Biomedical subjects

Michael J Bidochka

Publications and source records attributed to Michael J Bidochka.

18 recordsLinked to original sources

PubMed2006

Increased insect virulence in Beauveria bassiana strains overexpressing an engineered chitinase.

Entomopathogenic fungi are currently being used for the control of several insect pests as alternatives or supplements to chemical insecticides. Improvements in virulence and speed of kill can be achieved by understanding the mechanisms of fungal pathogenesis and genetically modifying targeted genes, thus improving the commercial efficacy of these biocontrol agents. Entomopathogenic fungi, such as Beauveria bassiana, penetrate the insect cuticle utilizing a plethora of hydrolytic enzymes, including chitinases, which are important virulence factors. Two chitinases (Bbchit1 and Bbchit2) have previously been characterized in B. bassiana, neither of which possesses chitin-binding domains. Here we report the construction and characterization of several B. bassiana hybrid chitinases where the chitinase Bbchit1 was fused to chitin-binding domains derived from plant, bacterial, or insect sources. A hybrid chitinase containing the chitin-binding domain (BmChBD) from the silkworm Bombyx mori chitinase fused to Bbchit1 showed the greatest ability to bind to chitin compared to other hybrid chitinases. This hybrid chitinase gene (Bbchit1-BmChBD) was then placed under the control of a fungal constitutive promoter (gpd-Bbchit1-BmChBD) and transformed into B. bassiana. Insect bioassays showed a 23% reduction in time to death in the transformant compared to the wild-type fungus. This transformant also showed greater virulence than another construct (gpd-Bbchit1) with the same constitutive promoter but lacking the chitin-binding domain. We utilized a strategy where genetic components of the host insect can be incorporated into the fungal pathogen in order to increase host cuticle penetration ability.

Amino Acid Sequence↗

PubMed2006

Expression of genes involved in germination, conidiogenesis and pathogenesis in Metarhizium anisopliae using quantitative real-time RT-PCR.

Characterization of genes involved in germination, conidiogenesis and insect pathogenesis is an important step in identifying methods to increase the efficacy of Metarhizium anisopliae, a commercially important entomopathogenic fungus. Real-time RT-PCR is a sensitive, reproducible and quantitative method to study gene expression. However, it requires reliable reference gene transcripts for normalization. In this study, six putative housekeeping genes (act, gpd, 18sRNA, tef, try and ubi) were investigated as reliable reference genes. Transcripts from tef, gpd and try were found to be the most suitable reference genes for real-time RT-PCR analysis of genes expressed during germination, conidiogenesis and pathogenesis. Using these as reference genes, the relative expression levels of a virulence gene, a subtilisin-like protease (pr1), a regulator of G protein signaling gene involved in conidiogenesis (cag8), the nitrogen response regulator gene (nrr1), and a hydrophobin gene (ssga) were studied. None of these transcripts could be detected in the early stages of insect pathogenesis. The nitrogen response regulator, nrr1, was consistently expressed during all developmental stages. Expression levels of cag8 increased significantly in the later stages of conidiogenesis on insect cadavers. The expression level of ssga during conidiogenesis was significantly higher than that in mycelia during vegetative growth in nutrient rich media. The pr1 gene was expressed during fungal conidiation on the insect cadaver. This study acts as a foundation for investigating the transcriptional levels of genes expressed during germination, conidiogenesis and pathogenesis of M. anisopliae using real-time RT-PCR.

Animals↗

PubMed2006

The host acts as a genetic bottleneck during serial infections: an insect-fungal model system.

