RHO-3 plays a significant role in hyphal extension rate, conidiation, and the integrity of the Spitzenkörper in Neurospora crassa.

The Rho family of monomeric GTPases act as signaling proteins to establish and maintain cell polarity and other essential cellular processes. Rho3 is a GTPase of the Rho family that is exclusive of fungi that regulate cell polarity in yeast. However, studies have yet to explore its function in filamentous fungi. In this work, we investigated the role of RHO-3 in the model organism Neurospora crassa. Confocal microscopy analysis revealed that RHO-3 localizes in the outer region of the Spitzenkörper (Spk), in the plasma membrane from region II to the beginning of region III, and in the septa of mature hyphae. The phenotypic effect of the rho-3 deletion was analyzed. The results revealed that the rho-3 null strain showed severe defects in growth rate, aerial hyphae length, and conidia production. The organization of the Spk is also affected in the absence of RHO-3. Co-expression analysis of GFP-RHO-3 with glucan synthase 1 (GS-1-mChFP) and chitin synthase 1 (CHS-1-mChFP) revealed that RHO-3 localizes in the external region of the Spk in the macrovesicles zone. In summary, our results suggest that RHO-3 is not essential for the polarized growth of hyphae but plays a significant role in hyphal extension rate, conidiation, sexual reproduction and the integrity of the Spk, possibly regulating the delivery of macrovesicles to the apical dome.

Biosynthesis of gold nanoparticles by fungi and its potential in SERS.

Surface enhanced Raman spectroscopy (SERS) by using gold nanoparticles (AuNPs) has gained relevance for the identification of biomolecules and some cancer cells. Searching for greener NPs synthesis alternatives, we evaluated the SERS properties of AuNPs produced by using different filamentous fungi. The AuNPs were synthesized utilizing the supernatant of Botrytis cinerea, Trichoderma atroviride, Trichoderma asperellum, Alternaria sp. and Ganoderma sessile. The AuNPs were characterized by ultraviolet-visible spectroscopy (UV-Vis) to identify its characteristic surface plasmon resonance, which was located at 545 nm (B. cinerea), 550 nm (T. atroviride), 540 nm (T. asperellum), 530 nm (Alternaria sp.), and 525 nm (G. sessile). Morphology, size and crystal structure were characterized through transmission electron microscopy (TEM); colloidal stability was assessed by Z-potential measurements. We found that, under specific incubation conditions, it was possible to obtain AuNPs with spherical and quasi-spherical shapes, which mean size range depends on the fungal species supernatant with 92.9 nm (B. cinerea), 24.7 nm (T. atroviride), 16.4 nm (T. asperellum), 9.5 nm (Alternaria sp.), and 13.6 nm (G. sessile). This, as it can be expected, has an effect on Raman amplification. A micro-Raman spectroscopy system operated at a wavelength of 532 nm was used for the evaluation of the SERS features of the AuNPs. We chose methylene blue as our target molecule since it has been widely used for such a purpose in the literature. Our results show that AuNPs synthesized with the supernatant of T. atroviride, T. asperellum and Alternaria sp. produce the stronger SERS effect, with enhancement factor (EF) of 20.9, 28.8 and 35.46, respectively. These results are promising and could serve as the base line for the development of biosensors through a facile, simple, and low-cost green alternative.

The small Ras-like GTPase BUD-1 modulates conidial germination and hyphal growth guidance in the filamentous fungus Neurospora crassa.

