Inhalation of Stachybotrys chartarum Fragments Induces Pulmonary Arterial Remodeling.

Stachybotrys chartarum is a fungal contaminant within the built environment and a respiratory health concern in the United States. The objective of this study was to characterize the mechanisms influencing pulmonary immune responses to repeatedly inhaled S. chartarum. Groups of B6C3F1/N mice repeatedly inhaled viable trichothecene-producing S. chartarum conidia (strain A or strain B), heat-inactivated conidia, or high-efficiency particulate absolute-filtered air twice per week for 4 and 13 weeks. Strain A was found to produce higher amounts of respirable fragments than strain B. Lung tissue, serum, and BAL fluid were collected at 24 and 48 hours after final exposure and processed for histology, flow cytometry, and RNA and proteomic analyses. At 4 weeks after exposure, a T-helper cell type 2-mediated response was observed. After 13 weeks, a mixed T-cell response was observed after exposure to strain A compared with a T-helper cell type 2-mediated response after strain B exposure. After exposure, both strains induced pulmonary arterial remodeling at 13 weeks; however, strain A-exposed mice progressed more quickly than strain B-exposed mice. BAL fluid was composed primarily of eosinophils, neutrophils, and macrophages. Both the immune response and the observed pulmonary arterial remodeling were supported by specific cellular, molecular, and proteomic profiles. The immunopathological responses occurred earlier in mice exposed to high fragment-producing strain A. The rather striking induction of pulmonary remodeling by S. chartarum appears to be related to the presence of fungal fragments during exposure.

Suppression subtractive hybridization and comparative expression of a pore-forming toxin and glycosyl hydrolase genes in Rhizoctonia solani during potato sprout infection.

Rhizoctonia solani is a plant pathogenic fungus that causes black scurf on tubers and stem and stolon canker on underground parts of potato plant. Early in the season, the fungus attacks germinating sprouts underground before they emerge from the soil. Damage at this stage results in delayed emergence of weakened plants with poor and uneven stands. The mechanism underlying this phenomenon has been investigated in this study by coupling a cDNA-suppression subtractive hybridization (SSH) library to differential screening to identify transcripts of R. solani that are down-regulated during infection of potato sprouts. We report on the identification of 33 unique genes with functions related to carbohydrate binding, vitamin synthesis, pathogenicity, translation, ATP and nucleic acid binding and other categories. RACE-PCR was used to clone and characterize the first full-length cDNA clones, RSENDO1 and RSGLYC1 that encode for an eukaryotic delta-endotoxin CytB protein and an intracellular glycosyl hydrolase, respectively. Quantitative real-time PCR revealed the down-regulation of RSENDO1 during infection of potato sprouts and the up-regulation of RSGLYC1 when the fungus was grown on a cellulose-based nutrient medium. In contrast, additional experiments have highlighted the down-regulation of RSENDO1 when R. solani was co-cultured with the mycoparasite Stachybotrys elegans and the bacterial antagonist Bacillus subtilis B26. These results advance our understanding of R. solani-potato interaction in subterranean parts of the plant. Such approaches could be considered in building an efficient integrated potato disease management program.

Characterization and transcriptional regulation of Stachybotrys elegans mitogen-activated-protein kinase gene smkA following mycoparasitism and starvation conditions.

Mitogen-activated protein kinase (MAPK) signaling pathways play an important role in the development and conidiation of fungal pathogens on their hosts and the sensing of host-derived cues. Mycoparasitism is a fungus-fungus interaction comprising host-pathogen cross talk. Until now, only little information is available on the role of the MAPK signaling pathway during this interaction. Here, we report on the differential expression of a MAPK/ERK gene in the mycoparasite Stachybotrys elegans in response to direct parasitism of different vegetative structures of the plant pathogen Rhizoctonia solani (i.e., carbon-rich condition) and to nutrient starvation (i.e., carbon-poor condition). Western blot analysis against ERK1/2 highlighted an increase in their phosphorylated forms when S. elegans was grown under starvation condition compared to that detected in response to mycoparasitism. A higher abundance of phosphorylated ERK1/2 at the third day of interaction compared to that estimated under starvation condition was detected applying LC-MS/MS. At the transcriptional level, smkA, a YERK1 class member, was significantly induced in response to hyphal parasitism compared to parasitized sclerotia at 3, 4, and 5 days of interaction. However, under starvation condition, smkA levels were significantly induced after 7 days of growth. Southern blot analysis revealed that smkA is member of a small gene family. Collectively, these results suggest that smkA could be implicated in the mycoparasitic process in S. elegans as well as in stress-activated pathways. These results may be of wider significance in other fungus-fungus interactions.

