63. Uriel, Y; Gries, R; Tu, L; Carroll, C; Zhai, HM; Moore, M; Gries, G. (2020) The fly factor phenomenon is mediated by interkingdom signaling between bacterial symbionts and their blow fly hosts.Insect Sci. 27: 256-265 The fly factor phenomenon is mediated by interkingdom signaling between bacterial symbionts and their blow fly hosts
blow flies; enteric bacteria; fly factor; interkingdom communication; microbial symbionts; semiochemical attractants
We tested the recent hypothesis that the "fly factor" phenomenon (food currently or previously fed on by flies attracts more flies than the same type of food kept inaccessible to flies) is mediated by bacterial symbionts deposited with feces or regurgitated by feeding flies. We allowed laboratory-reared black blow flies, Phormia regina (Meigen), to feed and defecate on bacterial Luria-Bertani medium solidified with agar, and isolated seven morphologically distinct bacterial colonies. We identified these using matrix-assisted laser desorption/ionization mass spectrometry and sequencing of the 16S rRNA gene. In two-choice laboratory experiments, traps baited with cultures of Proteus mirabilis Hauser, Morganella morganii subsp. sibonii Jensen, or Serratia marcescens Bizio, captured significantly more flies than corresponding control jars baited with tryptic soy agar only. A mixture of seven bacterial strains as a trap bait was more attractive to flies than a single bacterial isolate (M. m. sibonii). In a field experiment, traps baited with agar cultures of P. mirabilis and M. m. sibonii in combination captured significantly more flies than traps baited with either bacterial isolate alone or the agar control. As evident by gas chromatography-mass spectrometry, the odor profiles of bacterial isolates differ, which may explain the additive effect of bacteria to the attractiveness of bacterial trap baits. As "generalist bacteria," P. mirabilis and M. m. sibonii growing on animal protein (beef liver) or plant protein (tofu) are similarly effective in attracting flies. Bacteria-derived airborne semiochemicals appear to mediate foraging by flies and to inform their feeding and oviposition decisions. DOI PubMed
62. Babcock, T; Borden, JH; Gries, R; Carroll, C; Lafontaine, JP; Moore, M; Gries, G. (2019) Inter-kingdom signaling - symbiotic yeasts produce semiochemicals that attract their yellowjacket hosts.Entomol. Exp. Appl. 167 Inter-kingdom signaling - symbiotic yeasts produce semiochemicals that attract their yellowjacket hosts
Vespula pensylvanica; yeast symbiosis; Hanseniaspora uvarum; Lachancea thermotolerans; Hymenoptera; Vespidae
In recent studies, the yeast species Hanseniaspora uvarum and Lachancea thermotolerans were isolated from the digestive tract of four North American yellowjacket species (Hymenoptera: Vespidae), and attraction of yellowjackets to brewer's yeast, Saccharomyces cerevisiae (all Saccharomycetaceae), growing on fruit powder was demonstrated. We tested the hypothesis that Vespula spp. are attracted to cultures of H. uvarum and L. thermotolerans and their respective volatiles. In field experiments, we found that H. uvarum and L. thermotolerans are attractive to three species of yellowjacket, but only when grown on grape juice-infused yeast peptone dextrose (YPD) agar. Using gas chromatography-mass spectrometry, we analyzed the headspace volatiles produced by these yeasts, and field tested an 18-component yeast synthetic semiochemical blend. This synthetic blend attracted western yellowjackets, Vespula pensylvanica (Saussure), but no other yellowjacket species. Acetic acid or ethanol added to the synthetic blend at biologically relevant doses either had no effect or significantly lowered trap captures. Our results demonstrate that yeast symbionts isolated from the digestive tract of yellowjackets are attractive to their hosts. Further research is needed to identify the volatiles mediating attraction of species other than V. pensylvanica to the yeast cultures. DOI
61. Culibrk, L; Croft, CA; Toor, A; Yang, SJ; Singhera, GK; Dorscheid, DR; Moore, MM; Tebbutt, SJ. (2019) Phagocytosis of Aspergillus fumigatus by Human Bronchial Epithelial Cells Is Mediated by the Arp2/3 Complex and WIPF2.Front. Cell. Infect. Microbiol. 9 Phagocytosis of Aspergillus fumigatus by Human Bronchial Epithelial Cells Is Mediated by the Arp2/3 Complex and WIPF2
phagocytosis; fungi; aspergillus; cytoskeleton; invasion; host; airway; epithelium
Aspergillus fumigatus is an opportunistic fungal pathogen capable of causing severe infection in humans. One of the limitations in our understanding of how A. fumigatus causes infection concerns the initial stages of infection, notably the initial interaction between inhaled spores or conidia and the human airway. Using publicly-available datasets, we identified the Arp2/3 complex and the WAS-Interacting Protein Family Member 2 WIPF2 as being potentially responsible for internalization of conidia by airway epithelial cells. Using a cell culture model, we demonstrate that RNAi-mediated knockdown of WIPF2 significantly reduces internalization of conidia into airway epithelial cells. Furthermore, we demonstrate that inhibition of Arp2/3 by a small molecule inhibitor causes similar effects. Using super-resolution fluorescence microscopy, we demonstrate that WIPF2 is transiently localized to the site of bound conidia. Overall, we demonstrate the active role of the Arp2/3 complex and WIPF2 in mediating the internalization of A. fumigatus conidia into human airway epithelial cells. DOI PubMed
60. Ramakrishnan, G; Perez, NM; Carroll, C; Moore, MM; Nakarnoto, RK; Fox, TE. (2019) Citryl Ornithine Is an Intermediate in a Three-Step Biosynthetic Pathway for Rhizoferrin in Francisella.ACS Chem. Biol. 14: 1760-1766 Citryl Ornithine Is an Intermediate in a Three-Step Biosynthetic Pathway for Rhizoferrin in Francisella
The Gram-negative bacterium Francisella tularensis secretes the siderophore rhizoferrin to scavenge necessary iron from the environment. Rhizoferrin, also produced by a variety of fungi and bacteria, comprises two citrate molecules linked by amide bonds to a central putrescine (diaminobutane) moiety. Genetic analysis has determined that rhizoferrin production in F. tularensis requires two enzymes: FslA, a siderophore synthetase of the nonribosomal peptide synthetase-independent siderophore synthetase (NIS) family, and FslC, a pyridoxal-phosphate-dependent decarboxylase. To discern the steps in the biosynthetic pathway, we tested F. tularensis strain LVS and its Delta fslA and Delta fslC mutants for the ability to incorporate potential precursors into rhizoferrin. Unlike putrescine supplementation, supplementation with ornithine greatly enhanced siderophore production by LVS. Radioactivity from L-[U-C-14] ornithine, but not from L-[1-C-1(4)] ornithine, was efficiently incorporated into rhizoferrin by LVS. Although neither the Delta fslA nor the Delta fslC mutant produced rhizoferrin, a putative siderophore intermediate labeled by both [U-C-14] ornithine and [1-C-1(4)] ornithine was secreted by the Delta fslC mutant. Rhizoferrin was identified by liquid chromatography and mass spectrometry in LVS culture supernatants, while citryl-ornithine was detected as the siderophore intermediate in the culture supernatant of the Delta fslC mutant. Our findings support a three-step pathway for rhizoferrin production in Francisella; unlike the fungus Rhizopus delemar, where putrescine functions as a primary precursor for rhizoferrin, biosynthesis in Francisella preferentially starts with ornithine as the substrate for FslA-mediated condensation with citrate. Decarboxylation of this citryl ornithine intermediate by FslC is necessary for a second condensation reaction with citrate to produce rhizoferrin. DOI PubMed
59. Babcock, T; Borden, J; Gries, R; Carroll, C; Moore, M; Gries, G. (2018) Lachancea thermotolerans, a Yeast Symbiont of Yellowjackets, Enhances Attraction of Three Yellowjacket Species (Hymenoptera: Vespidae) to Fruit Powder.Environ. Entomol. 47 Lachancea thermotolerans, a Yeast Symbiont of Yellowjackets, Enhances Attraction of Three Yellowjacket Species (Hymenoptera: Vespidae) to Fruit Powder
yellowjackets; Lachancea thermotolerans; semiochemical attractant; fruit bait; symbiotic yeast
Previously, we showed that the symbiotic yeast Lachancea thermotolerans (Filippov) (Saccharomycetales: Saccharomycetaceae) is attractive to its Vespula (Hymenoptera: Vespidae) yellowjacket hosts when grown on media supplemented with grape juice. We hypothesized that "Concerto': a commercial strain of this yeast, could be combined with fruit powder to form a shelf-stable bait for trapping yellowjackets. Using molecular techniques, we first confirmed that Concerto yeast is indeed the species L. thermotolerans. We then tested whether: 1) Concerto yeast produces volatiles similar to those produced by L. thermotolerans isolated from yellowjackets, 2) Concerto yeast enhances attraction of yellowjackets to fruit powder, 3) a Concerto yeast/fruit powder bait interacts synergistically with a yellowjacket semiochemical lure, and 4) a synthetic analog blend of Concerto-produced volatiles attracts yellowjackets. Using gas chromatography-mass spectrometry, we demonstrated that the chemical composition of Concerto-produced volatiles closely resembles that produced by a yellowjacket-isolated strain of L. thermotolerans. In field experiments, addition of Concerto to fruit powder doubled its attractiveness to yellowjackets. Addition of the Concerto/fruit powder bait to a heptyl butyrate-based wasp lure revealed a weak additive effect. A three-component synthetic analog blend of volatiles identified from the Concerto/fruit powder bait attracted Vespula pensylvanica (Saussure), but no other yellowjacket species. Our results suggest that commercial L. thermotolerans in combination with fruit powder could be used as a yellowjacket bait, and that addition of yeast-produced volatiles to a commercial wasp lure may improve its attractiveness to V. pensylvanica. Further research should determine why the synthetic volatile blend failed to attract Vespula species other than V. pensylvanica. DOI PubMed
58. Carroll, CS; Moore, MM. (2018) Ironing out siderophore biosynthesis: a review of non-ribosomal peptide synthetase (NRPS)-independent siderophore synthetases.Crit. Rev. Biochem. Mol. Biol. 53 Ironing out siderophore biosynthesis: a review of non-ribosomal peptide synthetase (NRPS)-independent siderophore synthetases
NRPS-independent siderophore (NIS); adenylating enzymes; carboxylate siderophore; virulence; halobacteria; siderophore biosynthesis
Iron is required for microbial growth and proliferation. To survive in low-iron environments, some microorganisms secrete ferric iron chelators called siderophores. Siderophore biosynthesis occurs via two pathways: the non-ribosomal peptide synthetase (NRPS) pathway and the NRPS-independent siderophore (NIS) synthetase pathway. NIS enzymes function by adenylating a carboxylic acid substrate, typically citrate, or a derivative, followed by nucleophilic capture of an amine or alcohol and displacement of a citryl intermediate. In this review, we summarize recent advances in NIS biochemistry with a particular focus on structural biology and confirm the classification of NIS enzymes into Types A, A', B, and C based on substrate specificity. Based on a phylogenetic analysis, we also propose a new subclass of NIS enzymes, Type C', responsible for dimerization and macrocyclization of complex and substituted amine or amide intermediates. Finally, we describe the role of NIS enzymes in virulence of pathogenic microbes and discuss NIS inhibitors as potential anti-microbial agents. DOI PubMed
57. Nesbitt, JR; Steves, EY; Schonhofer, CR; Cait, A; Manku, SS; Yeung, JHF; Bennet, AJ; McNagny, KM; Choy, JC; Hughes, MR; Moore, MM. (2018) The Aspergillus fumigatus Sialidase (Kdnase) Contributes to Cell Wall Integrity and Virulence in Amphotericin B-Treated Mice.Front. Microbiol. 8 The Aspergillus fumigatus Sialidase (Kdnase) Contributes to Cell Wall Integrity and Virulence in Amphotericin B-Treated Mice
invasive aspergillosis; sialidase; cell wall integrity; chitin; Kdn
Aspergillus fumigatus is a filamentous fungus that can cause a life-threatening invasive pulmonary aspergillosis (IPA) in immunocompromised individuals. We previously characterized an exo-sialidase from A. fumigatus that prefers the sialic acid substrate, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (Kdn); hence it is a Kdnase. Sialidases are known virulence factors in other pathogens; therefore, the goal of our study was to evaluate the importance of Kdnase in A. fumigatus. A kdnase knockout strain (Delta kdnase) was unable to grow on medium containing Kdn and displayed reduced growth and abnormal morphology. Delta kdnase was more sensitive than wild type to hyperosmotic conditions and the antifungal agent, amphotericin B. In contrast, Delta kdnase had increased resistance to nikkomycin, Congo Red and Calcofluor White indicating activation of compensatory cell wall chitin deposition. Increased cell wall thickness and chitin content in Delta kdnase were confirmed by electron and immunofluorescence microscopy. In a neutropenic mouse model of invasive aspergillosis, the Delta kdnase strain had attenuated virulence and a significantly lower lung fungal burden but only in animals that received liposomal amphotericin B after spore exposure. Macrophage numbers were almost twofold higher in lung sections from mice that received the Delta kdnase strain, possibly related to higher survival of macrophages that internalized the Delta kdnase conidia. Thus, A. fumigatus Kdnase is important for fungal cell wall integrity and virulence, and because Kdnase is not present in the host, it may represent a potential target for the development of novel antifungal agents. DOI
56. Toor, A; Culibrk, L; Singhera, GK; Moon, KM; Prudova, A; Foster, LJ; Moore, MM; Dorscheid, DR; Tebbutt, SJ. (2018) Transcriptomic and proteomic host response to Aspergillus fumigatus conidia in an air-liquid interface model of human bronchial epithelium.PLoS One 13 Transcriptomic and proteomic host response to Aspergillus fumigatus conidia in an air-liquid interface model of human bronchial epithelium