In filamentous fungi, the hypha orientation is essential for polarized growth and morphogenesis. The ability to re-orient tip growth in response to environmental cues is critical for the colony survival. Therefore, hyphal tip orientation and tip extension are distinct mechanisms that operate in parallel during filamentous growth. In yeast, the axial growth orientation requires a pathway regulated by Rsr1p/Bud1p, a Ras-like GTPase protein, which determines the axial budding pattern. However, in filamentous fungi the function of the Rsr1/Bud1p gene (krev-1 homolog) has not been completely characterized. In this work, we characterized the phenotype of a homokaryon mutant Bud1p orthologous in Neurospora crassa (△bud-1) and tagged BUD-1 with the green fluorescent protein (GFP) to determine its localization and cell dynamics under confocal microscopy. During spore germination BUD-1 was localized at specific points along the plasma membrane and during germ tube emergence it was located at the tip of the germ tubes. In mature hyphae BUD-1 continued to be located at the cell tip and was also present at sites of branch emergence and at the time of septum formation. The △bud-1 mutant showed a delayed germination, and the orientation of hyphae was somewhat disrupted. Also, the hypha diameter was reduced approximately 37 % with respect to the wild type. The lack of BUD-1 affected the Spitzenkörper (Spk) formation, trajectory, the localization of polarisome components BNI-1 and SPA-2, and the actin cytoskeleton polarization. The results presented here suggest that BUD-1 participates in the establishment of a new polarity axis. It may also mediate the delivery of secretory vesicles for the efficient construction of new plasma membrane and cell wall.

Enzymatic characterization of agmatinase (AGM-1) from the filamentous fungus Neurospora crassa.

Agmatinase is a metallohydrolase involved in the hydrolysis of agmatine to produce urea and putrescine. Although its role in organisms is still under study, there are no reports of this family of enzymes in filamentous fungi. Recently, a protein showing agmatinase activity was reported in Neurospora crassa. Therefore, the aim of this work is to determine if the protein (AGM-1) found in the filamentous fungus N. crassa is a true agmatinase. The protein AGM-1was purified directly from N. crassa cultures, and its enzymatic characterization was carried out. The catalytic parameters such as optimum pH, thermostability, transformation kinetics, and activity in the presence of a cofactor were determined. The results show that AGM-1 can use manganese as a cofactor for its enzymatic activity, showing a transformation rate constant (kcat) of 77 s-1 and an affinity constant (KM) of 50.5 mM. The protein loses 50% of its activity when incubated 15 min at 30 °C and reaches maximal enzymatic activity at a pH range of 8-8.5. Our results indicate that the AGM-1 from N. crassa shows similar characteristics to true agmatinases already reported in other organisms. Thus, our findings strongly support that the protein annotated as hypothetical agmatinase in N. crassa is a true agmatinase.

The role of GYP-3 in cellular morphogenesis of Neurospora crassa: Analyzing its relationship with the polarisome.

In fungal hyphae multiple protein complexes assemble at sites of apical growth to maintain cell polarity. The polarisome, which in Saccharomyces cerevisiae consists of Spa2, Pea2, Bud6 and Bni1 is described as a small network of functionally related proteins that regulate polarized growth. In yeast Msb3 and Msb4 are considered polarisome components since both proteins interact directly with Spa2 and are involved in Bni1-nucleated actin assembly in vivo. Additionally they regulate exocytosis through their GAP activity towards Sec4 and perhaps other Rab GTPases. In filamentous fungi the role of these proteins has not been investigated, and in the genome of Neurospora crassa only the gene gyp-3 (NCU04514) was found to correlate with MSB3 and MSB4 of S. cerevisiae. Therefore in this work the role of GYP-3 and its relationship with the polarisome in N. crassa was analyzed. The results show that GYP-3 is required for normal colony development and cell morphology since the Δgyp-3 strain displayed a substantial reduction in colony diameter and hyphae showed a distorted morphology expressed as a general pattern of bulging areas in the distal region and hyphae were thinner at the active growing zone. The lack of GYP-3 had no effects on the localization of the polarisome components SPA-2 and BNI-1. Likewise, GYP-3 was not necessary for the normal localization of the F-actin population, however the dynamics of the Spitzenkörper (Spk) and the actin population at the apical region seemed to be destabilized. Additionally, the lack of GYP-3 strongly affects the localization and dynamics of SEC-4; which no longer accumulates at the tip of hyphae. The results presented here strongly suggest that GYP-3 is not part of the polarisome; however it requires the scaffold protein SPA-2 for arriving at the tip of hyphae. Although GYP-3 is not essential for cell survival, it has an important role in maintaining normal cell growth and morphology in N. crassa.