[Interstitial lung disease due to domestic moulds]. / Pneumopathie interstitielle diffuse secondaire aux moisissures domestiques.

Identifying the role of fungi present in the domestic environment in the development of interstitial pneumonia can be a difficult clinical problem. We report a case of interstitial lung disease case occurring in a 53-year-old patient. He presented with profound hypoxemia (PaO(2) 54mmHg). Chest CT showed diffuse ground glass opacities. Initial blood tests for allergy and autoimmune disease were negative. Faced with a worsening of his clinical status after returning home he was hospitalized several times. At fibreoptic bronchoscopy, multiple white deposits were observed. Bronchoalveolar lavage with differential cell count was performed, revealing a 23% lymphocytosis. Serology for specific household molds showed moderate reaction to various molds found in homes, especially Stachybotrys chartarum. Pulmonary function tests revealed a moderate restrictive pattern with impaired diffusion of carbon monoxide and a bronchiolocentric interstitial pneumonia was found at lung biopsy. After a permanent move to a new residence, clinical parameters, radiological, biological and functional normalized. The final diagnosis was interstitial lung disease related to mycotoxins of S. Chartarum. The diagnosis of hypersensitivity pneumonitis to domestic mold or interstitial lung disease secondary to mycotoxins should be considered in patients presenting with interstitial pneumonia and requires specific investigations to ensure that an environmental cause with an allergic or toxic role is not missed.

Expression of genes of Rhizoctonia solani and the biocontrol Stachybotrys elegans during mycoparasitism of hyphae and sclerotia.

Knowledge of mycoparasitism has been focused on how antagonists affect pathogens in relation to mechanisms, metabolites and gene expression. Just as microbial antagonists use a diverse arsenal of mechanisms to dominate interactions with hosts, hosts also have diverse responses to counteract antagonism. In this study differential gene expression of eight mycoparasitism-induced genes and eight host-response genes was monitored during in vivo interactions between the mycoparasite Stachybotrys elegans and hyphae and sclerotia of the host, Rhizoctonia solani over 5 d of interaction. Using real time reverse transcription polymerase chain reaction, comparative analyses demonstrated that hyphal and sclerotial structures triggered different expression patterns. These results indicated that multiple regulatory mechanisms might be involved. The high elevated expression of some genes belonging to the mycoparasite and the host suggest that these genes play an important role during the mycoparasitic process and host defense respectively.

Pulmonary responses to Stachybotrys chartarum and its toxins: mouse strain affects clearance and macrophage cytotoxicity.

We investigated differences in the pulmonary and systemic clearance of Stachybotrys chartarum spores in two strains of mice, BALB/c and C57BL/6J. To evaluate clearance, mice were intratracheally instilled with a suspension of radiolabeled S. chartarum spores or with unlabeled spores. The lungs of C57BL/6J mice showed more rapid spore clearance than the lungs of BALB/c mice, which correlated with increased levels of spore-associated radioactivity in the GI tracts of C57BL/6J as compared with BALB/c mice. To identify mechanisms responsible for mouse strain differences in spore clearance and previously described lung inflammatory responses, we exposed alveolar macrophages (AMs) lavaged from BALB/c and C57BL/6J mice to S. chartarum spores, S. chartarum spore toxin (SST), and satratoxin G (SG) in vitro. The S. chartarum spores were found to be highly toxic with most cells from either mouse strain being killed within 24 h when exposed to a spore:cell ratio of 1:75. The spores were more lethal to AMs from C57BL/6J than those from BALB/c mice. In mice, the SST elicited many of the same inflammatory responses as the spores in vivo, including AM recruitment, pulmonary hemorrhage, and cytokine production. Our data suggest that differences in pulmonary spore clearance may contribute to the differences in pulmonary responses to S. chartarum between BALB/c and C57BL/6J mice. Enhanced AM survival and subsequent macrophage-mediated inflammation may also contribute to the higher susceptibility of BALB/c mice to S. chartarum pulmonary effects. Analogous genetic differences among humans may contribute to reported variable sensitivity to S. chartarum.

Molds, mycotoxins, and sick building syndrome.

