Publications by authors named "Edward Sionov"

35 Publications

The Effect of Environmental pH during (Pers.:Fr.) Link Inoculation of Apple Fruits on the Host Differential Reactive Oxygen Species Metabolism.

Antioxidants (Basel) 2021 Apr 28;10(5). Epub 2021 Apr 28.

College of Food Science and Engineering, Gansu Agricultural University, Lanzhou 730070, China.

is an important postharvest pathogen, belonging to an alkalizing group of pathogens secreting ammonia during fungal growth and colonization of apple fruits. Fungal pH modulation is usually considered a factor for improving fungal gene expression, contributing to its pathogenicity. However, the effects of inoculation with spore suspensions at increasing pH levels from pH 3 up to pH 7, on the reactive oxygen species (ROS) production and scavenging capability of the apple fruits, affecting host susceptibility, indicate that the pH regulation by the pathogens also affects host response and may contribute to colonization. The present results indicate that the inoculation of spores at pH 3 caused the lowest cell membrane permeability, and reduced malondialdehyde content, NADPH oxidases activity, O and HO production in the colonized fruit. Observations of the colonized area on the 9th day after inoculation at pH 3, showed that the rate of O production and HO content was reduced by 57% and 25%, compared to their activities at pH 7. In contrast, antioxidative activities of superoxide dismutase, catalase and peroxidases of fruit tissue inoculated with spores' suspension in the presence of a solution at pH 3.0 showed their highest activity. The catalase and peroxidases activities in the colonized tissue at pH 3 were higher by almost 58% and 55.9%, respectively, on the 6th day after inoculation compared to inoculation at pH 7. The activities of key enzymes of the ascorbate-glutathione (AsA-GSH) cycle and their substrates and products by the 9th day after fruit inoculation at pH 3 showed 150%, 31%, 16%, and 110% higher activities of ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase, respectively, compared to pH 7. A similar pattern of response was also observed in the accumulation of ascorbic acid and dehydroascorbate which showed a higher accumulation at pH 3 compared to the colonization at pH 7. The present results indicate that the metabolic regulation of the pH environment by the not only modulates the fungal pathogenicity factors reported before, but it induces metabolic host changes contributing both together to fungal colonization.
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http://dx.doi.org/10.3390/antiox10050692DOI Listing
April 2021

Special Issue "Interplay between Fungal Pathogens and Harvested Crops and Fruits".

Microorganisms 2021 Mar 8;9(3). Epub 2021 Mar 8.

Department of Food Sciences, Institute of Postharvest and Food Sciences, Agricultural Research Organization-The Volcani Center, Rishon LeZion 7505101, Israel.

The interplay between fungal pathogens and harvest crops is important in determining the extent of food losses following the storage and transport of crops to consumers. The specific factors modulating the activation of colonization are of key importance to determining the initiation of fungal colonization and host losses. It is clear nowadays from the wide number of transcription studies in colonized fruits that pathogenicity in postharvest produce is not only the result of activation of fungal pathogenicity factors but is significantly contributed to fruit maturity and ripening. In this editorial summary of the Special Issue "Interplay between Fungal Pathogens and Harvested Crops and Fruits", we present a short summary of future research directions on the importance of the interplay between fruit and pathogens and nine published papers (one review and eight original research papers), covering a wide range of subjects within the mechanism of pathogenicity by postharvest pathogens, including transcriptome analysis of pathogenesis, pathogenicity factors, new antifungal compounds and food toxin occurrence by pathogens. This summary may lead the reader to understand the key factors modulating pathogenicity in fruits.
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http://dx.doi.org/10.3390/microorganisms9030553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998692PMC
March 2021

Host Factors Modulating Ochratoxin A Biosynthesis during Fruit Colonization by .

J Fungi (Basel) 2020 Dec 28;7(1). Epub 2020 Dec 28.

Institute of Postharvest and Food Sciences, The Volcani Center, Agricultural Research Organization, Rishon LeZion 7528809, Israel.

is a strong and consistent ochratoxin A (OTA) producer and considered to be the main source of this toxic metabolite in grapes and grape products such as wine, grape juice and dried vine fruit. OTA is produced under certain growth conditions and its accumulation is affected by several environmental factors, such as growth phase, substrate, temperature, water activity and pH. In this study, we examined the impact of fruit host factors on regulation and accumulation of OTA in colonized grape berries, and assessed in vitro the impact of those factors on the transcriptional levels of the key genes and global regulators contributing to fungal colonization and mycotoxin synthesis. We found that limited sugar content, low pH levels and high malic acid concentrations activated OTA biosynthesis by , both in synthetic media and during fruit colonization, through modulation of global regulator of secondary metabolism, and OTA gene cluster expression. These findings indicate that fruit host factors may have a significant impact on the capability of to produce and accumulate OTA in grapes.
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http://dx.doi.org/10.3390/jof7010010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823970PMC
December 2020

Functional roles of LaeA, polyketide synthase, and glucose oxidase in the regulation of ochratoxin A biosynthesis and virulence in Aspergillus carbonarius.

Mol Plant Pathol 2021 01 10;22(1):117-129. Epub 2020 Nov 10.

Institute of Postharvest and Food Sciences, The Volcani Center, Agricultural Research Organization, Rishon LeZion, Israel.

Aspergillus carbonarius is the major producer of ochratoxin A (OTA) among Aspergillus species, but the contribution of this secondary metabolite to fungal virulence has not been assessed. We characterized the functions and addressed the roles of three factors in the regulation of OTA synthesis and pathogenicity in A. carbonarius: LaeA, a transcriptional factor regulating the production of secondary metabolites; polyketide synthase, required for OTA biosynthesis; and glucose oxidase (GOX), regulating gluconic acid (GLA) accumulation and acidification of the host tissue during fungal growth. Deletion of laeA in A. carbonarius resulted in significantly reduced OTA production in colonized nectarines and grapes. The ∆laeA mutant was unable to efficiently acidify the colonized tissue, as a direct result of diminished GLA production, leading to attenuated virulence in infected fruit compared to the wild type (WT). The designed Acpks-knockout mutant resulted in complete inhibition of OTA production in vitro and in colonized fruit. Interestingly, physiological analysis revealed that the colonization pattern of the ∆Acpks mutant was similar to that of the WT strain, with high production of GLA in the colonized tissue, suggesting that OTA accumulation does not contribute to A. carbonarius pathogenicity. Disruption of the Acgox gene inactivated GLA production in A. carbonarius, and this mutant showed attenuated virulence in infected fruit compared to the WT strain. These data identify the global regulator LaeA and GOX as critical factors modulating A. carbonarius pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.
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http://dx.doi.org/10.1111/mpp.13013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749749PMC
January 2021

Fungi in sands of Mediterranean Sea beaches of Israel-Potential relevance to human health and well-being.