The genetic variation of a pathogen population is a pivotal component of pathogen evolution, having important implications for emerging diseases, nosocomial infections, and laboratory subculturing practices. Furthermore, it is undoubtedly altered during infection of a host. We address this issue using an insect-fungal model system to examine the influence of serial host passage on the genetic variation of a pathogen population. Using amplified fragment length polymorphism, a strain of the opportunistic fungus, Aspergillus flavus, showing initially 98% genetic similarity, was assessed for changes in genetic diversity during repeated passage through Galleria mellonella larvae and compared to that of a parallel population serially subcultured on artificial media. In two independent trials, the genetic diversity of the population passed through the insect dropped significantly, while the genetic variation of the population subcultured on media increased or remained unchanged. However, there were no changes in virulence or the production of protease or aflatoxin, indicating an apparent lack of selection. We suggest that the insect acted as a genetic bottleneck, reducing the genetic diversity of the A. flavus population. The ability of a host to produce a genetic bottleneck in a pathogen population impacts our understanding of emerging diseases, nosocomial infections, and laboratory subculturing practices.

Animals↗

PubMed2006

Psc-AFP, an antifungal protein with trypsin inhibitor activity from Psoralea corylifolia seeds.

An antifungal protein designated as Psc-AFP, with an apparent molecular mass of 18kDa, was isolated from a traditional Chinese herb, malaytea scurfpea (Psoralea corylifolia L.). The isolation procedure entailed extraction, cation exchange chromatography on CM FF, gel filtration chromatography on Superdex 75 and reversed-phase high performance liquid chromatography on SOURCE 5RPC column. Automated Edman degradation determined the partial N-terminal sequence of Psc-AFP to be NH2-EWEPVQNGGSSYYMVPRIWA, which displayed homology with plant trypsin inhibitors. The protease inhibitor activity of Psc-AFP was then confirmed by the inhibition on trypsin. Psc-AFP at 10 microM inhibited the mycelial growth of Alternari brassicae, Aspergillus niger, Fusarium oxysporum and Rhizoctonia cerealis, suggesting that Psc-AFP has a role in the defense against pathogens.

Amino Acid Sequence↗

PubMed2006

A cysteine/methionine auxotroph of the opportunistic fungus Aspergillus flavus is associated with host-range restriction: a model for emerging diseases.

The evolution of host specialization in pathogens is a topic of considerable interest, particularly since it can represent a decisive step in the emergence of infectious diseases. Aspergillus flavus is an opportunistic fungus capable of infecting a wide variety of hosts, including plants, insects and mammals, although with low virulence. Here the derivation of an A. flavus strain that exhibits severe host restriction is reported. This strain exhibited a severe diminution or a complete lack of conidial production on a variety of standard agar media and on various plant species. However, it retained its ability to infect insects from various orders and to re-emerge from and adequately conidiate on the insect cadavers as a culmination of the pathogenic life cycle. This strain, demonstrating insect-dependent conidiation, was discovered to be a cysteine/methionine auxotroph due to an inability to reduce sulfate to sulfite. However, other A. flavus auxotrophs tested for plant and insect host range failed to show insect-dependent conidiation. An association between this specific auxotroph and a decreased host range is shown, emphasizing the role of nutrition in the host-pathogen relationship with respect to host restriction and evolution towards obligate pathogenesis.

Animals↗

PubMed2006

Developing insect models for the study of current and emerging human pathogens.

The study of human diseases requires the testing of microorganisms in model systems. Although mammals are typically used, we argue the validity of using insects as models in order to examine human diseases, particularly the growing number of opportunistic microorganisms. Insects can be used in large numbers, are easily manipulated, and are not subject to the same ethical concerns as mammalian systems. Insects and mammals have many parallels with respect to microbial pathogenesis, from proteinaceous integuments that require breaching before infection to similarities in their innate immune responses. Reactions of insects to Candida and Pseudomonas spp. infections show good correlation with mouse models, providing precedent-setting examples of the study of human pathogens using insects. Insects as pathogen hosts also warrant study because they may act as reservoirs for emerging human pathogens. Finally, insect models may be used to examine the evolutionary processes involved in the acquisition of virulence factors and host-jumping mechanisms indispensable to emerging pathogens. Insect models may be used in 'niche' investigations where large sample sizes can facilitate rapid, informative screening of opportunistic diseases and provide insights into pathogen evolution, while reducing the cost and ethical concerns associated with mammalian models.

Animals↗

PubMed2006

Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens.