Aspergillus fumigatus (A. fumigatus) is a wide-spread fungus that is a potent allergen in hypersensitive individuals but also an opportunistic pathogen in immunocompromised patients. It reproduces asexually by releasing airborne conidiospores (conidia). Upon inhalation, fungal conidia are capable of reaching the airway epithelial cells (AECs) in bronchial and alveolar tissues. Previous studies have predominantly used submerged monolayer cultures for studying this host-pathogen interaction; however, these cultures do not recapitulate the mucocililary differentiation phenotype of the in vivo epithelium in the respiratory tract. Thus, the aim of this study was to use well-differentiated primary human bronchial epithelial cells (HBECs) grown at the air-liquid interface (ALI) to determine their transcriptomic and proteomic responses following interaction with A. fumigatus conidia. We visualized conidial interaction with HBECs using confocal laser scanning microscopy (CLSM), and applied NanoString nCounter and shotgun proteomics to assess gene expression changes in the human cells upon interaction with A. fumigatus conidia. Western blot analysis was used to assess the expression of top three differentially expressed proteins, CALR, SET and NUCB2. CLSM showed that, unlike submerged monolayer cultures, well-differentiated ALI cultures of primary HBECs were estimated to internalize less than 1% of bound conidia. Nevertheless, transcriptomic and proteomic analyses revealed numerous differentially expressed host genes; these were enriched for pathways including apoptosis/autophagy, translation, unfolded protein response and cell cycle (up-regulated); complement and coagulation pathways, iron homeostasis, nonsense mediated decay and rRNA binding (down-regulated). CALR and SET were confirmed to be up-regulated in ALI cultures of primary HBECs upon exposure to A. fumigatus via western blot analysis. Therefore, using transcriptomics and proteomics approaches, ALI models recapitulating the bronchial epithelial barrier in the conductive zone of the respiratory tract can provide novel insights to the molecular response of bronchial epithelial cells upon exposure to A. fumigatus conidia. DOI PubMed
55. Trowell, JJ; Gobas, FAPC; Moore, MM; Kennedy, CJ. (2018) Estimating the Bioconcentration Factors of Hydrophobic Organic Compounds from Biotransformation Rates Using Rainbow Trout Hepatocytes.Arch. Environ. Contam. Toxicol. 75 Estimating the Bioconcentration Factors of Hydrophobic Organic Compounds from Biotransformation Rates Using Rainbow Trout Hepatocytes
Determining the biotransformation potential of commercial chemicals is critical for estimating their persistence in the aquatic environment. In vitro systems are becoming increasingly important as screening methods for assessing the potential for chemical metabolism. Depletion rate constants (k (d)) for several organic chemicals with high octanol-water partition coefficient (K (ow)) values (9-methylanthracene, benzo(a)pyrene, chrysene, and PCB-153) in rainbow trout hepatocytes were determined to estimate biotransformation rate constants (k (MET)) that were used in fish bioconcentration factor (BCF) models. Benzo[a]pyrene was rapidly biotransformed when incubated singly; however, its depletion rate constant (k (d)) declined 79% in a mixture of all four chemicals. Chrysene also exhibited significant biotransformation and its depletion rate constant declined by 50% in the mixture incubation. These data indicate that biotransformation rates determined using single chemicals may overestimate metabolism in environments containing chemical mixtures. Incubations with varying cell concentrations were used to determine whether cell concentration affected k (d) estimates. No statistically significant change in depletion rate constants were seen, possibly due to an increase in nonspecific binding of hydrophobic chemicals as cell density increased, decreasing overall biotransformation. A new model was used to estimate BCFs from k (MET) values calculated from empirically derived k (d) values. The inclusion of k (MET) in models resulted in significantly lower BCF values (compared k (MET) = 0). Modelled BCF values were consistent with empirically derived BCF values from the literature. DOI PubMed
54. Jimenez, SI; Carroll, C; Babcock, T; Derstine, N; Hadwin, A; Moore, M; Gries, G. (2017) Yeasts Harbored by Vespine Wasps in the Pacific Northwest.Environmental Entomology 46: 217-225 Yeasts Harbored by Vespine Wasps in the Pacific Northwest
Lachancea; Hanseniaspora; vespine wasp; yeast-wasp interaction
The ecological role of social wasps has been extensively studied, but little is known about symbiotic relationships of these wasps with microbes. Recently, it was shown that vespid wasps in Europe carry yeasts, predominantly Saccharomyces cerevisiae, in their gastrointestinal (GI) tract. Interestingly, this niche allowed for sexual recombination of yeasts to occur and the formation of novel hybrid species. Our goals were 1) to survey the GI tract of eusocial wasps in the Pacific Northwest for the presence of yeasts and 2) to compare the diversity of such yeasts to that described for wasps in Europe. The GI tracts of 19 individual wasps from five species were plated, and 27 yeast-like colonies were identified to the species level. Yeasts in the genera Lachancea and Hanseniaspora each comprised similar to 30% of the isolates; similar to 25% were identified as Metschnikowia spp., with the remaining 10% belonging to Rhodotorula. Four bacterial isolates were identified as Escherichia coli, Enterococcus faecalis, and two isolates of Stenotrophomonas maltophilia. Yeasts were present at all life stages of the wasps except for two unfed gynes of Dolichovespula maculata (L.) that contained only bacteria. The presence of a particular yeast species was not correlated with any wasp species. Furthermore, S. cerevisiae was not found in any wasp species. This highlights an interesting difference in the life cycle of both S. cerevisiae and wasps in Europe and the Pacific Northwest, and prompts further studies on the interactions of these microbes with their host wasps. DOI
53. Lee, YS; Lo, JC; Otton, SV; Moore, MM; Kennedy, CJ; Gobas, FAPC. (2017) IN VITRO TO IN VIVO EXTRAPOLATION OF BIOTRANSFORMATION RATES FOR ASSESSING BIOACCUMULATION OF HYDROPHOBIC ORGANIC CHEMICALS IN MAMMALS.Environmental Toxicology and Chemistry 36: 1934-1946 IN VITRO TO IN VIVO EXTRAPOLATION OF BIOTRANSFORMATION RATES FOR ASSESSING BIOACCUMULATION OF HYDROPHOBIC ORGANIC CHEMICALS IN MAMMALS
Biotransformation; Bioaccumulation; In vitro to in vivo extrapolation; Bioaccumulation modeling
Incorporating biotransformation in bioaccumulation assessments of hydrophobic chemicals in both aquatic and terrestrial organisms in a simple, rapid, and cost-effective manner is urgently needed to improve bioaccumulation assessments of potentially bioaccumulative substances. One approach to estimate whole-animal biotransformation rate constants is to combine in vitro measurements of hepatic biotransformation kinetics with in vitro to in vivo extrapolation (IVIVE) and bioaccumulation modeling. An established IVIVE modeling approach exists for pharmaceuticals (referred to in the present study as IVIVE-Ph) and has recently been adapted for chemical bioaccumulation assessments in fish. The present study proposes and tests an alternative IVIVE-B technique to support bioaccumulation assessment of hydrophobic chemicals with a log octanol-water partition coefficient (K-OW) >= 4 in mammals. The IVIVE-B approach requires fewer physiological and physiochemical parameters than the IVIVE-Ph approach and does not involve interconversions between clearance and rate constants in the extrapolation. Using in vitro depletion rates, the results show that the IVIVE-B and IVIVE-Ph models yield similar estimates of rat whole-organism biotransformation rate constants for hypothetical chemicals with log K-OW >= 4. The IVIVE-B approach generated in vivo biotransformation rate constants and biomagnification factors (BMFs) for benzo[a] pyrene that are within the range of empirical observations. The proposed IVIVE-B technique may be a useful tool for assessing BMFs of hydrophobic organic chemicals in mammals. (C) 2016 SETAC DOI
52. Carroll, CS; Amankwa, LN; Pinto, LJ; Fuller, JD; Moore, MM. (2016) Detection of a Serum Siderophore by LC-MS/MS as a Potential Biomarker of Invasive Aspergillosis.PLoS One 11 Detection of a Serum Siderophore by LC-MS/MS as a Potential Biomarker of Invasive Aspergillosis
Invasive aspergillosis (IA) is a life-threatening systemic mycosis caused primarily by Aspergillus fumigatus. Early diagnosis of IA is based, in part, on an immunoassay for circulating fungal cell wall carbohydrate, galactomannan (GM). However, a wide range of sensitivity and specificity rates have been reported for the GM test across various patient populations. To obtain iron in vivo, A. fumigatus secretes the siderophore, N, N', N ''-triacetylfusarinine C (TAFC) and we hypothesize that TAFC may represent a possible biomarker for early detection of IA. We developed an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for TAFC analysis from serum, and measured TAFC in serum samples collected from patients at risk for IA. The method showed lower and upper limits of quantitation (LOQ) of 5 ng/ml and 750 ng/ml, respectively, and complete TAFC recovery from spiked serum. As proof of concept, we evaluated 76 serum samples from 58 patients with suspected IA that were investigated for the presence of GM. Fourteen serum samples obtained from 11 patients diagnosed with probable or proven IA were also analyzed for the presence of TAFC. Control sera (n = 16) were analyzed to establish a TAFC cut-off value (>= 6 ng/ml). Of the 36 GM-positive samples (>= 0.5 GM index) from suspected IA patients, TAFC was considered positive in 25 (69%). TAFC was also found in 28 additional GM-negative samples. TAFC was detected in 4 of the 14 samples (28%) from patients with proven/probable aspergillosis. Log-transformed TAFC and GM values from patients with proven/probable IA, healthy individuals and SLE patients showed a significant correlation with a Pearson r value of 0.77. In summary, we have developed a method for the detection of TAFC in serum that revealed this fungal product in the sera of patients at risk for invasive aspergillosis. A prospective study is warranted to determine whether this method provides improved early detection of IA. DOI
51. Croft, CA; Culibrk, L; Moore, MM; Tebbutt, SJ. (2016) Interactions of Aspergillus fumigatus Conidia with Airway Epithelial Cells: A Critical Review.Frontiers in Microbiology 7 Interactions of Aspergillus fumigatus Conidia with Airway Epithelial Cells: A Critical Review
Aspergillus fumigatus; conidia; airway epithelial cell; aspergillosis; virulence; immunity; host-pathogen interactions
Aspergillus fumigatus is an environmental filamentous fungus that also acts as an opportunistic pathogen able to cause a variety of symptoms, from an allergic response to a life-threatening disseminated fungal infection. The infectious agents are inhaled conidia whose first point of contact is most likely to be an airway epithelial cell (AEC). The interaction between epithelial cells and conidia is multifaceted and complex, and has implications for later steps in pathogenesis. Increasing evidence has demonstrated a key role for the airway epithelium in the response to respiratory pathogens, particularly at early stages of infection; therefore, elucidating the early stages of interaction of conidia with AECs is essential to understand the establishment of infection in cohorts of at-risk patients. Here, we present a comprehensive review of the early interactions between A. fumigatus and AECs, including bronchial and alveolar epithelial cells. We describe mechanisms of adhesion, internalization of conidia by AECs, the immune response of AECs, as well as the role of fungal virulence factors, and patterns of fungal gene expression characteristic of early infection. A clear understanding of the mechanisms involved in the early establishment of infection by A. furnigatus could point to novel targets for therapy and prophylaxis. DOI
50. Freschi, L; Jeukens, J; Kukavica-Ibrulj, I; Boyle, B; Dupont, MJ; Laroche, J; Larose, S; Maaroufi, H; Fothergill, JL; Moore, M; Winsor, GL; Aaron, SD; Barbeau, J; Bell, SC; Burns, JL; Camara, M; Cantin, A; Charette, SJ; Dewar, K; Deziel, E; Grimwood, K; Hancock, REW; Harrison, JJ; Heebs, S; Jelsbak, L; Jia, BF; Kenna, DT; Kidd, TJ; Klockgether, J; Lam, JS; Lamont, IL; Lewenza, S; Loman, N; Malouin, F; Manos, J; McArthur, AG; McKeown, J; Milot, J; Naghra, H; Nguyen, D; Pereira, SK; Perron, GG; Pirnay, JP; Rainey, PB; Rousseau, S; Santos, PM; Stephenson, A; Taylor, V; Turton, JF; Waglechner, N; Williams, P; Thrane, SW; Wright, GD; Brinkman, FSL; Tucker, NP; Tummler, B; Winstanley, C; Levesque, RC. (2015) Clinical utilization of genomics data produced by the international Pseudomonas aeruginosa consortium.Frontiers in Microbiology 6 Clinical utilization of genomics data produced by the international Pseudomonas aeruginosa consortium
Pseudomonas aeruginosa; next-generation sequencing; bacterial genome; phylogeny; database; cystic fibrosis; antibiotic resistance; clinical microbiology
The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consortium Database (http://ipcd.ibis.ulaval.ca/). Here, we present our strategy and the results that emerged from the analysis of the first 389 genomes. With as yet unmatched resolution, our results confirm that P. aerugihosa strains can be divided into three major groups that are further divided into subgroups, some not previously reported in the literature. We also provide the first snapshot of P aeruginosa strain diversity with respect to antibiotic resistance. Our approach will allow us to draw potential links between environmental strains and those implicated in human and animal infections, understand how patients become infected and how the infection evolves over time as well as identify prognostic markers for better evidence-based decisions on patient care. DOI
49. Lee, YS; Lee, DHY; Delafoulhouze, M; Otton, SV; Moore, MM; Kennedy, CJ; Gobas, FAPC. (2014) IN VITRO BIOTRANSFORMATION RATES IN FISH LIVER S9: EFFECT OF DOSING TECHNIQUES.Environmental Toxicology and Chemistry 33: 1885-1893 IN VITRO BIOTRANSFORMATION RATES IN FISH LIVER S9: EFFECT OF DOSING TECHNIQUES
In vitro biotransformation; Bioaccumulation; Sorbent-phase dosing; Ethylene vinyl acetate thin film
