Paul J. Szaniszlo

MECHANISMS OF IRON ACQUISITION FROM SIDEROPHORES BY MICROORGANISMS AND PLANTS

Most bacteria, fungi, and some plants respond to Fe stress by the induction of high-affinity Fe transport systems that utilize biosyrthetic chelates called siderophores. To competitively acquire Fe, some microbes have transport systems that enable them to use other siderophore types in addition to their own. Bacteria such as Escherichia coli achieve this ability by using a combination of separate siderophore receptors and transporters, whereas other microbial species, such as Streptomyces pilosus, use a low specificity, high-affinity transport system that recognizes more than one siderophore type. By either strategy, such versatility may provide an advantage under Fe-limiting conditions; allowing use of siderophores produced at another organisms expense, or Fe acquisition from siderophores that could otherwise sequester Fe in an unavailable form.Plants that use microbial siderophores may also be more Fe efficient by virtue of their ability to use a variety of Fe sources under different soil conditions. Results of our research examining Fe transport by oat indicate parity in plant and microbial requirements for Fe and suggest that siderophores produced by root-colonizing microbes may provide Fe to plants that can use the predominant siderophore types. In conjunction with transport mechanisms, ecological and soil chemical factors can influence the efficacy of siderophores and phytosiderophores. A model presented here attempts to incorporate these factors to predict conditions that may govern competition for Fe in the plant rhizosphere. Possibly such competition has been a factor in the evolution of broad transport capabilities for different siderophores by microorganisms and plants.

Pathogenicity and virulence of wild-type and melanin-deficient Wangiella dermatitidis

Wild-type, dematiaceous Wangiella dermatitidis (DMD 368) and melanin-deficient mutant (Mel 3) strains derived therefrom were compared for pathogenic and virulent effects in Swiss albino mice following intravenous infection. Parameters examined were mouse survival and central nervous system signs of infection, time-course cultures of fungus from brains, lungs, livers, spleens and kidneys, and histopathology of brains. Over a range of concentrations, DMD 368 produced 100% mortality while one Mel 3 strain, DMD 369, produced no mortality by 21 days after inoculation. However, in chronic infections with DMD 369, mice developed ataxia and torticollis. These signs of disease were indistinguishable from those produced by low concentrations of DMD 368. The brain was the most severely affected organ where both DMD 368 and 369 grew exponentially. Histological responses to the two strains appeared to be indistinguishable. However, the mutant appeared not to form the invasive hyphal forms of growth associated with the acute, fatal infections caused by the wild type. Thus, although the absence of melanin was associated with decreased mortality in mice, the chronic neurological signs of mouse phaeohyphomycosis appeared to be unrelated to melanin.

Molecular Cloning and Characterization of WdPKS1, a Gene Involved in Dihydroxynaphthalene Melanin Biosynthesis and Virulence in Wangiella (Exophiala) dermatitidis

ABSTRACT 1,8-Dihydroxynaphthalene (1,8-DHN) is a fungal polyketide that contributes to virulence when polymerized to 1,8-DHN melanin in the cell walls of Wangiella dermatitidis, an agent of phaeohyphomycosis in humans. To begin a genetic analysis of the initial synthetic steps leading to 1,8-DHN melanin biosynthesis, a 772-bp PCR product was amplified from genomic DNA using primers based on conserved regions of fungal polyketide synthases (Pks) known to produce the first cyclized 1,8-DHN-melanin pathway intermediate, 1,3,6,8-tetrahydroxynaphthalene. The cloned PCR product was then used as a targeting sequence to disrupt the putative polyketide synthase gene, WdPKS1, in W. dermatitidis. The resultingwdpks1Δ disruptants showed no morphological defects other than an albino phenotype and grew at the same rate as their black wild-type parent. Using a marker rescue approach, the intactWdPKS1 gene was then successfully recovered from two plasmids. The WdPKS1 gene was also isolated independently by complementation of the mel3 mutation in an albino mutant of W. dermatitidis using a cosmid library. Sequence analysis substantiated that WdPKS1 encoded a putative polyketide synthase (WdPks1p) in a single open reading frame consisting of three exons separated by two short introns. This conclusion was supported by the identification of highly conserved Pks domains for a β-ketoacyl synthase, an acetyl-malonyl transferase, two acyl carrier proteins, and a thioesterase in the deduced amino acid sequence. Studies using a neutrophil killing assay and a mouse acute-infection model confirmed that all wdpks1Δ strains were less resistant to killing and less virulent, respectively, than their wild-type parent. Reconstitution of 1,8-DHN melanin biosynthesis in awdpks1Δ strain reestablished its resistance to killing by neutrophils and its ability to cause fatal mouse infections.

Production of Hydroxamate Siderophore Iron Chelators by Ectomycorrhizal Fungi

Numerous ectomycorrhizal fungi were surveyed for their ability to produce iron-chelating, hydroxamate siderophores (HS). Production of HS was determined by a bioassay based on the stimulation of th...

Hydroxamate siderophores in the iron nutrition of plants

Abstract Most fungi and some bacteria respond to Fe deprivation by producing high affinity, ferric‐specific Fe transport ligands called hydroxamate siderophores (HSs). These secondary hydroxamic acids have molecular weights between 500 and 1200 daltons and typically contain three 6‐N‐hydroxyornithine moieties that combine to form ferric chelates with stability constants of approximately 1030. Higher plants under iron‐stressed conditions have been reported to assimilate Fe supplied as Fe(III)‐HSs. This has been confirmed in our laboratories in preliminary studies which demonstrated the uptake and translocation of 55Fe supplied to monooot seedlings as the HS, ferrichrome. Monocot seedlings were also able to absorb and translocate a mixture of fungal HS ligands. These results suggested that the presence of HSs in the vicinity of the root would enhance Fe availability in soil systems. The presence of HSs in a variety of soils was established. Soils from 49 sites representing 22 different taxonomic subgroups w...

