Joanna Tannous

Sequencing, physical organization and kinetic expression of the patulin biosynthetic gene cluster from Penicillium expansum

Patulin is a polyketide-derived mycotoxin produced by numerous filamentous fungi. Among them, Penicillium expansum is by far the most problematic species. This fungus is a destructive phytopathogen capable of growing on fruit, provoking the blue mold decay of apples and producing significant amounts of patulin. The biosynthetic pathway of this mycotoxin is chemically well-characterized, but its genetic bases remain largely unknown with only few characterized genes in less economic relevant species. The present study consisted of the identification and positional organization of the patulin gene cluster in P. expansum strain NRRL 35695. Several amplification reactions were performed with degenerative primers that were designed based on sequences from the orthologous genes available in other species. An improved genome Walking approach was used in order to sequence the remaining adjacent genes of the cluster. RACE-PCR was also carried out from mRNAs to determine the start and stop codons of the coding sequences. The patulin gene cluster in P. expansum consists of 15 genes in the following order: patH, patG, patF, patE, patD, patC, patB, patA, patM, patN, patO, patL, patI, patJ, and patK. These genes share 60-70% of identity with orthologous genes grouped differently, within a putative patulin cluster described in a non-producing strain of Aspergillus clavatus. The kinetics of patulin cluster genes expression was studied under patulin-permissive conditions (natural apple-based medium) and patulin-restrictive conditions (Eagles minimal essential medium), and demonstrated a significant association between gene expression and patulin production. In conclusion, the sequence of the patulin cluster in P. expansum constitutes a key step for a better understanding of the mechanisms leading to patulin production in this fungus. It will allow the role of each gene to be elucidated, and help to define strategies to reduce patulin production in apple-based products.

Patulin is a cultivar-dependent aggressiveness factor favouring the colonization of apples by Penicillium expansum

The blue mould decay of apples is caused by Penicillium expansum and is associated with contamination by patulin, a worldwide regulated mycotoxin. Recently, a cluster of 15 genes (patA-patO) involved in patulin biosynthesis was identified in P. expansum. blast analysis revealed that patL encodes a Cys6 zinc finger regulatory factor. The deletion of patL caused a drastic decrease in the expression of all pat genes, leading to an absence of patulin production. Pathogenicity studies performed on 13 apple varieties indicated that the PeΔpatL strain could still infect apples, but the intensity of symptoms was weaker compared with the wild-type strain. A lower growth rate was observed in the PeΔpatL strain when this strain was grown on nine of the 13 apple varieties tested. In the complemented PeΔpatL:patL strain, the ability to grow normally in apple and the production of patulin were restored. Our results clearly demonstrate that patulin is not indispensable in the initiation of the disease, but acts as a cultivar-dependent aggressiveness factor for P. expansum. This conclusion was strengthened by the fact that the addition of patulin to apple infected by the PeΔpatL mutant restored the normal fungal colonization in apple.

A study on the physicochemical parameters for Penicillium expansum growth and patulin production: effect of temperature, pH, and water activity

Abstract Penicillium expansum is among the most ubiquitous fungi disseminated worldwide, that could threaten the fruit sector by secreting patulin, a toxic secondary metabolite. Nevertheless, we lack sufficient data regarding the growth and the toxigenesis conditions of this species. This work enables a clear differentiation between the favorable conditions to the P. expansum growth and those promising for patulin production. A mathematical model allowing the estimation of the P. expansum growth rate according to temperature, a W, and pH, was also developed. An optimal growth rate of 0.92 cm/day was predicted at 24°C with pH level of 5.1 and high a W level of 0.99. The models predictive capability was tested successfully on artificial contaminated apples. This model could be exploited by apple growers and the industrialists of fruit juices in order to predict the development of P. expansum during storage and apple processing.