A simple, highly efficient, and reliable Agrobacterium tumefaciens-mediated transformation method was developed for the insect pathogenic fungus Metarhizium anisopliae. Expression of the green fluorescent protein gene, egfp, and the benomyl resistance gene, benA3, were used as markers in transformed M. anisopliae. Transformation efficiencies were dependent on the strain of A. tumefaciens used. With strain AGL-1, 17.0 +/- 1.4 transformants per plate could be obtained using conidial concentrations of 10(6) conidia/mL and a 2 day co-cultivation in the presence of 200 micromol/L acetosyringone. On the other hand, transformations using strain LBA4404 were unsuccessful. Ten transformants were tested by Southern analysis and found to contain a single copy T-DNA. Twenty transformants were subcultured for five generations on nonselective media, and 95% of the transformants were mitotically stable. Agrobacterium tumefaciens-mediated transformation of M. anisopliae can serve as a useful tool to investigate genes involved in insect pathogenicity.

Agrobacterium tumefaciens↗

PubMed2005

Up-regulation of Prl, a subtilisin-like protease, during conidiation in the insect pathogen Metarhizium anisopliae.

Metarhizium anisopliae exhibits two different developmental patterns under nutrient-deprived conditions: appressorium formation in early stages and conidiation in late stages of pathogenesis in its insect hosts. In this study we isolated genes enriched during mature conidial production under nutrient-deprived conditions in M. anisopliae by using the method of suppression subtractive hybridization. We sequence-identified seven conidiation-associated genes (cag) in M. anisopliae. One of the genes, cag7, encoded an extracellular subtilisin-like protease, Pr1, that plays a fundamental role in cuticular protein degradation. Reverse-transcription polymerase chain reaction (RT-PCR) analysis confirmed that cag cDNAs are expressed during the development of mature conidia under nutrient-deprived conditions. RT-PCR analysis was also performed for Pr1 during infection of greater wax moth larvae (Galleria mellonella). Results showed up-regulation of Pr1 in the infected insect as the mycelia emerge and produce conidia on the surface of the cadaver. It is well documented that Pr1 is produced during the initial stages of transcuticular penetration by M. anisopliae. Here we show that Pr1 is also up-regulated during the final stages of pathogenesis as the fungus emerges from the dead host and subsequently conidiates on the cadaver.

Animals↗

PubMed2005

Recombination within sympatric cryptic species of the insect pathogenic fungus Metarhizium anisopliae.

Metarhizium anisopliae is an insect pathogenic fungus with a worldwide distribution. It is being developed and used as a biocontrol agent against a wide range of insect pests but relatively little is known of the life history of this fungus. We tested hypotheses concerning reproductive isolation and recombination in a sample of heat-active (ability to grow at 37 degrees C) and cold-active (ability to grow at 8 degrees C) sympatrically occurring isolates of M. anisopliae from Ontario, Canada by assaying nucleotide sequence variation at six polymorphic loci: the internally transcribed spacer (ITS) region of the nuclear ribosomal DNA repeat, and portions of calmodulin (CAL), chitin synthase (CHS), subtilisin-like protease (PR1), neutral trehalase (NTL) and actin (ACT)-encoding genes. The most parsimonious trees constructed showed a topology consistent with the heat-active and cold-active isolates as two monophyletic groups. We then applied Genealogical Concordance Phylogenetic Species Recognition (GCPSR) to the genealogical trees and concluded that the transition from concordance among branches to incongruity among branches delimited two species of M. anisopliae within Ontario. The GCPSR of two species was supported by intraspecific incongruity within each species when tested using the Partition Homogeneity test, indicating recombination. The GCPSR of two species also corresponded to the heat-active and cold-active groups. As the groups are morphologically indistinguishable we applied the term 'cryptic species'. Therefore, the sympatrically occurring heat-active and cold-active isolates represent different cryptic species with a history of recombination among isolates within each species.

DNA, Fungal↗

PubMed2005

Serial passage of the opportunistic pathogen Aspergillus flavus through an insect host yields decreased saprobic capacity.

