Surface-enhanced Raman spectroscopy and ultrastructural analysis of penicillin-producing Penicillium rubens strains.

Raman spectroscopy, transmission electron microscopy (TEM) and atomic force microscopy (AFM) techniques can perform chemical analyses and acquire high-resolution images of cell samples. For this reason, in this study, semi-thin sections of a single Penicillium rubens cell were analysed by Raman enhanced surface spectroscopy. The spectra showed peaks corresponding to the macromolecules that make up the cellular components. In addition, the various organelles were analysed by TEM and AFM to observe the cellular nanostructures. With the use of these techniques, it is possible to identify molecules in semi-thin sections, which provides a wide potential for biomedical applications and for the analysis of cell dynamics. The observation of the most detailed possible structure of cells is used as a starting point in numerous studies to identify and localise some biochemical processes. Given that the function of eukaryotic cells depends on the location, shape, structure and function of the subcellular organelles (and on the interaction between them), the sum of the data obtained allows a complete analysis of what happens in the cell. This article addresses, from a multidisciplinary point of view, what happens in a single cell of a filamentous fungus (Penicillium rubens) while it is in a physiological moment (secondary metabolism) that allows the biosynthesis of an antibiotic (penicillin). For this purpose, different types of microscopies were used (TEM: transmission electron microscopy, and AFM: atomic force microscopy, which allow visualising small details in the cell) and a spectroscopy method (Raman, which allows detecting certain characteristics of the macromolecules and some stretching bonds). Regarding the results, during the synthesis of penicillin, the antibiotic-producing Penicillium rubens cells showed significant changes compared to the non-producing cells: the cell wall is observed to be significantly thickened in the production phase, organelles such as peroxisomes grow in number and size since it is known that the final route of metabolite synthesis takes place in them. When penicillin is released from peroxisomes, they must be degraded to release the load from the cell; this is done by vacuoles, which are active and engulf peroxisomes. The newly synthesised penicillin is found within secretory vesicles that travel towards the cell membrane and both membranes fuse creating ripples. On the other hand, and given that a single cell is being studied, it is essential to increase the signal to detect biomolecules employing the Raman-SERS technique, using a silver substrate to obtain the increased signal.

Blistering1 Modulates Penicillium expansum Virulence Via Vesicle-mediated Protein Secretion.

The blue mold fungus, Penicillium expansum, is a postharvest apple pathogen that contributes to food waste by rotting fruit and by producing harmful mycotoxins (e.g. patulin). To identify genes controlling pathogen virulence, a random T-DNA insertional library was created from wild-type P. expansum strain R19. One transformant, T625, had reduced virulence in apples, blistered mycelial hyphae, and a T-DNA insertion that abolished transcription of the single copy locus in which it was inserted. The gene, Blistering1, encodes a protein with a DnaJ domain, but otherwise has little homology outside the Aspergillaceae, a family of fungi known for producing antibiotics, mycotoxins, and cheese. Because protein secretion is critical for these processes and for host infection, mass spectrometry was used to monitor proteins secreted into liquid media during fungal growth. T625 failed to secrete a set of enzymes that degrade plant cell walls, along with ones that synthesize the three final biosynthetic steps of patulin. Consequently, the culture broth of T625 had significantly reduced capacity to degrade apple tissue and contained 30 times less patulin. Quantitative mass spectrometry of 3,282 mycelial proteins revealed that T625 had altered cellular networks controlling protein processing in the endoplasmic reticulum, protein export, vesicle-mediated transport, and endocytosis. T625 also had reduced proteins controlling mRNA surveillance and RNA processing. Transmission electron microscopy of hyphal cross sections confirmed that T625 formed abnormally enlarged endosomes or vacuoles. These data reveal that Blistering1 affects internal and external protein processing involving vesicle-mediated transport in a family of fungi with medical, commercial, and agricultural importance.

Vesicular transport and secretion of penicillin G in Penicillium rubens P2-32-T.

The compartmentalization of penicillin G biosynthesis in Penicillium rubens has been extensively studied. However, how this compound is secreted has not been completely elucidated, although its transport could be of the vesicular type. This work was aimed at observing vesicles and penicillin secretion and proposing a hypothetical model for their compartmentalization and secretion. For this purpose, a high-penicillin-producing strain (P. rubens P2-32-T) was compared by transmission electron microscopy (TEM) and atomic force microscopy (AFM) with a null-producing strain (P. rubens npe10) in 24- and 48-h cultures. The results showed multivesicular bodies and secretory vesicles, suggesting that P. rubens transports and secretes penicillin G through vesicular excretion.