LaeA regulation of secondary metabolism modulates virulence in Penicillium expansum and is mediated by sucrose

Penicillium expansum, the causal agent of blue mould rot, is a critical health concern because of the production of the mycotoxin patulin in colonized apple fruit tissue. Although patulin is produced by many Penicillium species, the factor(s) activating its biosynthesis are not clear. Sucrose, a key sugar component of apple fruit, was found to modulate patulin accumulation in a dose-responsive pattern. An increase in sucrose culture amendment from 15 to 175 mm decreased both patulin accumulation and expression of the global regulator laeA by 175- and five-fold, respectively, whilst increasing expression of the carbon catabolite repressor creA. LaeA was found to regulate several secondary metabolite genes, including the patulin gene cluster and concomitant patulin synthesis in vitro. Virulence studies of ΔlaeA mutants of two geographically distant P. expansum isolates (Pe-21 from Israel and Pe-T01 from China) showed differential reduction in disease severity in freshly harvested fruit, ranging from no reduction for Ch-Pe-T01 strains to 15%-25% reduction for both strains in mature fruit, with the ΔlaeA strains of Is-Pe-21 always showing a greater loss in virulence. The results suggest the importance of abiotic factors in LaeA regulation of patulin and other secondary metabolites that contribute to pathogenicity.

Patulin transformation products and last intermediates in its biosynthetic pathway, E- and Z-ascladiol, are not toxic to human cells

Patulin is the main mycotoxin contaminating apples. During the brewing of alcoholic beverages, this mycotoxin is degraded to ascladiol, which is also the last precursor of patulin. The present study aims (1) to characterize the last step of the patulin biosynthetic pathway and (2) to describe the toxicity of ascladiol. A patE deletion mutant was generated in Penicillium expansum. In contrast to the wild strain, this mutant does not produce patulin but accumulates high levels of E-ascladiol with few traces of Z-ascladiol. This confirms that patE encodes the patulin synthase involved in the conversion of E-ascladiol to patulin. After purification, cytotoxicities of patulin and E- and Z-ascladiol were investigated on human cell lines from liver, kidney, intestine, and immune system. Patulin was cytotoxic for these four cell lines in a dose-dependent manner. By contrast, both E- and Z-ascladiol were devoid of cytotoxicity. Microarray analyses on human intestinal cells treated with patulin and E-ascladiol showed that the latter, unlike patulin, did not alter the whole human transcription. These results demonstrate that E- and Z-ascladiol are not toxic and therefore patulin detoxification strategies leading to the accumulation of ascladiol are good approaches to limit the patulin risk.

NRPS-Derived Isoquinolines and Lipopetides Mediate Antagonism between Plant Pathogenic Fungi and Bacteria

Bacterial-fungal interactions are presumed to be mediated chiefly by small-molecule signals; however, little is known about the signaling networks that regulate antagonistic relationships between pathogens. Here, we show that the ralstonins, lipopeptides produced by the plant pathogenic bacteria Ralstonia solanacearum, interfere with germination of the plant-pathogenic fungus Aspergillus flavus by down-regulating expression of a cryptic biosynthetic gene cluster (BGC), named imq. Comparative metabolomic analysis of overexpression strains of the transcription factor ImqK revealed imq-dependent production of a family of tripeptide-derived alkaloids, the imizoquins. These alkaloids are produced via a nonribosomal peptide synthetase- (NRPS-)derived tripeptide and contain an unprecedented tricyclic imidazo[2,1-a]isoquinoline ring system. We show that the imizoquins serve a protective role against oxidative stress that is essential for normal A. flavus germination. Supplementation of purified imizoquins restored wildtype germination to a ΔimqK A. flavus strain and protected the fungus from ROS damage. Whereas the bacterial ralstonins retarded A. flavus germination and suppressed expression of the imq cluster, the fungal imizoquins in turn suppressed growth of R. solanacearum. We suggest such reciprocal small-molecule-mediated antagonism is a common feature in microbial encounters affecting pathogenicity and survival of the involved species.

