Persistence versus escape: Aspergillus terreus and Aspergillus fumigatus employ different strategies during interactions with macrophages

Invasive bronchopulmonary aspergillosis (IBPA) is a life-threatening disease in immunocompromised patients. Although Aspergillus terreus is frequently found in the environment, A. fumigatus is by far the main cause of IBPA. However, once A. terreus establishes infection in the host, disease is as fatal as A. fumigatus infections. Thus, we hypothesized that the initial steps of disease establishment might be fundamentally different between these two species. Since alveolar macrophages represent one of the first phagocytes facing inhaled conidia, we compared the interaction of A. terreus and A. fumigatus conidia with alveolar macrophages. A. terreus conidia were phagocytosed more rapidly than A. fumigatus conidia, possibly due to higher exposure of β-1,3-glucan and galactomannan on the surface. In agreement, blocking of dectin-1 and mannose receptors significantly reduced phagocytosis of A. terreus, but had only a moderate effect on phagocytosis of A. fumigatus. Once phagocytosed, and in contrast to A. fumigatus, A. terreus did not inhibit acidification of phagolysosomes, but remained viable without signs of germination both in vitro and in immunocompetent mice. The inability of A. terreus to germinate and pierce macrophages resulted in significantly lower cytotoxicity compared to A. fumigatus. Blocking phagolysosome acidification by the v-ATPase inhibitor bafilomycin increased A. terreus germination rates and cytotoxicity. Recombinant expression of the A. nidulans wA naphthopyrone synthase, a homologue of A. fumigatus PksP, inhibited phagolysosome acidification and resulted in increased germination, macrophage damage and virulence in corticosteroid-treated mice. In summary, we show that A. terreus and A. fumigatus have evolved significantly different strategies to survive the attack of host immune cells. While A. fumigatus prevents phagocytosis and phagolysosome acidification and escapes from macrophages by germination, A. terreus is rapidly phagocytosed, but conidia show long-term persistence in macrophages even in immunocompetent hosts.     

Effects of chloroperoxidase and hydrogen peroxide on the viabilities of Aspergillus flavus conidiospores

The effects of chloroperoxidase [EC 1.1.1.10] and hydrogen peroxide on the viabilities of quiescent and germinating conidiospores of an aflatoxigenic fungus, Aspergillus flavus, were determined. Hydrogen peroxide was found moderately lethal and chloroperoxidase produced a 30-fold increase in the lethality of hydrogen peroxide to germinating conidia, which were 75-fold more susceptible to chloroperoxidase than were quiescent conidia. According to infrared examinations of fungal corpses, mortality occurred by oxidation rather than peroxidative chlorination.     

Protoplast fusion between Aspergillus oryzae and Aspergillus niger in relation to their multinuclear nature

Protoplasts of cycloheximide-resistant strains from Aspergillus oryzae IFO 5239 were fused with those of kabicidin-resistant strains from Aspergillus niger IFO 4407. By nuclear staining in conidia, it appeared that all of the fusant conidia had two kinds of nuclei. Small nuclei seemed to be derived from A. oryzae and large nuclei seemed to be derived from A. niger. However, three types of antibiotic resistance were shown among the conidia of fusants. Almost all were kabicidin resistant. Conidia of fusants were multinuclear and had various DNA contents and various sizes. By the comparison with the growth rates of parental strains, the growth rates of A. niger were superior to those of A. oryzae. The inclination in the distribution of antibiotic resistance of fusant conidia seemed to owe more to the differences of growth rates between parental strains than the influence of the multinucleate nature of a parental strain.     

