A Developmentally Regulated Gene Cluster Involved in Conidial Pigment Biosynthesis in Aspergillus fumigatus

Aspergillus fumigatus, a filamentous fungus producing bluish-green conidia, is an important opportunistic pathogen that primarily affects immunocompromised patients. Conidial pigmentation of A. fumigatus significantly influences its virulence in a murine model. In the present study, six genes, forming a gene cluster spanning 19 kb, were identified as involved in conidial pigment biosynthesis in A. fumigatus. Northern blot analyses showed the six genes to be developmentally regulated and expressed during conidiation. The gene products of alb1 (for "albino 1"), arp1 (for "aspergillus reddish-pink 1"), and arp2 have high similarity to polyketide synthases, scytalone dehydratases, and hydroxynaphthalene reductases, respectively, found in the dihydroxynaphthalene (DHN)-melanin pathway of brown and black fungi. The abr1 gene (for "aspergillus brown 1") encodes a putative protein possessing two signatures of multicopper oxidases. The abr2 gene product has homology to the laccase encoded by the yA gene of Aspergillus nidulans. The function of ayg1 (for "aspergillus yellowish-green 1") remains unknown. Involvement of the six genes in conidial pigmentation was confirmed by the altered conidial color phenotypes that resulted from disruption of each gene in A. fumigatus. The presence of a DHN-melanin pathway in A. fumigatus was supported by the accumulation of scytalone and flaviolin in the arp1 deletant, whereas only flaviolin was accumulated in the arp2 deletants. Scytalone and flaviolin are well-known signature metabolites of the DHN-melanin pathway. Based on DNA sequence similarity, gene disruption results, and biochemical analyses, we conclude that the 19-kb DNA fragment contains a six-gene cluster which is required for conidial pigment biosynthesis in A. fumigatus. However, the presence of abr1, abr2, and ayg1 in addition to alb1, arp1, and arp2 suggests that conidial pigment biosynthesis in A. fumigatus is more complex than the known DHN-melanin pathway.     

Phagocytosis of Aspergillus fumigatus conidia by murine macrophages involves recognition by the dectin-1 beta-glucan receptor and Toll-like receptor 2

Aspergillus fumigatus is a fungal pathogen causing severe infections in immunocompromised patients. For clearance of inhaled conidia, an efficient response of the innate immune system is required. Macrophages represent the first line of defence and ingest and kill conidia. C-type lectins represent a family of receptors, which recognize pathogen-specific carbohydrates. One of them is beta1-3 glucan, a major component of the fungal cell wall. Here we provide evidence that beta1-3 glucan plays an important role for the elimination of A. fumigatus conidia. Laminarin, a soluble beta1-3 glucan and antibodies to dectin-1, a well known beta1-3 glucan receptor, significantly inhibited conidial phagocytosis. On resting conidia low amounts of surface accessible beta1-3 glucan were detected, whereas high amounts were found on small spores that appear early during germination and infection as well as on resting conidia of a pksP mutant strain. Swollen conidia also display larger quantities of beta1-3 glucan, although in an irregular spotted pattern. Resting pksP mutant conidia and swollen wild-type conidia are phagocytosed with high efficiency thereby confirming the relevance of beta1-3 glucans for conidial phagocytosis. Additionally we found that TLR2 and the adaptor protein MyD88 are required for efficient conidial phagocytosis, suggesting a link between the TLR2-mediated recognition of A. fumigatus and the phagocytic response.     

Structure and role of sialic acids on the surface of Aspergillus fumigatus conidiospores