Mycoses 2020 Aug 23. Epub 2020 Aug 23.

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

Background: Sand of sea harbour bacteria that may cause enteric and other infections in humans, and are controlled by regulatory measures. Data on fungi in sea sand are scarce. Thus, an international group of mycologists was formed to explore fungal flora in sand of various waterbodies.

Objectives: The aim was to explore fungal sand contamination in beaches of the Israeli Mediterranean Sea Coast, regarding possible impact on human health in three aspects: (a) faecal contamination, as judged by presence of the human enteric fungi; (b) contamination by fungi, causing dermal infections; (c) and the presence of moulds, causing respiratory allergies and pose a risk for infection in immunocompromised individuals.

Methods: The study included sand screen of six urban beaches from north to south of the Israeli Mediterranean Coast. Sand samples were extracted by water, and the water wash was cultured and quantitated. The fungi were identified phenotypically, by MALDI-TOF MS system and ITS sequencing.

Results: The screen revealed that about 80% of the isolates were moulds and about 20% yeasts. The mould species included opportunistic pathogens and potential allergens: Aspergillus fumigatus, Fusarium and Mucorales species. Yeast isolates included Candida, Cryptococcus and Rhodotorula species.

Conclusions: (a) Fungi are contaminating Israeli Mediterranean sand beaches; (b) the contaminating fungi include various yeast and mould species; (c) some of the yeasts and mould species found in sand are known opportunistic pathogens, or respiratory allergens; (d) the data could serve as basis for initiating regulatory measures to control fungal contamination of sand for the benefit of public health.
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http://dx.doi.org/10.1111/myc.13144DOI Listing
August 2020

New Insight Into Pathogenicity and Secondary Metabolism of the Plant Pathogen Through Deletion of the Epigenetic Reader SntB.

Front Microbiol 2020 9;11:610. Epub 2020 Apr 9.

Department of Medical Microbiology and Immunology, University of Wisconsin - Madison, Madison, WI, United States.

is one of the most harmful post-harvest pathogens of pomaceous fruits and the causal agent of blue rot disease. During infection, produces the toxic secondary metabolites patulin and citrinin that can impact virulence and, further, render the fruit inedible. Several studies have shown that epigenetic machinery controls synthesis of secondary metabolites in fungi. In this regard, the epigenetic reader, SntB, has been reported to govern the production of multiple toxins in species, and impact virulence of plant pathogenic fungi. Here we show that deletion of in results in several phenotypic changes in the fungus including stunted vegetative growth, reduced conidiation, but enhanced germination rates as well as decreased virulence on Golden Delicious apples. In addition, a decrease in both patulin and citrinin biosynthesis and patulin in apples, was observed. SntB positively regulates expression of three global regulators of virulence and secondary metabolism (LaeA, CreA, and PacC) which may explain in part some of the phenotypic and virulence defects of the PeΔ strain. Lastly, results from this study revealed that the controlled environmental factors (low temperatures and high CO levels) to which is commonly exposed during fruit storage, resulted in a significant reduction of expression and consequent patulin and citrinin reduction. These data identify the epigenetic reader SntB as critical factor regulated in post-harvest pathogens under storage conditions and a potential target to control fungal colonization and decaying of stored fruit.
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http://dx.doi.org/10.3389/fmicb.2020.00610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160234PMC
April 2020

The pH-Responsive Transcription Factor PacC Governs Pathogenicity and Ochratoxin A Biosynthesis in .

Front Microbiol 2020 13;11:210. Epub 2020 Feb 13.

Institute of Postharvest and Food Sciences, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel.

Pathogenic fungi must respond effectively to changes in environmental pH for successful host colonization, virulence and toxin production. is a mycotoxigenic pathogen with the ability to colonize many plant hosts and secrete ochratoxin A (OTA). In this study, we characterized the functions and addressed the role of PacC-mediated pH signaling in pathogenicity using designed gene knockout mutant. Δ mutant displayed an acidity-mimicking phenotype, which resulted in impaired fungal growth at neutral/alkaline pH, accompanied by reduced sporulation and conidial germination compared to the wild type (WT) strain. The Δ mutant was unable to efficiently acidify the growth media as a direct result of diminished gluconic and citric acid production. Furthermore, loss of resulted in a complete inhibition of OTA production at pH 7.0. Additionally, Δ mutant exhibited attenuated virulence compared to the WT toward grapes and nectarine fruits. Reintroduction of gene into Δ mutant restored the WT phenotype. Our results demonstrate important roles of PacC of in OTA biosynthesis and in pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.
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http://dx.doi.org/10.3389/fmicb.2020.00210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031272PMC
February 2020

Synergistic Inhibition of Mycotoxigenic Fungi and Mycotoxin Production by Combination of Pomegranate Peel Extract and Azole Fungicide.

Front Microbiol 2019 20;10:1919. Epub 2019 Aug 20.

Department of Food Quality and Safety, Institute for Postharvest and Food Sciences, Agricultural Research Organization, The Volcani Center, Rishon LeZion, Israel.

