Insect antimicrobial peptides
Insects do not have a complement system as vertebrates thus mostly AMPs are likely to be responsible for bactericidal effect. Most of the AMPs detectable in the
haemolymph upon microbial infection are produced within a few hours by the fat body, haemocytes and other specific tissues (Lemaitre & Hoffmann, 2007). Apart
from induced AMPs synthesis there is also constitutive level of AMPs present in haemolymph. These peptides are synthesised by either the haemocytes or the fat
body. In Lepidoptera linear α-helical (cecropins and moricins), cysteine-stabilized (defensins), proline-rich and glycine-rich inducible AMPs have been identified;
moreover the peptides cooperate with lysozyme which is naturally occurring in haemolymph. AMPs are attracted by electrostatic forces to negatively charged
groups on the surface of bacteria e.g. lipopolysaccharide or teichoic acid. After attachment to bacterial membrane AMPs interact with phospholipids double layer
which usually leads to pore creation and cell lysis.
Bioluminescence determination of antibacterial activity
of Bombyx mori and Galleria mellonella haemolymph
Libor Vojtek1, Pavel Dobeš1, Ender Büyükgüzel2, Pavel Hyršl1
1 Department of Animal Physiology and Immunology, Institute of Experimental Biology, Faculty of Science,
Masaryk University, Kotlářská 2, 61137 Brno, Czech Republic
2 Department of Biology, Faculty of Arts and Science, Bülent Ecevit University, 67100, Zonguldak, Turkey
e-mail: libor.vojtek@mail.muni.cz
Introduction
Bioluminescence is the production and emission of light by living organisms. Genus Photorhabdus includes terrestrial Gram negative bacteria, which are mainly
found in association with entomopathogenic nematodes Heterorhabditis spp. Upon entering an insect host nematodes release bacterial cells from their intestinal
tract which quickly establish a lethal septicaemia in the host (ffrench-Constant et al., 2003). Similarly to P. luminescens, transformed Escherichia coli K12 is
capable of light production. Insect immunity involves both humoral and cellular aspects. Cellular activities in the insect rely on haemocytes which perform
phagocytosis, encapsulation and nodulation. Humoral factors include especially highly potent antimicrobial peptides (AMPs) and the enzyme phenoloxidase. The
aim of this study was to analyse antibacterial activity of insect haemolymph using direct real time measurement of changes in bioluminescence produced by P.
luminescens or E. coli K12.
Atosuo J, Lehtinen J, Vojtek L, Lilius EM: Luminescence doi:10.1002/bio.2435, 2012.
ffrench-Constant R, Waterfield N, Daborn P, Joyce S, Bennet H, Au C, Dowling A, Boundy S, Reynolds S,
Clarke D: FEMS Microbiol Rev, 26, 433-56, 2003.
Escherichia coli K12
As well as P. luminescens, E. coli K12 (Fig. 2) is
G- bacteria. It was geneticaly transformed with
luxABCDEamp operon (Fig. 3). Genes luxA
and B codes two subunits of bacterial luciferase;
luxC, D and E codes the fatty acid reductase
complex needed for aldehyde synthesis. This
operon is originaly from genus Photorhabdus
thus the principle of light emition is the same for
both species (Atosuo et al., 2012).
Photorhabdus luminescensr
P. luminescens (Fig. 1) is the only terrestrial
bacteria capable of bioluminescence. It is Gbacteria
which can produce light because of the
expression of bacterial luciferase (Lux) and its
substrate. This unique enzyme catalyses the
oxidation of long-chain aldehyde (substrate)
and reduced flavin mononucleotid (FMNH2)
produced only by living cells followed by the
emission of light (Hakkila et al., 2002).
Refferences: Hakkila K., Maksimow M., Karp M., Virta M.: Anal Biochem, 301, 235-242, 2002.
Lemaitre, B., Hoffmann J.: Annu Rev Immunol 25, 697-743, 2007.
Conclusion
Bioluminescent bacteria assay can be used as a new, fast and real-time method for assessment of insect haemolymph antibacterial activity. Dose
dependence in antibacterial activity was found in both B. mori and G. mellonella. Results with haemocyte free haemolymph and haemolymph stored in
-20°C confirmed that antibacterial activity is of humoral origin. Measurements done on pricked larvae showed that constitutive level of AMPs which are
present in haemolymph can be increased by their induction with both E. coli and P. luminescens.