Aspergillus fumigatus is an opportunistic fungal pathogen that causes a life-threatening invasive fungal disease (invasive aspergillosis, IA) in immunocompromised individuals. The first step of pathogenesis is thought to be the attachment of conidia to proteins in lung tissue. Previous studies in our laboratory have shown that conidia adhere to basal lamina proteins via negatively charged sugars on their surface, presumably sialic acids. Sialic acids are a family of more than 50 substituted derivatives of a nine-carbon monosaccharide, neuraminic acid. The purpose of this study was 2-fold: (1) to determine the structure of sialic acids and the glycan acceptor on A. fumigatus oligosaccharides and (2) to determine the effect on the removal of sialic acids from conidia on conidial binding to the extracellular matrix protein fibronectin and phagocytosis of conidia by cultured macrophages and type 2 pneumocytes. Surface sialic acids were removed using Micromonospora viridifaciens sialidase or using acetic acid, mild acid hydrolysis. Lectin binding studies revealed that the majority of conidial sialic acids are alpha2,6-linked to a galactose residue. High-pressure liquid chromatography of derivatized sialic acids released from conidia revealed that unsubstituted N-acetylneuraminic acid is the predominant sialic acid on the surface of conidia. Enzymatic removal of sialic acid significantly decreased the binding of conidia to fibronectin by greater than 65% when compared with sham-treated controls. In addition, removal of sialic acids decreased conidial uptake by cultured murine macrophages and Type 2 pneumocytes by 33% and 53%, respectively. Hence, sialylated molecules on A. fumigatus conidia are ligands for both professional and nonprofessional phagocytes.     

Effect of nutrition on growth and virulence of the entomopathogenic fungus Beauveria bassiana

Three isolates of the entomopathogen Beauveria bassiana along with one strain of Metarhizium anisopliae were cultured on seven media with different carbon/nitrogen (C/N) ratios. The effect of nutrition on virulence of the isolates was evaluated via measurement of colony growth, spore yield, germination speed, conidial C/N ratio and Pr1 (a serine protease) activity. 'Osmotic stress' medium produced the lowest colony growth with low numbers of conidia in all isolates. However, these conidia showed a high germination rate and virulence. However, conidial Pr1 activity was low in some isolates. In most but not in all cases conidia from 1% yeast extract, 2% peptone and low (10 : 1) C/N medium had higher Pr1 activity compared with conidia from other media. However, in some instances we could not conclude that there was a relationship among germination rate, conidial Pr1 activity and virulence. C/N ratio of conidia was statistically different among various media and fungal isolates. Conidia with lower C/N ratio generally produced lower LT(50) (lowest median lethal time) values (more virulent). Insect-passaged conidia from different media had lower C/N ratio compared with similar conidia from artificial cultures. Therefore, they should be more virulent than in vitro produced conidia. As germination rate, conidial Pr1 activity and C/N ratio are independent of host, it seems that host-related determinants such as insect cuticle and physiology and environmental conditions may influence host susceptibility and therefore fungal isolate virulence towards host insects.     

CYTOLOGICAL INVESTIGATIONS OF KABATIELLA CAULIVORA

Cole , Herbert , Jr ., and Houston B. Couch . (Penn. State U., University Park.) Cytological investigations of Kabatiella caulivora. Amer. Jour. Bot. 46(1) : 12-16. Illus. 1959.—Initial growth of K. caulivora on artificial media is characterized by budding, yeast-like, conidia, exclusive of mycelia. After 14 days growth, at 20°C., mycelial growth becomes macroscopically evident. A study of the germination behavior of a total of 5500 conidia from the initial yeast-like growth stage showed 59 to germinate by the production of mycelia, while the balance germinated by budding. Five thousand conidia of the mycelial growth type were studied in a successive, single-spore transfer series, and, in all cases, conidial germination continued to be by means of germ tubes. Conidia of both growth forms were studied by means of bright field and phase contrast microscopy, and found to be multinucleate—possessing variable nuclear numbers, ranging from 1-8 per cell. Mean nuclear number for both the mycelial and conidial types was 2.8. All nuclei of both growth types appeared to contain the same chromosome complement. The cultural variability exhibited by K. caulivora cannot be reconciled with the concept of dual phenomenon. It is suggested, rather, that the mycelial homotype probably arises as the result of a unidirectional mutation within the conidial growth form.     

Immune evasion of Moraxella catarrhalis involves ubiquitous surface protein A-dependent C3d binding