Fungal plant pathogens cause considerable losses in yield and quality of field crops worldwide. In addition, under specific environmental conditions, many fungi, including such as some and spp., are further able to produce mycotoxins while colonizing their host, which accumulate in human and animal tissues, posing a serious threat to consumer health. Extensive use of azole fungicides in crop protection stimulated the emergence of acquired azole resistance in some plant and human fungal pathogens. Combination treatments, which become popular in clinical practice, offer an alternative strategy for managing potentially resistant toxigenic fungi and reducing the required dosage of specific drugs. In the current study we tested the effect of pomegranate peel extract (PPE) on the growth and toxin production of the mycotoxigenic fungi and , both alone and in combination with the azole fungicide prochloraz (PRZ). Using time-lapse microscopy and quantitative image analysis we demonstrate significant delay of conidial germination and hyphal elongation rate in both fungi following PPE treatment in combination with PRZ. Moreover, PPE treatment reduced aflatoxin production by up to 97%, while a combined treatment with sub-inhibitory doses of PPE and PRZ resulted in complete inhibition of toxin production over a 72 h treatment. These findings were supported by qRT-PCR analysis, showing down-regulation of key genes involved in the aflatoxin biosynthetic pathway under combined PPE/PRZ treatment al low concentrations. Our results provide first evidence for synergistic effects between the commercial drug PRZ and natural compound PPE. Future application of these findings may allow to reduce the required dosage of PRZ, and possibly additional azole drugs, to inhibit mycotoxigenic fungi, ultimately reducing potential concerns over exposure to high doses of these potentially harmful fungicides.
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http://dx.doi.org/10.3389/fmicb.2019.01919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6710344PMC
August 2019

Identification and Toxigenic Potential of Fungi Isolated from Peppers.

Microorganisms 2019 Aug 30;7(9). Epub 2019 Aug 30.

Department of Food Quality and Safety, Institute for Postharvest and Food Sciences, The Volcani Center, Agricultural Research Organization, Rishon LeZion 7528809, Israel.

peppers are among the most popular horticultural crops produced and consumed worldwide. This study aimed to assess the occurrence of spoilage fungi responsible for post-harvest losses in the most common varieties of peppers collected from retail markets in Nigeria and Ghana. Forty fungal isolates belonging to 7 families, 8 genera, and 17 species were identified on the basis of morphology, culture characteristics, and DNA sequencing of the internal transcribed spacer (ITS) region. spp. (42.5%), spp. (22.5%), and spp. (15%) were found to be the predominant fungal pathogens. Furthermore, potential ability of the isolated mycotoxigenic fungi to produce some major mycotoxins was analyzed using high-performance liquid chromatography (HPLC). Among the 22 isolates analyzed, 11 strains belonging to the genera of , and were found to be able to produce mycotoxins, such as aflatoxin B1, gliotoxin, deoxynivalenol, and citrinin. A better understanding of the role of fungal contaminants in pepper fruits, especially the prevalence of mycotoxigenic fungi and their associated mycotoxigenic potential, will assist in the development of management strategies to control mycotoxin contamination and to reduce toxicological risks related to pepper consumption by humans and animals.
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http://dx.doi.org/10.3390/microorganisms7090303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6780498PMC
August 2019

Pulmonary Iron Limitation Induced by Exogenous Type I IFN Protects Mice from Cryptococcus gattii Independently of T Cells.

mBio 2019 06 18;10(3). Epub 2019 Jun 18.

Molecular Microbiology Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA

causes deadly mycosis primarily in AIDS patients, whereas infects mostly non-HIV patients, even in regions with high burdens of HIV/AIDS and an established environmental presence of As HIV induces type I IFN (t1IFN), we hypothesized that t1IFN would differentially affect the outcome of and infections. Exogenous t1IFN induction using stabilized poly(I·C) (pICLC) improved murine outcomes in either cryptococcal infection. In infected mice, pICLC activity was associated with containment and classical Th1 immunity. In contrast, pICLC activity against did not require any immune factors previously associated with immunity: T, B, and NK cells, IFN-γ, and macrophages were all dispensable. Interestingly, pICLC activity depended on β-2-microglobulin, which impacts iron levels among other functions. Iron supplementation reversed pICLC activity, suggesting pICLC activity requires iron limitation. Also, pICLC induced a set of iron control proteins, some of which were directly inhibitory to cryptococcus , suggesting t1IFN regulates iron availability in the pulmonary air space fluids. Thus, exogenous induction of t1IFN significantly improves the outcome of murine infection by and but by distinct mechanisms; the effect was mediated by iron limitation, while the effect on infection was through induction of classical T-cell-dependent immunity. Together this difference in types of T-cell-dependent t1IFN immunity for different species suggests a possible mechanism by which HIV infection may select against but not and cause fatal infection in immunodeficient and immunocompetent individuals. While these fungi are sibling species, infects very few AIDS patients, while infection is an AIDS-defining illness, suggesting that the host response to HIV selects over We used a viral mimic molecule (pICLC) to stimulate the immune response, and pICLC treatment improved mouse outcomes from both species. pICLC-induced action against was due to activation of well-defined immune pathways known to deter , whereas these immune pathways were dispensable for pICLC treatment of Since these immune pathways are eventually destroyed by HIV/AIDS, our data help explain why the antiviral immune response in AIDS patients is unable to control infection but is protective against Furthermore, pICLC induced tighter control of iron in the lungs of mice, which inhibited , thus suggesting an entirely new mode of nutritional immunity activated by viral signals.
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http://dx.doi.org/10.1128/mBio.00799-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581853PMC
June 2019

Shifts in the Composition of the Microbiota of Stored Wheat Grains in Response to Fumigation.

Front Microbiol 2019 17;10:1098. Epub 2019 May 17.

Institute of Postharvest and Food Sciences, Agricultural Research Organization - The Volcani Center, Rishon LeZion, Israel.