In vitro biotransformation assays are currently being explored to improve estimates of bioconcentration factors of potentially bioaccumulative organic chemicals in fish. The present study compares thin-film and solvent-delivery dosing techniques as well as single versus multiple chemical dosing for measuring biotransformation rates of selected polycyclic aromatic hydrocarbons in rainbow trout (Oncorhynchus mykiss) liver S9. The findings show that biotransformation rates of very hydrophobic substances can be accurately measured in thin-film sorbent-dosing assays from concentration-time profiles in the incubation medium but not from those in the sorbent phase because of low chemical film-to-incubation-medium mass-transfer rates at the incubation temperature of 13.5 degrees C required for trout liver assays. Biotransformation rates determined by thin-film dosing were greater than those determined by solvent-delivery dosing for chrysene (octanol-water partition coefficient [K-OW] = 10(5.60)) and benzo[a]pyrene (K-OW = 10(6.04)), whereas there were no statistical differences in pyrene (K-OW = 10(5.18)) biotransformation rates between the 2 methods. In sorbent delivery-based assays, simultaneous multiple-chemical dosing produced biotransformation rates that were not statistically different from those measured in single-chemical dosing experiments for pyrene and benzo[a]pyrene but not for chrysene. In solvent-delivery experiments, multiple-chemical dosing produced biotransformation rates that were much smaller than those in single-chemical dosing experiments for all test chemicals. While thin-film sorbent-phase and solvent delivery-based dosing methods are both suitable methods for measuring biotransformation rates of substances of intermediate hydrophobicity, thin-film sorbent-phase dosing may be more suitable for superhydrophobic chemicals. (c) 2014 SETAC DOI PubMed
48. Koch, I; Reimer, KJ; Bakker, MI; Basta, NT; Cave, MR; Denys, S; Dodd, M; Hale, BA; Irwin, R; Lowney, YW; Moore, MM; Paquin, V; Rasmussen, PE; Repaso-Subang, T; Stephenson, GL; Siciliano, SD; Wragg, J; Zagury, GJ. (2013) Variability of bioaccessibility results using seventeen different methods on a standard reference material, NIST 2710.Journal of Environmental Science and Health Part A-Toxic/Hazardous Substances & Environmental Engineering 48: 641-655 Variability of bioaccessibility results using seventeen different methods on a standard reference material, NIST 2710
VITRO DIGESTION MODELS; CONTAMINATED SOILS; IN-VIVO; RELATIVE BIOAVAILABILITY; ARSENIC BIOACCESSIBILITY; LEAD BIOACCESSIBILITY; VALIDATION; CADMIUM; DUST; METHODOLOGIES
Bioaccessibility is a measurement of a substance's solubility in the human gastro-intestinal system, and is often used in the risk assessment of soils. The present study was designed to determine the variability among laboratories using different methods to measure the bioaccessibility of 24 inorganic contaminants in one standardized soil sample, the standard reference material NIST 2710. Fourteen laboratories used a total of 17 bioaccessibility extraction methods. The variability between methods was assessed by calculating the reproducibility relative standard deviations (RSDs), where reproducibility is the sum of within-laboratory and between-laboratory variability. Whereas within-laboratory repeatability was usually better than (<) 15% for most elements, reproducibility RSDs were much higher, indicating more variability, although for many elements they were comparable to typical uncertainties (e.g., 30% in commercial laboratories). For five trace elements of interest, reproducibility RSDs were: arsenic (As), 2244%; cadmium (Cd), 1141%; Cu, 1530%; lead (Pb), 4583%; and Zn, 1856%. Only one method variable, pH, was found to correlate significantly with bioaccessibility for aluminum (Al), Cd, copper (Cu), manganese (Mn), Pb and zinc (Zn) but other method variables could not be examined systematically because of the study design. When bioaccessibility results were directly compared with bioavailability results for As (swine and mouse) and Pb (swine), four methods returned results within uncertainty ranges for both elements: two that were defined as simpler (gastric phase only, limited chemicals) and two were more complex (gastric + intestinal phases, with a mixture of chemicals). DOI
47.Moore, Margo M. (2013) The crucial role of iron uptake in Aspergillus fumigatus virulence.Current Opinion in Microbiology 16: 692-699 The crucial role of iron uptake in Aspergillus fumigatus virulence
Aspergillus fumigatus is an opportunistic fungal pathogen that causes life-threatening infections in immunocompromised individuals. Siderophore-mediated iron acquisition has been shown to be essential for virulence. New studies have revealed that enzymes involved in siderophore biosynthesis and uptake are compartmentalized in peroxisomes and endosome-like vesicles, respectively. Gene and protein expression studies have revealed coordinated regulation of siderophore and sterol metabolism linked to the common precursor mevalonate. Several A. fumigatus transcription factors have been identified that are unexpectedly involved in the regulation of iron homeostasis. New diagnostic and drug treatments are being developed that exploit the requirement of A. fumigatus for extracellular siderophores.Website
46. Woodbury, N; Moore, M; Gries, G. (2013) Horizontal transmission of the microbial symbionts Enterobacter cloacae and Mycotypha microspora to their firebrat host.Entomologia Experimentalis et Applicata 147: 160-166 Horizontal transmission of the microbial symbionts Enterobacter cloacae and Mycotypha microspora to their firebrat host
THERMOBIA-DOMESTICA; SCHISTOCERCA-GREGARIA; MIDGUT EPITHELIUM; DESERT LOCUST; BACTERIA; PROTECTION; MUTUALISM; INSECTS; ANTS
The firebrat, Thermobia domestica (Packard) (Thysanura: Lepismatidae), aggregates in response to the faeces of conspecifics. This aggregation response is mediated by two microbial symbionts, the bacterium Enterobacter cloacae (Jordan) Hormaeche & Edwards (Enterobacteriaceae) and the fungus Mycotypha microspora Fenner (Mucorales). Our objective was to determine how these microbes are transmitted between firebrats. We produced fluorescently labelled E.cloacae and M.microspora and presented them to firebrats. Firebrats consumed large quantities of these labelled microbes and deposited them with their faeces where they proliferated rapidly. Firebrats did not harbour E.cloacae or M.microspora within their ovarioles or eggs, and thus cannot transmit them transovarially. Instead, firebrats acquired them horizontally whenever they fed on microbe-contaminated material, such as faeces, faeces-contaminated paper, or egg surfaces. Firebrats moult throughout their life, and with each moult they shed the cuticular lining of their digestive tract and likely any microbes residing therein. Because firebrats remain in close contact and live in groups of mixed age and gender, newly moulted individuals can readily re-acquire E.cloacae or M.microspora from group members. This ensures the perpetuation of their microbial aggregation and arrestment signal. DOI
45. Yeung, JHF; Telford, JC; Shidmoossavee, FS; Bennet, AJ; Taylor, GL; Moore, MM. (2013) Kinetic and Structural Evaluation of Selected Active Site Mutants of the Aspergillus fumigatus KDNase (Sialidase).Biochemistry 52: 9177-9186 Kinetic and Structural Evaluation of Selected Active Site Mutants of the Aspergillus fumigatus KDNase (Sialidase)
Aspergillus fumigatus is an airborne fungal pathogen. We previously cloned and characterized an exo-sialidase from A. fumigatus and showed that it preferred 2-keto-3-deoxynononic acid (KDN) as a substrate to N-acetylneuraminic acid (Neu5Ac). The purpose of this study was to investigate the structure function relationships of critical catalytic site residues. Site-directed mutagenesis was used to create three mutant recombinant enzymes: the catalytic nucleophile (Y358H), the general acid/base catalyst (D84A), and an enlargement of the binding pocket to attempt to accommodate the N-acetyl group of Neu5Ac (R171L). Crystal structures for all enzymes were determined. The D84A mutation had an effect in decreasing the activity of AfKDNase that was stronger than that of the same mutation in the structurally similar sialidase from the bacterium Micromonospora viridifaciens. These data suggest that the catalytic acid is more important in the reaction of AfKDNase and that catalysis is less dependent on nucleophilic or electrostatic stabilization of the developing positive charge at the transition state for hydrolysis. Removal of the catalytic nucleophile (Y358H) significantly lowered the activity of the enzyme, but this mutant remained a retaining glycosidase as demonstrated by nuclear magnetic resonance spectroscopic analysis. This is a novel finding that has not been shown with other sialidases. Kinetic activity measured at pH 5.2 revealed that R171L had higher activity on a Neu5Ac-based substrate than wild-type KDNase; hence, leucine in place of arginine in the binding pocket improved catalysis toward Neu5Ac substrates. Hence, whether a sialidase is primarily a KDNase or a neuraminidase is due in part to the presence of an amino acid that creates a steric clash with the N-acetyl group.Website DOI PubMed
44. Kickham, P; Otton, SV; Moore, MM; Ikonomou, MG; Gobas, FAPC. (2012) Relationship between biodegradation and sorption of phthalate esters and their metabolites in natural sediments.Environmental Toxicology and Chemistry 31: 1730-1737 Relationship between biodegradation and sorption of phthalate esters and their metabolites in natural sediments
Phthalate ester; Biodegradation; Sorption; Sediment; Persistence
Regulatory evaluations of commercial chemicals in Canada, the United States, the European Union, and other countries aim to identify biodegradation rates of chemicals in natural soils and sediments. However, commonly used biodegradation testing methods are limited in their capacity to determine biodegradation rates under natural environmental conditions. As a result, widely varying biodegradation rates have been reported for many very hydrophobic substances. This variability causes difficulties in regulatory evaluations, potentially leading to chemical misclassification. In the present study, the authors developed a model of the relationship between biodegradation, sorption, and hydrophobicity, and tested the model in experiments that measured the biodegradation rates of a range of di-phthalate esters (DPEs) and mono-phthalate esters (MPEs) in natural sediments. The results indicate that DPEs and MPEs have the inherent capacity to be quickly degraded by microbes in sediments at a common rate, but that DPE biodegradation rates in natural sediments decrease with increasing phthalate ester sorption to sediments. The results show that inherently biodegradable substances that are subject to a high degree of sorption can be expected to exhibit long half-lives in natural sediments. The model provides a potential methodology for assessing biodegradation rates in natural sediments from inherent biodegradation rates measured in screening tests by accounting for chemical sorption. The present study indicates that a reduced rate of biodegradation is due to a reduced fraction of freely dissolved chemical concentration in the interstitial water, and that the environmental significance of sorption-reduced biodegradation rates needs to be viewed in the context of risk in chemical evaluations. Environ. Toxicol. Chem. 2012; 31: 17301737. (c) 2012 SETAC DOI
43. Lee, YS; Otton, SV; Campbell, DA; Moore, MM; Kennedy, CJ; Gobas, FAPC. (2012) Measuring In Vitro Biotransformation Rates of Super Hydrophobic Chemicals in Rat Liver S9 Fractions Using Thin-Film Sorbent-Phase Dosing.Environmental Science & Technology 46: 410-418 Measuring In Vitro Biotransformation Rates of Super Hydrophobic Chemicals in Rat Liver S9 Fractions Using Thin-Film Sorbent-Phase Dosing
Methods for rapid and cost-effective assessment of the biotransformation potential of very hydrophobic and potentially bioaccumulative chemicals in mammals are urgently needed for the ongoing global evaluation of the environmental behavior of commercial chemicals. We developed and tested a novel solvent-free, thin-film sorbent-phase in vitro dosing system to measure the in vitro biotransformation rates of very hydrophobic chemicals in male Sprague-Dawley rat liver S9 homogenates and compared the rates to those measured by conventional solvent-delivery dosing. The thin-film sorbent-phase dosing system using ethylene vinyl acetate coated vials was developed to eliminate the incomplete dissolution of very hydrophobic substances in largely aqueous liver homogenates, to determine biotransformation rates at low substrate concentrations, to measure the unbound fraction of substrate in solution, and to simplify chemical analysis by avoiding the difficult extraction of test chemicals from complex biological matrices. Biotransformation rates using sorbent-phase dosing were 2-fold greater than those measured using solvent-delivery dosing. Unbound concentrations of very hydrophobic test chemicals were found to decline with increasing S9 and protein concentrations, causing measured biotransformation rates to be independent of S9 or protein concentrations. The results emphasize the importance of specifying both protein content and unbound substrate fraction in the measurement and reporting of in vitro biotransformation rates of very hydrophobic substances, which can be achieved in a thin-film sorbent-phase dosing system. DOI PubMed
42. Raymond-Bouchard, I; Carroll, CS; Nesbitt, JR; Henry, KA; Pinto, LJ; Moinzadeh, M; Scott, JK; Moore, MM. (2012) Structural Requirements for the Activity of the MirB Ferrisiderophore Transporter of Aspergillus fumigatus.Eukaryotic Cell 11: 1333-1344 Structural Requirements for the Activity of the MirB Ferrisiderophore Transporter of Aspergillus fumigatus
Siderophores have been identified as virulence factors in the opportunistic fungal pathogen Aspergillus fumigatus. The 14-pass transmembrane protein MirB is postulated to function as a siderophore transporter, responsible for uptake of the hydroxamate siderophore N,N', N ''-triacetylfusarinine C (TAFC). Our aim was to identify amino acids of A. fumigatus MirB that are crucial for uptake of TAFC. Site-directed mutagenesis was used to create MirB mutants. Expression of wild-type and mutant proteins in the Saccharomyces cerevisiae strain PHY14, which lacks endogenous siderophore transporters, was confirmed by Western blotting. TAFC transport assays using Fe-55-labeled TAFC and growth assays with Fe-TAFC as the sole iron source identified alanine 125, tyrosine 577, loop 3, and the second half of loop 7 (Loop7Del2) as crucial for function, since their substitution or deletion abrogated uptake completely. Wild-type MirB transported ferricrocin and coprogen as well as TAFC but not ferrichrysin. MirB was localized by fluorescence microscopy using antisera raised against a MirB extracellular loop peptide. Immunofluorescence microscopy showed that in yeast, wild-type MirB had a punctate distribution under the plasma membrane, as did the A125D and Y577A strains, indicating that the defect in transport of these mutants was unlikely to be due to mislocalization or degradation. MirB immunolocalization in A. fumigatus showed that the transporter was found in vesicles which cycled between the cytoplasm and the plasma membrane and was concentrated at the hyphal tips. The location of MirB was not influenced by the presence of the siderophore TAFC but was sensitive to internal iron stores. DOI