The following is a review of some of the work we have done since 2004 regarding the importance of molds and their mycotoxins in the phenomenon of sick building syndrome (SBS). In these studies we showed that the macrocyclic trichothecene mycotoxins (MTM) of Stachybotrys chartarum (SC) are easily dissociated from the surface of the organism as it grows and could therefore be consequently spread in buildings as the fungus experiences additional water events. We then showed that SC and Penicillium chrysogenum (PC) colonies remain viable long after a water source has been removed, and the MTM produced by SC remain toxic over extended periods of time. We next showed that PC when inhaled, can release in vivo, a protease allergen that can cause a significant allergic inflammatory reaction in the lungs of mice. We then showed, in a laboratory study, that the MTM of SC can become airborne attached to spores or SC particulates smaller than spores. Following that study, we next showed that the same phenomenon actually occurred in SC infested buildings where people were complaining of health problems potentially associated with SBS. Finally, we were able to demonstrate the presence of MTM in the sera of individuals who had been exposed to SC in indoor environments. This last study was done with enough mold exposed individuals to allow for the statistical significance of SC exposure to be evaluated.

Mycoparasitism of endophytic fungi isolated from reed on soilborne phytopathogenic fungi and production of cell wall-degrading enzymes in vitro.

Antagonism of three endophytic fungi isolated from common reed (Phragmites australis) against eight soilborne pathogenic fungi was investigated on potato dextrose agar by light microscopy, scanning electron microscopy, and transmission electron microscopy. Inhibitory zones were not observed. The microscopical studies suggested that the endophytes inhibit growth of soilborne pathogens by means of coiling around hyphae and, after penetration, the degradation of hyphal cytoplasm. Since penetration of hyphae seems to play a major role in parasitism, we studied the production of cell wall degrading enzymes by the three endophytes. Choiromyces aboriginum produced higher activities of beta-1,3-glucanases compared to Stachybotrys elegans and Cylindrocarpon sp. For C. aboriginum and S. elegans, colloidal chitin was the best substrate for the induction of beta-1,3-glucanases and chitinases, respectively. This result suggests that mycoparasitism by endophytes on soilborne plant pathogens can be explained by their mycoparasitic activity.

Aerosolization of particulate (1-->3)-beta-D-glucan from moldy materials.

Mold-damaged building materials may contain biologically active agents, such as (1-->3)-beta-D-glucan, allergens, and mycotoxins, which have been associated with adverse health effects. The release of these components from contaminated surfaces into the air is not well understood. The purpose of this study was to characterize the release of particulate (1-->3)-beta-D-glucan from the surface of artificially mold-contaminated materials. Aspergillus versicolor and Stachybotrys chartarum were grown on malt extract agar (MEA), white ceiling tiles, and a wall-papered gypsum board for 1 and 6 months. The (1-->3)-beta-D-glucan on the surfaces of moldy materials and in air samples collected from these materials was analyzed by the Limulus amebocyte lysate assay. The aerosolization ratio was defined as the amount of (1-->3)-beta-D-glucan in the air divided by the amount on the surface. The results showed that the aerosolization of particulate (1-->3)-beta-D-glucan was influenced mainly by the type of material and the fungal species. For A. versicolor, the aerosolization ratios of particulate (1-->3)-beta-D-glucan released from the three types of material were not significantly different. However, the ratios for S. chartarum released from ceiling tiles and gypsum board were significantly higher than the ratios for this organism released from MEA (P < 0.001) and were comparable to those for A. versicolor. These findings indicate that the use of MEA in aerosolization experiments is likely to underestimate the release of S. chartarum particles from building materials. These results provide important background information for design of future laboratory or animal experiments, as well as for interpretation of field measurement data.

Biomechanics of conidial dispersal in the toxic mold Stachybotrys chartarum.

Conidial dispersal in Stachybotrys chartarum in response to low-velocity airflow was studied using a microflow apparatus. The maximum rate of spore release occurred during the first 5 min of airflow, followed by a dramatic reduction in dispersal that left more than 99% of the conidia attached to their conidiophores. Micromanipulation of undisturbed colonies showed that micronewton (microN) forces were needed to dislodge spore clusters from their supporting conidiophores. Calculations show that airspeeds that normally prevail in the indoor environment disturb colonies with forces that are 1000-fold lower, in the nanonewton (nN) range. Low-velocity airflow does not, therefore, cause sufficient disturbance to disperse a large proportion of the conidia of S. chartarum.

Taxonomic history and current status of Stachybotrys chartarum and related species.

