The nematophagous fungus Verticillium chlamydosporium: aspects of pathogenicity

Verticillium chlamydosporium is a fungal pathogen of eggs and females of plantparasitic nematodes. The fungus produced an alkaline serine protease in submerged culture. This enzyme, VCPI, was characterized as a class II subtilisin, based on amino acid sequenceh omology. Several of its characteristics, e.g. molecular mass (33 kDa), pI (ca 10) and broad substrate utilisation, are typical of fungal subtilisins. Although some immunological cross-reactivity existed with other enzymes of this class, an antigenic fingerprint was obtained that was distinct, even from the subtilisin that was its closest homologue based on amino acid sequence, PrI from the entomogenous fungus Metarhizium anisopliae. There was circumstantial evidence, suggesting that this fungal protease was involved in the infection of nematode eggs, which have a largely proteinaceous eggshell. First of all, the enzyme was able to remove the outer protein layer from eggs of the susceptible root-knot nematode, Meloidogyne incognita, exposing the underlying chitin layer. Scanning electron microscopy revealed that fungal hyphae on the egg surface left an imprint, presumably through enzymatic action. There was also evidence of the protease weakening the eggshell, as enzyme-treated nematode eggs were more easily lysed and infected by the fungus than those not pre-incubated in the enzyme. A polyclonal antibody against VCPI demonstrated protease production by the fungus, prior to, or concurrent with, penetration. The enzyme was associated with appressoria, i.e. fungal infection structures. In contrast to the susceptible root-knot nematode, VCPI had little impact on the egg shell of the potato cyst nematode Globodera rostochiensis. It is suggested that the limited in situ hydrolysis of G. rostochiensis egg shell proteins is a factor contributing to its relative resistance to the fungus. Regulation studies in batch culture showed that production of the protease VCPI was repressed by high carbon and nitrogen levels. Its basic regulatory mechanism was that of repression/derepression. However, the highest protease titre was obtained when M incognita eggs were present in the medium, suggesting induction by the host. Collagen and chitin were possibly responsible for this inductive effect. In conclusion, it is believed that VCPI is a protease with a dual role for V chlamydosporium. During saprotrophic growth, VCP1 would allow the fungus to scavenge nutrients from a wide range of protein sources. However, the enzyme also has a designated function in penetration of the host, which makes it a versatile tool for a fungus that can switch trophic modes during its life-cycle. The achievements of this research include the first demonstration in a nematode-attacking fungus of: -a well-characterized protease, including data on stability, kinetics and isoforms; -a subtilisin-like protease in an egg-parasitic nematophagous fungus; -a pathogenicity-related enzyme in V chlamydosporium; -a determinant of host specificity; - enzyme regulation in general, and induction by the host, in particular

The nematophagous fungus Verticillium chlamydosporium: aspects of pathogenicity

Verticillium chlamydosporium is a fungal pathogen of eggs and females of plantparasitic nematodes. The fungus produced an alkaline serine protease in submerged culture. This enzyme, VCPI, was characterized as a class II subtilisin, based on amino acid sequenceh omology. Several of its characteristics, e.g. molecular mass (33 kDa), pI (ca 10) and broad substrate utilisation, are typical of fungal subtilisins. Although some immunological cross-reactivity existed with other enzymes of this class, an antigenic fingerprint was obtained that was distinct, even from the subtilisin that was its closest homologue based on amino acid sequence, PrI from the entomogenous fungus Metarhizium anisopliae. There was circumstantial evidence, suggesting that this fungal protease was involved in the infection of nematode eggs, which have a largely proteinaceous eggshell. First of all, the enzyme was able to remove the outer protein layer from eggs of the susceptible root-knot nematode, Meloidogyne incognita, exposing the underlying chitin layer. Scanning electron microscopy revealed that fungal hyphae on the egg surface left an imprint, presumably through enzymatic action. There was also evidence of the protease weakening the eggshell, as enzyme-treated nematode eggs were more easily lysed and infected by the fungus than those not pre-incubated in the enzyme. A polyclonal antibody against VCPI demonstrated protease production by the fungus, prior to, or concurrent with, penetration. The enzyme was associated with appressoria, i.e. fungal infection structures. In contrast to the susceptible root-knot nematode, VCPI had little impact on the egg shell of the potato cyst nematode Globodera rostochiensis. It is suggested that the limited in situ hydrolysis of G. rostochiensis egg shell proteins is a factor contributing to its relative resistance to the fungus. Regulation studies in batch culture showed that production of the protease VCPI was repressed by high carbon and nitrogen levels. Its basic regulatory mechanism was that of repression/derepression. However, the highest protease titre was obtained when M incognita eggs were present in the medium, suggesting induction by the host. Collagen and chitin were possibly responsible for this inductive effect. In conclusion, it is believed that VCPI is a protease with a dual role for V chlamydosporium. During saprotrophic growth, VCP1 would allow the fungus to scavenge nutrients from a wide range of protein sources. However, the enzyme also has a designated function in penetration of the host, which makes it a versatile tool for a fungus that can switch trophic modes during its life-cycle. The achievements of this research include the first demonstration in a nematode-attacking fungus of: -a well-characterized protease, including data on stability, kinetics and isoforms; -a subtilisin-like protease in an egg-parasitic nematophagous fungus; -a pathogenicity-related enzyme in V chlamydosporium; -a determinant of host specificity; - enzyme regulation in general, and induction by the host, in particular