Role and dynamics of an agmatinase-like protein (AGM-1) in Neurospora crassa.

Agmatinase is known as a metalloenzyme which hydrolyzes agmatine to produce urea and putrescine, being crucial in the alternative pathway to produce polyamines. In this study, an agmatinase-like protein (AGM-1) (NCU 01348) in the filamentous fungus Neurospora crassa is reported. Purified AGM-1 from N. crassa displays enzymatic activity hydrolyzing agmatine; therefore, it can be considered as an agmatinase-like protein. However, its role in the alternative pathway to produce polyamines apparently is not its main function since only a slight reduction of polyamines concentration was detected in the Δagm-1 het strain. Moreover, the null mutant Δagm-1 (homokaryon strain) was unable to grow and the deficiency of agm-1 in the heterokaryon strain provoked a decrease in elongation rate, conidia and biomass production, despite of having de constitutive pathway via the ornithine decarboxylase (ODC). Additionally, mature hyphae of the Δagm-1 het strain presented unusual apical branching and a disorganized Spitzenkörper (Spk). Trying to reveal the role of AGM-1in N. crassa, the protein was tagged with GFP and interestingly the dynamics and intracellular localization of AGM-1 closely resembles the F-actin population. This finding was further examined in order to elucidate if AGM-1is in a close association with F-actin. Since polyamines, among them agmatine, have been reported to act as stabilizers of actin filaments, we evaluated in vitro G-actin polymerization in the presence of agmatine and the effect of purified AGM-1 from N. crassa on these polymerized actin filaments. It was found that polymerization of actin filaments increases in the presence of agmatine and the addition of purified AGM-1 from N. crassa depolymerizes these actin filaments. Also, it was determined that an intact substrate binding site of the enzyme is necessary for the localization pattern of the native AGM-1. These results suggest that in N. crassa AGM-1 has a close association with the F-actin population via its substrate agmatine, playing an essential role during cell development.

CDC-42 and RAC-1 regulate opposite chemotropisms in Neurospora crassa.

Cell polarization and fusion are crucial developmental processes that occur in response to intracellular and extracellular signals. Asexual spores (conidia) of the mold Neurospora crassa differentiate two types of polarized cell protrusions, germ tubes and conidial anastomosis tubes (CATs), which exhibit negative and positive chemotropism, respectively. We provide the first evidence that shared and separate functions of the Rho-type GTPases CDC-42 and RAC-1 regulate these opposite chemotropisms. We demonstrate that RAC-1 is essential for CAT formation and cell fusion, whereas CDC-42 is necessary and sufficient for normal germ tube development. Cdc42-Rac-interactive-binding (CRIB) reporters were constructed to exclusively label locally activated GTP-bound GTPases. Time course analyses showed that repositioning of these activated GTPase clusters within germ tube and CAT tip apices controls directional growth in the absence of a tip-localized vesicle supply center (Spitzenkörper). We propose a model in which the local assembly of a plasma-membrane-associated GTPase-PAK-MAPK signaling platform regulates chemoattractant perception and secretion in order to synchronize oscillatory cell-cell communication and directional CAT tip growth.

Hyperparasitism by the bacteriophage (Caudovirales) infecting Candidatus Xenohaliotis californiensis (Rickettsiales-like prokaryote) parasite of wild abalone Haliotis fulgens and Haliotis corrugata from the Peninsula of Baja California, Mexico.