Root-microbial effects on plant iron uptake from siderophores and phytosiderophores

Collaborative experiments were conducted to determine whether microbial populations associated with plant roots may artifactually affect the rates of Fe uptake and translocation from microbial siderophores and phytosiderophores. Results showed nonaxenic maize to have 2 to 34-fold higher Fe-uptake rates than axenically grown plants when supplied with 1 μM Fe as either the microbial siderophore, ferrioxamine B (FOB), or the barley phytosiderophore, epi-hydroxymugineic acid (HMA). In experiments with nonsterile plants, inoculation of maize or oat seedlings with soil microorganisms and amendment of the hydroponic nutrient solutions with sucrose resulted in an 8-fold increase in FOB-mediated Fe-uptake rates by Fe-stressed maize and a 150-fold increase in FOB iron uptake rates by Fe-stressed oat, but had no effect on iron uptake by Fe-sufficient plants. Conversely, Fe-stressed maize and oat plants supplied with HMA showed decreased uptake and translocation in response to microbial inoculation and sucrose amendment. The ability of root-associated microorganisms to affect Fe-uptake rates from siderophores and phytosiderophores, even in short-term uptake experiments, indicates that microorganisms can be an unpredictable confounding factor in experiments examining mechanisms for utilization of microbial siderophores or phytosiderophores under nonsterile conditions.

Wangiella (Exophiala) dermatitidis WdChs5p, a Class V Chitin Synthase, Is Essential for Sustained Cell Growth at Temperature of Infection

ABSTRACT The chitin synthase structural gene WdCHS5 was isolated from the black fungal pathogen of humans Wangielladermatitidis. Sequence analysis revealed that the gene has a myosin motor-like-encoding region at its 5′ end and a chitin synthase (class V)-encoding region at its 3′ end. Northern blotting showed that WdCHS5 is expressed at high levels under conditions of stress. Analysis of the 5′ upstream region of WdCHS5 fused to a reporter gene indicated that one or more of the potential regulatory elements present may have contributed to the high expression levels. Disruption of WdCHS5 produced mutants that grow normally at 25°C but have severe growth and cellular abnormalities at 37°C. Osmotic stabilizers, such as sorbitol and sucrose, rescued the wild-type phenotype, which indicated that the loss of WdChs5p causes cell wall integrity defects. Animal survival tests with a mouse model of acute infection showed that all wdchs5Δ mutants are less virulent than the parental strain. Reintroduction of the WdCHS5 gene into the wdchs5Δ mutants abolished the temperature-sensitive phenotype and reestablished their virulence. We conclude that the product of WdCHS5 is required for the sustained growth of W. dermatitidis at 37°C and is of critical importance to its virulence.

Fungal transformation of naphthalene

Eighty-six species of fungi belonging to sixty-four genera were examined for their ability to metabolize naphthalene. Analysis by thin-layer and high pressure liquid chromatography revealed that naphthalene metabolism occurred in forty-seven species belonging to thirty-four genera from the major fungal taxa. All organisms tested from the order Mucorales oxidized naphthalene with species of Cunninghamella, Syncephalastrum and Mucor showing the greatest activity. Significant metabolism was also observed with Neurospora crassa, Claviceps paspali and four species of Psilocybe. The predominant metabolite formed by most organisms was 1-naphthol. Other products identified were, 4-hydroxy-1-tetralone, trans-1,2-dihydroxy-1,2-dihydronaphthalene, 2-naphthol, 1,2-and 1,4-naphthoquinone.

Pentaketide metabolites of melanin synthesis in the dematiaceous fungus Wangiella dermatitidis

Melanin synthesis in the dematiaceous, polymorphic hyphomycete Wangiella dermatitidis, a human pathogen, was investigated by biochemical and physiological techniques. Mutants with a decrease or loss in melanin synthesis were induced and isolated. Melanin precursors were obtained from the mutants, purified, and then identified by comparison with authentic compounds from Verticillium dahliae. Isolation of scytalone, vermelone, flaviolin, and 1,8-dihydroxynaphthalene from the mutants of Wangiella dermatitidis, and cross-feeding of the mutants with those of Verticillium dahliae indicated that melanin synthesis in this organism took place by the pentaketide pathway. Melanin that formed in cell walls of an albino mutant treated with scytalone was identical in appearance to that in cell walls of the wild-type strain. This also suggested that pentaketide synthesis of melanin occurred in the fungus.

Melanized and non-melanized multicellular form mutants of Wangiella dermatitidis in mice : mortality and histopathology studies

Summary. One melanized (Mc3) and one non‐melanized (Mc3W) multicellular form mutant of W. dermatitidis were compared with parental wild type in NYLAR mice. Each mutant grows as multicellular (muriform‐like) forms in vitro at 37 °C and as yeasts at ≤ 30 °C. Yeast cells of all three strains were injected intravenously at concentrations of 1 times 104, 1 times 106, 1 times 107, 3 times 107 and 1 times 108 cells/mouse in groups of 10 mice. There was no virulence difference between wild type and Mc3, with 100% mortality obtained with each strain at ≥1 times 107 cells/mouse. In contrast, Mc3W was less virulent, with mortality being obtained only at 1 times 108 cells/mouse. Histopathological study of brains, lungs, livers and spleens of moribund mice revealed that both Mc3 and Mc3W persisted in tissue as muriform cells, and in some cases as yeast, pseudohyphal and hyphal forms. There was no major difference between Mc3 and Mc3W in terms of histopathological response. These data support the association between melanin and virulence in W. dermatitidis and provide a model for the study of muriform cells in vivo.