While the wheat-associated microbiome is of major agricultural importance, little is known about the alterations in wheat grain microbial community composition during storage. Characterization of the bacterial and fungal communities in stored wheat grains revealed the impact of phosphine fumigation, one of the most effective methods to eliminate insects in stored commodities, on the composition of the wheat grain microbiome. High-throughput amplicon sequencing of the bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS) region was used to analyze the wheat grain microbiome at different times over as 6 months period of storage. Higher bacterial diversity was found across the samples during the first (immediately after harvest) and second (3 months later) time points, with a predominance of , , , and . A two-fold decrease in the number of bacterial operational taxonomic units (OTUs) was observed in wheat grains at the last time point (6 months later), following phosphine treatment. In contrast to the effect of phosphine on bacteria, it did not affect fungal diversity in stored grains. The majority of fungal sequences were assigned to , followed by , , and unidentified fungi, which were evenly distributed throughout the storage period. Alpha and beta diversity analyses were confirmed by examination of the cultured microbial taxa obtained from the stored wheat grains. Mycotoxin analysis of wheat grains collected after phosphine fumigation revealed the presence of toxins, primarily deoxynivalenol (DON). Several mycotoxigenic spp. were also detected in the same samples. Results of the present study indicate that microbiome of stored, whole wheat grains was strongly affected by phosphine fumigation, which changed the structure of the microbial community leading to shifts in species composition toward mycotoxigenic strains. A better understanding of the complex interactions within the microbial communities of stored grains will assist in the development of novel biocontrol strategies to overcome mycotoxin contamination.
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http://dx.doi.org/10.3389/fmicb.2019.01098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533538PMC
May 2019

Apple Intrinsic Factors Modulating the Global Regulator, LaeA, the Patulin Gene Cluster and Patulin Accumulation During Fruit Colonization by .

Front Plant Sci 2018 27;9:1094. Epub 2018 Jul 27.

Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel.

The mycotoxin patulin is produced in colonized tissue by 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, 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 and the patulin biosynthesis gene cluster ( genes) and patulin accumulation, suggesting a complexity of host factors contribution to patulin accumulation during colonization. To start elucidating these apple intrinsic factors, we examined their impact on and gene expression concomitant with patulin synthesis. Increasing sucrose concentrations from 15 to 175 mM repressed and 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 and gene expression, the decrease in pH from 3.5 to 2.5 reduced their expression. Also the increased and 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 .
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http://dx.doi.org/10.3389/fpls.2018.01094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6073165PMC
July 2018

Fungal attack and host defence pathways unveiled in near-avirulent interactions of Penicillium expansum creA mutants on apples.

Mol Plant Pathol 2018 12 22;19(12):2635-2650. Epub 2018 Oct 22.

Department of Medical Microbiology and Immunology, University of Wisconsin - Madison, Madison 53706, WI, USA.

Amongst the universal diseases affecting apples, blue mould caused by Penicillium expansum is a major concern, resulting in yield and quality losses as a result of the production of the mycotoxin patulin. Despite the characterization of the patulin biosynthetic gene cluster at both the molecular and chemical levels, the underlying regulation of patulin biosynthesis in P. expansum and the mechanisms of apple colonization remain largely obscure. Recent work has indicated that sucrose, a carbon catabolite repressive metabolite, is a critical factor in the regulation of patulin synthesis. Here, CreA, the global carbon catabolite regulator, was assessed for virulence both in vitro and in vivo. We showed that loss-of-function creA strains were nearly avirulent and did not produce patulin in apples. On the basis of RNA-sequencing (RNA-seq) analysis and physiological experimentation, these mutants were unable to successfully colonize apples for a multitude of potential mechanisms including, on the pathogen side, a decreased ability to produce proteolytic enzymes and to acidify the environment and impaired carbon/nitrogen metabolism and, on the host side, an increase in the oxidative defence pathways. Our study defines CreA and its downstream signalling pathways as promising targets for the development of strategies to fight against the development and virulence of this post-harvest pathogen.
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http://dx.doi.org/10.1111/mpp.12734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638163PMC
December 2018

Roles of Three Cryptococcus neoformans and Cryptococcus gattii Efflux Pump-Coding Genes in Response to Drug Treatment.

Antimicrob Agents Chemother 2018 04 27;62(4). Epub 2018 Mar 27.

Molecular Microbiology Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

and species complexes are the etiologic agents of cryptococcosis. We have deciphered the roles of three ABC transporters, Afr1, Afr2, and Mdr1, in the representative strains of the two species, H99 and R265. Deletion of in H99 and R265 drastically reduced the levels of resistance to three xenobiotics and three triazoles, suggesting that Afr1 is the major drug efflux pump in both strains. Fluconazole susceptibility was not affected when or was deleted in both strains. However, when these genes were deleted in combination with , a minor additive effect in susceptibility toward several drugs was observed. Deletion of all three genes in both strains caused further increases in susceptibility toward fluconazole and itraconazole, suggesting that Afr2 and Mdr1 augment Afr1 function in pumping these triazoles. Intracellular accumulation of Nile Red significantly increased in mutants of both strains, but rhodamine 6G accumulation increased only in the mutant of H99. Thus, the three efflux pumps play different roles in the two strains when exposed to different azoles and xenobiotics. and expression was upregulated in H99 and R265 when treated with fluconazole. However, expression was upregulated only in R265 under the same conditions. We screened a library of transcription factor mutants and identified several mutants that manifested either altered fluconazole sensitivity or an increase in the frequency of fluconazole heteroresistance. Gene expression analysis suggests that the three efflux pumps are regulated independently by different transcription factors in response to fluconazole exposure.
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http://dx.doi.org/10.1128/AAC.01751-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5913978PMC
April 2018

Rapid Detection and Identification of Mycotoxigenic Fungi and Mycotoxins in Stored Wheat Grain.

Toxins (Basel) 2017 09 25;9(10). Epub 2017 Sep 25.

Department of Food Quality and Safety, Institute for Postharvest and Food Sciences, Agricultural Research Organization, The Volcani Center, Rishon LeZion 7528809, Israel.