Our research is supported by grant from Ministry of Agriculture of the Czech
Republic NAZV – KUS (QJ1210047)
Lux
FMN + RCO2H + H2O + lightFMNH2 + RCHO + 02
luxABCD
Eamp
luxA
luxB
luxC
luxD
luxEamp
Fig 3:
Fig 2: E. coli culture
under phase contrast.
Fig 1: P. l. culture
under phase contrast.
E.coliE.coliE.coli
P.luminescens
200
400
600
800
1000
1200
1400
1600
1800
2000
0 10 20 30 40 50 60
Bioluminescence(RLU)
Time (min)
control (E. coli suspension)
E. coli + 10 % haemolymph
E. coli + 20 % haemolymph
E. coli + 30 % haemolymph
E. coli + 40 % haemolymph
Fig 5: Antibacterial activity of different concentrations
of B. mori haemolymph against E. coli
Concentrations of
haemolymph ranging from
10 % to 40 % were tested
against both E. coli (Fig 5)
and P. luminescens (Fig 4).
For subsequent
experiments 20 % was
selected as an optimal
dilution of haemolymph.
This concentration
suppresses approximately
50 % of bacteria in 30
minutes.
Fig 4: Antibacterial activity of different concentrations
of B. mori haemolymph against P. luminescens
25
50
75
100
125
150
175
0 10 20 30 40 50 60
Bioluminescence(RLU)
Time (min)
control (P. luminescens
suspension)
P. luminescens + 20 % fresh
haemolymph
P. luminescens + 20 % frozen
haemolymph
P. luminescens + 20 %
centrifuged haemolymph
Fig 6: Antibacterial activity of fresh, frozen and
centrifuged B. mori haemolymph against P.
luminescens
1600
2000
2400
2800
3200
3600
4000
4400
4800
0 10 20 30 40 50 60
Bioluminescence(RLU)
Time (min)
control (E. coli suspension)
E. coli + 20 % fresh
haemolymh
E. coli + 20 % frozen
haemolymph
E.coli + 20 % centrifged
haemolymph
Fig 7: Antibacterial activity of fresh, frozen and
centrifuged B. mori haemolymph against E. coli
Freshly collected
haemolymph was
compared to haemocytefree
haemolymph and
haemolymph stored at
–20 °C for 30 days. All
tested samples showed
comparable antibacterial
activity against E. coli (Fig
7) and P. luminescens (Fig
6) antibacterial activity is
caused by humoral factors.
Fig 8: Antibacterial activity of 20 % G. mellonella
haemolymph against P. luminescens
1500
3000
4500
6000
7500
9000
10500
12000
0 10 20 30 40 50 60
Bioluminescence(RLU)
Time (min)
control (E. coli suspension)
E. coli + 20 % fresh
haemolymph (B. mori pricked
with physiological solution)
E. coli + 20 % fresh
haemolymph (B. mori pricked
with E. coli suspension)
E. coli + 20 % fresh
haemolymph (B. mori pricked
with P. luminescens
suspension)
Fig 9: Antibacterial activity against E. coli of 20 %
B. mori haemolymph after septic injurySimilarly to B. mori,
haemolymph from G.
mellonella showed strong
antibacterial activity against
both E. coli and P.
luminescens, approximately
50 % decrease in bacterial
bioluminescence signal in
30 minutes was observed
(Fig 8).
P.luminescens
25
50
75
100
125
150
175
0 10 20 30 40 50 60
Bioluminescence(RLU)
Time (min)
control (P. luminescens
suspension)
P. luminescens + 10 %
haemolymph
P. luminescens + 20 %
haemolymph
P. luminescens + 30 %
haemolymph
P. luminescens + 40 %
haemolymph
P.luminescens
0
100
200
300
400
500
0 10 20 30 40 50 60
Bioluminescence(RLU)
Time (min)
control (P. luminescens
suspension)
P. luminescens + 20 % fresh
haemolymph
B. mori larvae pricked by E. coli or P. luminescens showed higher antibacterial activity five hours after pricking (Fig 9). The increase of antibacterial
activity was reflected in decreased E. coli bioluminescence, significantly different compared to untreated control or larvae pricked with insect saline.
Insects treated with E. coli had stronger antibacterial response than larvae treated by natural insect pathogen P. luminescens.
P.luminescens
E.coliE.coliE.coli