Moricin-like peptide B

We investigated the effects of extracellular proteinases of two Pseudomonas aeruginosa clinical isolates on the essential humoral immune response parameters in hemolymph of the insect model organism Galleria mellonella in vitro. Two culture media, rich LB and minimal M9, known to induce synthesis of different sets of proteinases secreted by P. aeruginosa were used. Changes in lysozyme, antibacterial and antifungal activities, as well as protein and peptide patterns in hemolymph treated with proteolytic fractions were evaluated. The effect of the proteolytic fractions on the apoLp-III level in hemolymph was determined by immunoblotting with antibodies against G. mellonella apolipophorin III (apoLp-III). We found that apoLp-III is hardly degraded by the proteinases of the proteolytic fractions of both clinical P. aeruginosa strains, in contrast to the high susceptibility of the protein to the proteinases of the entomopathogenic strain. The detected differences, together with the changes in the hemolymph protein and peptide patterns caused by the studied fractions, reflected the distinct composition of secreted proteinases of the entomopathogenic P. aeruginosa strain and the clinical strains tested. Our results also suggest the involvement of alkaline protease, the main proteinase of proteolytic fractions of P. aeruginosa grown in minimal medium, in the degradation of G. mellonella antimicrobial factors, such as lysozyme, antibacterial polypeptides, and proteins with antifungal activity. The diverse effects of the P. aeruginosa proteolytic fractions studied on the parameters of G. mellonella immune response indicate that this model insect may be useful in the analysis of the virulence factors of different P. aeruginosa strains.

We report that Galleria mellonella larvae exposed to heat shock was more resistant to infection with entomopathogenic bacteria Bacillus thuringiensis. The insects were exposed to a temperature of 40°C for 30 min directly before injection of vegetative bacterial cells. It appeared that the kinetics of the immune response was affected in heat-shocked animals. The infection-induced antimicrobial activity of larval hemolymph was stronger in shocked animals in comparison to the non-shocked ones. Hemolymph proteins of molecular weight below 10 kDa, corresponding to the size of antimicrobial peptides, were responsible for this activity. Furthermore, the transcription level of genes encoding antimicrobial peptides: cecropin, gallerimycin, and galiomycin was increased in the fat bodies of insects exposed to heat shock before infection. On the contrary, the heat-shock treatment did not enhance expression of the metalloproteinase inhibitor-IMPI in the infected animals. The difference in the amount of antimicrobial peptides and, consequently, in the defense activity of insect hemolymph, persisted after the action of bacterial metalloproteinases, which are well-known virulence factors. Furthermore, peptides with antimicrobial activity in the hemolymph of infected larvae pre-exposed to heat shock appeared to be more resistant to proteolytic degradation both in vitro and in vivo. Our results point to the mechanism of cross-protection of thermal stress toward innate immune response.

In numerous studies, the greater wax moth Galleria mellonella has been exploited as an alternative model host for investigating virulence factors of different pathogenic bacteria. In the present paper, we provide evidence that G. mellonella constitutes a useful and convenient model for analysis of the pathogenicity of Pseudomonas aeruginosa clinical strains. In this in vivo study on the G. mellonella-P. aeruginosa interaction, a bidirectional analysis comprising evaluation of humoral immune response of the bacteria-infected larvae and determination of P. aeruginosa proteinases synthesized during the infection was performed. The effects of G. mellonella infection by two clinical strains (PA C124/9 and PA 02/18) and one entomopathogenic strain (ATCC 27853) cultured in a rich LB and minimal M9 medium, known to induce synthesis of different sets of extracellular proteinases, were evaluated. Both clinical isolates were able to establish infection in G. mellonella caterpillars after intrahemocelic injection. However, although the final effect of the larvae infection by each P. aeruginosa strain was their death within ca. 48 h, considerable strain and medium-dependent differences in the immune response of the insects were detected. The results indicated that G. mellonella larvae distinguished between the three P. aeruginosa strains, which was well reflected by the diverse humoral immune response. The significant differences concerned, among others, the level of phenoloxidase, lysozyme, and antibacterial activity in the hemolymph of the infected insects. An analysis of proteinases performed using specific activity tests, zymography and immunoblotting, revealed that elastase B and alkaline protease were synthesized by each P. aeruginosa strain during the infection. In contrast, a high level of elastase A activity was detected only in the larvae infected by the P. aeruginosa ATCC 27853 strain. It can be postulated that the three P. aeruginosa strains exploit different strategies to avoid and overcome insect immunity. Our results provided further evidence on G. mellonella usefulness as a model for analysis of P. aeruginosa virulence factors and their involvement in pathogenicity.