Pexophagy modes during penicillin biosynthesis in Penicillium rubens P2-32-T.

Pexophagy is a peroxisome degradation process. The last two steps of penicillin biosynthesis in Penicillium rubens are carried out in peroxisomes. These organelles proliferate in large numbers during this process, so that after the penicillin secretion, their removal is essential as a regulatory mechanism. In this work, two pexophagy modes are described for the high-penicillin producing strain P. rubens P2-32-T, by transmission electron microscopy (TEM) on 24- and 48-h cultures (when maximum penicillin production is achieved). The obtained images show peroxisome phagocytosis by vacuoles in three different ways: macropexophagy, micropexophagy, and a new proposed model: unipexophagy.

Antifungal activity of ß-carbolines on Penicillium digitatum and Botrytis cinerea.

ß-carbolines (ßCs) are alkaloids widely distributed in nature that have demonstrated antimicrobial properties. Here, we tested in vitro six ßCs against Penicillium digitatum and Botrytis cinerea, causal agents of postharvest diseases on fruit and vegetables. Full aromatic ßCs (harmine, harmol, norharmane and harmane) exhibited a marked inhibitory effect on conidia germination at concentrations between 0.5 and 1 mM, while dihydro-ßCs (harmalina and harmalol) only caused germination delay. Harmol showed the highest inhibitory effect on both fungal pathogens. After 24 h of exposure to 1 mM harmol, conidia revealed a severe cellular damage, exhibiting disorganized cytoplasm and thickened cell wall. Harmol antimicrobial effect was fungicidal on B. cinerea, while it was fungistatic on P. digitatum. Conidia membrane permeabilization was detected in treatments with harmol at sub-inhibitory and inhibitory concentrations, for both pathogens. In addition, residual infectivity of P. digitatum on lemons and B. cinerea on blueberries was significantly reduced after exposure to this alkaloid. It also inhibited mycelial growth, preventing sporulation at the highest concentration tested. These results indicate that harmol might be a promising candidate as a new antifungal molecule to control causal agents of fruit diseases.

Effect of octanal on the mycelial growth of Penicillium italicum and P. digitatum.

The present study investigated the antifungal activity of octanal against Penicillium italicum and P. digitatum. Results showed that octanal exhibited strong antifungal activity against the test pathogens in a dose-dependent manner. Scanning electron microscopy observation revealed that octanal obviously altered the morphology of P. italicum and P. digitatum hyphae by causing the loss of cytoplasm and distortion of mycelia. A rapid increase in the membrane permeability of P. italicum and P. digitatum was observed after treated with octanal at minimum inhibitory concentration or minimum fungicidal concentration, evidenced by the release of cell constituents, the extracellular conductivity and the extracellular potential of hydrogen. In addition, octanal apparently induced a decrease in total lipid contents of P. italicum and P. digitatum cells. These results suggested that the antifungal activity of octanal against P. italicum and P. digitatum can be attributed to the disruption of the cell membrane integrity and the leakage of cell components.

Penicillium simile sp. nov. revealed by morphological and phylogenetic analysis.

The morphology of three phenetically identical Penicillium isolates, collected from the bioaerosol in a restoration laboratory in Italy, displayed macro- and microscopic characteristics that were similar though not completely ascribable to Penicillium raistrickii. For this reason, a phylogenetic approach based on DNA sequencing analysis was performed to establish both the taxonomic status and the evolutionary relationships of these three peculiar isolates in relation to previously described species of the genus Penicillium. We used four nuclear loci (both rRNA and protein coding genes) that have previously proved useful for the molecular investigation of taxa belonging to the genus Penicillium at various evolutionary levels. The internal transcribed spacer region (ITS1-5.8S-ITS2), domains D1 and D2 of the 28S rDNA, a region of the tubulin beta chain gene (benA) and part of the calmodulin gene (cmd) were amplified by PCR and sequenced. Analysis of the rRNA genes and of the benA and cmd sequence data indicates the presence of three isogenic isolates belonging to a genetically distinct species of the genus Penicillium, here described and named Penicillium simile sp. nov. (ATCC MYA-4591(T)  = CBS 129191(T)). This novel species is phylogenetically different from P. raistrickii and other related species of the genus Penicillium (e.g. Penicillium scabrosum), from which it can be distinguished on the basis of morphological trait analysis.

Peroxisomes: flexible and dynamic organelles.