The complement system plays an important role in eliminating invading pathogens. Activation of complement results in C3b deposition (opsonization), phagocytosis, anaphylatoxin (C3a, C5a) release, and consequently cell lysis. Moraxella catarrhalis is a human respiratory pathogen commonly found in children with otitis media and in adults with chronic obstructive pulmonary disease. The species has evolved multiple complement evasion strategies, which among others involves the ubiquitous surface protein (Usp) family consisting of UspA1, A2, and A2 hybrid. In the present study, we found that the ability of M. catarrhalis to bind C3 correlated with UspA expression and that C3 binding contributed to serum resistance in a large number of clinical isolates. Recombinantly expressed UspA1 and A2 inhibit both the alternative and classical pathways, C3b deposition, and C3a generation when bound to the C3 molecule. We also revealed that the M. catarrhalis UspA-binding domain on C3b was located to C3d and that the major bacterial C3d-binding domains were within UspA1(299-452) and UspA2(165-318). The interaction with C3 was not species specific since UspA-expressing M. catarrhalis also bound mouse C3 that resulted in inhibition of the alternative pathway of mouse complement. Taken together, the binding of C3 to UspAs is an efficient strategy of Moraxella to block the activation of complement and to inhibit C3a-mediated inflammation.     

The snpA, a temperature-sensitive suppressor of npgA1, encodes the eukaryotic translation release factor, eRF1, in Aspergillus nidulans

The npgA1 mutation causes defects in the outer layer of the cell wall resulting in a colorless colony. In this study, a temperature-sensitive suppressor of npgA1 named snpA was isolated by UV mutagenesis. The suppressing mutant showed pleiotropic phenotypes in cellular structure and developmental processes when incubated at a temperature of 37 degrees C or above. At 37 degrees C, multiple germ tubes emerged from germinating conidia. Moreover, at 42 degrees C conidia germination was delayed more than 12h and hyphal growth was strongly inhibited. The suppressor allele, snpA6, is recessive and maps to the linkage group III. A gene complementing the mutation was identified employing the chromosome III-specific cosmid library. Sequencing analysis revealed that the snpA gene encodes the eukaryotic polypeptide release factor, eRF1. The snpA6 allele contains a G-A mutation resulting in SnpA(E117K), which may allow read-through of the nonsense mutation in the npgA1 allele in a similar manner to the yeast omni-potent suppressor SUP45 and SUP35.     

In vitro complement binding on cytoplasmic structures in normal human skin: immunoelectronmicroscopic studies

We have previously provided evidence that suggests that exposure of cryostat skin sections to normal human serum (NHS) results in the antibody-independent Clq binding to cytoplasmic structures of various cell types, leading to classical complement pathway activation as evidenced by cytoplasmic C3 deposition. In the present study, we have employed immunoelectronmicroscopic methods to clarify the exact nature of cytoplasmic C3 binding structures. Incubation of cryostat skin sections with NHS followed by peroxidase-labeled rabbit anti-human C3 serum (HRP-R/Hu C3) revealed that intracytoplasmic binding of C3 occurred in suprabasal keratinocytes, melanocytes, fibroblasts, smooth muscle cells, endothelial cells, pericytes, Schwann cells, and nerve axons, but not in basal keratinocytes, Langerhans cells, and other cellular constituents of the skin. C3 binding, as revealed by the deposition of HRP reaction product, was exclusively confined to intermediate-sized filaments (ISF), which can therefore be considered to represent the subcellular site for classical complement pathway activation. Under experimental conditions that do not allow classical complement pathway activation, ISF were not decorated. Our observation that ISF of ontogenetically different cell types share the capacity of complement fixation is in accordance with the recent finding that different ISF types, despite their biochemical and antigenic heterogeneity, have common alpha-helical domains and may provide a clue to the mechanism and site of interaction between complement components and ISF.     

Nonspecific Factors Involved in Attachment of Entomopathogenic Deuteromycetes to Host Insect Cuticle

The attachment of the conidia of the insect-pathogenic fungi Nomuraea rileyi, Beauveria bassiana, and Metarrhizium anisopliae to insect cuticle was mediated by strong binding forces. The attachment was passive and nonspecific in that the conidia adhered readily to both host and nonhost cuticle preparations. The hydrophobicity of the conidial wall and the insect epicuticle appeared to mediate the adhesion process. Detergents, solvents, and high-molecular-weight proteins known to neutralize hydrophobicity reduced conidial binding when added to conidium-cuticle preparations. However, these chemicals did not remove the hydrophobic components from the epicuticle or from conidial preparations. The outer surface of the conidium consists of a resilient layer of well-organized fascicles of rodlets. Intact rodlets extracted from B. bassiana conidia bound to insect cuticle and exhibited the hydrophobicity expressed by intact conidia. Both electrostatic charges and various hemagglutinin activities were also present on the conidial surface. However, competitive-inhibition studies indicated that these forces played little, if any, role in the adhesion process.     