This study aimed to assess the occurrence of toxigenic fungi and mycotoxin contamination in stored wheat grains by using advanced molecular and analytical techniques. A multiplex polymerase chain reaction (PCR) strategy was established for rapid identification of mycotoxigenic fungi, and an improved analytical method was developed for simultaneous multi-mycotoxin determination in wheat grains by liquid chromatography-tandem mass spectrometry (LC/MS/MS) without the need for any clean-up. The optimized multiplex PCR method was highly specific in detecting fungal species containing species-specific and mycotoxin metabolic pathway genes. The method was applied for evaluation of 34 wheat grain samples collected from storage warehouses for the presence of mycotoxin-producing fungi, and a few samples were found positive for and species. Further chemical analysis revealed that 17 samples contained mycotoxins above the level of detection, but only six samples were found to be contaminated over the EU regulatory limits with at least one mycotoxin. Aflatoxin B₁, fumonisins, and deoxynivalenol were the most common toxins found in these samples. The results showed a strong correlation between the presence of mycotoxin biosynthesis genes as analyzed by multiplex PCR and mycotoxin detection by LC/MS/MS. The present findings indicate that a combined approach might provide rapid, accurate, and sensitive detection of mycotoxigenic species and mycotoxins in wheat grains.
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http://dx.doi.org/10.3390/toxins9100302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666349PMC
September 2017

Does the Host Contribute to Modulation of Mycotoxin Production by Fruit Pathogens?

Toxins (Basel) 2017 09 12;9(9). Epub 2017 Sep 12.

Department of Postharvest Science of Fresh Produce, The Volcani Center, Bet Dagan 50250, Israel.

Storage of freshly harvested fruit is a key factor in modulating their supply for several months after harvest; however, their quality can be reduced by pathogen attack. Fruit pathogens may infect their host through damaged surfaces, such as mechanical injuries occurring during growing, harvesting, and packing, leading to increased colonization as the fruit ripens. Of particular concern are fungal pathogens that not only macerate the host tissue but also secrete significant amounts of mycotoxins. Many studies have described the importance of physiological factors, including stage of fruit development, biochemical factors (ripening, C and N content), and environmental factors (humidity, temperature, water deficit) on the occurrence of mycotoxins. However, those factors usually show a correlative effect on fungal growth and mycotoxin accumulation. Recent reports have suggested that host factors can induce fungal metabolism, leading to the synthesis and accumulation of mycotoxins. This review describes the new vision of host-factor impact on the regulation of mycotoxin biosynthetic gene clusters underlying the complex regulation of mycotoxin accumulation in ripening fruit.
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http://dx.doi.org/10.3390/toxins9090280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5618213PMC
September 2017

Type I IFN Induction via Poly-ICLC Protects Mice against Cryptococcosis.

PLoS Pathog 2015 Aug 7;11(8):e1005040. Epub 2015 Aug 7.

Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, United States of America.

Cryptococcus neoformans is the most common cause of fungal meningoencephalitis in AIDS patients. Depletion of CD4 cells, such as occurs during advanced AIDS, is known to be a critical risk factor for developing cryptococcosis. However, the role of HIV-induced innate inflammation in susceptibility to cryptococcosis has not been evaluated. Thus, we sought to determine the role of Type I IFN induction in host defense against cryptococci by treatment of C. neoformans (H99) infected mice with poly-ICLC (pICLC), a dsRNA virus mimic. Unexpectedly, pICLC treatment greatly extended survival of infected mice and reduced fungal burdens in the brain. Protection from cryptococcosis by pICLC-induced Type I IFN was mediated by MDA5 rather than TLR3. PICLC treatment induced a large, rapid and sustained influx of neutrophils and Ly6Chigh monocytes into the lung while suppressing the development of eosinophilia. The pICLC-mediated protection against H99 was CD4 T cell dependent and analysis of CD4 T cell polyfunctionality showed a reduction in IL-5 producing CD4 T cells, marginal increases in Th1 cells and dramatic increases in RORγt+ Th17 cells in pICLC treated mice. Moreover, the protective effect of pICLC against H99 was diminished in IFNγ KO mice and by IL-17A neutralization with blocking mAbs. Furthermore, pICLC treatment also significantly extended survival of C. gattii infected mice with reduced fungal loads in the lungs. These data demonstrate that induction of type I IFN dramatically improves host resistance against the etiologic agents of cryptococcosis by beneficial alterations in both innate and adaptive immune responses.
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http://dx.doi.org/10.1371/journal.ppat.1005040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4529209PMC
August 2015

Recognition of seven species in the Cryptococcus gattii/Cryptococcus neoformans species complex.

Fungal Genet Biol 2015 May 23;78:16-48. Epub 2015 Feb 23.

CBS-KNAW Fungal Biodiversity Centre, Basidiomycete and Yeast Research, Utrecht, The Netherlands; Shanghai Key Laboratory of Molecular Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China; Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. Electronic address:

Phylogenetic analysis of 11 genetic loci and results from many genotyping studies revealed significant genetic diversity with the pathogenic Cryptococcus gattii/Cryptococcus neoformans species complex. Genealogical concordance, coalescence-based, and species tree approaches supported the presence of distinct and concordant lineages within the complex. Consequently, we propose to recognize the current C. neoformans var. grubii and C. neoformans var. neoformans as separate species, and five species within C. gattii. The type strain of C. neoformans CBS132 represents a serotype AD hybrid and is replaced. The newly delimited species differ in aspects of pathogenicity, prevalence for patient groups, as well as biochemical and physiological aspects, such as susceptibility to antifungals. MALDI-TOF mass spectrometry readily distinguishes the newly recognized species.
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http://dx.doi.org/10.1016/j.fgb.2015.02.009DOI Listing
May 2015

Activity, reduced toxicity, and scale-up synthesis of amphotericin B-conjugated polysaccharide.

Biomacromolecules 2014 Jun 16;15(6):2079-89. Epub 2014 May 16.

Institute of Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem , Jerusalem 91120, Israel.

Amphotericin B (AMB) arabinogalactan (AG) conjugate was synthesized by the conjugation of AMB to oxidized AG by reductive amination. The conjugate was evaluated for in vitro antifungal activity and in vivo toxicity. Optimization of the conjugation process was investigated using large batches of 100 g, which are 20 times larger than previously reported for AMB-AG conjugation. The efficacy of AMB-AG conjugates was studied as a function of reaction conditions and time, aldehyde/reducing agent mole ratio, and purification procedure. The most potent AMB-AG conjugate having low minimal inhibitory concentration (MIC) and high maximal tolerated dose (MTD) was obtained following reduction with NaBH4 at 1:2 mol ratio (AG units/NaBH4) at 25 °C for 24 h. AMB-AG conjugate prepared under these conditions demonstrated MIC of 0.5 mg/L (equiv of AMB) in Candida albicans, and an MTD of 60 mg/kg (equiv of AMB) in mice, while AMB clinical formulation (Fungizone) demonstrated high toxicity (MTD = 3 mg/kg). These findings confirm the simplicity and reproducibility of the conjugation allowing this method to be applied on larger scale production.
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http://dx.doi.org/10.1021/bm5002125DOI Listing
June 2014

Anti-Candida albicans biofilm effect of novel heterocyclic compounds.