Secondary metabolism in Penicillium expansum: Emphasis on recent advances in patulin research

ABSTRACT The plant pathogenic fungus Penicillium expansum is a major concern of the global food industry due to its wide occurrence and ability to produce various mycotoxins, of which the most significant is patulin. Relatively less highlighted in the literature, in comparison with the other food-borne mycotoxins, patulin is one of the main factors in economic losses of vegetables and fruits. Otherwise, patulin is a health hazard which results in both short-term and long-term risks. This review includes knowledge on the biosynthetic mechanisms used for secondary metabolite production in P. expansum, with special emphasis on patulin biosynthesis. The abiotic factors triggering the production of patulin and the strategies developed to reduce or prevent the contamination by this mycotoxin are comprehensively discussed. The database presented in this review would be useful for the prioritization and development of future research.

Apple intrinsic factors modulating the global regulator, laeA, the patulin gene cluster and patulin accumulation during fruit colonization by Penicillium expansum

The mycotoxin patulin is produced in colonized tissue by Penicillium expansum during storage of apples and is significantly affected by environmental factors that contribute to its accumulation. Few reports have, however, examined the effect of natural intrinsic factors associated with the fruit on the production of patulin. Here, we find that with advancing maturity, Golden Delicious apples show increased concentrations of total soluble solids (TSS) from 14 to 17% associated with the increased expression of the global transcription factor involved in regulation of secondary metabolite biosynthesis in filamentous fungi, laeA expression and patulin accumulation. However, the apple cultivar Granny Smith, with similar TSS values but differing in pH levels and malic acid concentrations, showed reduced expression levels of laeA and the patulin biosynthesis gene cluster (pat genes) and patulin accumulation, suggesting a complexity of host factors contribution to patulin accumulation during P. expansum colonization. To start elucidating these apple intrinsic factors, we examined their in vitro impact on laeA and pat gene expression concomitant with patulin synthesis. Increasing sucrose concentrations from 15 to 175 mM repressed laeA and pat gene expression and patulin production. However, this affect was modified and often reversed and sometimes accentuated by changes in pH, or the addition of malic acid or the major apple phenolic compounds, chlorogenic acid and epicatechin. While the increase in malic acid from 0 to 1% increased laeA and pat gene expression, the decrease in pH from 3.5 to 2.5 reduced their expression. Also the increased laeA and pat genes expressions at increasing epicatechin concentrations from 0 to 1 mM, was reversed by increasing sucrose concentrations, all together suggesting the complexity of the interactions in vivo.

Contribution of ATPase copper transporters in animal but not plant virulence of the crossover pathogen Aspergillus flavus

ABSTRACT The ubiquitous fungus Aspergillus flavus is notorious for contaminating many important crops and food-stuffs with the carcinogenic mycotoxin, aflatoxin. This fungus is also the second most frequent Aspergillus pathogen after A. fumigatus infecting immunosuppressed patients. In many human fungal pathogens including A. fumigatus, the ability to defend from toxic levels of copper (Cu) is essential in pathogenesis. In A. fumigatus, the Cu-fist DNA binding protein, AceA, and the Cu ATPase transporter, CrpA, play critical roles in Cu defense. Here, we show that A. flavus tolerates higher concentrations of Cu than A. fumigatus and other Aspergillus spp. associated with the presence of two homologs of A. fumigatus CrpA termed CrpA and CrpB. Both crpA and crpB are transcriptionally induced by increasing Cu concentrations via AceA activity. Deletion of crpA or crpB alone did not alter high Cu tolerance, suggesting they are redundant. Deletion of both genes resulted in extreme Cu sensitivity that was greater than that following deletion of the regulatory transcription factor aceA. The ΔcrpAΔcrpB and ΔaceA strains were also sensitive to ROI stress. Compared to wild type, these mutants were impaired in the ability to colonize maize seed treated with Cu fungicide but showed no difference in virulence on non-treated seed. A mouse model of invasive aspergillosis showed ΔcrpAΔcrpB and to a lesser degree ΔaceA to be significantly reduced in virulence, following the greater sensitivity of ΔcrpAΔcrpB to Cu than ΔaceA.