Aspergillus terreus Accessory Conidia Are Unique in Surface Architecture,Cell Wall Composition and Germination Kinetics

Infection with Aspergillus terreus is more likely to result in invasive, disseminated disease when compared to other Aspergillus species; importantly this species appears to be less susceptible to the antifungal drug amphotericin B. Unique to this species is the ability to produce specialized structures denoted as accessory conidia (AC) directly on hyphae both in vitro and in vivo. With the hypothesis that production of AC by A. terreus may enhance virulence of this organism, we analyzed the phenotype, structure and metabolic potential of these conidia. Comparison of A. terreus phialidic conidia (conidia that arise from conidiophores, PC) and AC architecture by electron microscopy revealed distinct morphological differences between the two conidial forms; AC have a smoother, thicker outer cell surface with no apparent pigment-like layer. Further, AC germinated rapidly, had enhanced adherence to microspheres, and were metabolically more active compared to PC. Additionally, AC contained less cell membrane ergosterol, which correlated with decreased susceptibility to AMB as determined using a flow cytometry based analysis. Furthermore, AC exhibited surface patches of β1-3 glucan, suggestive of attachment scarring. Collectively, the findings of this study suggest a possible role for AC in A. terreus pathogenesis.     

Transfer Ribonucleic Acid Methylases During the Germination of Neurospora crassa

Transfer ribonucleic acid (tRNA) methylases were studied during the germination of spores in Neurospora crassa. The total methylase capacity and base specific tRNA methylase activities were determined in extracts from cells harvested at various stages of germination. Germinated conidia have a 65% higher methylase capacity than ungerminated conidia. Three predominant methylase activities were found in the extracts, and the relative amount of each activity was different at the various stages. Enzymes from vegetative cells catalyzed significant hypermethylation of tRNA from conidia, whereas conidial enzymes were much less active on tRNA from vegetative cells. The results indicate differences in the tRNA methylase content and tRNA species of conidia and vegetative cells.     

Effect of amphotericin B on the metabolism of Aspergillus fumigatus

Action of amphotericin B on the growth and metabolism of Aspergillus fumigatus has been investigated. The fungus proved to be very sensitive to amphotericin B, showing complete inhibition of growth at 0.5 units/ml. Amphotericin B suppressed the exogenous and endogenous respiration and glycolysis of A. fumigatus as well as the assimilation of various glycolysis and TCA cycle intermediates. Addition of cations and cholesterol failed to reverse the action of amphotericin B. The treated mycelium released a variety of cellular constituents and it is inferred that the antibiotic effects the permeability of A. fumigatus cells. In experiments with ³²P labelled mycelium phosphorus compounds leached out in concentrations which were dependent on the antibiotic dose, period of contact, incubation temperature and metabolic state of the fungus.     

曲霉生物膜的形成过程与结构特征

目的 研究曲霉生物膜的形成过程和结构特征.方法 我们利用一个曲霉生物膜体外模型研究其形成过程和结构特征.将200 μL浓度为1×10<'5>孢子/mL的受试曲霉(烟曲霉AF293,黄曲霉BMU03940,土曲霉BMU00802,黑曲霉BMU04689)的孢子悬液加到24孔组织培养板中的无菌塑料细胞培养盖玻片上,37℃孵育不同时间(0、2、4、8、10、12、16、18、24、48、72 h),加入25 μmol/L的FUN-1室温避光染色后,用波长488 nm激光激发,通过共聚焦激光扫描显微镜观察曲霉生物膜的形成过程;再用波长为488 am和633 am激光同时激发,将两个波长下的图像叠加后观察曲霉生物膜的活力;利用:XYZ轴成像观察其结构特征.在上述不同的时间点用钙荧光白染色后,用波长为405 nm的紫外光激发,观察曲霉生物膜细胞外基质的产生.结果 烟曲霉AF293在第4 h即开始有散在的孢子黏附于盖玻片上;8 h时孢子开始萌芽,10～12 h菌丝延长形成单细胞层;16～20 h菌丝缠绕形成多层立体结构;24 h形成一个具有复杂的三维立体结构特征的多细胞菌落,菌丝有序排列,细胞外基质弥散的分布在菌丝的周围;48～72 h生物膜逐渐成熟.成熟的烟曲霉生物膜是由细胞外基质包裹的有序排列的菌丝形成的复杂立体结构.黄曲霉BMU03940、土曲霉BMU00802、黑曲霉BMU04689与烟曲霉AF293有类似的生物膜发育阶段,包括黏附、孢子萌芽、菌丝延长、菌丝有序排列形成三维立体结构.结论 烟曲霉、黄曲霉、土曲霉和黑曲霉在体外都能形成典型的生物膜,它的形成过程和结构特征与其他真菌生物膜类似.     

