Atomic resolution crystal structure of Sapp2p, a secreted aspartic protease from Candida parapsilosis

The virulence of theCandidapathogens is enhanced by the production of secreted aspartic proteases, which therefore represent possible targets for drug design. Here, the crystal structure of the secreted aspartic protease Sapp2p fromCandida parapsilosiswas determined. Sapp2p was isolated from its natural source and crystallized in complex with pepstatin A, a classical aspartic protease inhibitor. The atomic resolution of 0.83 Å allowed the protonation states of the active-site residues to be inferred. A detailed comparison of the structure of Sapp2p with the structure of Sapp1p, the most abundantC. parapsilosissecreted aspartic protease, was performed. The analysis, which included advanced quantum-chemical interaction-energy calculations, uncovered molecular details that allowed the experimentally observed equipotent inhibition of both isoenzymes by pepstatin A to be rationalized.</jats:p

Pathogen quantitation in complex matrices : a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition

Background: Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year. Methodology/Principal Findings: We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pretreatment step, the FastDNA® Spin kit, the UltraCleanTM and PowerSoilTM soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities. Conclusions: M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25 x 105 cells g-1 using Griffiths and 4.25 x 106 cells g-1 using FastDNA® Spin kit