To study the early stages of the effect of host restriction on pathogen evolution, we subjected the opportunistic fungus Aspergillus flavus to a serial propagation scheme, whereby insect-virulent conidia were selected for repeated passage through an insect host (Galleria mellonella larvae) for 5 generations. Of the 35 lineages promulgated through this scheme, there were no consistent changes in virulence, which was measured by percent mortality of the larvae. There were, however, increases in the number of conidia on the insect cadavers (9 of 35 lineages) and decreases in the number of days between death and the appearance of fungal growth on the cadavers (4 of 35 lineages). Notably, most of the lineages (28 of 35 lineages) demonstrated a statistically significant decrease in the diameter of the colonies subcultured onto artificial media, indicating a decreased ability to grow saprobically. Conversely, most of the A. flavus cultures successively grown on agar media (9 of 10 lineages) exhibited no change in colony diameter after 15 rounds of subculturing. Propagation of the opportunist A. flavus through the insect host G. mellonella resulted in a diminished capacity to grow on an alternate substrate, while maintaining or increasing its ability to use the host as a nutrient supply.

Animals↗

PubMed2004

Nucleotide sequence variation does not relate to differences in kinetic properties of neutral trehalase from the insect pathogenic fungus Metarhizium anisopliae.

Genetic variability in a putative virulence factor, the neutral trehalase ( Ntl) gene, was examined in strains of the insect pathogenic fungi Metarhizium anisopliae and Metarhizium flavoviride by restriction fragment length polymorphism (RFLP). The Ntl gene was sequenced from four of these strains that showed dissimilar RFLP patterns. Enzyme kinetic experiments were also performed on the partially purified neutral trehalase in order to assess whether nucleotide changes in these strains related to differences in enzyme catalytic function (i.e., Km, Vmax, and Kcat). Finally, the Metarhizium strains were assessed in bioassays against waxworm larvae in order to relate nucleotide variation with Ntl enzyme kinetics and insect virulence. The greatest RFLP variation was observed with Rsa1. M. flavoviride was found to be most dissimilar in RFLP patterns when compared with the M. anisopliae strains. RFLP patterns for Ntl were diagnostic markers for previously studied genetic groups of M. anisopliae. Comparisons of Ntl sequences showed that the introns were found to be more variable (6.2%) than the exons (3.1%). Comparisons of the translated nucleotide codons showed high levels (91%) of synonymous sequence variation between strains. Another fraction of the remaining mutations was neutral, resulting in amino acid substitutions with similar functions. The neutral trehalase was partially purified by preparative isoelectric focus, revealing a single band of enzyme activity as assessed by analytical isoelectric focusing (pI ca. 5). Kinetic properties of the neutral trehalases revealed no differences between the M. anisopliae strains, while the M. flavovoride had a lower Kcat/Km. However, there was lower virulence in one strain that showed Ntl enzyme kinetic properties that were similar to the other strains, suggesting that factors other than neutral trehalase may be responsible for delimiting virulence in this insect pathogenic fungi. Although there is nucleotide variation in genes involved in pathogenicity, this variation is mostly neutral in nature, and there is strong stabilizing selection to maintain enzyme function.

Amino Acid Substitution↗

PubMed2004

West Nile virus infection rates in pooled and individual mosquito samples.

The detection of West Nile virus (WNV) in mosquitoes by real-time RT-PCR provides valuable information on the epidemiology of the virus and identifies mosquito species that are potential vectors. Testing sets of pooled mosquitoes of the same species is logistically the easiest and most cost-effective approach for WNV testing; however, little information is available on how the results of small pooled sets relate to those of testing individual mosquitoes. During the 2002 outbreak, we compared pooled and individual samples of two mosquito species (Culex pipiens and Culex restuans) collected from three Health Unit regions in Ontario, Canada. Significantly more Cx. restuans were infected with WNV compared to Cx. pipiens. We show that with pool sizes of five individuals both MIR (minimum infection rates) and MLE (maximum likelihood estimation) values were acceptable in estimating infection rates.