Use of the Weibull model to describe inactivation of dry‐harvested conidia of different Penicillium species by ethanol vapours

Aims: This study aimed at modelling the effect of ethanol vapours, in the range 0·7–7·5 kPa, on the inactivation of dry‐harvested conidia of Penicillium chrysogenum, Penicillium digitatum and Penicillium italicum. Methods and Results: Survival curves were modelled by a Weibull model: log (N/N₀) = ?1/2·303 (t/α)^β. The shape parameter β was different from one in all cases, indicating that the classical first‐order kinetics approach is the exception rather than the rule. Survival curves exhibited upward concavity (β < 1) with the notable exception of P. chrysogenum at ethanol vapour pressures 0·7 and 1·5 kPa. The scale parameter α (h) varied greatly depending on the ethanol vapour pressure and on the species. Conclusions: For safety reasons, it is recommended not to exceed an ethanol vapour pressure of 3·3 kPa. At 2·8 kPa, more than 4 log₁₀ reductions in viable conidia were achieved for all the species after 24‐h exposure. Significance and Impact of the Study: Ethanol has GRAS status in the USA and represents an interesting alternative to fungicides. The effectiveness of ethanol vapours to inactivate dry‐harvested conidia of some Penicillium was demonstrated in this study.     

Phenol oxidase activity and pigment synthesis in conidiospores of Penicillium cyclopium.

Phenol oxidase (PO) of Penicillium cyclopium belongs to the laccases (E.C. 1.14.18.1). By PAA-gel electrophoresis three forms of the enzyme were detected. Most of the PO in emerged sporulating cultures accumulates in the cell wall of the conidiospores. However, a high percentage is not bound to the wall constituents and can be easily removed with aqueous solutions. This soluble fraction was 570-fold enriched by (NH4)2SO4-precipitation, followed by chromatography on sephadex G 200 and on hydroxylapatite-cellulose, The necessity of PO for conidiospore pigmentation was shown by means of suspended spores whose rates of pigment accumulation resemble those of conidia maturing in situ. Pigmentation is stopped if PO is inhibited by diethyldithiocarbamate. This corresponds to the fact that in strains with heavily pigmented conidiospores PO activity is much higher than in strains with weakly pigmented or unpigmented spores. Also the time course of PO accumulation in the latter strains is different from that of the former. Furthermore, in mutant res-eth 1 which has unpigmented conidiospores, PO isoenzyme no. 3 could not be detected.     

Culture supernatants of patient-derived Aspergillus isolates have toxic and lytic activity towards neurons and glial cells

Infection of the central nervous system by the ubiquitous fungi Aspergillus spp. is a life-threatening disease. Therefore we investigated the mechanism of brain damage by fungal infection. To examine whether secretory factors of Aspergillus isolates derived from patients can induce death of different brain cells, culture supernatants of Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus and Aspergillus niger were added to different astrocytes as well as to the neuroblastoma cell line SK-N-SH, and to the microglial cell line CHME. All four fungal species were shown to secrete toxic factors with neurons being most sensitive against these factors. Very low amounts and short incubation times are sufficient to induce irreversible cell damage, indicating that secreted factors might also affect distant brain regions. Further characterization of the toxic factors revealed that A. fumigatus and A. terreus produced small, heat-stable components whereas the toxic activity of A. niger filtrates was triggered by a high molecular mass factor which could be inactivated by heat. The active component of A. flavus had a molecular mass similar to that of A. niger but was heat-stable and had a significantly lower activity. Taken together these results indicate that secretion of different necrotizing factors might contribute to brain lesions in patients with cerebral aspergillosis.     

