Limits...
Functional characterization of two defensin isoforms of the hard tick Ixodes ricinus.

Chrudimská T, Slaninová J, Rudenko N, Růžek D, Grubhoffer L - Parasit Vectors (2011)

Bottom Line: In addition to antimicrobial activities, the hemolysis effect of def1 and def2 on human erythrocytes was also established.Although there is nothing known about the realistic concentration of defensins in I. ricinus tick body, these results suggest that defensins play an important role in defence against different pathogens.Moreover this is a first report of a one amino acid substitution in a defensins molecule and its impact on antimicrobial activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of South Bohemia, Faculty of Science, České Buděovice, Czech Republic. terezam@paru.cas.cz

ABSTRACT

Background: The immune system of ticks is stimulated to produce many pharmacologically active molecules during feeding and especially during pathogen invasion. The family of cationic peptides - defensins - represents a specific group of antimicrobial compounds with six conserved cysteine residues in a molecule.

Results: Two isoforms of the defensin gene (def1 and def2) were identified in the European tick Ixodes ricinus. Expression of both genes was induced in different tick organs by a blood feeding or pathogen injection. We have tested the ability of synthetic peptides def1 and def2 to inhibit the growth or directly kill several pathogens. The antimicrobial activities (expressed as minimal inhibition concentration and minimal bactericidal concentration values) against Gram positive bacteria were confirmed, while Gram negative bacteria, yeast, Tick Borne Encephalitis and West Nile Viruses were shown to be insensitive. In addition to antimicrobial activities, the hemolysis effect of def1 and def2 on human erythrocytes was also established.

Conclusions: Although there is nothing known about the realistic concentration of defensins in I. ricinus tick body, these results suggest that defensins play an important role in defence against different pathogens. Moreover this is a first report of a one amino acid substitution in a defensins molecule and its impact on antimicrobial activity.

Show MeSH

Related in: MedlinePlus

Expression patterns of I. ricinus defensins isoforms def1 (A) and def2 (B) after various stimuli in comparison to house-keeping gene actin (C). Before (NF) or after a completed blood feeding (BF), ticks were inoculated intrahemocoelly with a suspension of bacteria (E. coli, M. luteus, O.D.600 = 0.2; Borrelia burgdorferisensu stricto, 4.8 × 107spirochetes/ml; volume 0.5 μl). As a negative control one group of ticks was injected with the same volume of sterile water. RNA from all groups of ticks was isolated the next day. Expression of genes for def1(A) and def2(B) were determined by semi-quantitative two steps RT-PCR (30 cycles for BF ticks, 33 cycles for NF ticks) in salivary glands (SG), midgut (MG) and ovaries (OV). Gene for tick β-actin(C) was used as a positive control (27 cycles).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3102633&req=5

Figure 3: Expression patterns of I. ricinus defensins isoforms def1 (A) and def2 (B) after various stimuli in comparison to house-keeping gene actin (C). Before (NF) or after a completed blood feeding (BF), ticks were inoculated intrahemocoelly with a suspension of bacteria (E. coli, M. luteus, O.D.600 = 0.2; Borrelia burgdorferisensu stricto, 4.8 × 107spirochetes/ml; volume 0.5 μl). As a negative control one group of ticks was injected with the same volume of sterile water. RNA from all groups of ticks was isolated the next day. Expression of genes for def1(A) and def2(B) were determined by semi-quantitative two steps RT-PCR (30 cycles for BF ticks, 33 cycles for NF ticks) in salivary glands (SG), midgut (MG) and ovaries (OV). Gene for tick β-actin(C) was used as a positive control (27 cycles).

Mentions: Semi-quantitative two step RT-PCR was performed to determine defensin gene expression in all developmental stages and in selected tissues before and after blood feeding or hemocel injection of different substances. Expression of def1 mRNA was induced by blood intake at all life stages - larva, nymph and imago, while def2 mRNA expression was detected only in adult females with a significant increase after a blood meal (Figure 2). Def1 was expressed in mitgut in both cases-before and after blood meal, while its expression in Malphigian tubes and ovaries was induce after blood intake or injection of different compounds into blood fed female, respectively (see Figure 2, 3a). Results of tissue specific expression patterns analysis for I. ricinus def2 isoform in response to blood feeding, injection of bacteria or sterile water (Figure 2, 3a,) showed that expression of def2 isoform was present in midgut and ovaries of unfed female after injection and blood feeding was a big stimulus for def2 expression in all tested organs (Figure 2, 3b) House-keeping gene, β-actin, was expressed at the same expression levels in all samples (Figure 2, 3c).


