No abstract

The influence of infection with an entomopathogenic strain of Pseudomonas aeruginosa on Galleria mellonella hemocytes was investigated. Extensive bacteriaemia developed 18 h after infection. This was correlated with significant changes in morphology, viability and the spreading ability of immunocompetent hemocytes, namely granulocytes and plasmatocytes. Since bacteriaemia developed, membrane blebbing, cytoplasm vacuolization, cell and organelle swelling, and chromatin condensation were observed among others. These features are typical for apoptotic and autophagal cell death. A gradually increasing level of procaspase and its activation as well as lack of DNA degradation were also detected. Propidium iodide and acridine orange staining indicated that hemocytes become dead ultimately. Infection of G. mellonella larvae with P. aeruginosa also caused significant changes in the arrangement of the actin cytoskeleton in the hemocytes, which might be correlated with their restricted spreading ability.

The proteolytic activity of three Pseudomonas aeruginosa strains, ATCC 27853 - a reference strain, and two clinical isolates was tested. The activity was examined after culturing the bacteria in two different growth media: the minimal M9 medium and rich Luria-Bertani broth (LB). Based on zymograms and protease activity specific assays, it was concluded that the reference strain produced three proteolytic enzymes in the LB medium: protease IV, elastase B and elastase A, while alkaline protease was only produced in the M9 medium. The clinical isolates of P. aeruginosa produced elastase B and alkaline protease when grown in the LB medium and the minimal M9 medium, respectively. PCR analysis confirmed the presence of both the lasB gene encoding elastase B and aprA coding for alkaline protease in the genomes of the three P. aeruginosa strains analyzed. The expression of these genes coding for two important P. aeruginosa virulence factors was dependent on the growth conditions in all the strains studied. The contribution of the extracellular proteinases to the virulence of P. aeruginosa strains used in this study was investigated using an insect model, the greater wax moth Galleria mellonella.

Our results demonstrated that Pseudomonas aeruginosa serine protease IV degraded apolipophorin III from the haemolymph of Galleria mellonella larvae. ApoLp-III protein was degraded in a stepwise manner. Four intermediate forms of 15, 13.3, 11.9 and 9.5 kDa were detected after 30 min digestion while only one of 5.6 kDa was released after 1-h incubation time. N-terminal amino acid sequence analysis of 5.6 kDa peptide revealed that it was released from apoLp-III after cleavage between lysine 70 and 71. ApoLp-III degradation by protease IV was inhibited by 1 mM TLCK but not 1 mM EDTA, additionally demonstrating that digestion was catalysed by a serine protease. Our data also indicated apoLp-III degradation in vivo during P. aeruginosa infection of G. mellonella larvae.

No abstract