Animals↗

PubMed2002

Genetic groups of the insect-pathogenic fungus Beauveria bassiana are associated with habitat and thermal growth preferences.

A persistent paradigm in insect pathology is one that relates the insect host to certain genetic groups of insect-pathogenic fungi. This paradigm assumes that the genotype of an insect-pathogenic fungus coevolves with a certain taxon of insect host that it infects. The insect-pathogenic fungus Beauveria bassiana shows a wide host range and is considered to be a facultative insect pathogen. In this study, a population genetics analysis of B. bassiana from forested and agricultural habitats as well as from the Canadian Arctic showed distinct genetic groups associated with the three different habitats. Within each group, recombining population structures and clonally reproducing lineages were observed. The B. bassiana isolates were also assessed for their abilities to grow at 8, 15, 25 and 37 degrees C and for their tolerances to UV exposure. The genetic groups from the Arctic and from the forested habitats grew at lower temperatures, while the genetic group from the agricultural habitat grew at 37 degrees C and was tolerant to UV exposure. There were no clear associations between the genetic group and the ability to infect coleopteran or lepidopteran insect larvae. There is increasing evidence that such studies represent a significant paradigm shift; habitat selection, not insect host selection, drives the population structure of deuteromycetous insect-pathogenic fungi. We suggest that adaptation to a certain habitat type is an important criterion for identifying insect-pathogenic fungal strains for use in insect biocontrol efforts.

Adaptation, Psychological↗

PubMed2002

Attenuation of fungal infection in thermoregulating Locusta migratoria is accompanied by changes in hemolymph proteins.

Hemolymph proteins in the locust, Locusta migratoria migratorioides infected with the fungus Metarhizium anisopliae var acridum were analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under conditions that allowed locusts to thermoregulate, 2 proteins, ITB1 (ca. 18kDa) and ITB2 (ca. 13kDa) were induced 48h post inoculation. In contrast, under non-thermoregulating conditions, only 1 band, INTB1 (ca. 18kDa) was induced with similar molecular mass to ITB1. ITB1 and ITB2 were N-terminally sequenced but showed little homology to known proteins. The induction of hemolymphal proteins in infected, thermoregulating locusts and implication in insect immune defence are discussed.

Animals↗

PubMed2002

Microsatellite analysis of environmental and clinical isolates of the opportunist fungal pathogen Aspergillus fumigatus.

Microsatellite analysis was used to examine the genetic relatedness of 111 clinical and environmental isolates of the opportunist human pathogenic fungus Aspergillus fumigatus from Ontario, Canada. Forty-three A. fumigatus isolates were from clinical sources and 68 from environmental sources. Phylogenetic analysis of the genotypes revealed that there were no geographical or temporal associations of clinical or environmental genotypes. In fact, several of the environmental and clinical isolates showed identical (clonal) genotypes from disparate geographical areas. However, a locus by locus examination revealed that there were several significant differences in allele frequencies between clinical and environmental isolates. There may be linkage of certain microsatellite loci with genes affecting virulence in A. fumigatus. A susceptible individual may be equally predisposed to infection by any isolate of A. fumigatus. However, under transient selection as a pathogen, genes encoding alleles for enhanced virulence may not assort independently from microsatellite loci. A dynamic equilibrium may exist between random recombination of loci in the natural environment and selection for virulence factors during host infection cycles.

Aspergillosis↗

PubMed2002

Protein analysis in a pleomorphically deteriorated strain of the insect-pathogenic fungus Metarhizium anisopliae.

Pleomorphic deterioration is a process where a fungal isolate loses the ability to produce conidia during repeated subculturing. We have previously isolated strains of the entomopathogenic fungus Metarhizium anisopliae that have irreversibly lost the ability to produce conidia and only produce mycelia when grown on agar. Gel electrophoresis was used to examine differences in intracellular protein patterns (urea-soluble proteins and urea-insoluble proteins (i.e., hydrophobins)) in conidiating and mycelial cultures of M. anisopliae. Two major proteins present in a conidiating culture and one from a mycelial culture were N-terminally sequenced but showed no homologies to known proteins. The presence of hydrophobins in conidiating and mycelial cultures was also examined, and it was shown that these proteins were abundant in conidiating cultures but not in mycelial cultures. We also used primers designed from regulatory genes involved in conidiation in Aspergillus nidulans. The amplified fragments were not homologous to A. nidulans genes.

