Cathelicidin antimicrobial peptides in cod and salmon

132 Pages·2012·6.75 MB·English

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Daniela C. Broekman

2
0 Faculty of Life an d Environmental
1
2
Sciences

University of Iceland
2012
Cathelicidin antimicrobial
peptides in cod and salmon

Daniela C. Broekman

Dissertation submitted in partial fulfillment of a
Philosophiae Doctor degree in Biology

Advisor
Guðmundur H. Guðmundsson

PhD Committee
Ólafur S. Andrésson
Valerie H. Maier

Opponents
Bergljót Magnadóttir
Henk P. Haagsman

Life and Environmental Sciences
School of Engineering and Natural Sciences
University of Iceland
Reykjavik, October 2012
Cathelicidin antimicrobial
peptides in cod and salmon

Daniela C. Broekman

Dissertation submitted in partial fulfillment of a
Philosophiae Doctor degree in Biology

Advisor
Guðmundur H. Guðmundsson

PhD Committee
Ólafur S. Andrésson
Valerie H. Maier

Opponents
Bergljót Magnadóttir
Henk P. Haagsman

Life and Environmental Sciences
School of Engineering and Natural Sciences
University of Iceland
Reykjavik, October 2012
Philosophiae
CDaotchtoelricidin antimicrobial peptides in cod and salmon
Dissertation submitted in partial fulfillment of a
degree in Biology

Copyright © 2012 Daniela C. Broekman
All rights reserved
Life and Environmental Sciences
School of Engineering and Natural Sciences
University of Iceland
Askja, Sturlugötu 7
101, Reykjavik
Iceland

Telephone: [phone]

Cathelicidin antimicrobial peptides in
cBoibdl aiongdr asaplhmico innformation:
Daniela C. Broekman, 2012,
PhD dissertation, Department of Life and
Environmental Sciences, University of Iceland.

ISBN 978-9935-9064-4-1

Printing: büttner-druck
Nordhorn, Germany, October 2012
Philosophiae
CDaotchtoelricidin antimicrobial peptides in cod and salmon
Dissertation submitted in partial fulfillment of a
degree in Biology

Copyright © 2012 Daniela C. Broekman
All rights reserved
Life and Environmental Sciences
School of Engineering and Natural Sciences
University of Iceland
Askja, Sturlugötu 7
101, Reykjavik
Iceland

Telephone: [phone]

Cathelicidin antimicrobial peptides in
cBoibdl aiongdr asaplhmico innformation:
Daniela C. Broekman, 2012,
PhD dissertation, Department of Life and
Environmental Sciences, University of Iceland.