UNLABELLED: The fungus Stachybotrys chartarum is the type species of the genus Stachybotrys. It is a cellulolytic saprophyte with a worldwide distribution and is frequently recovered in water-damaged buildings. Three isolates of S. chartarum were studied morphologically from single-spore isolations. Significant differences were found with the sizes, lengths, width, and L/W ratio of conidia and phialides among the isolates. QPCR analysis on S. chartarum, S. yunnanensis, S. chlorohalonata, S. elegans, S. microspora, and S. nephrospora showed that the primers and probe for detecting S. chartarum used by commercial laboratories were not able to differentiate S. chartarum from S. chlorohalonata and S. yunnanensis. Results suggested that S. chartarum may not be well delineated even after S. chlorohalonata was recently segregated from the species complex. Further study on the taxonomic status of the epithet S. chartarum is necessary. PRACTICAL IMPLICATIONS: Six species of Stachybotrys are present indoors. Differentiation of Stachybotrys chartarum from S. chlorohalonata, and S. yunnanensis can be challenging using either morphological or QPCR methods. Caution should be taken to identify S. chartarum and closely related species and to explain their health effects implication for indoor air quality investigations.

Prevalence and airborne spore levels of Stachybotrys spp. in 200 houses with water incursions in Houston, Texas.

Two hundred homes with a history of water incursion were sampled for fungi to determine the prevalence and airborne spore levels of Stachybotrys spp. Sampling methods included room air, surface, and wall cavity air sampling. Stachybotrys spp. were detected with at least one of the methods in 58.5% of the houses tested, but only 9.6% of the room air samples contained Stachybotrys spores. Aerosolization of Stachybotrys spores was correlated with both wall cavity and surface contamination. However, after adjustment for the surface effect, Stachybotrys spores detected in wall cavities were not a significant factor contributing to spores detected in room air samples. We conclude that Stachybotrys spp. are commonly found on water-damaged building materials. In addition, the observations made in this study suggest that the impact on the living space air is low if the fungal spores are contained within a wall cavity.

Ultraviolet germicidal irradiation disinfection of Stachybotrys chartarum.

The ultraviolet germicidal irradiation (UVGI) dose necessary to inactivate fungal spores on an agar surface and the efficacy of UVGI were determined for cultures of Stachybotrys chartarum (ATCC 208877). This study employed a UVGI testing unit consisting of four chambers with a 9-W, Phillips, low pressure, mercury UVGI lamp in each chamber. The testing unit's apertures were adjusted to provide 50, 100, 150, and 200 microW/cm2 of uniform flux to the Petri dish surfaces, resulting in a total UVGI surface dose ranging from 12 to 144 mJ/cm2. The UVGI dose necessary to inactivate 90% of the S. chartarum was greater than the maximum dose of 144 mJ/cm2 evaluated in this study. While UVGI has been used to inactivate several strains of culturable fungal spores, S. chartarum was not susceptible to an appropriate dose of UVGI. The results of this study may not correlate directly to the effect of UVGI on airborne fungal spores. However, they indicate that current technology may not be efficacious as a supplement to ventilation unless it can provide higher doses of UVGI to kill spores, such as S. chartarum, traveling through the irradiated zone.

Stachybotrys chartarum alters surfactant-related phospholipid synthesis and CTP:cholinephosphate cytidylyltransferase activity in isolated fetal rat type II cells.

Stachybotry chartarum, a fungal contaminant of water-damaged buildings commonly grows on damp cellulose-containing materials. It produces a complex array of mycotoxins. Their mechanisms of action on the pulmonary system are not entirely clear. Previous studies suggest spore products may depress formation of disaturated phosphatidylcholine (DSPC), the major surface-active component of pulmonary surfactant (PS). If S. chartarum can indeed affect formation of this phospholipid, then mold exposure may be a significant issue for pulmonary function in both mature lung and developing fetal lung. To address this possibility, fetal rat type II cells, the principal source of DSPC, were used to assess effects of S. chartarum extract on formation of DSPC. Isolated fetal rat lung type II cells prelabeled with 3H-choline and incubated with spore extract showed decreased incorporation of 3H-choline into DSPC. The activity of CTP:cholinephosphate cytidylyltransferase (CPCT), the rate-limiting enzyme in phosphatidylcholine synthesis was reduced by approximately 50% by a 1:10 dilution of spore extract. Two different S. chartarum extracts (isolates from S. chartarum (Cleveland) and S. chartarum (Hawaiian)) were used to compare activity of CPCT in the presence of phosphatidylglycerol (PG), a known activator. PG produced an approximate two-fold increase in CPCT activity. The spore isolate from Hawaii did not alter enzyme activity. S. chartarum (Cleveland) eliminated the PG-induced activation of CPCT. These results support previous observations that mold products alter PS metabolism and may pose a risk in developing lung, inhibiting surfactant synthesis. Different isolates of the same species of fungus are not equivalent in terms of potential exposure risks.

