could have significant clinical repercussions, as sexual reproduction might produce offspring with increased virulence and/or resistance to antifungal agents. In this sense, the results of our investigations suggest the existence of an association between the MAT1-1 mating type and the invasiveness of A. fumigatus isolates. CONCLUSIONS: The study of the sexual reproduction of the fungal pathogens and its possible relationship with virulence will continue to be a topic of interest during the next years, not only because of its basic interest, but also for the possible clinical repercussions.

Neosartorya udagawae (Aspergillus udagawae), an emerging agent of aspergillosis: how different is it from Aspergillus fumigatus?

A recent report on several cases of invasive aspergillosis caused by Neosartorya udagawae suggested distinctive patterns of disease progression between N. udagawae and Aspergillus fumigatus. This prompted us to characterize N. udagawae in comparison to A. fumigatus. Our findings showed that both species exist in two mating types at similar ratios and produce gliotoxin. However, the thermotolerance of the two species differs: while A. fumigatus is able to grow at 55 degrees C but not at 10 degrees C, N. udagawae is able to grow at 10 degrees C but fails to grow at >42 degrees C. Furthermore, compared to A. fumigatus, the conidia of N. udagawae require longer incubation periods to germinate at 37 degrees C and are more susceptible to neutrophil attack as well as hydrogen peroxide; N. udagawae is also less virulent in gp91(phox-/-) mice. These findings suggest that growth and susceptibility to the host response might account for the reduced virulence of N. udagawae and the subtle distinction in the progression of the disease caused by the two species.