The effect of Galleria mellonella hemolymph polypeptides on Legionella gormanii

Among Legionella species, which are recognized to be pathogenic for humans, L. gormanii is the second prevalent causative agent of community-acquired pneumonia after L. pneumophila. Anti-L. gormanii activity of Galleria mellonella hemolymph extract and apolipophorin III (apoLp-III) was examined. The extract and apoLp-III at the concentration 0.025 mg/ml caused 75% and 10% decrease of the bacteria survival rate, respectively. The apoLp-III-induced changes of the bacteria cell surface were analyzed for the first time by atomic force microscopy. Our studies demonstrated the powerful anti-Legionella effects of the insect defence polypeptides, which could be exploited in drugs design against these pathogens

Anti-Legionella dumoffii activity of Galleria mellonella defensin and apolipophorin III

The gram-negative bacterium Legionella dumoffii is, beside Legionella pneumophila, an etiological agent of Legionnaires’ disease, an atypical form of pneumonia. The aim of this study was to determine the antimicrobial activity of Galleria mellonella defense polypeptides against L. dumoffii. The extract of immune hemolymph, containing a mixture of defense peptides and proteins, exhibited a dose-dependent bactericidal effect on L. dumoffii. The bacterium appeared sensitive to a main component of the hemolymph extract, apolipophorin III, as well as to a defense peptide, Galleria defensin, used at the concentrations 0.4 mg/mL and 40 μg/mL, respectively. L. dumoffii cells cultured in the presence of choline were more susceptible to both defense factors analyzed. A transmission electron microscopy study of bacterial cells demonstrated that Galleria defensin and apolipophorin III induced irreversible cell wall damage and strong intracellular alterations, i.e., increased vacuolization, cytoplasm condensation and the appearance of electron-white spaces in electron micrographs. Our findings suggest that insects, such as G. mellonella, with their great diversity of antimicrobial factors, can serve as a rich source of compounds for the testing of Legionella susceptibility to defense-related peptides and proteins

Diversity of antimicrobial peptides in invertebrates

Peptydy przeciwdrobnoustrojowe są kluczowymi efektorami odporności wrodzonej. Wykazują działanie przeciwbakteryjne, przeciwgrzybowe, przeciwpierwotniacze, a często przeciwwirusowe i przeciwnowotworowe. Wiele z nich zaangażowanych jest w neutralizację endotoksyn patogenów oraz ma właściwości immunomodulacyjne, stąd określane są również jako peptydy odpornościowe. O bogactwie i różnorodności naturalnie występujących peptydów przeciwdrobnoustrojowych świadczy ich liczba zgromadzona w bazie Antimicrobial Peptide Database (aps.unmc.edu/AP/main.php), która zawiera dane dotyczące ponad 2100 peptydów zidentyfikowanych u zwierząt. Spośród nich ponad 570 to peptydy odpornościowe bezkręgowców, w tym 495 opisanych u stawonogów. Niezwykle szerokie występowanie peptydów przeciwdrobnoustrojowych u przedstawicieli wszystkich królestw jednoznacznie wskazuje na ich fundamentalną rolę w sukcesie ewolucyjnym złożonych organizmów wielokomórkowych. Ich zasadnicze znaczenie w odporności bezkręgowców podkreśla ponadto fakt, że większość opisanych pod tym względem gatunków wytwarza zestaw peptydów zróżnicowanych pod względem struktury przestrzennej, właściwości biochemicznych, mechanizmu działania oraz spektrum aktywności przeciwdrobnoustrojowej.Antimicrobial peptides (AMPs) are the key effectors of innate immunity. They exhibit antimicrobial, antifungal, antiprotozoal, and often antiviral and anticancer activities. Many of them are involved in neutralization of pathogen endotoxins, have immunomodulatory properties, and are therefore referred to as defense peptides (host defense peptides). The wealth and diversity of naturally occurring AMPs is evidenced by their numbers in the Antimicrobial Peptide Database (aps.unmc.edu/AP/main.php), which contains data on over 2100 peptides identified in animals. Of these, over 570 are invertebrate peptides, including 495 described in arthropods. The unusually widespread presence of AMPs in all kingdoms clearly indicates their fundamental role in the evolutionary success of complex multicellular organisms. Their essential role in invertebrate immunity further emphasizes the fact that most species produce a set of defense peptides varied in terms of spatial structure, biochemical properties, mechanism of action and spectrum of antimicrobial activity

The effect of vanadate on Pichia pastoris growth, protein kinase A activity and ribosomal protein phosphorylation.

It was found that wild type yeast Pichia pastoris can tolerate vanadate concentration as high as 25 mM in the growth medium. Moreover, four vanadate-resistant P. pastoris strains designated JC100/1, JC100/3, JC100/9 and JC100/15 exhibiting tolerance up to 150 mM vanadate were selected. Growth of P. pastoris was correlated with vanadate to vanadyl reduction and its accumulation in the growth medium. In two selected strains, JC100/9 and JC100/15, protein kinase A activity was much higher in comparison to the wild type strain even without vanadate addition to the growth medium. Moreover, in the presence of vanadate, protein kinase A activity was significantly increased in the wild type and the vanadate-resistant JC100/1 and JC100/3 strains. It was also found that phosphorylation of a 40 kDa protein associated with ribosomes occured in all vanadate-resistant strains from the logarithmic, while in the wild type strain from the stationary growth phase. From the presented results it can be concluded that a protein kinase A signalling pathway(s) might be involved in the mechanism of P. pastoris vanadate resistance. The results also indicate a possible role of the 40 kDa protein in protection of P. pastoris against vanadate toxicity

The effect of cAMP and cGMP on the activity and substrate specificity of protein kinase A from methylotrophic yeast Pichia pastoris.

Cyclic AMP dependent protein kinase (PKA) from Pichia pastoris yeast cells was found to be activated by either cAMP or cGMP. Analogs of cAMP such as 8-chloro-cAMP and 8-bromo-cAMP were as potent as cAMP in PKA activation while N6,2'-O-dibutyryl-cAMP did not stimulate the enzyme activity. It was shown that protamine sulfate was almost equally phosphorylated in the presence of 1-2 × 10-6 M cAMP or cGMP while other substrates such as Kemptide, ribosomal protein S6, were phosphorylated to a lower extent in the presence of cGMP. It was demonstrated that pyruvate kinase is a substrate of PKA which co-purified with the P. pastoris enzyme. Moreover, pyruvate kinase was phosphorylated by PKA in the presence of cAMP and cGMP to comparable levels

The involvement of protein kinase A in the immune response of Galleria mellonella larvae to bacteria

The role of protein kinase A (PKA) in the humoral immune response of the greater wax moth Galleria mellonella larvae to live Gram-positive bacteria Micrococcus lysodeikticus and Gram-negative bacteria Escherichia coli was investigated. The immune challenge of larvae with both kinds of bacteria caused an increase in fat body PKA activity depending on the injected bacteria. Gram-positive M. lysodeikticus was a much better inducer of the enzyme activity than Gram-negative E. coli. The PKA activity was increased about 2.5-fold and 1.5-fold, after M. lysodeikticus and E. coli injection, respectively. The in vivo inhibition of the enzyme activity by a cell permeable selective PKA inhibitor, Rp-8-Br-cAMPS, was correlated with considerable changes of fat body lysozyme content and hemolymph antimicrobial activity in bacteria-challenged insects. The kinetics of changes were different and dependent on the bacteria used for the immune challenge of G. mellonella larvae