ISBN 978-9935-9064-4-1

Printing: büttner-druck
Nordhorn, Germany, October 2012
receptor mediated signaling and is involved in the signaling
cascade to cathelicidin upregulation in the salmon cell line.
Abstract
In conclusion, a novel cathelicidin from a teleost fish was isolated
and inducibility of expression by microbial PAMPs as well as
antimicrobial activity of the peptide indicate that cathelidin in cod
Cathelicidins are a family of antimicrobial peptides that have been
plays a role in immunity.
well researched in mammals, where they have been described to
have an important role in immunity. Cathelicidins are ubiquitous
among vertebrates having been found in fish, amphibia, reptiles,
bGiardduss a mndo rmhuaammals including marsupialia, thereby demonstrating
their conserved position throughout evolution. The Atlantic cod
is an economically important fish in Iceland. In this
thesis the focus is on the cod cathelicidin „codCath“. It was in the
course of this project isolated from cod and then characterized
regarding its activity and regulation.
The cathelicidin antimicrobial peptide was isolated from cod head
kidney by high-performance liquid chromatography. The mature
peptide was sequenced at the N-terminus and a total size of 67
amino acids was deduced. The peptide has a predominance of the
amino acids serine, arginine and glycine, a positive charge of +15
and is considered a novel and unique cathelicidin. Both the
isolated and the synthetic peptide demonstrated antibacterial
activity. The synthetic peptide helped to identify a salt sensitivity
of this peptide with reduced activity at high salt concentrations.
Our results indicated that the mode of activity of codCath was
rapid lysis of bacterial cells, while it was selectively active towards
microbial but not eukaryotic membranes. The peptide was
degraded and inactivated by extracellular products of pathogenic
bacteria.
Cod cathelicidin transcripts were found in eggs and larvae of
Atlantic cod and in larvae levels were responsive to
environmental stimuli. In a salmonid cell lLinacet ocbaathciellluicsi din was
significantly upregulated through Gram-negative bacteria and
flagellin and in a gadoid cell line through sp. and
poly(I:C). The responsiveness through microbial PAMPs indicates
signaling through TLRs or other receptors. The inhibition of the
PI3K further indicated that this protein has an inhibitory role on
vi
vii
receptor mediated signaling and is involved in the signaling
cascade to cathelicidin upregulation in the salmon cell line.
Abstract
In conclusion, a novel cathelicidin from a teleost fish was isolated
and inducibility of expression by microbial PAMPs as well as
antimicrobial activity of the peptide indicate that cathelidin in cod
Cathelicidins are a family of antimicrobial peptides that have been
plays a role in immunity.
well researched in mammals, where they have been described to
have an important role in immunity. Cathelicidins are ubiquitous
among vertebrates having been found in fish, amphibia, reptiles,
bGiardduss a mndo rmhuaammals including marsupialia, thereby demonstrating
their conserved position throughout evolution. The Atlantic cod
is an economically important fish in Iceland. In this
thesis the focus is on the cod cathelicidin „codCath“. It was in the
course of this project isolated from cod and then characterized
regarding its activity and regulation.
The cathelicidin antimicrobial peptide was isolated from cod head
kidney by high-performance liquid chromatography. The mature
peptide was sequenced at the N-terminus and a total size of 67
amino acids was deduced. The peptide has a predominance of the
amino acids serine, arginine and glycine, a positive charge of +15
and is considered a novel and unique cathelicidin. Both the
isolated and the synthetic peptide demonstrated antibacterial
activity. The synthetic peptide helped to identify a salt sensitivity
of this peptide with reduced activity at high salt concentrations.
Our results indicated that the mode of activity of codCath was
rapid lysis of bacterial cells, while it was selectively active towards
microbial but not eukaryotic membranes. The peptide was
degraded and inactivated by extracellular products of pathogenic
bacteria.
Cod cathelicidin transcripts were found in eggs and larvae of
Atlantic cod and in larvae levels were responsive to
environmental stimuli. In a salmonid cell lLinacet ocbaathciellluicsi din was
significantly upregulated through Gram-negative bacteria and
flagellin and in a gadoid cell line through sp. and
poly(I:C). The responsiveness through microbial PAMPs indicates