41. Christians, JK; Cheema, MS; Vergara, IA; Watt, CA; Pinto, LJ; Chen, NS; Moore, MM. (2011) Quantitative Trait Locus (QTL) Mapping Reveals a Role for Unstudied Genes in Aspergillus Virulence.PLOS One 6 Quantitative Trait Locus (QTL) Mapping Reveals a Role for Unstudied Genes in Aspergillus Virulence
Infections caused by the fungus Aspergillus are a major cause of morbidity and mortality in immunocompromised populations. To identify genes required for virulence that could be used as targets for novel treatments, we mapped quantitative trait loci (QTL) affecting virulence in the progeny of a cross between two strains of A. nidulans (FGSC strains A4 and A91). We genotyped 61 progeny at 739 single nucleotide polymorphisms (SNP) spread throughout the genome, and constructed a linkage map that was largely consistent with the genomic sequence, with the exception of one potential inversion of similar to 527 kb on Chromosome V. The estimated genome size was 3705 cM and the average intermarker spacing was 5.0 cM. The average ratio of physical distance to genetic distance was 8.1 kb/cM, which is similar to previous estimates, and variation in recombination rate was significantly positively correlated with GC content, a pattern seen in other taxa. To map QTL affecting virulence, we measured the ability of each progeny strain to kill model hosts, larvae of the wax moth Galleria mellonella. We detected three QTL affecting in vivo virulence that were distinct from QTL affecting in vitro growth, and mapped the virulence QTL to regions containing 7-24 genes, excluding genes with no sequence variation between the parental strains and genes with only synonymous SNPs. None of the genes in our QTL target regions have been previously associated with virulence in Aspergillus, and almost half of these genes are currently annotated as "hypothetical". This study is the first to map QTL affecting the virulence of a fungal pathogen in an animal host, and our results illustrate the power of this approach to identify a short list of unknown genes for further investigation. DOI
40. Oosthuizen, JL; Gomez, P; Ruan, J; Hackett, TL; Moore, MM; Knight, DA; Tebbutt, SJ. (2011) Dual Organism Transcriptomics of Airway Epithelial Cells Interacting with Conidia of Aspergillus fumigatus.PLOS One 6 Dual Organism Transcriptomics of Airway Epithelial Cells Interacting with Conidia of Aspergillus fumigatus
Background: Given the complex nature of the responses that can occur in host-pathogen interactions, dual transcriptomics offers a powerful method of elucidating these interactions during infection. The gene expression patterns of Aspergillus fumigatus conidia or host cells have been reported in a number of previous studies, but each focused on only one of the interacting organisms. In the present study, we profiled simultaneously the transcriptional response of both A. fumigatus and human airway epithelial cells (AECs). Methodology: 16HBE14o-transformed bronchial epithelial cells were incubated with A. fumigatus conidia at 37 degrees C for 6 hours, followed by genome-wide transcriptome analysis using human and fungal microarrays. Differentially expressed gene lists were generated from the microarrays, from which biologically relevant themes were identified. Human and fungal candidate genes were selected for validation, using RT-qPCR, in both 16HBE14o- cells and primary AECs co-cultured with conidia. Principal Findings: We report that ontologies related to the innate immune response are activated by co-incubation with A. fumigatus condia, and interleukin-6 (IL-6) was confirmed to be up-regulated in primary AECs via RT-qPCR. Concomitantly, A. fumigatus was found to up-regulate fungal pathways involved in iron acquisition, vacuolar acidification, and formate dehydrogenase activity. Conclusion: To our knowledge, this is the first study to apply a dual organism transcriptomics approach to interactions of A. fumigatus conidia and human airway epithelial cells. The up-regulation of IL-6 by epithelia and simultaneous activation of several pathways by fungal conidia warrants further investigation as we seek to better understand this interaction in both health and disease. The cellular response of the airway epithelium to A. fumigatus is important to understand if we are to improve host-pathogen outcomes. DOI
39. Telford, JC; Yeung, JHF; Xu, GG; Kiefel, MJ; Watts, AG; Hader, S; Chan, J; Bennet, AJ; Moore, MM; Taylor, GL. (2011) The Aspergillus fumigatus Sialidase Is a 3-Deoxy-D-glycero-D-galacto-2-nonulosonic Acid Hydrolase (KDNase) STRUCTURAL AND MECHANISTIC INSIGHTS.J. Biol. Chem. 286: 10783-10792 The Aspergillus fumigatus Sialidase Is a 3-Deoxy-D-glycero-D-galacto-2-nonulosonic Acid Hydrolase (KDNase) STRUCTURAL AND MECHANISTIC INSIGHTS
Aspergillus fumigatus is a filamentous fungus that can cause severe respiratory disease in immunocompromised individuals. A putative sialidase from A. fumigatus was recently cloned and shown to be relatively poor in cleaving N-acetylneuraminic acid (Neu5Ac) in comparison with bacterial sialidases. Here we present the first crystal structure of a fungal sialidase. When the apo structure was compared with bacterial sialidase structures, the active site of the Aspergillus enzyme suggested that Neu5Ac would be a poor substrate because of a smaller pocket that normally accommodates the acetamido group of Neu5Ac in sialidases. A sialic acid with a hydroxyl in place of an acetamido group is 2-keto-3-deoxynononic acid (KDN). We show that KDN is the preferred substrate for the A. fumigatus sialidase and that A. fumigatus can utilize KDN as a sole carbon source. A 1.45-angstrom resolution crystal structure of the enzyme in complex with KDN reveals KDN in the active site in a boat conformation and nearby a second binding site occupied by KDN in a chair conformation, suggesting that polyKDN may be a natural substrate. The enzyme is not inhibited by the sialidase transition state analog 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) but is inhibited by the related 2,3-didehydro-2,3dideoxy-D-glycero-D-galacto-nonulosonic acid that we show bound to the enzyme in a 1.84-angstrom resolution crystal structure. Using a fluorinated KDN substrate, we present a 1.5-angstrom resolution structure of a covalently bound catalytic intermediate. The A. fumigatus sialidase is therefore a KDNase with a similar catalytic mechanism to Neu5Ac exosialidases, and this study represents the first structure of a KDNase. DOI PubMed
38. Gomez, P; Hackett, TL; Moore, MM; Knight, DA; Tebbutt, SJ. (2010) Functional genomics of human bronchial epithelial cells directly interacting with conidia of Aspergillus fumigatus.BMC Genomics 11: Article 358 Functional genomics of human bronchial epithelial cells directly interacting with conidia of Aspergillus fumigatus
Background: Aspergillus fumigatus (A. fumigatus) is a ubiquitous fungus which reproduces asexually by releasing abundant airborne conidia (spores), which are easily respirable. In allergic and immunocompromised individuals A. fumigatus can cause a wide spectrum of diseases, including allergic bronchopulmonary aspergillosis, aspergilloma and invasive aspergillosis. Previous studies have demonstrated that A. fumigatus conidia are internalized by macrophages and lung epithelial cells; however the exact transcriptional responses of airway epithelial cells to conidia are currently unknown. Thus, the aim of this study was to determine the transcriptomic response of the human bronchial epithelial cell line (16HBE14o-) following interaction with A. fumigatus conidia. We used fluorescence-activated cell sorting (FACS) to separate 16HBE14o-cells having bound and/or internalized A. fumigatus conidia expressing green fluorescent protein from cells without spores. Total RNA was then isolated and the transcriptome of 16HBE14o-cells was evaluated using Agilent Whole Human Genome microarrays. Results: Immunofluorescent staining and nystatin protection assays demonstrated that 16HBE14o-cells internalized 30-50% of bound conidia within six hrs of co-incubation. After FAC-sorting of the same cell culture to separate cells associated with conidia from those without conidia, genome-wide analysis revealed a set of 889 genes showing differential expression in cells with conidia. Specifically, these 16HBE14o-cells had increased levels of transcripts from genes associated with repair and inflammatory processes (e. g., matrix metalloproteinases, chemokines, and glutathione S-transferase). In addition, the differentially expressed genes were significantly enriched for Gene Ontology terms including: chromatin assembly, G-protein-coupled receptor binding, chemokine activity, and glutathione metabolic process (up-regulated); cell cycle phase, mitosis, and intracellular organelle (down-regulated). Conclusions: We demonstrate a methodology using FACs for analyzing the transcriptome of infected and uninfected cells from the same cell population that will provide a framework for future characterization of the specific interactions between pathogens such as A. fumigatus with human cells derived from individuals with or without underlying disease susceptibility. DOI
37. Warwas, ML; Yeung, JHF; Indurugalla, D; Mooers, AO; Bennet, AJ; Moore, MM. (2010) Cloning and characterization of a sialidase from the filamentous fungus, Aspergillus fumigatus.Glycoconjugate Journal 27: 533-548 Cloning and characterization of a sialidase from the filamentous fungus, Aspergillus fumigatus
Sialidase; Sialic acid; Phylogeny; Fungi N-acetylneuraminic acid; Expression
A gene encoding a putative sialidase was identified in the genome of the opportunistic fungal pathogen, Aspergillus fumigatus. Computational analysis showed that this protein has Asp box and FRIP domains, it was predicted to have an extracellular localization, and a mass of 42 kDa, all of which are characteristics of sialidases. Structural modeling predicted a canonical 6-bladed beta-propeller structure with the model's highly conserved catalytic residues aligning well with those of an experimentally determined sialidase structure. The gene encoding the putative Af sialidase was cloned and expressed in Escherichia coli. Enzymatic characterization found that the enzyme was able to cleave the synthetic sialic acid substrate, 4-methylumbelliferyl alpha-D-N-acetylneuraminic acid (MUN), and had a pH optimum of 3.5. Further kinetic characterization using 4-methylumbelliferyl alpha-D-N-acetylneuraminylgalactopyranoside revealed that Af sialidase preferred alpha 2-3-linked sialic acids over the alpha 2-6 isomers. No trans-sialidase activity was detected. qPCR studies showed that exposure to MEM plus human serum induced expression. Purified Af sialidase released sialic acid from diverse substrates such as mucin, fetuin, epithelial cell glycans and colominic acid, though A. fumigatus was unable to use either sialic acid or colominic acid as a sole source of carbon. Phylogenetic analysis revealed that the fungal sialidases were more closely related to those of bacteria than to sialidases from other eukaryotes. DOI PubMed
36. Lam, K; Thu, K; Tsang, M; Moore, M; Gries, G. (2009) Bacteria on housefly eggs, Musca domestica, suppress fungal growth in chicken manure through nutrient depletion or antifungal metabolites.Naturwissenschaften 96: 1127-1132 Bacteria on housefly eggs, Musca domestica, suppress fungal growth in chicken manure through nutrient depletion or antifungal metabolites
DROSOPHILA; LARVAE; COMPETITION; SYMBIONTS; DIPTERA; FLIES; PLANT; FLY
Female houseflies, Musca domestica (Diptera: Muscidae), lay their eggs in ephemeral resources such as animal manure. Hatching larvae compete for essential nutrients with fungi that also colonize such resources. Both the well-known antagonistic relationship between bacteria and fungi and the consistent presence of the bacterium Klebsiella oxytoca on housefly eggs led us to hypothesize (1) that K. oxytoca, and possibly other bacteria on housefly eggs, help curtail the growth of fungal resource competitors and (2) that such fungi indeed adversely affect the development of housefly larvae. Bacteria washed from housefly eggs significantly reduced the growth of fungi in chicken manure. Nineteen bacterial strains and ten fungal strains were isolated from housefly eggs or chicken manure, respectively. Co-culturing each of all the possible bacterium-fungus pairs revealed that the bacteria as a group, but no single bacterium, significantly suppressed the growth of all fungal strains tested. The bacteria's adverse effect on fungi is due to resource nutrient depletion and/or the release of antifungal chemicals. Well-established fungi in resources significantly reduced the number of larval offspring that completed development to adult flies. DOI
34. Pinto, LJ; Moore, MM. (2009) Screening method to identify inhibitors of siderophore biosynthesis in the opportunistic fungal pathogen, Aspergillus fumigatus.Letters in Applied Microbiology 49: 8-13 Screening method to identify inhibitors of siderophore biosynthesis in the opportunistic fungal pathogen, Aspergillus fumigatus
INFECTIONS; VIRULENCE; IRON
Aims: Aspergillus fumigatus is the most common cause of airborne mould infections in immunocompromised patients worldwide. Our aim was to develop a method to identify agents that inhibit siderophore biosynthesis because this pathway is unique to the fungus and is essential for virulence. Methods and Results: A high-throughput two-step screening assay was developed using 96-well plates in which fungal growth and siderophore production is assessed spectrophotometrically. If a compound inhibits growth only in iron-limited medium (screen 1), its effect on siderophore production is then determined (screen 2). The proof of concept was demonstrated using a known antifungal agent, amphotericin B, and a strain of A. fumigatus deficient in siderophore production. Conclusions: The two-stage screening method clearly identified growth defects in A. fumigatus related specifically to siderophore biosynthesis. Significance and Impact of the Study: The increasing incidence of life-threatening fungal infections has produced an urgent need for novel antifungal agents. The method described in this report will facilitate the identification of novel antifungal compounds that inhibit a pathway critical for A. fumigatus virulence and have a reduced probability of affecting host metabolism. DOI