Multigene phylogeny reveals new lineage for Stachybotrys chartarum, the indoor air fungus.

Stachybotrys chartarum is an asexually reproducing fungus commonly isolated from soil and litter that is also known to occur in indoor environments and is implicated as the cause of serious illness and even death in humans. Despite its economic importance, higher level phylogenetic relationships of Stachybotrys have not been determined nor has a sexual state for S. chartarum been reported. DNA sequences from four nuclear and one mitochondrial gene were analyzed to determine the ordinal and familial placement of Stachybotrys within the Euascomycota. These data reveal that species of Stachybotrys including S. chartarum, S. albipes, for which the sexual state Melanopsamma pomiformis is reported, species of Myrothecium, and two other tropical hypocrealean species form a previously unknown monophyletic lineage within the Hypocreales. These results suggest that Stachybotrys and Myrothecium are closely related and share characteristics with other hypocrealean fungi. In addition, S. chartarum may have a sexual state in nature that consists of small, black, fleshy perithecia similar to Melanopsamma.

[A comparative analysis of Stachybotrys chartarum strains isolated in Russia]. / Sravnitel'nyi analiz rossiiskikh shtammov Stachybotrys chartarum.

This work deals with a comparative analysis of Stachybotrys chartarum strains isolated from various artificial cellulose-containing materials and natural substrates in the geographically distant regions of Russia. The analysis included the determination of the spore size, the strain toxicity to Paramecium caudatum, the strain resistance to the fungicides Benomil, Olilen, and Tilt, and the PCR study of the genome structure with the aid of a primer that was complementary to the core sequence of the SINE retrotransposon. It was found that some of the strains that were isolated from different areas and from different substrates differ in their toxicity, fungicide resistance, and genome structure. The PCR analysis showed the absence of any correlation between the genome structure, the strain properties, the geographic area, and the substrates from which the strains were isolated. The pheno- and genotypic diversity of the strains and their different vegetative compatibility suggest the existence of an intraspecies diversity of the S. chartarum strains that were isolated in different geographic areas. The absence of any correlation between the pheno- and genotypic properties of the strains and the substrates from which they were isolated implies that the colonization of artificial substrates by S. chartarum occurred occasionally from natural habitats. The S. chartarum populations that live on artificial substrates are unlikely to have their own evolutionary history.

Comparison of inflammatory and cytotoxic lung responses in mice after intratracheal exposure to spores of two different Stachybotrys chartarum strains.

Stachybotrys chartarum is an important toxigenic fungus that has been associated with respiratory disease onset in animals and humans. While it can be separated into macrocyclic trichothecene- and atranone-producing chemotypes based on secondary metabolite production, effects of spores of the two chemotypes on lungs are poorly understood. In this study we used bronchoalveolar lavage fluid (BALF) to investigate dose-response (30, 300, 3000 spores/g body weight [BW]) and time-course (3, 6, 24, 48, 96 h post instillation [PI]) relationships in mice to exposure of macrocyclic trichothecene- (JS 58-17) and atranone-producing (JS 58-06) S. chartarum strains, as well as Cladosporium cladosporioides spores. BALF total protein, albumin, pro-inflammatory cytokine (IL-1beta, IL-6, and tumor necrosis factor-alpha [TNF-alpha]), and lactate dehydrogenase (LDH) concentrations showed significant (p < 0.05) fungal species (S. chartarum vs. C. cladosporioides) and strain (58-17 vs. 58-06), spore dose and time dependent changes. The no adverse effect level (NOAEL) due to exposure to spores of JS 58-17 and JS 58-06 was < 30 spores/g BW; for C. cladosporioides it was < 300 spores/g BW. At moderate and high S. chartarum doses, BALF composition reflects differences in strain toxicity while at the lowest dose, BALF composition of either S. chartarum strain were similar. This suggests that at low spore doses, it is spore sequestered factors common to both strains not strain dependent toxins that are contributing to lung disease onset.