J Antimicrob Chemother 2014 Feb 26;69(2):416-27. Epub 2013 Sep 26.

Department of Clinical Microbiology and Infectious Diseases, Hadassah-Hebrew University Medical Center, PO Box 12000, Jerusalem 91120, Israel.

Objectives: The aims of this study were to develop new anti-biofilm drugs, examine their activity against Candida albicans biofilm and investigate their structure-activity relationship and mechanism of action.

Methods: A series of thiazolidinedione and succinimide derivatives were synthesized and their ability to inhibit C. albicans biofilm formation and destroy pre-formed biofilm was tested. The biofilms' structure, metabolic activity and viability were determined by XTT assay and propidium iodide and SYTO 9 live/dead stains combined with confocal microscopic analysis. The effect of the most active compounds on cell morphology, sterol distribution and cell wall morphology and composition was then determined by specific fluorescent stains and transmission electron microscopy.

Results: Most of the compounds were active at sub-MICs. Elongation of the aliphatic side chain resulted in reduced anti-biofilm activity and the sulphur atom contributed to biofilm killing, indicating a structure-activity relationship. The compounds differed in their effects on biofilm viability, yeast-to-hyphal form transition, hyphal morphology, cell wall morphology and composition, and sterol distribution. The most effective anti-biofilm compounds were the thiazolidinedione S8H and the succinimide NA8.

Conclusions: We developed novel anti-biofilm agents that both inhibited and destroyed C. albicans biofilm. With some further development, these agents might be suitable for therapeutic purposes.
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http://dx.doi.org/10.1093/jac/dkt365DOI Listing
February 2014

Azole heteroresistance in Cryptococcus neoformans: emergence of resistant clones with chromosomal disomy in the mouse brain during fluconazole treatment.

Antimicrob Agents Chemother 2013 Oct 8;57(10):5127-30. Epub 2013 Jul 8.

Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

We have previously reported that Cryptococcus neoformans strains are innately heteroresistant to fluconazole in vitro, producing minor, highly resistant subpopulations due to adaptive formation of disomic chromosomes. Using a mouse model, we assessed the emergence of heteroresistant clones in the brain during fluconazole treatment and found that the occurrence of heteroresistant clones in vivo with chromosomal disomy is strain dependent. Interestingly, emergence of heteroresistant clones in vivo was unrelated to the strain's MIC to fluconazole.
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http://dx.doi.org/10.1128/AAC.00694-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811407PMC
October 2013

Toxicity mechanisms of amphotericin B and its neutralization by conjugation with arabinogalactan.

Antimicrob Agents Chemother 2012 Nov 20;56(11):5603-11. Epub 2012 Aug 20.

Department of Clinical Microbiology and Infectious Diseases, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.

Amphotericin B (AMB) is an effective antifungal agent. However, its therapeutic use is hampered by its toxicity, mainly due to channel formation across kidney cell membranes and the disruption of postendocytic trafficking. We previously described a safe injectable AMB-arabinogalactan (AG) conjugate with neutralized toxicity. Here we studied the mechanism of the toxicity of free AMB and its neutralization by conjugation with AG. AMB treatment of a kidney cell line modulated the trafficking of three receptors (C-X-C chemokine receptor type 4 [CXCR4], M1 receptor, and human transferrin receptor [hTfnR]) due to an increase in endosomal pH. Similar data were also obtained in yeast but with an increase in vacuolar pH and the perturbation of Hxt2-green fluorescent protein (GFP) trafficking. The conjugation of AMB with AG neutralized all elements of the toxic activity of AMB in mammalian but not in fungal cells. Based on these results, we provide an explanation of how the conjugation of AMB with AG neutralizes its toxicity in mammalian cells and add to the knowledge of the mechanism of action of free AMB in both fungal and mammalian cells.
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http://dx.doi.org/10.1128/AAC.00612-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486601PMC
November 2012

The primary target organ of Cryptococcus gattii is different from that of Cryptococcus neoformans in a murine model.

mBio 2012 8;3(3). Epub 2012 May 8.

Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, NIAID, NIH, Bethesda, Maryland, USA.

Unlabelled: Cryptococcosis is caused by the opportunistic pathogen Cryptococcus neoformans or by the primary pathogen Cryptococcus gattii. Epidemiological studies suggest that patients infected with C. gattii mainly present with pulmonary disease, while those infected with C. neoformans commonly manifest meningoencephalitis. We compared the pathogenesis of the two species using the C. neoformans H99 and C. gattii R265 strains in a murine inhalation model. C. neoformans grew faster in the brain and caused death by meningoencephalitis, while C. gattii grew faster in the lungs and caused death without producing fulminating meningoencephalitis. Despite the consistent failure to recover R265 cells from blood, a fraction of the R265 population was detected in the extrapulmonary organs, including the brain. Upon intravenous (i.v.) inoculation of 10(4) cells via the tail vein, however, C. gattii produced severe meningoencephalitis, demonstrating that C. gattii cells can efficiently cross the blood-brain barrier. Interestingly, i.v. inoculation with five cells caused brain infection in only 10% of C. gattii-infected mice, compared to 60% of mice infected with C. neoformans. In mice that had been initially inoculated via the pulmonary route and subsequently challenged intravenously, a protective effect was observed only in mice infected with C. gattii. C. neoformans cells grew 10 to 100 times faster than C. gattii cells in blood or serum collected from naive mice. The paucity of meningoencephalitis upon inhalation of C. gattii, therefore, may be partly due to an unknown factor(s) in the host's blood coupled with immune protection that reduces dissemination to the brain and fosters lung infection.