33. Vasiluk, L; Pinto, LJ; Tsang, WS; Gobas, FAPC; Eickhoff, C; Moore, MM. (2008) The uptake and metabolism of benzo[a]pyrene from a sample food substrate in an in vitro model of digestion.Food and Chemical Toxicology 46: 610-618 The uptake and metabolism of benzo[a]pyrene from a sample food substrate in an in vitro model of digestion
benzo[a]pyrene; bioavailability; Caco-2 cells; food; in vitro test
Food ingestion is the major route of exposure to many hydrophobic organic contaminants (HOCs) such as benzo[a]pyrene (BaP). It has been proposed that food-bound HOCs may become bioavailable after their mobilization by gastrointestinal fluids. The purpose of this research was to measure the uptake efficiency of [C-14]-BaP bound to skim milk powder using an in vitro model of gastrointestinal digestion followed by sorption to human enterocytes (Caco-2 cells). Neutralization of intestinal fluids released [C-14]-BaP into the soluble fraction. Ageing of benzo[a]pyrene onto skim milk for 6 months significantly decreased the mobilized fraction but did not affect the amount of benzo[a]pyrene taken up into Caco-2 cells. Hence, significant differences in aqueous phase concentrations may not always be reflected in significant differences in uptake. We obtained evidence that the digestion/uptake of skim milk lipids is accompanied by the diffusive uptake of BaP (the fat flush hypothesis) as trans-cellular transfer of BaP was favoured in the apical to basolateral direction. These data support the theory that non-polar substances including HOCs are preferentially transferred from the lumen into the bloodstream and provide indirect evidence that the uptake is related to the fugacity gradient created by the unidirectional uptake of dietary lipids. (C) 2007 Elsevier Ltd. All rights reserved. DOI
32. Enick, OV; Moore, MM. (2007) Assessing the assessments: Pharmaceuticals in the environment.Environmental Impact Assessment Review 27: 707-729 Assessing the assessments: Pharmaceuticals in the environment
pharmaceuticals; wastewater; values; risk assessment; precautionary principle
The relatively new issue of pharmaceutical contamination of the environment offers the opportunity to explore the application of values to the construction, communication and management of risk. The still-developing regulatory policies regarding environmental contamination with pharmaceuticals provide fertile ground for the introduction of values into the definition and management of risk. In this report, we summarize the current knowledge regarding pharmaceutical contamination of the environment and discuss specific attributes of pharmaceuticals that require special consideration. We then present an analysis showing that if values are incorporated into assessing, characterizing and managing risk, the results of risk assessments will more accurately reflect the needs of various stakeholders. Originating from an acknowledgement of the inherent uncertainty and value-laden nature of risk assessment, the precautionary principle (and later, the multi-criteria, integrated risk assessment), provides a direction for further research and policy development. (c) 2007 Elsevier Inc. All rights reserved. DOI
31. Lam, K; Babor, D; Duthie, B; Babor, EM; Moore, M; Gries, G. (2007) Proliferating bacterial symbionts on house fly eggs affect oviposition behaviour of adult flies.Animal Behaviour 74: 81-92 Proliferating bacterial symbionts on house fly eggs affect oviposition behaviour of adult flies
bacterial symbiont; communication ecology; house fly; microhabitat management; Musca domestica; reproductive strategy; signalling
Animals commonly leave scent messages by depositing pheromones, faeces, or urine. The intensity of a chemical message may fade over time, but the 'intention' remains the same. We argue that house flies, Musca domestica (Diptera: Muscidae), require a message with evolving (sensu changing over time) information content. Gravid females reportedly deploy a pheromone that induces concerted oviposition so that many even-aged larvae ameliorate the resource, such as animal manure. However, continued oviposition by late-arriving females may result in age disparity and cannibalism of larval offspring. Thus, we predicted that house flies have a type of cue that evolves from oviposition induction to inhibition some time after eggs are deposited on a resource. Here we show (1) the existence of such evolving ovipositional cues, (2) the adverse fitness consequences that accrue from ignoring the inhibitory cues and (3) the mechanism by which these cues evolve. The evolving cues depend upon a key bacterial strain, Klebsiella oxytoca, which originates with female M. domestica and which proliferates over time on the surface of deposited eggs. At a threshold density of this strain, further oviposition is inhibited. By deploying such evolving cues, female M. domestica can visit an oviposition site just once and deposit cues that will mediate immediate oviposition induction followed by delayed inhibition, thereby ensuring conditions conducive for offspring development. (C) 2007 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. DOI
29. Vasiluk, L; Pinto, LJ; Walji, ZA; Tsang, WS; Gobas, FAPC; Eickhoff, C; Moore, MM. (2007) Benzo[a]pyrene bioavailability from pristine soil and contaminated sediment assessed using two in vitro models.Environmental Toxicology and Chemistry 26: 387-393 Benzo[a]pyrene bioavailability from pristine soil and contaminated sediment assessed using two in vitro models
benzo[a]pyrene; bioavailability; Caco-2 cells; soil; in vitro tests
A major route of exposure to hydrophobic organic contaminants (HOCs), such as benzo[a]pyrene (BaP), is ingestion. Matrix-bound HOCs may become bioavailable after mobilization by the gastrointestinal fluids followed by sorption to the intestinal epithelium. The purpose of this research was to measure the bioavailability of [C-14] -BaP bound to pristine soils or field-contaminated sediment using an in vitro model of gastrointestinal digestion followed by sorption to human enterocytes (Caco-2 cells) or to a surrogate membrane, ethylene vinyl acetate (EVA) thin film. Although Caco-2 cells had a twofold higher lipid-normalized fugacity capacity than EVA, [C-14]-BaP uptake by Caco-2 lipids and EVA thin film demonstrated a linear relationship within the range of BaP concentrations tested. These results suggest that EVA thin film is a good membrane surrogate for passive uptake of BaP The in vitro system provided enough sensitivity to detect matrix effects on bioavailability; after 5 h, significantly lower concentrations of [C-14]-BaP were sorbed into Caco-2 cells from soil containing a higher percentage of organic matter compared to soil with a lower percentage of organic matter. The [C-14]-BaP desorption rate from Caco-2 lipids consistently was twofold higher than from EVA thin film for all matrices tested. The more rapid kinetics observed with Caco-2 cells probably were due to the greater surface area available for absorption/desorption in the cells. After 5 h, the uptake of BaP into Caco-2 lipid was similar in live and metabolically inert Caco-2 cells, suggesting that the primary route of BaP uptake is by passive diffusion. Moreover, the driving force for uptake is the fugacity gradient that exists between the gastrointestinal fluid and the membrane.
28. Warwas, ML; Watson, JN; Bennet, AJ; Moore, MM. (2007) Structure and role of sialic acids on the surface of Aspergillus fumigatus conidiospores.Glycobiology 17: 401-410 Structure and role of sialic acids on the surface of Aspergillus fumigatus conidiospores
adhesion; fibronectin; high-pressure liquid chromatography; phagocytosis; virulence factor
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 alpha 2,6-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. DOI PubMed
27. Del Rio, LF; Hadwin, AKM; Pinto, LJ; MacKinnon, MD; Moore, MM. (2006) Degradation of naphthenic acids by sediment micro-organisms.J Appl Microbiol 101: 1049-1061 Degradation of naphthenic acids by sediment micro-organisms
cyclohexane carboxylic acid; degradation; GC-MS; naphthenic acids; oil sands; wetlands
Aims: Naphthenic acids (NAs) are naturally occurring, linear and cyclic carboxylic surfactants associated with the acidic fraction of petroleum. NAs account for most of the acute aquatic toxicity of oil sands process-affected water (OSPW). The toxicity of OSPW can be reduced by microbial degradation. The aim of this research was to determine the extent of NA degradation by sediment microbial communities exposed to varying amounts of OSPW. Methods and Results: Eleven wetlands, both natural and process-affected, and one tailings settling pond in Northern Alberta were studied. The natural wetlands and process-affected sites fell into two distinct groups based on their water chemistry. The extent of degradation of a C-14-labelled monocyclic NA surrogate [C-14-cyclohexane carboxylic acid (CCA)] was relatively uniform in all sediments (approximately 30%) after 14 days. In contrast, degradation of a bicyclic NA surrogate [C-14-decahydronaphthoic acid (DHNA)]was significantly lower in non process-affected sediments. Enrichment cultures, obtained from an active tailings settling pond, using commercially available NAs as the sole carbon source, resulted in the isolation of a co-culture containing Pseudomonas putida and Pseudomonas fluorescens. Quantitative GC-MS analysis showed that the co-culture removed > 95% of the commercial NAs, and partially degraded the process NAs from OSPW with a resulting NA profile similar to that from 'aged wetlands'. Conclusions: Exposure to NAs induced and/or selected micro-organisms capable of more effectively degrading bicyclic NAs. Native Pseudomonas spp. extensively degraded fresh, commercial NA. The recalcitrant NAs resembled those found in process-affected wetlands. Significance and Impact of the Study: These results suggest that it may be possible to manipulate the existing environmental conditions to select for a microbial community exhibiting higher rates of NA degradation. This will have significant impact on the design of artificial wetlands for water treatment.
26. Del Rio, LF; Hadwin, AKM; Pinto, LJ; MacKinnon, MD; Moore, MM. (2006) Degradation of naphthenic acids by sediment micro-organisms.J. Appl. Microbiol. 101: 1049-1061 Degradation of naphthenic acids by sediment micro-organisms
cyclohexane carboxylic acid; degradation; GC-MS; naphthenic acids; oil sands; wetlands
Aims: Naphthenic acids (NAs) are naturally occurring, linear and cyclic carboxylic surfactants associated with the acidic fraction of petroleum. NAs account for most of the acute aquatic toxicity of oil sands process-affected water (OSPW). The toxicity of OSPW can be reduced by microbial degradation. The aim of this research was to determine the extent of NA degradation by sediment microbial communities exposed to varying amounts of OSPW. Methods and Results: Eleven wetlands, both natural and process-affected, and one tailings settling pond in Northern Alberta were studied. The natural wetlands and process-affected sites fell into two distinct groups based on their water chemistry. The extent of degradation of a C-14-labelled monocyclic NA surrogate [C-14-cyclohexane carboxylic acid (CCA)] was relatively uniform in all sediments (approximately 30%) after 14 days. In contrast, degradation of a bicyclic NA surrogate [C-14-decahydronaphthoic acid (DHNA)]was significantly lower in non process-affected sediments. Enrichment cultures, obtained from an active tailings settling pond, using commercially available NAs as the sole carbon source, resulted in the isolation of a co-culture containing Pseudomonas putida and Pseudomonas fluorescens. Quantitative GC-MS analysis showed that the co-culture removed > 95% of the commercial NAs, and partially degraded the process NAs from OSPW with a resulting NA profile similar to that from 'aged wetlands'. Conclusions: Exposure to NAs induced and/or selected micro-organisms capable of more effectively degrading bicyclic NAs. Native Pseudomonas spp. extensively degraded fresh, commercial NA. The recalcitrant NAs resembled those found in process-affected wetlands. Significance and Impact of the Study: These results suggest that it may be possible to manipulate the existing environmental conditions to select for a microbial community exhibiting higher rates of NA degradation. This will have significant impact on the design of artificial wetlands for water treatment. DOI PubMed
25. Hadwin, AKM; Del Rio, LF; Pinto, LJ; Painter, M; Routledge, R; Moore, MM. (2006) Microbial communities in wetlands of the Athabasca oil sands: genetic and metabolic characterization.Fems Microbiol Ecol 55: 68-78 Microbial communities in wetlands of the Athabasca oil sands: genetic and metabolic characterization
naphthenic acids; microbial community; PFLA; BIOLOG; DGGE
Naphthenic acids are a complex family of naturally occurring cyclic and acyclic carboxylic acids that are present in the acidic fraction of petroleum. Naphthenic acids are acutely toxic to aquatic organisms. Previous studies showed that wetland sediments exposed to oil sands process water containing naphthenic acids had higher rates of naphthenic acid degradation in vitro compared with unexposed wetlands. In this study we compare the microbial community structures in sediments from wetlands exposed to different amounts of oil sands process water using BIOLOG, phospholipid fatty acid analysis and denaturing gradient gel electrophoresis of total bacterial DNA. Community profiles were compared using cluster analysis. BIOLOG profiles were primarily influenced by seasonal trends rather than naphthenic acids content. In contrast, phospholipid fatty acid analysis comparisons clustered communities that had higher levels of residual oil, although this association was not strong. In contrast, cluster diagrams produced from the denaturing gradient gel electrophoresis data clearly separated bacterial communities according to naphthenic acids concentrations, indicating that naphthenic acids content was a major influence on the composition of the bacterial community. In addition, denaturing gradient gel electrophoresis profiles indicated that naphthenic acids-exposed bacterial communities were homogeneous on a scale of meters, whereas unexposed (off-site) wetlands were less homogeneous.