Candidatus Xenohaliotis californiensis (CXc) is a Rickettsiales-like prokaryote that is considered the causal agent of Withering Syndrome (WS), a chronic disease of abalone, from the west coast of North America and it is listed by the International Organization for Animal Health (OIE) as a reportable agent due to its pathogenicity. This bacterium in red abalone Haliotis rufescens, black abalone Haliotis cracherodii, and yellow abalone Haliotis corrugata from California, US and Baja California, Mexico has been found to be infected by a bacteriophage. To date, there is no information on the epizootiology of CXc and its bacteriophage in natural populations of abalone; furthermore, it is unknown if the bacteriophage was also present in CXc infecting blue abalone Haliotis fulgens. The objective of this study was to determine the distribution, prevalence and intensity of CXc, as well as to determine the distribution and prevalence of the bacteriophage and to study interactions between host sex and hyperparasitism in blue abalone and yellow abalone. Tissue samples were obtained from seven localities where the commercial capture of wild abalone is carried out. Samplings were conducted throughout the 2012-2013 capture seasons and a total of 182 blue abalone and 170 yellow abalone were obtained. The prevalence and intensity of CXc and the prevalence of the bacteriophage were determined by histology. The identity of CXc was confirmed by PCR, product sequence analysis and in situ hybridization while the identity of the bacteriophage was corroborated by TEM. The prevalence of CXc infected and uninfected by the bacteriophage was 80% in blue abalone and 62% in yellow abalone. Low infection intensities were found in 86% of blue abalone and 82% of yellow abalone. Infection intensity was significantly higher in undifferentiated yellow abalone. The bacteriophage in CXc showed a prevalence of 22% and 31% in blue abalone and yellow abalone respectively. These results show that CXc and its bacteriophage are widely distributed in the peninsula of Baja California and that they are well established in natural populations of blue abalone and yellow abalone. Additionally, this data constitutes the first record of a bacteriophage in blue abalone.

Antimicrobial Activity of Green Synthesized Silver and Copper Oxide Nanoparticles against the Foodborne Pathogen Campylobacter jejuni.

Campylobacter jejuni is a major cause of global foodborne illnesses. To develop alternative antimicrobial strategies against C. jejuni, this study designed and optimized the green synthesis of metallic nanoparticles (NPs) with intracellular components of the medicinal fungus Ganoderma sessile to provide the needed reducing and stabilizing agents. NPs were characterized by transmission electron microscopy and dynamic light scattering, and the quasi-spherical NPs had sizes of 2.9 ± 0.9 nm for the copper oxide NPs and 14.7 ± 0.6 nm for the silver NPs. Surface charge assessment revealed zeta potentials of -21.0 ± 6.5 mV and -24.4 ± 7.9 mV for the copper oxide and silver NPs, respectively. The growth inhibition of C. jejuni by the NPs occurred through attachment to the outer cell membrane and subsequent intracellular internalization and resulted in minimum inhibitory concentrations of the silver NPs at 6 µg/mL and copper oxide NPs at 10 µg/mL. On the other hand, a differential ROS production caused by silver and copper NPs was observed. In summary, this research presents the first demonstration of using green synthesis with the medicinal fungus G. sessile to produce metallic NPs that effectively inhibit C. jejuni growth, providing a sustainable and effective approach to the traditional use of antimicrobials.

Potential application of a fungal co-culture crude extract for the conservation of post-harvest fruits.

Fungal plant pathogens are responsible for serious losses in many economically important crop species worldwide. Due to the use of fungicides and the fungi genome plasticity, multi-drug resistant strains are emerging as a new generation of pathogens, causing an expansive range of superficial and systemic plant infections, or new opportunistic fungal pathogens for humans. The group of antagonistic fungi Trichoderma spp. has been widely used to enhance plant growth and for the control of different pathogens affecting crops. Although Neurospora crassa is not a mycoparasitic fungus, its secretion of secondary metabolites with antimicrobial activity has been described. In this work, the effect of crude extract of the monoculture of Trichoderma asperellum T8a or the co-culture with N. crassa as an inhibitory treatment against the fungal pathogens Botrytis cinerea and Fusarium solani was evaluated. The findings demonstrate that the secondary metabolites contained in the T. asperellum crude extract have a clear fungistatic activity against B. cinerea and F. solani. Interestingly, this fungistatic activity highly increases when T. asperellum is co-cultivated with the non-pathogenic fungus N. crassa. Moreover, the co-culture crude extract also showed antifungal activity on post-harvest fruits, and no toxic effects on Murine fibroblast L929 (CCL-1) and murine macrophages RAW 264.7 (TIB-71) were observed. All these results together are solid evidence of the potential of the co-culture crude extract of T. asperellum and N. crassa, as an antifungal agent against phytopathogenic fungi, or post-harvest fruits during the transportation or commercialization time.