Importance: While Cryptococcus neoformans is the most common cause of fatal meningoencephalitis, especially in HIV patients, Cryptococcus gattii causes disease mainly in non-HIV patients. Clinical studies revealed that most patients infected with C. gattii VGII strains have lung infections with minimal brain involvement. Despite extensive clinicopathological studies on cryptococcosis in animal models, only a few have included C. gattii. We compared the pathogenesis of the two species in mice using an inhalation model. Similar to infection in humans, even though C. gattii can cross the blood-brain barrier, it failed to cause fatal meningoencephalitis but caused fatal lung infection. We show that growth of C. gattii in mouse blood is significantly slower than that of C. neoformans and that a secondary protective phenomenon, though weak, manifests itself only in C. gattii infection. Our study provides a model for understanding the clinicopathological differences between these two closely genetically related pathogens.
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http://dx.doi.org/10.1128/mBio.00103-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3350374PMC
August 2012

Identification of a Cryptococcus neoformans cytochrome P450 lanosterol 14α-demethylase (Erg11) residue critical for differential susceptibility between fluconazole/voriconazole and itraconazole/posaconazole.

Antimicrob Agents Chemother 2012 Mar 12;56(3):1162-9. Epub 2011 Dec 12.

Laboratory of Clinical Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

Cryptococcus neoformans strains resistant to azoles due to mutations causing alterations in the ERG11 gene, encoding lanosterol 14α-demethylase, have rarely been reported. In this study, we have characterized a C. neoformans serotype A strain that is resistant to high concentrations of fluconazole (FLC). This strain, which was isolated from an FLC-treated patient, contained five missense mutations in the ERG11 gene compared to the sequence of reference strain H99. Molecular manipulations of the ERG11 gene coupled with susceptibility to triazole revealed that a single missense mutation resulting in the replacement of tyrosine by phenylalanine at amino acid 145 was sufficient to cause the high FLC resistance of the strain. Importantly, this newly identified point mutation in the ERG11 gene of C. neoformans afforded resistance to voriconazole (VRC) but increased susceptibility to itraconazole (ITC) and posaconazole (PSC), which are structurally similar to each other but distinct from FLC/VRC. The in vitro susceptibility/resistance of the strains with or without the missense mutation was reflected in the therapeutic efficacy of FLC versus ITC in the animals infected with the strains. This study shows the importance of the Y145F alteration of Erg11 in C. neoformans for manifestation of differential susceptibility toward different triazoles. It underscores the necessity of in vitro susceptibility testing for each FLC-resistant C. neoformans clinical isolate against different groups of azoles in order to assist patient management.
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http://dx.doi.org/10.1128/AAC.05502-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294891PMC
March 2012

Prevalence of the VNIc genotype of Cryptococcus neoformans in non-HIV-associated cryptococcosis in the Republic of Korea.

FEMS Yeast Res 2010 Sep 19;10(6):769-78. Epub 2010 May 19.

National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

PCR fingerprinting and multilocus sequence typing were applied to determine the major molecular types of the Cryptococcus neoformans/Cryptococcus gattii species complex in the Republic of Korea. Of the 78 strains isolated from patients diagnosed with cryptococcosis between 1990 and 2008, 96% were C. neoformans serotype A, mating type MATalpha and molecular type VNI. The remaining 4% were C. gattii, serotype B, mating type MATalpha and either molecular type VGIIb or VGIII. Of the 62 strains with known HIV status, only 14 (22.6%) were isolated from HIV-positive patients and belonged to molecular type VNI. Remarkably, 93% of the C. neoformans isolates had identical PCR fingerprint profiles with the VNIc genotype that has been identified recently as the major genotype among C. neoformans strains in China. Most strains (81.8%) of the VNIc genotype were associated with non-HIV patients compared with strains of the non-VNIc genotype (20%) (P=0.009). Unlike the Chinese strains, a majority (60%) of the non-HIV patients infected with strains of the VNIc genotype in the Republic of Korea had serious underlying conditions, with cancer and liver disease being the most common. This study affirms VNIc to be the most prevalent genotype of C. neoformans isolated from non-HIV patients with cryptococcosis.
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http://dx.doi.org/10.1111/j.1567-1364.2010.00648.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2920376PMC
September 2010

Cryptococcus neoformans overcomes stress of azole drugs by formation of disomy in specific multiple chromosomes.

PLoS Pathog 2010 Apr 1;6(4):e1000848. Epub 2010 Apr 1.

Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, United States of America.

Cryptococcus neoformans is a haploid environmental organism and the major cause of fungal meningoencephalitis in AIDS patients. Fluconazole (FLC), a triazole, is widely used for the maintenance therapy of cryptococcosis. Heteroresistance to FLC, an adaptive mode of azole resistance, was associated with FLC therapy failure cases but the mechanism underlying the resistance was unknown. We used comparative genome hybridization and quantitative real-time PCR in order to show that C. neoformans adapts to high concentrations of FLC by duplication of multiple chromosomes. Formation of disomic chromosomes in response to FLC stress was observed in both serotype A and D strains. Strains that adapted to FLC concentrations higher than their minimal inhibitory concentration (MIC) contained disomies of chromosome 1 and stepwise exposure to even higher drug concentrations induced additional duplications of several other specific chromosomes. The number of disomic chromosomes in each resistant strain directly correlated with the concentration of FLC tolerated by each strain. Upon removal of the drug pressure, strains that had adapted to high concentrations of FLC returned to their original level of susceptibility by initially losing the extra copy of chromosome 1 followed by loss of the extra copies of the remaining disomic chromosomes. The duplication of chromosome 1 was closely associated with two of its resident genes: ERG11, the target of FLC and AFR1, the major transporter of azoles in C. neoformans. This adaptive mechanism in C. neoformans may play an important role in FLC therapy failure of cryptococcosis leading to relapse during azole maintenance therapy.
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http://dx.doi.org/10.1371/journal.ppat.1000848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2848560PMC
April 2010

Induced production of antifungal naphthoquinones in the pitchers of the carnivorous plant Nepenthes khasiana.