Studies on the prolonged storage of Metarhizium anisopliae conidia: effect of growth substrate on conidial survival and virulence against mosquitoes

Metarhizium anisopliae was grown on six complex mycological media and on three types of rice at three moisture levels to determine the effect of growth substrate on conidial yield, viability, and virulence against mosquitoes immediately after spore maturation and after the storage of conidia at four different temperature-relative humidity (RH) combinations over a 1-year period. Conidial yields varied with the mycological media, but the viability and virulence of conidia against mosquitoes produced on all substrates were similar when spores were stored under the same conditions. The storage conditions were more critical to spore survival and virulence than the substrate upon which conidia were produced. The comparison of rice types for conidial production indicated that conidial yield, viability, and virulence to mosquitoes were more dependent upon the moisture level during growth and on the storage conditions that upon the rice used. The best storage conditions among those tested for the retention of both spore viability and virulence against mosquitoes were 19°C–97% RH and 4°C–0% RH.     

Ulex europaeus agglutinin II (UEA-II) is a novel, potent inhibitor of complement activation

Complement is an important mediator of vascular injury following oxidative stress. We recently demonstrated that complement activation following endothelial oxidative stress is mediated by mannose-binding lectin (MBL) and activation of the lectin complement pathway. Here, we investigated whether nine plant lectins which have a binding profile similar to that of MBL competitively inhibit MBL deposition and subsequent complement activation following human umbilical vein endothelial cell (HUVEC) oxidative stress. HUVEC oxidative stress (1% O(2), 24 hr) significantly increased Ulex europaeus agglutinin II (UEA-II) binding by 72 +/- 9% compared to normoxic cells. UEA-II inhibited MBL binding to HUVEC in a concentration-dependent manner following oxidative stress. Further, MBL inhibited UEA-II binding to HUVEC in a concentration-dependent manner following oxidative stress, suggesting a common ligand. UEA-II (< or = 100 micromol/L) did not attenuate the hemolytic activity, nor did it inhibit C3a des Arg formation from alternative or classical complement pathway-specific hemolytic assays. C3 deposition (measured by ELISA) following HUVEC oxidative stress was inhibited by UEA-II in a concentration-dependent manner (IC(50) = 10 pmol/L). UEA-II inhibited C3 and MBL co-localization (confocal microscopy) in a concentration-dependent manner on HUVEC following oxidative stress (IC(50) approximately 1 pmol/L). Finally, UEA-II significantly inhibited complement-dependent neutrophil chemotaxis, but failed to inhibit fMLP-mediated chemotaxis, following endothelial oxidative stress. These data demonstrate that UEA-II is a novel, potent inhibitor of human MBL deposition and complement activation following human endothelial oxidative stress.     

Human pentraxin 3 binds to the complement regulator c4b-binding protein

The long pentraxin 3 (PTX3) is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP). A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.     

The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.     

Antifungal activity of the local complement system in cerebral aspergillosis

Dissemination of aspergillosis into the central nervous system is associated with nearly 100% mortality. To study the reasons for the antifungal immune failure we analyzed the efficacy of cerebral complement to combat the fungus Aspergillus. Incubation of Aspergillus in non-inflammatory cerebrospinal fluid (CSF) revealed that complement levels were sufficient to obtain a deposition on the surface, but opsonization was much weaker than in serum. Consequently complement deposition from normal CSF on fungal surface stimulated a very low phagocytic activity of microglia, granulocytes, monocytes and macrophages compared to stimulation by conidia opsonized in serum. Similarly, opsonization of Aspergillus by CSF was not sufficient to induce an oxidative burst in infiltrating granulocytes, whereas conidia opsonized in serum induced a clear respiratory signal. Thus, granulocytes were capable of considerably reducing the viability of serum-opsonized Aspergillus conidia, but not of conidia opsonized in CSF. The limited efficacy of antifungal attack by cerebral complement can be partly compensated by enhanced synthesis, leading to elevated complement concentrations in CSF derived from a patient with cerebral aspergillosis. This inflammatory CSF was able to induce (i) a higher complement deposition on the Aspergillus surface than non-inflammatory CSF, (ii) an accumulation of complement activation products and (iii) an increase in phagocytic and killing activity of infiltrating granulocytes. However, levels and efficacy of the serum-derived complement were not reached. These data indicate that low local complement synthesis and activation may represent a central reason for the insufficient antifungal defense in the brain and the high mortality rate of cerebral aspergillosis.     