24. Hadwin, AKM; Del Rio, LF; Pinto, LJ; Painter, M; Routledge, R; Moore, MM. (2006) Microbial communities in wetlands of the Athabasca oil sands: genetic and metabolic characterization.FEMS Microbiol. Ecol. 55: 68-78 Microbial communities in wetlands of the Athabasca oil sands: genetic and metabolic characterization
naphthenic acids; microbial community; PFLA; BIOLOG; DGGE
Naphthenic acids are a complex family of naturally occurring cyclic and acyclic carboxylic acids that are present in the acidic fraction of petroleum. Naphthenic acids are acutely toxic to aquatic organisms. Previous studies showed that wetland sediments exposed to oil sands process water containing naphthenic acids had higher rates of naphthenic acid degradation in vitro compared with unexposed wetlands. In this study we compare the microbial community structures in sediments from wetlands exposed to different amounts of oil sands process water using BIOLOG, phospholipid fatty acid analysis and denaturing gradient gel electrophoresis of total bacterial DNA. Community profiles were compared using cluster analysis. BIOLOG profiles were primarily influenced by seasonal trends rather than naphthenic acids content. In contrast, phospholipid fatty acid analysis comparisons clustered communities that had higher levels of residual oil, although this association was not strong. In contrast, cluster diagrams produced from the denaturing gradient gel electrophoresis data clearly separated bacterial communities according to naphthenic acids concentrations, indicating that naphthenic acids content was a major influence on the composition of the bacterial community. In addition, denaturing gradient gel electrophoresis profiles indicated that naphthenic acids-exposed bacterial communities were homogeneous on a scale of meters, whereas unexposed (off-site) wetlands were less homogeneous. DOI PubMed
23. Minhas, JK; Vasiluk, L; Pinto, LJ; Gobas, FAPC; Moore, MM. (2006) Mobilization of chrysene from soil in a model digestive system.Environmental Toxicology and Chemistry 25: 1729-1737 Mobilization of chrysene from soil in a model digestive system
chrysene; bioavailability; ethylene vinyl acetate; caco-2; mobilization
Accurate estimates for the oral bioavailability of hydrophobic contaminants bound to solid matrices are challenging to obtain because of sorption to organic matter. The purpose of this research was to measure the bioavailability of [C-14]chrysene sorbed to soil using an in vitro model of gastrointestinal digestion and absorption to a surrogate intestinal membrane, ethylene vinyl acetate (EVA) thin film. The [C-14]chrysene moved rapidly from soil into the aqueous compartment and reached steady state within 2 h. Equilibrium was reached in the EVA film within 32 h. Aging the spiked soil for 6 or 12 months had no effect on chrysene mobilization. This was supported by the finding that the data best fit a one-compartment model. Despite significant decreases in [C-14] chrysene mobilization when water or nonneutralized gastrointestinal fluids were used in place of the complete medium, the equilibrium concentration of [C-14]chrysene in EVA film remained the same in all conditions. Thus, the driving force for uptake was the fugacity gradient between the aqueous phase and the EVA film. Cultured human enterocytes (human colorectal carcinoma cell line [Caco-2 cells]) had a higher lipid-normalized fugacity capacity than EVA film, but the elimination rate constants were the same, suggesting that the rate was controlled by the resistance of the unstirred aqueous layer at the membrane-water interface.
22. Hissen, AHT; Moore, MM. (2005) Site-specific rate constants for iron acquisition from transferrin by the Aspergillus fumigatus siderophores N ',N '',N '''-triacetylfusarinine C and ferricrocin.Journal of Biological Inorganic Chemistry 10: 211-220 Site-specific rate constants for iron acquisition from transferrin by the Aspergillus fumigatus siderophores N ',N '',N '''-triacetylfusarinine C and ferricrocin
N ',N '',N '''-triacetylfusarinine C; ferricrocin; transferrin; kinetics; Aspergillus fumigatus
Aspergillus fumigatus is an opportunistic fungal pathogen that causes life-threatening infections in immunocompromised patients. Despite low levels of free iron, A. fumigatus grows in the presence of human serum in part because it produces high concentrations of siderophores. The most abundant siderophores produced by A. fumigatus are N',N&DPRIME;,N'''-triacetylfusarinine C (TAF) and ferricrocin, both of which have thermodynamic iron binding constants that theoretically allow them to remove transferrin (Tf)-bound iron. Urea-polyacrylamide gel electrophoresis was used to measure the change in concentration of Tf species incubated with TAF or ferricrocin. The rate of removal of iron from diferric Tf by both siderophores was measured, as were the individual microscopic rates of iron removal from each Tf species (diferric Tf, N-terminal monoferric Tf and C-terminal monoferric Tf). TAF removed iron from all Tf species at a faster rate than ferricrocin. Both siderophores showed a preference for removing C-terminal iron, evidenced by the fact that k(1C) and k(2C) were much larger than k(1N) and k(2N). Cooperativity in iron binding was observed with TAF, as the C-terminal iron was removed by TAF much faster from monoferric than from diferric Tf. With both siderophores, C-terminal monoferric Tf concentrations remained below measurable levels during incubations. This indicates that k(2C) and k(1C) are much larger than k(1N). TAF and ferricrocin both removed Tf-bound iron with second-order rate constants that were comparable to those of the siderophores of several bacterial pathogens, indicating they may play a role in iron uptake in vivo and thereby contribute to the virulence of A. fumigatus.
21. Hissen, AHT; Wan, ANC; Warwas, ML; Pinto, LJ; Moore, MM. (2005) The Aspergillus fumigatus siderophore biosynthetic gene sidA, encoding L-ornithine N-5-oxygenase, is required for virulence.Infection and Immunity 73: 5493-5503 The Aspergillus fumigatus siderophore biosynthetic gene sidA, encoding L-ornithine N-5-oxygenase, is required for virulence
Aspergillus fumigatus is the leading cause of invasive mold infection and is a serious problem in immuno-compromised populations worldwide. We have previously shown that survival of A. fumigatus in serum may be related to secretion of siderophores. In this study, we identified and characterized the sidA gene of A. fumigatus, which encodes L-ornithine N-5-oxygenase, the first committed step in hydroxamate siderophore biosynthesis. A. fumigatus sidA codes for a protein of 501 amino acids with significant homology to other fungal L-ornithine N-5-oxygenases. A stable Delta sidA strain was created by deletion of A. fumigatus sidA. This strain was unable to synthesize the siderophores N',N",N'''-triacetylfusarinine C (TAF) and ferricrocin. Growth of the Delta sidA strain was the same as that of the wild type in rich media; however, the Delta sidA strain was unable to grow in low-iron defined media or media containing 10% human serum unless supplemented with TAF or ferricrocin. No significant differences in ferric reduction activities were observed between the parental strain and the Delta sidA strain, indicating that blocking siderophore secretion did not result in upregulation of this pathway. Unlike the parental strain, the Delta sidA strain was unable to remove iron from human transferrin. A rescued strain (Delta sidA + sidA) was constructed; it produced siderophores and had the same growth as the wild type on iron-limited media. Unlike the wild-type and rescued strains, the Delta sidA strain was avirulent in a mouse model of invasive aspergillosis, indicating that sidA is necessary for A. fumigatus virulence.
20. Vasiluk, L; Pinto, LJ; Moore, MM. (2005) Oral bioavailability of glyphosate: Studies using two intestinal cell lines.Environmental Toxicology and Chemistry 24: 153-160 Oral bioavailability of glyphosate: Studies using two intestinal cell lines
glyphosate; oral bioavailability; caco-2; ileum epithelial cells-18
Glyphosate is a commonly used nonselective herbicide that inhibits plant growth through interference with the production of essential aromatic amino acids. In vivo studies in mammals with radiolabeled glyphosate have shown that 34% of radioactivity was associated with intestinal tissue 2 h after oral administration. The aim of our research was to investigate the transport, binding, and toxicity of glyphosate to the cultured human intestinal epithelial cell line, Caco-2, and the rat small intestinal crypt-derived cell line, ileum epithelial cells-18 (IEC-18). An in vitro analysis of the transport kinetics of [C-14]-glyphosate showed that 4 h after exposure, approximately 8% of radiolabeled glyphosate moved through the Caco-2 monolayer in a dose-dependent manner. Binding of glyphosate to cells was saturable and approximately 4 x 10(11) binding sites/cell were estimated from bound [C-14]. Exposure of Caco-2 cells to greater than or equal to10 mg/ml glyphosate reduced transmembrane electrical resistance (TEER) by 82 to 96% and increased permeability to [H-3]-mannitol, indicating that paracellular permeability increased in glyphosate-treated cells. At 10-mg/ml glyphosate, both IEC-18 and Caco-2 cells showed disruption in the actin cytoskeleton. In Caco-2 cells, significant lactate dehydrogenase leakage was observed when cells were exposed to 15 mg/ml of glyphosate. These data indicate that at doses > 10 mg/ml, glyphosate significantly disrupts the barrier properties of cultured intestinal cells.
19. Wasylnka, JA; Hissen, AHT; Wan, ANC; Moore, MM. (2005) Intracellular and extracellular growth of Aspergillus fumigatus.Medical Mycology 43: S27-S30 Intracellular and extracellular growth of Aspergillus fumigatus
Aspergillus fumigatus; endosomes; trafficking; siderophores
Aspergillus fumigatus is a filamentous fungus that can cause a life-threatening systemic mycosis in immunocompromised patients. We have studied the growth of A. fumigatus inside cultured cells, and the extracellular growth requirements (in serum). We measured the uptake of bound conidia by the cultured human type 11 pneumocyte cell line (A549) and a mouse macrophage cell line (J774). The extent of internalization was determined using a nystatin protection assay and by confocal microscopy. Both assays showed that A549 cells internalized 30% of bound conidia after three hours. In contrast, the value for J774 cells was 90%. In both J774 and A549 cells, conidia entered the endosomal pathway and ultimately co-localized with lysosomal markers. Lysosomes containing conidia were acidified. Internalized conidia germinated, and after 24-36 h of incubation with A549 cells, the hyphal tips of some intracellular germlings became exposed to the extracellular space. The importance of iron acquisition to extracellular growth was assessed by creating a strain of A. fumigatus in which the gene encoding the first step of hydroxamate siderophore biosynthesis, ornithine N-5-oxygenase (AfusidA), was inactivated by gene replacement. Mutant strains were avirulent in a mouse model of invasive aspergillosis indicating that siderophore biosynthesis is a virulence factor in A. fumigatus.
18. Hissen, AHT; Chow, JMT; Pinto, LJ; Moore, MM. (2004) Survival of Aspergillus fumigatus in serum involves removal of iron from transferrin: the role of siderophores.Infection and Immunity 72: 1402-1408 Survival of Aspergillus fumigatus in serum involves removal of iron from transferrin: the role of siderophores
Aspergillus fumigatus is a filamentous fungus which can cause invasive disease in immunocompromised individuals. A. fumigatus can grow in medium containing up to 80% human serum, despite very low concentrations of free iron. The purpose of this study was to determine the mechanism by which A. fumigatus obtains iron from the serum iron-binding protein transferrin. In iron-depleted minimal essential medium (MEM), A. fumigatus growth was supported by the addition of holotransferrin (holoTf) or FeCl3 but not by the addition of apotransferrin (apoTf). Proteolytic degradation of transferrin by A. fumigatus occurred in MEM-serum; however, transferrin degradation did not occur until late logarithmic phase. Moreover, transferrin was not degraded by A. fumigatus incubated in MEM-holoTf. Urea polyacrylamide gel electrophoresis showed that in MEM-holoTf, holoTf was completely converted to apoTf by A.fumigatus. In human serum, all of the monoferric transferrin was converted to apoTf within 8 h. Siderophores were secreted by A. fumigatus after 8 h of growth in MEM-sermn and 12 h in MEM-holoTf. The involvement of small molecules in iron acquisition was confirmed by the fact that transferrin was deferrated by A. fumigatus even when physically separated by a 12-kDa-cutoff membrane. Five siderophores were purified from A. fumigatus culture medium, and the two major siderophores were identified as triacetylfusarinine C and ferricrocin. Both triacetylfusarinine C and ferricrocin removed iron from holoTf with an affinity comparable to that of ferrichrome. These data indicate that A. fumigatus survival in human serum in vitro involves siderophore-mediated removal of iron from transferrin. Proteolytic degradation of transferrin may play a secondary role in iron acquisition.
17. Jurgensen, A; Widmeyer, JR; Gordon, RA; Bendell-Young, LI; Moore, MM; Crozier, ED. (2004) The structure of the manganese oxide on the sheath of the bacterium Leptothrix discophora: An XAFS study.American Mineralogist 89: 1110-1118 The structure of the manganese oxide on the sheath of the bacterium <i>Leptothrix discophora</i>: An XAFS study
In natural waters, manganese oxides (MnOx) are important in mediating the bioavailability of trace metals such as Ni, Cu, Zn, Cd, and Pb, as these metals readily adsorb to the MnOx surface. Manganese from a variety of anthropogenic sources usually enters the aquatic environment in dissolved form as Mn-2divided by. It is subsequently oxidized under oxic and neutral (pH = 6-7) conditions. Often this oxidation is catalyzed by bacteria, such as Leptothrix discophora, as part of their natural metabolic process. Mn K-edge X-ray Absorption Fine Structure Spectroscopy (XAFS) was used to investigate the local structure of manganese oxide on the sheath produced by the bacterium Leptothrix discophora SP-6. The features observed in the near edge region of the Mn K-edge spectrum indicate the presence of three oxidation states of manganese: Mn-2divided by, Mn-3divided by, and Mn-4divided by. Fitting the experimental XAFS data identifies the bacterial MnOx as being composed of single-layer microcrystals with layers similar to those occurring in Na-birnessite. Some MnO6 octahedra might lie outside the layer plane, sharing corners with those in the layer plane. X-ray diffraction results for the same samples are consistent with the single-layer structure.