Antibacterial Activity of Biosynthesized Copper Oxide Nanoparticles (CuONPs) Using Ganoderma sessile.

Copper oxide nanoparticles (CuONPs) were synthesized using an eco-friendly method and their antimicrobial and biocompatibility properties were determined. The supernatant and extract of the fungus Ganoderma sessile yielded small, quasi-spherical NPs with an average size of 4.5 ± 1.9 nm and 5.2 ± 2.1 nm, respectively. Nanoparticles were characterized by UV-Vis spectroscopy, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS), and zeta potential analysis. CuONPs showed antimicrobial activity against Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa). The half-maximal inhibitory concentration (IC50) for E. coli was 8.5 µg/mL, for P. aeruginosa was 4.1 µg/mL, and for S. aureus was 10.2 µg/mL. The ultrastructural analysis of bacteria exposed to CuONPs revealed the presence of small CuONPs all through the bacterial cells. Finally, the toxicity of CuONPs was analyzed in three mammalian cell lines: hepatocytes (AML-12), macrophages (RAW 264.7), and kidney (MDCK). Low concentrations (<15 µg/mL) of CuONPs-E were non-toxic to kidney cells and macrophages, and the hepatocytes were the most susceptible to CuONPs-S. The results obtained suggest that the CuONPs synthesized using the extract of the fungus G. sessile could be further evaluated for the treatment of superficial infectious diseases.

Highly Antifungal Activity of Biosynthesized Copper Oxide Nanoparticles against Candida albicans.

Candida albicans (ATCC SC5314) was exposed to biosynthesized copper oxide nanoparticles (CuONPs) to determine their inhibitory capacity. Nanoparticles were polydisperse of small size (5.8 ± 3.5 nm) with irregular shape. The minimum inhibitory concentration (MIC) against C. albicans was 35.5 µg/mL. The production of reactive oxygen species (ROS) of C. albicans was verified when exposed to different concentrations of CuONPs. Ultrastructural analysis of C. albicans revealed a high concentration of CuONPs in the cytoplasm and outside the cell; also, nanoparticles were detected within the cell wall. Cytotoxic analyses using fibroblasts (L929), macrophages (RAW 264.7), and breast (MCF-12) cell lines show good results of cell viability when exposed at the MIC. Additionally, a hemocompatibility analysis was carried out and was found to be below 5%, considered the threshold for biocompatibility. Therefore, it is concluded that the biosynthesized CuONPs have a high potential for developing a topical antifungal treatment.

Optimized Synthesis of Small and Stable Silver Nanoparticles Using Intracellular and Extracellular Components of Fungi: An Alternative for Bacterial Inhibition.