Amino Acid Sequence↗

PubMed1999

Nuclear rDNA phylogeny in the fungal genus Verticillium and its relationship to insect and plant virulence, extracellular proteases and carbohydrases.

Phylogenetic relationships among 18 isolates in the genus Verticillium, representing 13 species of diverse econutritional groups (pathogens of insects, plants, mushrooms, nematodes and spiders, and saprobes), were examined by using sequences from the internal transcribed spacer (ITS) and small nuclear (NS) rRNA regions. The isolates were also assessed for their abilities to infect insect larvae (Galleria mellonella) and to cause necrosis in alfalfa (Medicago sativa), and for their proteolytic, chitinolytic and pectinolytic activities. The phylogenetic data suggested that Verticillium is polyphyletic in origin and is therefore a form genus. However, the phylogenetic tree supported the plant pathogens (V. dahliae, V. albo-atrum and V. nigrescens) as a clade. The alfalfa isolate of V. albo-atrum (isolate 595) was an interesting outlier to the main body of plant pathogens as it clustered with the insect pathogen V. indicum. Strains of V. lecanii and V. indicum were able to infect insects and are present in divergent groups in the consensus tree, suggesting that the ability to infect insects may have evolved independently many times. Similarly, the nematophagous Verticillium species appear to have evolved independently along several different routes and one isolate, V. chlamydosporium, was able to infect insects. V. albo-atrum, V. nigrescens and V. dahliae all produced high levels of enzymes capable of degrading pectin, a major component of plant cell walls. The ability to excrete pectinase was a broad indicator of the ability to produce lesions on alfalfa. In the plant pathogens, the functions of a broad-spectrum protease were assumed by trypsins which degrade Bz-AA-AA-Arg-NA substrates (Bz, benzoyl; AA, various amino acids; NA, p-nitroanilide). The insect pathogens and mushroom pathogen (V. fungicola) were characterized by production of high levels of subtilisin-like proteases active against a chymotrypsin substrate (succinyl-Ala2-Pro-Phe-NA) and the inability to clear pectin. The insect and mushroom pathogens, and several nematode pathogens, were distinguishable from the plant pathogens in their ability to produce chitinases.

Animals↗

PubMed1995

An inner cell wall protein (cwp1) from conidia of the entomopathogenic fungus Beauveria bassiana.

Following the removal of the rodlet layer from aerial or submerged conidia of the entomopathogenic deuteromycetous fungus Beauveria bassiana, SDS-insoluble, formic-acid-extractable proteins were found in the residual cell wall material. Two major proteins (12.8 and 14.0 kDa) were extracted with formic acid from fractured aerial and submerged conidia but not from blastospores. Oxidation of the sample extracted by formic acid resulted in a single protein band (15.4 kDa) as judged by SDS-PAGE. Antibodies against this cell wall protein (cwp1) did not cross-react with cell wall extracts from the entomopathogenic deuteromycetous fungi Verticillium lecanii or Metarhizium anisopliae. Western blot analysis of two-dimensional gels revealed at least three acidic isoforms (pI 4.0-4.8) of cwp1. Immunohistological studies revealed that the cwp1 was primarily localized in the cell wall of aerial and submerged conidia but not in blastospores. Immunolocalization was possible only if the conidia were previously boiled in 5% (v/v) beta-mercaptoethanol. The N-terminal sequence of cwp1 showed no similarities with other published sequences. Our results suggest that at least two major species of SDS-insoluble, formic-acid-extractable proteins exist in cell walls of B. bassiana aerial or submerged conidia; one is the hydrophobin which occurs in the outermost rodlet layer and the other, cwp1, occurs primarily next to the rodlet layer.

Amino Acids↗