16. Widmeyer, JR; Crozier, ED; Moore, MM; Jurgensen, A; Bendell-Young, LI. (2004) Role of Leptothrix discophora in mediating metal uptake in the filter-feeding bivalve Mytilus trossulus (edulis).Environmental Science & Technology 38: 769-774 Role of Leptothrix discophora in mediating metal uptake in the filter-feeding bivalve Mytilus trossulus (edulis)
The potential for filter-feeding bivalves to accumulate metals from a wide range of food sources is an important consideration when examining trophic transfer of metals up the food chain. The objective of this study was to determine the role of Leptothrix discophora in mediating metal uptake in the filter-feeding bivalve Mytilus trossulus. The bacterium L. discophora SP-6 was cultured in the absence or presence of Mn, allowing for a naturally formed Mn oxide sheath to develop. Secondary metals (Cd and Pb) were then added to the cultures, allowing for potential Cd and Pb adsorption to the Mn oxide sheath. Resulting bacterial aggregates of known diameter were then fed to the bivalve M. trossulus using a flow-through system. Initial concentrations of both Pb and Cd on the bacterium did not differ significantly in the presence or absence of the Mn oxide; conversely both Pb (F = 7.39, p < 0.0001) and Cd (F = 33.65, P < 0.0001) were found at lower concentrations in the mussel tissue when the Mn oxide was present. To determine whether these differences in metal uptake could be attributed to sorting by the mussel based on food quality, nutritional analysis was performed. Bacterial food matrixes containing Mn oxides were found to have significantly lower levels of carbon (F = 256, p < 0.0001). Particle clearance rates for the various food matrixes were positively correlated with organic content (R-2 = 0.852, p > 0.008). The results of our study suggest that metal uptake in M. trossulus was significantly decreased for Cd with a similar trend for Pb when the SP-6 sheath contained Mn oxides. The mechanism mediating this differential uptake is best explained by food quality, in that a higher quality food source enhanced metal uptake due to an increased clearance rate of organic-rich particles by M. trossulus.
15. Wasylnka, JA; Moore, MM. (2003) Aspergillus fumigatus conidia survive and germinate in acidic organelles of A549 epithelial cells.J. Cell Sci. 116: 1579-1587 Aspergillus fumigatus conidia survive and germinate in acidic organelles of A549 epithelial cells
Aspergillus fumigatus; phagosome; germination; A549; J774
Aspergillus fumigatus is an environmental mould that can cause invasive disease in the immunocompromised host. Previous work has shown that conidia can be internalized by lung epithelial cells (A549) and murine macrophages (J774) in vitro. Therefore, the purpose of this study was to determine the fate of A. fumigatus conidia within the endosomal network of these cells. Co-localization of conidia expressing green fluorescent protein with proteins present in the early endosomal (CD71) and lysosomal (CD63, LAMP-1) membrane was assessed using confocal microscopy. In J774 cells, 75 % of internalized conidia were found in phagosomes containing LAMP-1 120 minutes post-infection. In A549 cells, 55 % and 58 % of internalized conidia were found to co-localize with LAMP-1 and CD63 by 24 hours. Cathepsin D also co-localized with internalized conidia in A549 cells. Phagosomes containing conidia were shown to be acidified in both cell types. Less than 1% of the initial inoculum survived in J774 cells by 12 hours post-infection. After 24 hours, 3 % of internalized conidia survived in A549 cells and 34% of these had germinated. By 36 hours, the germlings were able to escape the phagosome and form extracellular hyphae without lysis of the host cell. DOI PubMed
14. Gifford, AHT; Klippenstein, JR; Moore, MM. (2002) Serum stimulates growth of and proteinase secretion by Aspergillus fumigatus.Infection and Immunity 70: 19-26 Serum stimulates growth of and proteinase secretion by Aspergillus fumigatus
Serum contains iron-binding proteins, which inhibit the growth of most pathogenic microorganisms, including fungi. The purpose of this research was to investigate the effect of serum on growth of the opportunistic fungal pathogen Aspergillus fumigatus. Supplementing minimal essential medium (MEM) with up to 80% human serum or up to 80% fetal bovine serum (FBS) stimulated growth and increased the amount of A. fumigatus dry biomass approximately fourfold. In addition, a 100-fold increase in proteinase secretion, as measured by azocasein hydrolysis, was observed when 10% human serum or 10% FBS was added to MEM. The fungal proteinases secreted in serum-containing media were shown to degrade H-3-labeled basal lamina proteins. The factor in serum that stimulated proteinase secretion was larger than 10 kDa and was 85% inactivated when the serum was heated for 30 min at 66 degreesC. The proportions of proteinases of each catalytic class secreted by A. fumigatus in the presence of serum were different from the proportions secreted in media containing single proteins. Proteinase secretion did not result from increased protein concentration in the medium per se because bovine serum albumin (BSA) at a concentration equivalent to the concentration of serum produced only 20% of the proteinase activity per milligram (dry weight) that was produced by FBS. Addition of BSA plus 100 muM FeCl3 to MEM resulted in the same level of growth as addition of serum, indicating that a combination of nutritional factors in serum may stimulate growth. However, the level of proteinase secretion was still only 30% of the level observed with FBS. These data indicate that serum does not inhibit the growth of A. fumigatus and that the nutrients in serum result in high levels of proteinase secretion, potentially increasing the invasiveness of this species.
13. Wasylnka, JA; Moore, MM. (2002) Uptake of Aspergillus fumigatus conidia by phagocytic and nonphagocytic cells in vitro: Quantitation using strains expressing green fluorescent protein.Infect. Immun. 70: 3156-3163 Uptake of Aspergillus fumigatus conidia by phagocytic and nonphagocytic cells in vitro: Quantitation using strains expressing green fluorescent protein
Several pathogenic fungal organisms enter eukaryotic cells and manipulate the host cell environment to favor their own growth and survival. Aspergillus fumigatus is a saprophytic fungus that causes invasive lung disease in the immunocompromised host. To determine whether A. fumigatus could enter eukaryotic cells, we studied the uptake of two different GFP-expressing A. fumigatus strains into A549 lung epithelial cells, human umbilical vein endothelial (HUVE) cells, and J774 murine macrophages in vitro. A549 cells internalized 30% of the bound conidia whereas HUVE and J774 cells internalized 50 and 9011, respectively. Conidia within A549 cells remained viable for 6 h; however, 60 to 80% of conidia within J774 cells were killed after only 4 h, Live and heat-killed conidia were internalized to the same extent by A549 cells. After 6 h, almost none of the conidia inside A549 cells had germinated, whereas extracellular conidia had developed germ tubes. Internalization of conidia by A549 cells was a temperature-dependent process and required rearrangement of the underlying host cell cytoskeleton; uptake was inhibited by 75% with 0.5 muM cytochalasin D and by 65% with 5 muM colchicine. Fluorescent labeling of infected A549 cells with rhodamine phalloidin provided visible evidence of cytoskeletal alteration as many of the intracellular conidia were contained in actin-coated phagosomes. These data provide evidence that significant numbers of A. fumigatus conidia can be internalized by nonprofessional phagocytes in vitro and these cells may serve as reservoirs for immune cell evasion and dissemination throughout the host. DOI PubMed
12. Wasylnka, JA; Simmer, MI; Moore, MM. (2001) Differences in sialic acid density in pathogenic and non-pathogenic Aspergillus species.Microbiology-Uk 147: 869-877 Differences in sialic acid density in pathogenic and non-pathogenic Aspergillus species
Aspergillus fumigatus; invasive aspergillosis; lectin; flow cytometry; adhesion
Aspergillus fumigatus is a ubiquitous soil fungus that causes invasive lung disease in the immunocompromised host. The structure of the conidial wall has not keen well characterized although it is thought that adhesins present on the surface are involved in attachment of the conidia to host lung cells and proteins, which is a prerequisite for the establishment of infection. Negatively charged carbohydrates on the conidial surface have been previously identified as the molecules responsible for attachment of conidia to extracellular matrix proteins. The aim of this research was to identify carbohydrates on the conidial surface that contribute to its negative charge. Direct chemical analysis and indirect binding assays have demonstrated that A. fumigatus possesses sialic acids on the conidial surface. Pre-treatment of A. fumigatus conidia with sialidase decreased binding of a sialic acid-specific lectin, Limax flavus agglutinin (LFA), to the conidial surface and decreased adhesion of conidia to the positively charged polymer poly L-lysine. Two other sialic acid-specific lectins, Maackia amurensis agglutinin and Sambucus nigra agglutinin, exhibited negligible binding to A. fumigatus conidia indicating that 2,3-alpha- and 2,6-alpha -linked sialic acids are not the major structures found on the conidial surface. Mild acid hydrolysis and purification of conidial wall carbohydrates yielded a product that had the same R-F as the Neu5Ac standard when analysed by high-performance thin-layer chromatography. A density of 6.7 x 10(5) sialic acid residues per conidium was estimated using a colorimetric assay. Conidia grown on a minimal medium lacking sialic acid also reacted with LFA, indicating that sialic acid biosynthesis occurs de novo. Sialic acid biosynthesis was shown to be regulated by nutrient composition: the density of sialic acids on the surface of conidia grown in minimal media was tower than that observed when conidia were grown on rich, complex media. It has previously been shown that pathogenic Aspergillus species adhere to basal lamina proteins to a greater extent than non-pathogenic Aspergillus species. To determine whether the expression of sialic acid on the conidial surface was correlated with adhesion to basal lamina, conidia from other non-pathogenic Aspergillus species were tested for their reactivity towards LFA. Flow cytometric analysis demonstrated that A. fumigatus had a significantly greater sialic acid density than three non-pathogenic Aspergillus species. Sialic acids on the conidial wall may be involved in adhesion to fibronectin, a component of the basal lamina, as binding of A. fumigatus conidia to fibronectin was strongly inhibited in the presence of a sialylated glycoprotein.
11. Bendell-Young, LI; Bennett, KE; Crowe, A; Kennedy, CJ; Kermode, AR; Moore, MM; Plant, AL; Wood, A. (2000) Ecological characteristics of wetlands receiving an industrial effluent.Ecological Applications 10: 310-322 Ecological characteristics of wetlands receiving an industrial effluent
anthropogenic impacts; benthic community structure; ecosystem characteristics; fish acute lethality and stress; oil sands; wetlands
The primary objectives of this study were to evaluate the ecological characteristics of wetland ecosystems that had developed in response to oil sands effluent relative to reference wetland ecosystems and, from such an evaluation, to assess whether these wetlands were viable systems capable of integrating into the northern Canadian landscape. A secondary objective was to evaluate the use of several ecologically relevant endpoints as indicators of an ecosystem response to a known anthropogenic stress, in this case, wetlands receiving oil sands effluent. To achieve this, a suite of endpoints were compared between effluent-impacted wetlands and nonimpacted reference wetlands. Endpoints for comparison included: (1) benthic macroinvertebrate community structure, (2) chironomid density and biomass, (3) the incidence of chironomid mentum deformities, (4) the mutagenetic potential of sediment-dwelling chironomids, (5) growth and photosynthetic rare for the aquatic plant Typha latifolia (cattail), and (6) fish acute lethality and stress response as measured by changes in blood chemistry (percentage hematocrit [%hct], percentage leucocrit [%lct], and differential white blood cell count). Wetlands receiving oil sands effluent supported a low-diversity benthic community, dominated primarily by the Chironomidae and cattail. There was no evidence of mentum deformities or mutagenicity in chironomids sampled from the oil-impacted wetlands. Cattails grown in oil sands effluent and sediment demonstrated increased photosynthetic rates; however, these increased rates did not translate into increased plant growth. In contrast to the benthic community and the cattail, indigenous fish were unable to survive in wetlands containing oil sands effluent. Fish displayed altered blood chemistry (elevated %hct, depressed %lct) and ultimately death when held beyond 14 d in the oil-impacted wetlands. Of the various ecological endpoints measured, the macroinvertebrate community and changes in fish blood chemistry were the most sensitive indicators of an anthropogenic stress, demonstrating distinct differences in response between impacted and reference wetlands. To ensure that these wetlands can safely integrate into the northern Canadian landscape, future studies need to focus on their impacts at higher trophic levels indigenous to the wetland.
10. Launen, LA; Pinto, LJ; Percival, PW; Lam, SFS; Moore, MM. (2000) Pyrene is metabolized to bound residues by Penicillium janthinellum SFU403.Biodegradation 11: 305-312 Pyrene is metabolized to bound residues by Penicillium janthinellum SFU403
bioremediation; fungi; PAH; quinone; semiquinone; sorption
We have previously shown that the filamentous fungus, Penicillium janthinellum SFU403 (SFU403) oxidizes pyrene to pyrene 1,6- and 1,8-quinones and that the level of pyrenequinones (PQs) subsequently declines suggesting that PQs are not terminal metabolites. The purpose of this study was to determine the fate of PQs in SFU403. First, we compared the fate of C-14-pyrene in SFU403 and a non-pyrene-oxidizing fungus, a Paecilomyces sp.. After 7 days of incubation, more than 80% of the radioactivity was cell-associated in both fungi; however, while 90% of the C-14 could be extracted from the Paecilomyces sp. as unmetabolized pyrene, 65-80% of the bound radioactivity remained inextractable from SFU403. Further evidence that pyrene oxidation to PQs was required for irreversible binding was obtained by comparing the extent of C-14 bound to SFU403 when it was grown for 21 days under conditions that resulted in differing amounts of C-14-pyrene oxidation. The results showed that approximate to 40% of the inextractable products were bound residues derived from pyrene metabolites. The balance (60%) could be attributed to strong sorption of unreacted pyrene. We used electron paramagnetic resonance spectroscopy and oxygen consumption studies to demonstrate that both NADPH and glutathione can reduce PQs by one electron to their corresponding semiquinone anion radicals in vitro. These studies demonstrate that PQs are metabolized by SFU403 to bound residues, possibly via semiquinone intermediates. DOI PubMed
9. Melathopoulos, AP; Winston, ML; Whittington, R; Smith, T; Lindberg, C; Mukai, A; Moore, M. (2000) Comparative laboratory toxicity of neem pesticides to honey bees (Hymenoptera : Apidae), their mite parasites Varroa jacobsoni (Acari : Varroidae) and Acarapis woodi (Acari : Tarsonemidae), and brood pathogens Paenibacillus larvae and Ascophaera apis.Journal of Economic Entomology 93: 199-209 Comparative laboratory toxicity of neem pesticides to honey bees (Hymenoptera : Apidae), their mite parasites Varroa jacobsoni (Acari : Varroidae) and Acarapis woodi (Acari : Tarsonemidae), and brood pathogens Paenibacillus larvae and Ascophaera apis
Varroa; Acarapis; acaricide; bioassay; neem; oil; tau-fluvalinate
Laboratory bioassays were conducted to evaluate neem oil and neem extract for the management of key honey bee (Apis mellifera L.) pests. Neem pesticides inhibited the growth of Paenibacillus larvae (Ash, Priest & Collins) in vitro but had no effect on the growth of Ascophaera al,is (Olive & Spiltoir). Azadirachtin-rich extract (neem-aza) was 10 times more potent than crude neem oil (neem oil) against P. larvae suggesting that azadirachtin is a main antibiotic component in neem. Neem-aza, however, was ineffective at controlling the honey bee mite parasites Varroa jacobsoni (Ouduemans) and Acarapis woodi (Rennie). Honey bees also were deterred from feeding on sucrose syrup containing >0.01 mg/ml of neem-aza. However, neem oil applied topically to infested bees in the laboratory proved highly effective against both mite species. Approximately 50-90% V. jacobsoni mortality was observed 48 h after treatment with associated bee mortality lower than 10%. Although topically applied neem oil did not result in direct A. woodi mortality, it offered significant protection of bees from infestation by A. woodi. Other vegetable and petroleum-based oils also offered selective control of honey bee mites, suggesting neem oil has both a physical and a toxicological mode of action. Although oils are not as selective as the V. Jacobsoni acaricide tau-fluvalinate, they nonetheless hold promise for the simultaneous management of several honey bee pests.