Silver nanoparticles (AgNPs) represent an excellent option to solve microbial resistance problems to traditionally used antibiotics. In this work, we report optimized protocols for the production of AgNPs using extracts and supernatants of Trichoderma harzianum and Ganoderma sessile. AgNPs were characterized using UV-Vis spectroscopy and transmission electron microscopy, and the hydrodynamic diameter and Z potential were also determined. The obtained AgNPs were slightly larger using the fungal extract, and in all cases, a quasi-spherical shape was obtained. The mean sizes of AgNPs were 9.6 and 19.1 nm for T. harzianum and 5.4 and 8.9 nm for G. sessile using supernatant and extract, respectively. The AgNPs were evaluated to determine their in vitro antibacterial effect against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The minimum inhibitory concentration (MIC) was determined, and in all cases the AgNPs showed an antimicrobial effect, with a MIC varying from 1.26-5.0 µg/mL, depending on the bacterial strain and type of nanoparticle used. Cytotoxicity analyses of AgNPs were carried out using macrophages and fibroblast cell lines. It was determined that the cell viability of fibroblasts exposed for 24 h to different concentrations of AgNPs was more than 50%, even at concentrations of up to 20 µg/mL of silver. However, macrophages were more susceptible to exposure at higher concentrations of AgNPs as their viability decreased at concentrations of 10 µg/mL. The results presented here demonstrate that small AgNPs are obtained using either supernatants or extracts of both fungal strains. A remarkable result is that very low concentrations of AgNPs were necessary for bacterial inhibition. Furthermore, AgNPs were stable for more than a year, preserving their antibacterial properties. Therefore, the reported optimized protocol using fungal supernatants or extracts may be used as a fast method for synthesizing small AgNPs with high potential to use in the clinic.

Integrated omics approaches for deciphering antifungal metabolites produced by a novel Bacillus species, B. cabrialesii TE3T, against the spot blotch disease of wheat (Triticum turgidum L. subsp. durum).

Bipolaris sorokiniana is an important biotic constraint for global wheat production, causing spot blotch disease. In this work, we present a comprehensive characterization of the cell-free culture filtrate (CF) and precipitated fraction (PF) of Bacillus cabrialesii TE3T showing an effective inhibition of spot blotch. Our results indicated that CF produced by B. cabrialesii TE3T inhibits the growth of B. sorokiniana through stable metabolites (after autoclaving and proteinase K treatment). Antifungal metabolites in CF and PF were explored by an integrated genomic-metabolomic approach. Genome-mining revealed that strain TE3T contains the biosynthetic potential to produce wide spectrum antifungal (surfactin, fengycin, and rhizocticin A) and antibacterial metabolites (bacillaene, bacilysin, bacillibactin, and subtilosin A), and through bioactivity-guided LC-ESI-MS/MS approach we determined that a lipopeptide complex of surfactin and fengycin homologs was responsible for antifungal activity exhibited by B. cabrialesii TE3T against the studied phytopathogen. In addition, our results demonstrate that i) a lipopeptide complex inhibits B. sorokiniana by disrupting its cytoplasmatic membrane and ii) reduced spot blotch disease by 93 %. These findings show the potential application of metabolites produced by strain TE3T against B. sorokiniana and provide the first insight into antifungal metabolites produced by the novel Bacillus species, Bacillus cabrialesii.

Circadian rhythms in Neurospora crassa: dynamics of the clock component frequency visualized using a fluorescent reporter.

The frequency (frq) gene of Neurospora crassa has long been considered essential to the function of this organism's circadian rhythm. Increasingly, deciphering the coupling of core oscillator genes such as frq to the output pathways of the circadian rhythm has become a major focus of circadian research. To address this coupling it is critical to have a reporter of circadian activity that can deliver high resolution spatial and temporal information about the dynamics of core oscillatory proteins such as FRQ. However, due to the difficulty of studying the expression of circadian rhythm genes in aerobic N. crassa cultures, little is known about the dynamics of this gene under physiologically realistic conditions. To address these issues we report a fluorescent fusion to the frq gene using a codon optimized version of the mCherry gene. To trace the expression and accumulation of FRQ-mCherryNC (FRQ-mCh) during the circadian rhythm, growing vegetative hyphae were scanned every hour under confocal microscopy (100x). Fluorescence of FRQ-mCh was detected only at the growing edge of the colony, and located in the cytoplasm and nuclei of vegetative hyphae for a distance of approximately 150-200microm from the apices of leading hyphae. When driven by the frq promoter, apparently there was also a second FRQ entrance into the nucleus during the circadian cycle; however the second entrance had a lower accumulation level than the first entrance. Thus this fluorescent fusion protein has proven useful in tracking the spatial dynamics of the frq protein and has indicated that the dynamics of the FRQ protein's nuclear trafficking may be more complex than previously realized.