Effect of competition and adverse culture conditions on aflatoxin production by Aspergillus flavus through successive generations

Strains of Aspergillus flavus often degenerate with serial transfers on culture media, resulting in morphological changes and loss of aflatoxin production. However, degeneration does not readily occur in nature as indicated by the wild-type morphological characters of newly isolated strains and the high percentage of aflatoxigenic A. flavus from soil and crops in some geographic regions. In this study, three aflatoxin-producing strains of A. flavus were serially transferred using conidia for 20 generations (three independent generation lines per strain) on potato dextrose agar at 30 C. The rate of degeneration was compared to that of cultures grown in the presence of competing fungi (A. terreus, Penicillium funiculosum, and the yeast, Pichia guilliermondii) and under adverse conditions of elevated temperature, reduced water activity, low pH, and nutrient deprivation. Formation of morphological variants and the associated loss of aflatoxin production over generations varied considerably according to strain and the generation line within each strain. In the strain most sensitive to degeneration on potato dextrose agar, aflatoxin-producing ability was maintained to varying degrees under adverse culture conditions, but not when A. flavus was competing with other fungi.     

Conidial hydrophobins of Aspergillus fumigatus

The surface of Aspergillus fumigatus conidia, the first structure recognized by the host immune system, is covered by rodlets. We report that this outer cell wall layer contains two hydrophobins, RodAp and RodBp, which are found as highly insoluble complexes. The RODA gene was previously characterized, and DeltarodA conidia do not display a rodlet layer (N. Thau, M. Monod, B. Crestani, C. Rolland, G. Tronchin, J. P. Latgé, and S. Paris, Infect. Immun. 62:4380-4388, 1994). The RODB gene was cloned and disrupted. RodBp was highly homologous to RodAp and different from DewAp of A. nidulans. DeltarodB conidia had a rodlet layer similar to that of the wild-type conidia. Therefore, unlike RodAp, RodBp is not required for rodlet formation. The surface of DeltarodA conidia is granular; in contrast, an amorphous layer is present at the surface of the conidia of the DeltarodA DeltarodB double mutant. These data show that RodBp plays a role in the structure of the conidial cell wall. Moreover, rodletless mutants are more sensitive to killing by alveolar macrophages, suggesting that RodAp or the rodlet structure is involved in the resistance to host cells.     

Alteration of cell wall composition leads to amphotericin B resistance in Aspergillus flavus

An amphotericin B (AmB)-resistant mutant was isolated from a wild-type AmB-susceptible strain of Aspergillus flavus by serial transfer of conidia on agar plates containing stepwise increased concentrations of AmB up to 100 microg ml-1. The acquired resistance of mycelia was specific for polyene-antibiotics AmB, nystatin and trichomycin. Spheroplasts derived from the resistant mycelia were as susceptible to AmB as the wild-type. Chemical analysis of the cell wall revealed that levels of alkali-soluble and -insoluble glucans were significantly higher in the resistant mycelia as compared to those in the wild-type. When resistant mycelia were treated with SDS, they adsorbed as much AmB as wild-type mycelia. These results suggest that alterations in the cell wall components of mycelia, especially 1,3-alpha-glucan and protein complex in the outermost wall layer, lead to AmB resistance in A. flavus.     

Lethal effects of high-intensity violet 405-nm light on Saccharomyces cerevisiae,Candida albicans,and on dormant and germinating spores of Aspergillus niger

This study assessed the effects of high-intensity violet light on selected yeast and mould fungi. Cell suspensions of Saccharomyces cerevisiae, Candida albicans, and dormant and germinating spores (conidia) of the mould Aspergillus niger were exposed to high-intensity narrow band violet light with peak output at 405 nm generated from a light-emitting diode (LED) array. All three fungal species were inactivated by the 405-nm light without a requirement for addition of exogenous photosensitiser chemicals. Of the fungal species tested, S. cerevisiae was most sensitive and dormant conidia of A. niger were most resistant to 405-nm light exposure. Five-log₁₀ colony forming units per millilitre (CFU ml^?1) reductions of the tested species required exposure doses of 288 J cm^?2 for S. cerevisiae, 576 J cm^?2 for C. albicans, and a much higher value of 2.3 kJ cm^?2 for dormant conidia of A. niger. During germination, A. niger conidia became more sensitive to 405-nm light exposure and sensitivity increased as germination progressed over an 8 h test period. Light exposure under aerobic and anaerobic conditions, together with results obtained using ascorbic acid as a scavenger of reactive oxygen species, revealed that 405-nm light inactivation in fungi involved an oxygen-dependent mechanism, as previously described in bacteria. The inactivation results achieved with yeast cells and fungal spores together with operational advantages associated with the use of a visible (nonultraviolet (UV)) light source highlight the potential of 405-nm light for fungal decontamination applications.     