8. Pinto, LJ; Moore, MM. (2000) Release of polycyclic aromatic hydrocarbons from contaminated soils by surfactant and remediation of this effluent by Penicillium spp.Environmental Toxicology and Chemistry 19: 1741-1748 Release of polycyclic aromatic hydrocarbons from contaminated soils by surfactant and remediation of this effluent by Penicillium spp.
surfactant; bioremediation; soil; polycyclic aromatic hydrocarbons; Penicillium
Studies in which surfactants have been employed to increase the bioavailability of soil-bound polycyclic aromatic hydrocarbons (PAHs) have not yielded consistent results. Surfactant mobilization of high molecular weight (MV) PAHs from contaminated soils has not been extensively studied; therefore, the purpose of this research was to compare the extent of release of freshly added high MW C-14-PAH with aged PAM from four different PAH-contaminated soils using a nonionic detergent, Tween 80, and to determine whether Tween 80-solubilized C-14-PAH in soil washings could be degraded by indigenous microorganisms or by added Penicillium spp. Only very high concentrations of Tween 80 (>1,000 times the critical micelle concentration [CMC] for 3 of 4 soils) were able to mobilize bound C-14-pyrene, -chrysene, and -benzo[a]pyrene. The concentration of surfactant required to release 50% of bound C-14-PAH (the SC50 value) ranged from 5 to 30 gn depending on soil type; a modest correlation was found (0.512) between the fraction of organic carbon in the soil and the SC50 value. At 10(4) x CMC, Tween 80 released an average of 75% of bound C-14-PAH and 64% of the aged PAM, indicating that the C-14-PAH release only slightly overestimated PAM mobilization from weathered soil. An exception was one soil that had been previously remediated in which <30% of the PAHs were released. The PAM structure had a negligible effect on the mobilization by surfactant because the solubilization curves for all three PAHs were very similar. Tween 80-solubilized C-14-pyrene readsorbed to soil when the surfactant concentration dropped below 10(3) x CMC. Greater than 90% of the C-14-pyrene in the soil washing effluent could be removed by the addition of spores of active PAM-oxidizing Penicillium spp. plus nutrients. In contrast, <10% of C-14-pyrene was oxidized by the indigenous soil bacteria under the same conditions.
7. Wasylnka, JA; Moore, MM. (2000) Adhesion of Aspergillus species to extracellular matrix proteins: Evidence for involvement of negatively charged carbohydrates on the conidial surface.Infection and Immunity 68: 3377-3384 Adhesion of Aspergillus species to extracellular matrix proteins: Evidence for involvement of negatively charged carbohydrates on the conidial surface
Invasive lung disease caused by Aspergillus species is a potentially fatal infection in immunocompromised patients. The adhesion of Aspergillus fumigatus conidia to proteins in the basal lamina is thought to be an initial step in the development of invasive aspergillosis, The purpose of this study was to determine the mechanism of adhesion of A. fumigatus conidiospores to basal-lamina proteins and to determine whether conidia possess unique adhesins which allow them to colonize the host. We compared conidia from different Aspergillus species for the ability to bind to purified fibronectin and intact basal lamina. Adhesion assays using immobilized fibronectin or type II pneumocyte-derived basal lamina showed that A. fumigatus conidia bound significantly better than those of other Aspergillus species to both fibronectin and intact basal lamina. Neither desialylation nor complete deglycosylation of fibronectin decreased the binding of A. fumigatus conidia to fibronectin, suggesting that oligosaccharides on fibronectin were not involved in conidiospore binding. Further evidence for this hypothesis came from experiments using purified fragments of fibronectin; A. fumigatus conidia preferentially bound to the nonglycosylated 40-kDa fragment which contains the glycosaminoglycan (GAG) binding domain. Negatively charged carbohydrates, including dextran sulfate and heparin, as well as high-ionic-strength buffers, inhibited binding of A. fumigatus conidia to both fibronectin and intact basal lamina, suggesting that negatively charged carbohydrates on the surface of the conidium may bind to the GAG binding domain of fibronectin and other basal-lamina proteins. These data provide evidence for a novel mechanism of conidial attachment whereby adherence to fibronectin and other basal-lamina proteins is mediated via negatively charged carbohydrates on the conidial surface.
6. Launen, LA; Pinto, LJ; Moore, MM. (1999) Optimization of pyrene oxidation by Penicillium janthinellum using response-surface methodology.Applied Microbiology and Biotechnology 51: 510-515 Optimization of pyrene oxidation by Penicillium janthinellum using response-surface methodology
At present, there is little information on the optimization of the degradation of polycyclic aromatic hydrocarbons (PAH) by deuteromycete filamentous fungi, a reaction catalyzed by cytochrome P450 monooxygenases. We utilized response-surface methodology to determine the optimal growth conditions for the oxidation of the PAH pyrene by Penicillium janthinellum SFU403, with respect to the variables glucose concentration, nitrate concentration and bioconversion time. Models were derived for the relationship between the variables tested and the level of the pyrene oxidation products, 1-pyrenol (1-PY) and pyrenequinones (PQ). Production of 1-PY and PQ were optimized by the same glucose and nitrate concentrations: 2.5% glucose and 1.5% sodium nitrate. The optimized I-PY and PQ bioconversion times were 71 h and 73 h respectively. These conditions improved the yield of 1-PY by fivefold and PQ were more than 100-fold higher than the baseline levels obtained in this study. The optimized PQ yield represented 95% of the initial pyrene, thus the total optimised pyrene bioconversion to 1-PY and PQ was approximately 100%. Concentrations of glucose exceeding 4.0% repressed pyrene hydroxylation. Pyrene hydroxylation occurred almost exclusively during the deceleration phase of culture growth.
5. Roberts, K; Parameswaran, M; Moore, M; Muller, RS. (1999) A silicon microfabricated aperture for counting cells using the aperture impedance technique.Canadian Journal of Electrical and Computer Engineering-Revue Canadienne de Genie Electrique et Informatique 24: 109-113 A silicon microfabricated aperture for counting cells using the aperture impedance technique
Design. fabrication and testing of two distinct miniature blood-cell counters are presented. The first design utilizes a deep RIE silicon etch and anodic bonding technique to produce a compact flow cell capable of counting cells using the aperture impedance technique. The second design demonstrates a simpler aperture fabrication process that appears to be more suited for miniature blood-cell counters. Tests conducted indicate that the microfabricated aperture is capable of giving signals comparable to those of currently available desktop blood-cell counters.
4. Amaar, YG; Moore, MM. (1998) Mapping of the nitrate assimilation gene cluster (crnA-niiA-niaD) and characterization of the nitrite reductase gene (niiA) in the opportunistic fungal pathogen Aspergillus fumigatus.Current Genetics 33: 206-215 Mapping of the nitrate assimilation gene cluster (crnA-niiA-niaD) and characterization of the nitrite reductase gene (niiA) in the opportunistic fungal pathogen Aspergillus fumigatus
Aspergillus fumigatus; nitrate assimilation; nitrite reductase; fungal promoters
This study reports the molecular characterization of the nitrate-assimilation gene cluster from the opportunistic fungal pathogen Aspergillus fumigatus. A genomic fragment was isolated which contained the entire structural gene encoding nitrite reductase (niiA), plus segments of the nitrate reductase (niaD) and the nitrate transporter (crnA) genes. Nitrate-assimilation genes in A. fumigatus are physically linked and transcribed in the same direction as in A. nidulans. The nitrate-assimilation gene cluster is on the largest chromosome (5.3 Mb). The nitrite reductase (niiA) gene encodes a protein of 1110 amino ac ids that contains regions corresponding to FAD, NADPH, FeS and siroheme binding sites. Eight small introns interrupt the niiA open reading frame. The niaD-niiA intergenic regulatory region contains promoter consensus sequences including TATA, CAAT, and binding sites for the areA and nirA gene products. Northern analysis indicated that the expression of niaD, niiA and crnA are induced by nitrate and repressed by ammonium at the transcriptional level.
3. Kiehlmann, E; Pinto, L; Moore, M. (1996) The biotransformation of chrysene to trans-1,2-dihydroxy-1,2-dihydrochrysene by filamentous fungi.Canadian Journal of Microbiology 42: 604-608 The biotransformation of chrysene to trans-1,2-dihydroxy-1,2-dihydrochrysene by filamentous fungi
chrysene; polycyclic aromatic hydrocarbons; filamentous fungi; bioremediation; cytochrome P450
The purpose of this study was to assess the ability of filamentous fungi isolated from petroleum-contaminated soils to oxidize chrysene. Only 4 of the 17 isolates known to oxidize pyrene and benzo[a]pyrene were found to produce polar products when incubated in the presence of chrysene and Tween 80: Penicillium janthinellum, Syncephalastrum racemosum, and 2 Penicillium spp. Trans-1,2-dihydroxy-1,2-dihydrochrysene was identified by H-1-NMR as one of three fungal metabolites. The extent of bioconversion to diol was approximately 3% of chrysene in 6 days. Experiments to increase chrysene oxidation with other polycyclic aromatic hydrocarbons were not successful. To our knowledge, this is the first identification of a chrysene metabolite from any microorganism and the first report of fungal oxidation of chrysene.
2. Lai, JWS; Pinto, LJ; Kiehlmann, E; BendellYoung, LI; Moore, MM. (1996) Factors that affect the degradation of naphthenic acids in oil sands wastewater by indigenous microbial communities.Environmental Toxicology and Chemistry 15: 1482-1491 Factors that affect the degradation of naphthenic acids in oil sands wastewater by indigenous microbial communities
naphthenic acids; oil sands; carboxylic acids; biodegradation; mineralization
The acute toxicity of wastewater generated during the extraction of bitumen from oil sands is believed to be due to naphthenic acids (NAs). To determine the factors that affect the rate of degradation of representative NAs in microcosms containing wastewater and the acute toxicity of treated and untreated wastewater, the effects of temperature, dissolved oxygen concentration, and phosphate addition on the rate of (CO2)-C-14 release from two representative naphthenic acid substrates, (linear) U-C-14-palmitic acid (PA) and (bicyclic) decahydro-2-naphthoic acid 8-C-14 (DHNA), were monitored. Tailings pond water (TPW) contained microorganisms well adapted to mineralizing both PA and DHNA: PA was degraded more quickly (10-15% in 4 weeks) compared to DHNA (2-4% in 8 weeks). On addition of phosphate, the rate of NA degradation increased up to twofold in the first 4 weeks, with a concurrent increase in the rate of oxygen consumption by oil sands TPW. The degradation rate then declined to levels equivalent to those measured in flasks without phosphate. The observed plateau was not due to phosphate Limitation. Decreases in either the dissolved oxygen concentration or the temperature reduced the rate. Phosphate addition also significantly decreased the acute toxicity of TPW to fathead minnows. In contrast, Microtox(R) analyses showed no reduction in the toxicity of treated or untreated TPW after incubation for up to 8 weeks at 15 degrees C.
1. Milanova, R; Stoynov, N; Moore, M. (1996) The optimization of triptoquinone production by Cunninghamella elegans using factorial design.Enzyme and Microbial Technology 19: 86-93 The optimization of triptoquinone production by Cunninghamella elegans using factorial design
factorial design; Cunninghamella elegans; Tripterygium wilfordii; diterpenes; hydroxylation
Previous studies in our laboratory have shown that the synthetic abietane diterpene, triprophenolide is metabolized by the filamentous fungus, Cunninghamella elegans to three products: triptoquinone, 5 alpha,14-dihydroxybutenolide, and 14 beta-glucosyltriptophenolide in yields of 35, 12, and 5%, respectively. The purpose of this study was to increase the yield of the triptoquinone while simultaneously decreasing the production of the other metabolites. The effects of four factors (glucose concentration, nutrient broth concentration, malt extract concentration, and biotransformation lime) on the yield of triptoquinone were assessed using a sequential factorial design. Biotransformation time was critical for the production of triptoquinone whereas the concentration of the medium components affected the yield of triptoquinone to a lesser extent The optimal factor levels for its formation (41% yield) did not correspond to the settings for maximal biomass production. Similarly, the pH of the growth medium was nor correlated to the yield to triptoquinone. A second optimization experiment was performed using factor levels within a narrower range of the settings determined from the first experiment. The yield of triptoquinone predicted by the mathematical model increased to 70% and this value was confirmed experimentally.