The polarisome component SPA-2 localizes at the apex of Neurospora crassa and partially colocalizes with the Spitzenkörper.

In fungal hyphae multiple protein complexes assemble at sites of apical growth to maintain cell polarity and promote nucleation of actin. Polarity allows the directional traffic of vesicles to the Spitzenkörper (Spk) prior to fusing with the plasma membrane to provide precursors and enzymes required for cell extension and nutrition. One of these complexes is the polarisome, which in Saccharomyces cerevisiae contains Spa2p, Pea2p, Bud6p/Aip3p and Bni1p. To investigate the localization and role of the polarisome during Spk establishment in Neurospora crassa we tagged SPA-2 with the green fluorescent protein (GFP) and examined growing cells by laser scanning confocal microscopy in elongating germ tubes and mature hyphae. SPA-2-GFP accumulated gradually at the apex of germ tubes, when a FM4-64 stained Spk was not still detectable. When the germlings reached about 40microm in length, a FM4-64 stained Spk started to be apparent and from this point on SPA-2-GFP was observed in the apical region of both germ tubes and mature hyphae, as a hand fan shape with a brighter spot at the base. Fusion of the N. crassa SPA-2-GFP strain with a N. crassa strain expressing chitin synthase 1 (CHS-1) labeled with mCherryFP indicated only partial colocalization of the polarisome and the Spk core. N. crassa SPA-2-GFP was also found at the apex of forming branches but not in septa, suggesting that it participates only in areas of tip growth. A Deltaspa-2 strain displayed hyphae with uneven constrictions, apices with an unstable Spk, reduced growth rate and higher number of branches than the wild type strain, indicating that SPA-2 is required for the stability, behavior and morphology of the Spk and maintenance of regular apical growth in hyphae of N. crassa, although not for polarity or Spk establishment.

Dual function of EDTA with silver nanoparticles for root canal treatment-A novel modification.

The chelating and antimicrobial capacity of a novel modification of 17% EDTA with silver nanoparticles (AgNPs) (EDTA-AgNPs) was evaluated in-vitro for root canal treatment (RCT). The EDTA-AgNPs solution was characterized by UV-Vis spectroscopy, ζ-potential and high-resolution transmission electron microscopy (HRTEM). Antimicrobial capacity was evaluated against Candida albicans and Staphylococcus aureus in planktonic and biofilm cells by broth macrodilution (24 h) and XTT assays, (1, 10 and 30 min) respectively. The chelating capacity of EDTA-AgNPs was assessed indirectly (smear layer removal) and directly (demineralizing effect) in bovine dentin at two silver concentrations, 16 and 512 µg/ml at 1 and 10 minutes of exposure time. Smear layer removal was evaluated by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The demineralizing effect was determined by atomic absorption spectroscopy (AAS), microhardness test (MH) and X-ray diffractometer (XRD). Synthesized AgNPs were quasi-spherical in shape with an average size of 13.09 ± 8.05 nm. 17% EDTA-AgNPs was effective to inhibit C. albicans and S. aureus in planktonic and biofilm cultures. The smear layer removal and demineralizing effect were similar between 17% EDTA-AgNPs and 17% EDTA treatments. The 17% EDTA-AgNPs solution proved to be an effective antimicrobial agent, and has a similar chelating capacity to 17% EDTA alone. These in-vitro studies strongly suggest that EDTA-AgNPs could be used for effective smear layer removal, having an antimicrobial effect at the same time during RCT.

Ultrastructural analysis of Candida albicans when exposed to silver nanoparticles.