Peroxisome development is a dynamic process that may involve organelle fusion and fission events. Cells contain different types of peroxisomes that vary in protein composition and capacity to incorporate membrane and matrix proteins. The protein import machinery is highly flexible and includes a cycling receptor that passes the peroxisomal membrane.

Structural basis for substrate specificity of the peroxisomal acyl-CoA hydrolase MpaH' involved in mycophenolic acid biosynthesis.

Mycophenolic acid (MPA) is a fungal natural product and first-line immunosuppressive drug for organ transplantations and autoimmune diseases. In the compartmentalized biosynthesis of MPA, the acyl-coenzyme A (CoA) hydrolase MpaH' located in peroxisomes catalyzes the highly specific hydrolysis of MPA-CoA to produce the final product MPA. The strict substrate specificity of MpaH' not only averts undesired hydrolysis of various cellular acyl-CoAs, but also prevents MPA-CoA from further peroxisomal ß-oxidation catabolism. To elucidate the structural basis for this important property, in this study, we solve the crystal structures of the substrate-free form of MpaH' and the MpaH'S139A mutant in complex with the product MPA. The MpaH' structure reveals a canonical α/ß-hydrolase fold with an unusually large cap domain and a rare location of the acidic residue D163 of catalytic triad after strand ß6. MpaH' also forms an atypical dimer with the unique C-terminal helices α13 and α14 arming the cap domain of the other protomer and indirectly participating in the substrate binding. With these characteristics, we propose that MpaH' and its homologs form a new subfamily of α/ß hydrolase fold protein. The crystal structure of MpaH'S139A /MPA complex and the modeled structure of MpaH'/MPA-CoA, together with the structure-guided mutagenesis analysis and isothermal titration calorimetry (ITC) measurements, provide important mechanistic insights into the high substrate specificity of MpaH'.

Cellular damage induced by a sequential oxidative treatment on Penicillium digitatum.

AIM: To investigate the cellular damage on Penicillium digitatum produced by a sequential oxidative treatment (SOT), previously standardized in our laboratory, to prevent the conidia growth. Lethal SOT consists of 2-min preincubation with 10 ppm NaClO followed by 2-min incubation with 6 mmol l(-1) CuSO(4) and 100 mmol l(-1) H(2)O(2) at 25°C. METHODS AND RESULTS: After the application of lethal SOT or sublethal SOT (decreasing only the H(2)O(2) concentration), we analysed several conidia features such as germination, oxygen consumption, ultrastructure and integrity of the cellular wall and membrane. Also, we measured the production of reactive oxygen species (ROS) and the content of thiobarbituric acid-reactive species (TBARS). With the increase of H(2)O(2) concentration in the SOT, germination and oxygen consumption of conidia became inhibited, while the membrane permeability, ROS production and TBARS content of conidia increased. Several studies revealed ultrastructural disorganization in P. digitatum conidia after lethal SOT, showing severe cellular damage without apparent damage to the cell wall. In addition, mycelium of P. digitatum was more sensitive than conidia to the oxidative treatment, because growth ceased and permeability of the membranes increased after exposure of the mycelium to a SOT with only 50 mmol l(-1) H(2)O(2) compared to a SOT of 100 mmol l(-1) for these effects to occur on conidia. CONCLUSION: Our insights into cellular changes produced by the lethal SOT are consistent with the mode of action of the oxidant compounds, by producing both alteration of membrane integrity and intracellular damage. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results allow the understanding of SOT effects on P. digitatum, which will be useful to develop a reliable treatment to control postharvest diseases in view of its future application in packing houses.

Plasma membrane damage contributes to antifungal activity of silicon against Penicillium digitatum.

The antifungal activity of silicon (Si) on Penicillium digitatum, and the possible action mode involved were investigated. Spore germination, germ tube elongation, and mycelial growth of P. digitatum were strongly inhibited by Si in the form of sodium silicate. Using propidium iodide (PI) stain combined with fluorescent microscopy, it was found that the plasma membrane of Si-treated P. digitatum spores was obviously damaged, and the leakage of protein and sugar was significantly higher in Si-treated mycelia than that of control. These findings suggest that the damage on plasma membrane of P. digitatum played a crucial role in the antifungal effect of Si. Moreover, Si was effective in controlling green mold caused by P. digitatum in citrus fruit. These results have a beneficial impact on the application of Si in the control of postharvest diseases.