Functional characterization of two defensin isoforms of the hard tick Ixodes ricinus.

Chrudimská T, Slaninová J, Rudenko N, Růžek D, Grubhoffer L - Parasit Vectors (2011)

Expression patterns of I. ricinus defensins isoforms def1 (A) and def2 (B) after various stimuli in comparison to house-keeping gene actin (C). Before (NF) or after a completed blood feeding (BF), ticks were inoculated intrahemocoelly with a suspension of bacteria (E. coli, M. luteus, O.D.600 = 0.2; Borrelia burgdorferisensu stricto, 4.8 × 107spirochetes/ml; volume 0.5 μl). As a negative control one group of ticks was injected with the same volume of sterile water. RNA from all groups of ticks was isolated the next day. Expression of genes for def1(A) and def2(B) were determined by semi-quantitative two steps RT-PCR (30 cycles for BF ticks, 33 cycles for NF ticks) in salivary glands (SG), midgut (MG) and ovaries (OV). Gene for tick β-actin(C) was used as a positive control (27 cycles).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3102633&req=5

Figure 3: Expression patterns of I. ricinus defensins isoforms def1 (A) and def2 (B) after various stimuli in comparison to house-keeping gene actin (C). Before (NF) or after a completed blood feeding (BF), ticks were inoculated intrahemocoelly with a suspension of bacteria (E. coli, M. luteus, O.D.600 = 0.2; Borrelia burgdorferisensu stricto, 4.8 × 107spirochetes/ml; volume 0.5 μl). As a negative control one group of ticks was injected with the same volume of sterile water. RNA from all groups of ticks was isolated the next day. Expression of genes for def1(A) and def2(B) were determined by semi-quantitative two steps RT-PCR (30 cycles for BF ticks, 33 cycles for NF ticks) in salivary glands (SG), midgut (MG) and ovaries (OV). Gene for tick β-actin(C) was used as a positive control (27 cycles).
Mentions: Semi-quantitative two step RT-PCR was performed to determine defensin gene expression in all developmental stages and in selected tissues before and after blood feeding or hemocel injection of different substances. Expression of def1 mRNA was induced by blood intake at all life stages - larva, nymph and imago, while def2 mRNA expression was detected only in adult females with a significant increase after a blood meal (Figure 2). Def1 was expressed in mitgut in both cases-before and after blood meal, while its expression in Malphigian tubes and ovaries was induce after blood intake or injection of different compounds into blood fed female, respectively (see Figure 2, 3a). Results of tissue specific expression patterns analysis for I. ricinus def2 isoform in response to blood feeding, injection of bacteria or sterile water (Figure 2, 3a,) showed that expression of def2 isoform was present in midgut and ovaries of unfed female after injection and blood feeding was a big stimulus for def2 expression in all tested organs (Figure 2, 3b) House-keeping gene, β-actin, was expressed at the same expression levels in all samples (Figure 2, 3c).

Bottom Line: In addition to antimicrobial activities, the hemolysis effect of def1 and def2 on human erythrocytes was also established.Although there is nothing known about the realistic concentration of defensins in I. ricinus tick body, these results suggest that defensins play an important role in defence against different pathogens.Moreover this is a first report of a one amino acid substitution in a defensins molecule and its impact on antimicrobial activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of South Bohemia, Faculty of Science, České Buděovice, Czech Republic. terezam@paru.cas.cz

ABSTRACT

Background: The immune system of ticks is stimulated to produce many pharmacologically active molecules during feeding and especially during pathogen invasion. The family of cationic peptides - defensins - represents a specific group of antimicrobial compounds with six conserved cysteine residues in a molecule.

Results: Two isoforms of the defensin gene (def1 and def2) were identified in the European tick Ixodes ricinus. Expression of both genes was induced in different tick organs by a blood feeding or pathogen injection. We have tested the ability of synthetic peptides def1 and def2 to inhibit the growth or directly kill several pathogens. The antimicrobial activities (expressed as minimal inhibition concentration and minimal bactericidal concentration values) against Gram positive bacteria were confirmed, while Gram negative bacteria, yeast, Tick Borne Encephalitis and West Nile Viruses were shown to be insensitive. In addition to antimicrobial activities, the hemolysis effect of def1 and def2 on human erythrocytes was also established.

Conclusions: Although there is nothing known about the realistic concentration of defensins in I. ricinus tick body, these results suggest that defensins play an important role in defence against different pathogens. Moreover this is a first report of a one amino acid substitution in a defensins molecule and its impact on antimicrobial activity.

Show MeSH
Related in: MedlinePlus