Candida albicans is the most common fungal pathogen in humans, and recently some studies have reported the antifungal activity of silver nanoparticles (AgNPs) against some Candida species. However, ultrastructural analyses on the interaction of AgNPs with these microorganisms have not been reported. In this work we evaluated the effect of AgNPs on C. albicans, and the minimum inhibitory concentration (MIC) was found to have a fungicidal effect. The IC50 was also determined, and the use of AgNPs with fluconazole (FLC), a fungistatic drug, reduced cell proliferation. In order to understand how AgNPs interact with living cells, the ultrastructural distribution of AgNPs in this fungus was determined. Transmission electron microscopy (TEM) analysis revealed a high accumulation of AgNPs outside the cells but also smaller nanoparticles (NPs) localized throughout the cytoplasm. Energy dispersive spectroscopy (EDS) analysis confirmed the presence of intracellular silver. From our results it is assumed that AgNPs used in this study do not penetrate the cell, but instead release silver ions that infiltrate into the cell leading to the formation of NPs through reduction by organic compounds present in the cell wall and cytoplasm.

SERS properties of different sized and shaped gold nanoparticles biosynthesized under different environmental conditions by Neurospora crassa extract.

Surface-enhanced Raman scattering (SERS) is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces. It is known that metal nanoparticles, especially gold and silver nanoparticles, exhibit great SERS properties, which make them very attractive for the development of biosensors and biocatalysts. On the other hand, the development of ecofriendly methods for the synthesis of metallic nanostructures has become the focus of research in several countries, and many microorganisms and plants have already been used to biosynthesize metallic nanostructures. However, the majority of these are pathogenic to plants or humans. Here, we report gold nanoparticles with good SERS properties, biosynthesized by Neurospora crassa extract under different environmental conditions, increasing Raman signals up to 40 times using methylene blue as a target molecule. Incubation of tetrachloroauric acid solution with the fungal extract at 60°C and a pH value of a) 3, b) 5.5, and c) 10 resulted in the formation of gold nanoparticles of a) different shapes like triangles, hexagons, pentagons etc. in a broad size range of about 10-200 nm, b) mostly quasi-spheres with some different shapes in a main size range of 6-23 nm, and c) only quasi-spheres of 3-12 nm. Analyses included TEM, HRTEM, and EDS in order to corroborate the shape and the elemental character of the gold nanoparticles, respectively. The results presented here show that these 'green' synthesized gold nanoparticles might have potential applicability in the field of biological sensing.

Comparative live-cell imaging analyses of SPA-2, BUD-6 and BNI-1 in Neurospora crassa reveal novel features of the filamentous fungal polarisome.

A key multiprotein complex involved in regulating the actin cytoskeleton and secretory machinery required for polarized growth in fungi, is the polarisome. Recognized core constituents in budding yeast are the proteins Spa2, Pea2, Aip3/Bud6, and the key effector Bni1. Multicellular fungi display a more complex polarized morphogenesis than yeasts, suggesting that the filamentous fungal polarisome might fulfill additional functions. In this study, we compared the subcellular organization and dynamics of the putative polarisome components BUD-6 and BNI-1 with those of the bona fide polarisome marker SPA-2 at various developmental stages of Neurospora crassa. All three proteins exhibited a yeast-like polarisome configuration during polarized germ tube growth, cell fusion, septal pore plugging and tip repolarization. However, the localization patterns of all three proteins showed spatiotemporally distinct characteristics during the establishment of new polar axes, septum formation and cytokinesis, and maintained hyphal tip growth. Most notably, in vegetative hyphal tips BUD-6 accumulated as a subapical cloud excluded from the Spitzenkörper (Spk), whereas BNI-1 and SPA-2 partially colocalized with the Spk and the tip apex. Novel roles during septal plugging and cytokinesis, connected to the reinitiation of tip growth upon physical injury and conidial maturation, were identified for BUD-6 and BNI-1, respectively. Phenotypic analyses of gene deletion mutants revealed additional functions for BUD-6 and BNI-1 in cell fusion regulation, and the maintenance of Spk integrity. Considered together, our findings reveal novel polarisome-independent functions of BUD-6 and BNI-1 in Neurospora, but also suggest that all three proteins cooperate at plugged septal pores, and their complex arrangement within the apical dome of mature hypha might represent a novel aspect of filamentous fungal polarisome architecture.