Covariation of Ergot Severity and Alkaloid Content Measured by HPLC and One ELISA Method in Inoculated Winter Rye across Three Isolates and Three European Countries.

Ergot caused by Claviceps purpurea is a problem for food and feed security in rye due to the occurrence of toxic ergot alkaloids (EAs). For grain elevators and breeders, a quick, easy-to-handle, and cheap screening assay would have a high economic impact. The study was performed to reveal (1) the covariation of ergot severity (= percentage of sclerotia in harvested grain) and the content of 12 EAs determined by high performance liquid chromatography (HPLC) and (2) the covariation between these traits and results of one commercial enzyme linked immunosorbent assays (ELISA). In total, 372 winter rye samples consisting of a diverse set of genotypes, locations from Germany, Austria, and Poland over two years, and three isolates were analyzed. Ergocornine and α-ergocryptine were detected as major EAs. Ergocristinine occurred as a minor component. Claviceps isolates from different countries showed a similar EA spectrum, but different quantities of individual EAs. A moderate, positive covariation between ergot severity and EA content determined by HPLC was observed across two years (r = 0.53, p < 0.01), but large deviation from the regression was detected. ELISA values did neither correlate with the HPLC results nor with ergot severity. In conclusion, a reliable prediction of the EA content based on ergot severity is, at present, not possible.

Chlamydia pneumoniae GroEL1 protein is cell surface associated and required for infection of HEp-2 cells.

Chlamydia pneumoniae is an important obligate intracellular pathogen that replicates within an inclusion in the eukaryotic cell. The initial event of a chlamydial infection is the adherence to and subsequent uptake of the infectious elementary bodies (EBs) by the human cell. These processes require yet-unidentified bacterial and eukaryotic surface proteins. The GroEL1 protein, which exhibits a very strong antigenicity and in vitro can activate various eukaryotic cells, is a potential pathogenicity factor. We localized the protein during the infection process and found it in the inclusion but outside the chlamydial particles. GroEL1 was also localized on the surface of EBs, and the protein could be washed off the EBs. Latex beads coated with recombinantly produced GroEL1 (rGroEL1) bound in a dose-dependent manner to HEp-2 cells. Likewise, GroEL1, when expressed and displayed on the yeast cell surface, mediated adhesion to HEp-2 cells. Interestingly, the homologous GroEL2 and GroEL3 proteins showed no adhesive properties. Incubation of primary umbilical vein endothelial cells with soluble GroEL1 and GroEL1-coated latex beads activated the translocation of the general transcription factor NF-kappaB into the nucleus. Finally, preincubation of HEp-2 cells with rGroEL1 significantly reduced subsequent infection with C. pneumoniae, although adhesion of infectious bacteria to eukaryotic cells was not affected. Taken together, these data support a role for extracellular GroEL1 in the establishment of the chlamydial infection.

Mechanisms of Chlamydophila pneumoniae-mediated GM-CSF release in human bronchial epithelial cells.

Chlamydophila pneumoniae is an important respiratory pathogen. In this study we characterized C. pneumoniae strain TW183-mediated activation of human small airway epithelial cells (SAEC) and the bronchial epithelial cell line BEAS-2B and demonstrated time-dependent secretion of granulocyte macrophage colony-stimulating factor (GM-CSF) upon stimulation. TW183 activated p38 mitogen-activated protein kinase (MAPK) in epithelial cells. Kinase inhibition by SB202190 blocked Chlamydia-mediated GM-CSF release on mRNA and protein levels. In addition, the chemical inhibitor as well as dominant-negative mutants of p38 MAPK isoforms p38alpha, beta2, and gamma inhibited C. pneumoniae-related NF-kappaB activation. In contrast, blocking of MAPK ERK, c-Jun kinase/JNK, or PI-3 Kinase showed no effect on Chlamydia-related epithelial cell GM-CSF release. Ultraviolet-inactivated pathogens as compared with viable bacteria induced a smaller GM-CSF release, suggesting that viable Chlamydiae were only partly required for a full effect. Presence of an antichlamydial outer membrane protein-A (OmpA) antibody reduced and addition of recombinant heat-shock protein 60 from C. pneumoniae (cHsp60, GroEL-1)-enhanced GM-CSF release, suggesting a role of these proteins in epithelial cell activation. Our data demonstrate that C. pneumoniae triggers an early proinflammatory signaling cascade involving p38 MAPK-dependent NF-kappaB activation, resulting in subsequent GM-CSF release. C. pneumoniae-induced epithelial cytokine liberation may contribute significantly to inflammatory airway diseases like chronic obstructive pulmonary disease (COPD) or bronchial asthma.