Relationship between soil densities of Aspergillus species and colonization of wounded peanut seeds

Soil is a reservoir for Aspergillus flavus and A. parasiticus, fungi that commonly colonize peanut seeds and produce carcinogenic aflatoxins. Densities of these fungi in soil vary greatly among fields and may influence the severity of peanut infection. This study examined the relationship between soil density of Aspergillus species and the incidence of peanut seed colonization under laboratory conditions. Viable peanut seeds were wounded and inoculated with 20 soils differing in composition and density of Aspergillus species and were then incubated for 14 days at 37 degrees C (seed water activity = 0.92). The effect of soil density of individual section Flavi species (A. flavus strains L and S, A. parasiticus, A. caelatus, and A. tamarii), section Nigri, and A. terreus on the incidence of seed colonization was best expressed as a function of exponential rise to maximum. Exponential curves often rose to maximum percentages of seed colonization by section Flavi species that were well below 100% despite high species densities in some soils. Competition primarily among section Flavi species may explain the reduced incidences of seed colonization. An average of two or fewer propagules of each Aspergillus species in the soil at the wound site was required for colonization of 20% of peanut seeds. Other fungal species were capable of invading peanut seeds only when soil densities of sections Flavi and Nigri species were low.     

Maize ribosome-inactivating protein inhibits normal development of Aspergillus nidulans and Aspergillus flavus

The abundant maize kernel ribosome-inactivating protein 1 (RIP1) was tested for antifungal activity against Aspergillus nidulans and Aspergillus flavus. A microculture assay was developed to monitor fungal growth and development after treatment of conidia with RIP1 or control proteins. A striking decrease in hyphal proliferation was observed when conidia of A. nidulans, a genetically well-characterized nonpathogenic species, were treated with RIP1 protein. Treatment with a RIP1 mutant protein that lacked enzymatic ribosome-inactivating activity caused no observable effects. RIP1 treatment of conidia from the maize pathogen A. flavus resulted in increased hyphal branching. Examination of the branched hyphae after Congo red staining revealed only one growing hyphal tip per conidium. These results indicate that both fungi were affected by RIP1 treatment, but the lysis seen with treatment of A. nidulans was apparently avoided by A. flavus. A developmental time course revealed that both fungal species were affected by RIP1 at the postdivisional growth stage. The inhibitory activity of RIP1 against normal fungal growth is consistent with a biological function to protect the seed from fungal invasion.     

Quantitative PCR analysis of selected Aspergillus, Penicillium and Paecilomyces species

A total of 65 quantitative PCR (QPCR) assays, incorporating fluorigenic 5' nuclease (TaqMan) chemistry and directed at the nuclear ribosomal RNA operon, internal transcribed spacer regions (ITS1 or ITS2) was developed and tested for the detection of selected Aspergillus, Penicillium and Paecilomyces species. The assays varied in specificity from species or subspecies to closely related species groups, subject to the amount of nucleotide sequence variation in the different organisms. A generic assay for all target species of Aspergillus, Penicillium and Paecilomyces was also developed and tested. Using a previously reported DNA extraction method, estimated conidia detection limits for target species ranged from less than one to several hundred per sample for the different assays. Conidia detection limits for non-target species were at least 1,000 fold higher in nearly all instances. The assays were used to analyze ten HVAC dust samples from different sources around the US. Total quantities of Aspergillus, Penicillium and Paecilomyces conidia in the samples, determined by the generic assay and the summed totals from the specific assays, were in general agreement, suggesting that all of the numerically dominant species in the samples were accounted for by the specific assays. QPCR analyses of these samples after spiking them with selected target organisms indicated that the enumeration results were within approximately a one-half log range of the expected values 95% of the time. Evidence is provided that the commonly used practices of enumerating Aspergillus and Penicillium as a single group or only by genus can be misleading in understanding the indoor populations of these organisms and their potential health risks.