signaling through TLRs or other receptors. The inhibition of the
PI3K further indicated that this protein has an inhibitory role on
vi
vii
cathelicidin væri bæld af þessum megin kínasa frumunnar í
frumlínu laxfiskanna.
Útdráttur
Samantekið í meginatriðum var ný gerð varnarpeptíða einangruð
úr þorski og skilyrði fyrir örvaða tjáningu greind. Virkni peptíðins
og örvun bendir til mikilvægs hlutverks í ónæmiskerfi fiskanna.
Cathelicidins eru fjölskylda örverueyðandi peptíða sem hafa verið
rannsökuð mikið í spendýrum og gegna mikilvægu hlutverki í
ónæmiskerfinu. Þau hafa fundist meðal helstu flokka hryggdýra;
fiska, froskdýra, skriðdýra, fGuagdlau so g smpeonrdhuýraa) meðtalin pokadýr
sem gefur vísbendingu um varðveislu í þróun og mikilvægt
hlutverk. Þorskurinn ( hefur megin
efnahagsþýðingu fyrir Ísland. Í ritgerðinni og rannsóknunum var
áherslan á cathelicidin í þorski sem er nú kallað „codCath“. Í
verkefninu var virkt cathelicidin peptíð þorska einangrað, virkni
greind og tjáning athuguð.
Virka cathelidicin peptíð þorska var einangrað úr framnýra með
háþrýstivökvaskilju (HPLC). Amínosýruröð virka peptíðsins var
ákvörðuð með raðgreingu á amínóenda og þar með stærðin
ákvörðuð 67 amínósýrur. Áberandi amínósýrur í peptíðinu voru
serín, arginín og glýsín og við hlutlausar aðstæður er jákvæð
hleðsla +15. Peptíð líktist engu öðru einangruðu peptíði og er því
einstakt meðal cathelicidina. Bæði einangrað peptíð og
samsvarandi peptíð hannað við tilraunaaðstæður höfðu
bakteríudrepandi virkni. Hannaða peptíðið hjálpaði til við
virknigreiningu og sýnt var fram næmni fyrir salti sem minnkaði
virkni peptíðsins mikið. Peptíðið reyndist virka á himnu baktería
og framkalla rof en hafði ekki áhrif á himnu heilkjörnunga.
Bakteríusýklar reyndust seyta hindrum sem klipptu peptíðið og
óvirkjuðu.
Genið fyrir cathelicidin þorska er tjáð í hrognum og lirfum þorska
og er tjáningin háð umhverfisþáttum. Í frumulínu frá laxfiskum
reyndist tjáning á catheliciLdainct oböarvciullðu s af Gram-neikvæðum
bakteríum og flagellin prótíninu. Í frumulínu frá þorski reyndist
örvunin önnur og mest vegna sp. og poly(I:C). Þetta
munstur bendir til tjániga örvunar um viðtaka eins og TLR (Toll
líkir viðtakar) sem þekkja einmitt munstursameindir örvera sem
einkenna þær. Hindrun á PI3K kínasanum benti til að tjáningin á
viii
ix
cathelicidin væri bæld af þessum megin kínasa frumunnar í
frumlínu laxfiskanna.
Útdráttur
Samantekið í meginatriðum var ný gerð varnarpeptíða einangruð
úr þorski og skilyrði fyrir örvaða tjáningu greind. Virkni peptíðins
og örvun bendir til mikilvægs hlutverks í ónæmiskerfi fiskanna.
Cathelicidins eru fjölskylda örverueyðandi peptíða sem hafa verið
rannsökuð mikið í spendýrum og gegna mikilvægu hlutverki í
ónæmiskerfinu. Þau hafa fundist meðal helstu flokka hryggdýra;
fiska, froskdýra, skriðdýra, fGuagdlau so g smpeonrdhuýraa) meðtalin pokadýr
sem gefur vísbendingu um varðveislu í þróun og mikilvægt
hlutverk. Þorskurinn ( hefur megin
efnahagsþýðingu fyrir Ísland. Í ritgerðinni og rannsóknunum var
áherslan á cathelicidin í þorski sem er nú kallað „codCath“. Í
verkefninu var virkt cathelicidin peptíð þorska einangrað, virkni
greind og tjáning athuguð.
Virka cathelidicin peptíð þorska var einangrað úr framnýra með
háþrýstivökvaskilju (HPLC). Amínosýruröð virka peptíðsins var
ákvörðuð með raðgreingu á amínóenda og þar með stærðin
ákvörðuð 67 amínósýrur. Áberandi amínósýrur í peptíðinu voru
serín, arginín og glýsín og við hlutlausar aðstæður er jákvæð
hleðsla +15. Peptíð líktist engu öðru einangruðu peptíði og er því
einstakt meðal cathelicidina. Bæði einangrað peptíð og
samsvarandi peptíð hannað við tilraunaaðstæður höfðu
bakteríudrepandi virkni. Hannaða peptíðið hjálpaði til við
virknigreiningu og sýnt var fram næmni fyrir salti sem minnkaði
virkni peptíðsins mikið. Peptíðið reyndist virka á himnu baktería
og framkalla rof en hafði ekki áhrif á himnu heilkjörnunga.
Bakteríusýklar reyndust seyta hindrum sem klipptu peptíðið og
óvirkjuðu.
Genið fyrir cathelicidin þorska er tjáð í hrognum og lirfum þorska
og er tjáningin háð umhverfisþáttum. Í frumulínu frá laxfiskum
reyndist tjáning á catheliciLdainct oböarvciullðu s af Gram-neikvæðum
bakteríum og flagellin prótíninu. Í frumulínu frá þorski reyndist
örvunin önnur og mest vegna sp. og poly(I:C). Þetta
munstur bendir til tjániga örvunar um viðtaka eins og TLR (Toll
líkir viðtakar) sem þekkja einmitt munstursameindir örvera sem
einkenna þær. Hindrun á PI3K kínasanum benti til að tjáningin á
viii
ix
Dedicated to my grandmother Irma
Braakmann (1925-2012) and Bebi
(2010-2012).
x
xi