First morphomolecular identification of Penicillium griseofulvum and Penicillium aurantiogriseum toxicogenic isolates associated with blue mold on apple.

Postharvest blue mold decay caused by Penicillium spp. is the most important disease of fresh apple fruit in the world, which extend from the field to the store. Two new Penicillium spp. responsible for apple fruit decay were recovered. The morphological and molecular features of Penicillium griseofulvum and Penicillium aurantiogriseum isolated from apple fruits were characterized morphologically and molecularly. Pathogenicity test exhibited that both P. griseofulvum and P. aurantiogriseum were responsible for blue mold decay in storage apple fruits. Lesion diameter indicated that P. aurantiogriseum was more aggressive than P. griseofulvum. All tested isolates were able to synthesize citrinin in addition to patulin. Not all of the isolates belonging to the same species showed the same profile of secondary metabolites. Microsatellite-primed polymerase chain reaction was able to differentiate these isolates at the species level and divided the analyzed isolates into two genetically different groups. Little intraspecific variability was evident. Microsatellite-primed polymerase chain reaction analysis proved to be an objective, rapid, and reliable tool to identify Penicillium spp. involved in blue mold of apple. This is the first report of occurrence of P. griseofulvum and P. aurantiogriseum on imported apple fruits in Saudi Arabia.

Direct whole-mount imaging of fungal spores by energy-filtering transmission electron microscopy.

Whole-mount fungal spores were examined by energy-filtering transmission electron microscopy. Conidia of Penicillium species and Ustilaginoidea virens were suspended in distilled water and directly placed on a glow-discharged formvar-coated copper grid. Energy-filtered images were taken from 0 to 100eV loss regions. Due to their considerable inherent thickness, their globose morphology was evident. In zero-loss images, the fungal spores appeared to have higher contrast in general, showing darker periphery than unfiltered images. Most spores in zero-loss images exhibited almost homogeneous electron density across the spores. The contrast was partially inversed in low-loss images where more details of the outer cell wall ornamentations of spores could be discerned than zero-loss images. As obvious advantages of whole-mount spore imaging, it allows for ensuring two-dimensional images with higher spatial resolution than light microscopy and conventional scanning electron microscopy. If a higher resolution is needed to observe fungal surface structures such as fimbriae and rodlet layers, or discriminate an outer sheath enveloping spores, whole-mount spore imaging can be employed to unravel structural details.

A histidine kinase PmHHK1 regulates polar growth, sporulation and cell wall composition in the dimorphic fungus Penicillium marneffei.

Penicillium marneffei is an important opportunistic dimorphic fungal pathogen that can cause fatal systemic mycosis in AIDS patients. To find new ways of overcoming infection, candidate virulence associated genes and virulence mechanisms are under intensive investigation. In the present study, we have examined the function of a novel P. marneffei histidine kinase gene (PmHHK1) using dsRNAi mediated by Agrobacterium tumefaciens. Our results showed that reduction of PmHHK1 expression produces significant changes in morphogenesis (including polarized growth), sporulation and cell wall composition. Two-component signaling systems are widespread in the eukaryotes outside the animal kingdom, and could be potential drug targets for antifungal chemotherapy.

Ultrastructure, glutathione and low molecular weight proteins of Penicillium brevicompactum in response to cobalt.

Penicillium brevicompactum highly tolerated cobalt concentrations of 50, 200, 800 and 1000 ppm both through cell wall and intracellular sequestration- immobilization of the metal on/within the cell wall, cell wall thickness, presence of electron-dense deposits inside vacuoles (thiol peptides sequestering cobalt) and in the cytoplasm (cobalt), and presence of matrixed electron-dense deposits, only at 800 and 1000 ppm, were observed. Increased vacuole formation and plasmolysis were also observed. Fraction number 9 of the cell free extract showed maximum cobalt uptake for all the investigated cobalt concentrations. In this fraction, glutathione was only induced at 500, 800 and 1000 ppm. Maximum glutathione concentration supported maximum cobalt uptake at 800 ppm. Low molecular weight protein profiles of fraction number 9 revealed that the presence of cobalt induced the appearance of new proteins that were not detected in the same fraction of the control. These low molecular weight peptides (12-5 KDa) suggest the production of Co-metallothioneins. This is the first report of cobalt-induced glutathione by P. brevicompactum and suggests the possible production of phytochelatins.

Evaluation of chitosans and Pichia guillermondii as growth inhibitors of Penicillium digitatum.