Powdery mildew of tobacco (Erysiphe cichoracearum DC.): Some effects of methods of inoculation and air humidity on germination of conidia and growth of hyphae on leaves*

Germination of conidia of Erysiphe cichoracearum and hyphal growth in 3 days at 23° C. and 6 mb. saturation deficit was similar, whether conidia were applied dry or suspended for up to 1 hr. in glass-distilled water before being sprayed on tobacco leaves. Growth of single conidial colonies whose hyphae were more than 333 μ long, was positively correlated with numbers per cm.² of leaf (P < 0.01) on three of the eleven times tested, mostly when conidia were sparsely distributed; ten of the regressions were positive. A greater percentage of conidia germinated at 0–1.7 mb. saturation deficit than at 7–9 mb. Subsequent growth of hyphae was greater in the drier air.     

Culture of Metarhizium robertsii on salicylic-acid supplemented medium induces increased conidial thermotolerance

Salicylic acid (SA), a cell-signaling metabolite in plants, is involved in resistance of plants to pathogens and environmental stresses; however, there is little information available on the responses of fungi to SA. Conidia of Metarhizium robertsii (ARSEF 2575) (Hypocreales: Clavicipitaceae) were produced on potato dextrose agar medium plus yeast extract (PDAY) supplemented with 1, 2, 4, or 8 mM SA (pH adjusted to 6.9) and incubated under constant-dark conditions. Then the tolerance of conidia against wet heat (45 °C, 3 h) and UV-B radiation (7.0 kJ m(-2)) was tested. For comparison, conidia were also produced on minimal medium (MM) that contained no carbon source (carbon starvation), a condition known to induce elevated conidial tolerance to heat and UV-B radiation in M. robertsii. The heat tolerance of conidia produced on PDAY containing 1, 2, or 4 mM SA were two-fold higher than that of conidia produced on PDAY alone; which is the same level of thermotolerance induced by growth on MM. Conidia produced on PDAY with 8 mM SA, however, did not exhibit increased heat tolerance. Growth on PDAY + SA did not increase conidial UV-B tolerance at any of the SA concentrations tested. The conidial yields of M. robertsii produced on PDAY with all levels of SA were somewhat reduced in comparison to the yield on PDAY alone. Nevertheless, conidial yields on PDAY + SA were 20-40 times greater than that obtained on MM alone. In conclusion, M. robertsii conidia produced on PDAY medium containing low concentrations of SA demonstrated increased tolerance to heat, but not to UV-B radiation. In comparison to PDAY alone, SA-amended PDAY afforded somewhat reduced conidial yields; however, in a mass-production situation, yield reductions would be offset by the fact that the conidia obtained would have relatively high heat tolerance.     

A tritrophic effect of host plant on susceptibility of western flower thrips to the entomopathogenic fungus Beauveria bassiana

Adult female western flower thrips (Frankliniella occidentalis) were exposed 12-24h to bean (Phaseolus vulgaris) and impatiens (Impatiens wallerana) leaf disks treated with Beauveria bassiana conidia and then transferred to clean bean or impatiens at various times post-treatment. Significantly greater levels of fungal infection were observed when thrips were treated on bean versus impatiens, but exposure to impatiens following treatment had no effect on fungal infection (percent mortality). This result, combined with observations of no inhibition of germination of conidia exposed to intact or macerated impatiens foliage, indicated that the negative effect of the impatiens host plant was not due to plant chemical compounds (antibiosis). Further observations revealed that insects acquired (picked-up) 75% more conidia from treated bean disks than from treated impatiens disks. This difference in dose acquisition was determined to account for the observed difference in percent mortality (15%) following treatment on the two host plants. Median lethal doses (LD(50)) of B. bassiana were not significantly different on the two host plants, but median lethal concentrations were nearly 7-fold greater on impatiens. This difference was explained by disproportionate rates of conidial acquisition at measured rates of conidial deposition (an inverse relationship was observed between application rate expressed as conidia/mm(2) and the number of conidia acquired). The mechanism underlying the differential rates of conidial acquisition from bean versus impatiens was not determined.