J Exp Bot 2010 Mar 16;61(3):911-22. Epub 2009 Dec 16.

Department of Plant Sciences, The George S Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel.

Nepenthes spp. are carnivorous plants that have developed insect capturing traps, evolved by specific modification of the leaf tips, and are able to utilize insect degradation products as nutritional precursors. A chitin-induced antifungal ability, based on the production and secretion to the trap liquid of droserone and 5-O-methyldroserone, is described here. Such specific secretion uniquely occurred when chitin injection was used as the eliciting agent and probably reflects a certain kind of defence mechanism that has been evolved for protecting the carnivory-based provision of nutritional precursors. The pitcher liquid containing droserone and 5-O-methyldroserone at 3:1 or 4:1 molar ratio, as well as the purified naphthoquinones, exerted an antifungal effect on a wide range of plant and human fungal pathogens. When tested against Candida and Aspergillus spp., the concentrations required for achieving inhibitory and fungicidal effects were significantly lower than those causing cytotoxicity in cells of the human embryonic kidney cell line, 293T. These naturally secreted 1,4-naphthoquinone derivatives, that are assumed to act via semiquinone enhancement of free radical production, may offer a new lead to develop alternative antifungal drugs with reduced selectable pressure for potentially evolved resistance.
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http://dx.doi.org/10.1093/jxb/erp359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2814117PMC
March 2010

The fatal fungal outbreak on Vancouver Island is characterized by enhanced intracellular parasitism driven by mitochondrial regulation.

Proc Natl Acad Sci U S A 2009 Aug 27;106(31):12980-5. Epub 2009 Jul 27.

School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.

In 1999, the population of Vancouver Island, Canada, began to experience an outbreak of a fatal fungal disease caused by a highly virulent lineage of Cryptococcus gattii. This organism has recently spread to the Canadian mainland and Pacific Northwest, but the molecular cause of the outbreak remains unknown. Here we show that the Vancouver Island outbreak (VIO) isolates have dramatically increased their ability to replicate within macrophages of the mammalian immune system in comparison with other C. gattii strains. We further demonstrate that such enhanced intracellular parasitism is directly linked to virulence in a murine model of cryptococcosis, suggesting that this phenotype may be the cause of the outbreak. Finally, microarray studies on 24 C. gattii strains reveals that the hypervirulence of the VIO isolates is characterized by the up-regulation of a large group of genes, many of which are encoded by mitochondrial genome or associated with mitochondrial activities. This expression profile correlates with an unusual mitochondrial morphology exhibited by the VIO strains after phagocytosis. Our data thus demonstrate that the intracellular parasitism of macrophages is a key driver of a human disease outbreak, a finding that has significant implications for a wide range of other human pathogens.
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http://dx.doi.org/10.1073/pnas.0902963106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2722359PMC
August 2009

Heteroresistance to fluconazole in Cryptococcus neoformans is intrinsic and associated with virulence.

Antimicrob Agents Chemother 2009 Jul 4;53(7):2804-15. Epub 2009 May 4.

Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

In 1999, heteroresistance to triazoles was reported in Cryptococcus neoformans strains isolated from an azole therapy failure case of cryptococcosis in an AIDS patient and in a diagnostic strain from a non-AIDS patient. In this study, we analyzed 130 strains of C. neoformans isolated from clinical and environmental sources before 1979, prior to the advent of triazoles, and 16 fluconazole (FLC)-resistant strains isolated from AIDS patients undergoing FLC maintenance therapy during 1990 to 2000. All strains isolated prior to 1979 manifested heteroresistance (subset of a population that grows in the presence of FLC) at concentrations between 4 and 64 microg/ml, and all 16 FLC-resistant AIDS isolates manifested heteroresistance at concentrations between 16 and 128 microg/ml. Upon exposure to stepwise increases in the concentration of FLC, subpopulations that could grow at higher concentrations emerged. Repeated transfer on drug-free media caused the highly resistant subpopulations to revert to the original level of heteroresistance. The reversion pattern fell into four categories based on the number of transfers required. The strains heteroresistant at > or =32 microg/ml were significantly more resistant to other xenobiotics and were also more virulent in mice than were those heteroresistant at < or =8 microg/ml. During FLC treatment of mice infected by strains with low levels of heteroresistance, subpopulations exhibiting higher levels of heteroresistance emerged after a certain period of time. The ABC transporter AFR1, known to efflux FLC, was unrelated to the heteroresistance mechanism. Our study showed that heteroresistance to azole is universal and suggests that heteroresistance contributes to relapse of cryptococcosis during azole maintenance therapy.
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http://dx.doi.org/10.1128/AAC.00295-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2704677PMC
July 2009

Synthesis and antifungal activity of a novel series of alkyldimethylamine cyanoboranes and their derivatives.

J Med Chem 2006 Aug;49(16):4879-85

Department of Natural Products and Medicinal Chemistry, School of Pharmacy, Hebrew University of Jerusalem, Israel.

A series of new amine cyanoborane derivatives were synthesized and exhibited antifungal activity. A long alkyl chain attached to the nitrogen of the amine cyanoboranes and carboxyboranes enhances antifungal activity. An enhanced activity was also obtained upon the halogenation of the amine cyanoboranes as well as in the presence of C=C double bond at the end of the N-alkyl group. The lead compounds were dimethylundecylamine cyanoborane (C11H23N(CH3)2BH2CN), 9, and its dibromo derivative dimethylundecylamine dibromocyanoborane (C11H23N(CH3)2BBr2CN), 11. The MIC values for the lead compounds against the most important human pathogenic fungi ranged from 16.25 to 32.5 micromol/L and from 10.05 to 79 micromol/L, respectively. Both compounds were found to be relatively safe in intravenous injections to mice, (MTD = 121.9 and 73.1 micromol/kg, respectively) and active against strains that are resistant to fluconazole (a conventional antifungal medicine). These data indicate their potential to become antifungal agents.
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http://dx.doi.org/10.1021/jm060476eDOI Listing
August 2006