Chitosans were obtained by room-temperature-homogeneous-deacetylation (RTHD) and freeze-pump-out-thaw-heterogeneous-deacetylation (FPT) from chitins purified from fermentations. Commercial chitosan was deacetylated by three-FPT-cycles. Chitosans and Pichia guillermondii were evaluated on the growth of Penicillium digitatum. Medium molecular weight (M(W)) chitosans displayed higher inhibitory activity against the yeast than low M(W) biopolymers. Chitosans with low degree of acetylation (DA) were inhibitory for yeast and mould. Therefore, a low M(W) and high DA chitosan was selected for use against moulds combined with yeasts. Biopolymer and yeasts presented an additive effect, since chitosans were effective to delay spore germination, whereas yeast decreased apical fungal growth.

Inactivation of Penicillum expansum in sour cherry juice, peach and apricot nectars by pulsed electric fields.

Inhibitory effects of pulsed electric fields (PEF) on Penicillum expansum inoculated into sour cherry juice, apricot and peach nectars were determined based on germination tube elongation, spore germination rate, and light and scanning electron microscopy (SEM) observations in this study. After inoculation of juice/nectar samples with P. expansum spores at the level of 10(5)-10(6)cfu/mL, the samples were processed by bench scale PEF pulse generator as a function of differing electric field strengths (0, 13, 17, 20, 23, 27, 30 and 34kV/cm) and processing times (0, 62, 94, 123, 163, 198 and 218mus). Results revealed that with an increase in electric field strength and processing time, germination tube elongation and spore germination rate were completely inhibited. Light and SEM observations revealed considerable morphological alterations in fungal conidia such as cytoplasmic coagulation, vacuolations, shrinkage and protoplast leakage. PEF processing of juice/nectars was demonstrated to be effective in inactivating P. expansum. To our knowledge, this is the first study confirming the inhibitory effects of PEF on germination tube elongation and spore germination rate of P. expansum in fruit juice/nectars.

Scanning electron microscopy and energy-dispersive X-ray microanalysis of Penicillium brevicompactum treated with cobalt.

Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) were used to study the morphology and elemental composition of the conidia, phialids and hyphae of Penicillium brevicompactum grown in the presence of cobalt concentrations of 0, 50, 200, 500, 800 and 1000 ppm (mg/l). Cobalt uptake was through the hyphae, phialids and the conidia with maximum uptake being by the conidia at a concentration of 1000 ppm. EDX revealed the increase in the percentage of calcium and magnesium in the hyphae, conidia and phialids, compared to corresponding controls, accompanying the increase in cobalt uptake. Alternatively a decrease in sulfur percentage was observed. This study might reflect the possibility of using SEM-EDX as a new technique in understanding the mechanism of tolerance.

Fungal networks shape dynamics of bacterial dispersal and community assembly in cheese rind microbiomes.

Most studies of bacterial motility have examined small-scale (micrometer-centimeter) cell dispersal in monocultures. However, bacteria live in multispecies communities, where interactions with other microbes may inhibit or facilitate dispersal. Here, we demonstrate that motile bacteria in cheese rind microbiomes use physical networks created by filamentous fungi for dispersal, and that these interactions can shape microbial community structure. Serratia proteamaculans and other motile cheese rind bacteria disperse on fungal networks by swimming in the liquid layers formed on fungal hyphae. RNA-sequencing, transposon mutagenesis, and comparative genomics identify potential genetic mechanisms, including flagella-mediated motility, that control bacterial dispersal on hyphae. By manipulating fungal networks in experimental communities, we demonstrate that fungal-mediated bacterial dispersal can shift cheese rind microbiome composition by promoting the growth of motile over non-motile community members. Our single-cell to whole-community systems approach highlights the interactive dynamics of bacterial motility in multispecies microbiomes.

Scanning Electron Microscopy Sample Preparation and Imaging.

Scanning electron microscopes allow us to reach magnifications of 20-130,000× and resolve compositional and topographical images with intense detail. These images are created by bombarding a sample with electrons in a focused manner to generate a black and white image from the electrons that bounce off of the sample. The electrons are detected using positively charged detectors. Scanning electron microscopy permits three-dimensional imaging of desiccated specimens or wet cells and tissues by using variable pressure chambers. SEM ultrastructural analysis and intracellular imaging supplement light microscopy for molecular profiling of prokaryotes, plants, and mammals. This chapter demonstrates how to prepare and image samples that are (a) desiccated and conductive, (b) desiccated and nonconductive but coated with an electron conductive film using a gold sputter coater, and