Effekt van organische materialen op nematoden Case study : chitine versus meloidogyne goeldi (tylenchidae)

SIGLEKULeuven Campusbibliotheek Exacte Wetenschappen / UCL - Université Catholique de LouvainBEBelgiu

Accuracy of 4 different algorithms for the analysis of tomographic radionuclide ventriculography using a physical, dynamic 4-chamber cardiac phantom

Various automatic algorithms are now being developed to calculate left ventricular (LV) and right ventricular (RV) ejection fraction from tomographic radionuclide ventriculography. We tested the performance of 4 of these algorithms in estimating LV and RV volume and ejection fraction using a dynamic 4-chamber cardiac phantom. Methods: We developed a realistic physical, dynamic 4-chamber cardiac phantom and acquired 25 tomographic radionuclide ventriculography images within a wide range of end-diastolic volumes, end-systolic volumes, and stroke volumes. We assessed the ability of 4 algorithms (QBS, QUBE, 4D-MSPECT, and BP-SPECT) to calculate LV and RV volume and ejection fraction. Results: For the left ventricle, the correlations between reference and estimated volumes (0.93, 0.93, 0.96, and 0.93 for QBS, QUBE, 4D-MSPECT, and BP-SPECT, respectively; all with P < 0.001) and ejection fractions (0.90, 0.93, 0.88, and 0.92, respectively; all with P < 0.001) were good, although all algorithms underestimated the volumes (mean difference [+/-2 SDs] from Bland-Altman analysis: -39.83 +/- 43.12 mL, -33.39 +/- 38.12 mL, -33.29 +/- 40.70 mL, and -16.61 +/- 39.64 mL, respectively). The underestimation by QBS, QUBE, and 4D-MSPECT was greater for higher volumes. QBS, QUBE, and BP-SPECT could also be tested for the right ventricle. Correlations were good for the volumes (0.93, 0.95, and 0.97 for QBS, QUBE, and BP-SPECT, respectively; all with P < 0.001). In terms of absolute volume estimation, the mean differences (+/-2 SDs) from Bland-Altman analysis were -41.28 +/- 43.66 mL, 11.13 +/- 49.26 mL, and -13.11 +/- 28.20 mL, respectively. Calculation of RV ejection fraction correlated well with true values (0.84, 0.92, and 0.94, respectively; all with P < 0.001), although an overestimation was seen for higher ejection fractions. Conclusion: Calculation of LV and RV ejection fraction based on tomographic radionuclide ventriculography was accurate for all tested algorithms. All algorithms underestimated LV volume; estimation of RV volume seemed more difficult, with different results for each algorithm. The more irregular shape and inclusion of a relatively hypokinetic RV outflow tract in the right ventricle seemed to cause the greater difficulty with delineation of the right ventricle, compared with the left ventricle

Secondary and primary murine alveolar echinococcosis: combined albendazole/nitazoxanide chemotherapy exhibits profound anti-parasitic activity.

In this study, the efficacies of chemotherapy employing nitazoxanide (NTZ), albendazole (ABZ), and a NTZ/ABZ-combination against alveolar echinococcosis (AE) were investigated in an experimental murine model. Following secondary infection, meaning i.p. injection of 20 Echinococcus multilocularis metacestodes, the drugs were administered by intragastric inoculation on a daily bases for a period of 5 weeks. Treatment was started either immediately on the day of infection, or at 2 months p.i., respectively. Application of the NTZ/ABZ-combination starting at 2 months p.i. was proven to be most effective in terms of reducing parasite weight (from 4.42+/-1.03 to 1+/-0.05 g; P=0.01). Inspection of treated parasites by transmission electron microscopy showed that ABZ- and NTZ-treated metacestode tissues, respectively, were heterogeneous in that both largely intact parasites as well as severely altered metacestodes could be observed. NTZ/ABZ-combination treatment induced the most severe ultrastructural alterations, including massive reduction in length and number of microtriches, severely damaged tegumental architecture, and progressive loss of viability of the germinal layer, associated with encapsulation by host connective tissue. A comparative pharmacokinetic study in mice revealed that the application of ABZ and NTZ in combination resulted in a two- to four-fold increase of albendazole sulfoxide serum levels for the period of 4-8 h following drug uptake compared to application of ABZ alone. In a third experiment, mice were orally infected with E. multilocularis eggs, and treated with NTZ starting at 2 months p.i. This resulted in a significantly lower lesion number in treated versus untreated mice (P=0.01). This investigation indicates the potential value for NTZ and/or a combined ABZ/NTZ chemotherapy against AE

Neurotoxicity of Alzheimer's disease Aβ peptides is induced by small changes in the Aβ42 to Aβ40 ratio

The amyloid peptides Aβ40 and Aβ42 of Alzheimer's disease contribute differentially to the disease process. The findings show here that small changes in the Aβ42/40 ratio have significant effects on the neurotoxicity of Aβ mixtures