(Apis mellifera) Antimicrobial Peptides on Paenibacillus larvae

107 Pages·2016·14.05 MB·English

by  Jasmin Camille Khilnani

UNLV Theses, Dissertations, Professional Papers, and Capstones
8-1-2015
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Jasmin Camille Khilnani
University of Nevada, Las Vegas
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THE
 EFFECTS
 OF
 HONEYBEE
 (APIS
 MELLIFERA)
 ANTIMICROBIAL

PEPTIDES
 ON
 PAENIBACILLUS
 LARVAE

By

Jasmin
 Camille
 Khilnani

Bachelor
 of
 Science
 in
 General
 Biology

University
 of
 California,
 San
 Diego

2011

A
 thesis
 submitted
 in
 partial
 fulfillment

of
 the
 requirements
 for
 the

Master
 of
 Science
 -­‐
 Biological
 Sciences

School
 of
 Life
 Sciences

College
 of
 Sciences

The
 Graduate
 College

University
 of
 Nevada,
 Las
 Vegas

August
 2015
Thesis Approval
The Graduate College
The University of Nevada, Las Vegas

June 30, 2015
This thesis prepared by
Jasmin C. Khilnani
entitled
The Effects of Honeybee (Apis mellifera) Antimicrobial Peptides on Paenibacillus larvae
is approved in partial fulfillment of the requirements for the degree of
Master of Science – Biological Sciences
Department of Life Sciences

Helen Wing, Ph.D. Kathryn Hausbeck Korgan, Ph.D.
Examination Committee Chair Graduate College Interim Dean

Andrew Andres, Ph.D.
Examination Committee Member

Eduardo Robleto, Ph.D.
Examination Committee Member

Ernesto Abel-Santos, Ph.D.
Graduate College Faculty Representative
ii
ABSTRACT

The
 Effects
 of
 Honeybee
 (Apis
 mellifera)
 Antimicrobial

Peptides
 on
 Paenibacillus
 larvae

By

Jasmin
 Camille
 Khilnani

Dr.
 Helen
 J.
 Wing,
 Examination
 Committee
 Chair

Associate
 Professor
 of
 Microbiology

University
 of
 Nevada,
 Las
 Vegas

American
 Foulbrood
 Disease
 (AFB)
 is
 the
 most
 detrimental
 bacterial
 disease
 that
 affects

honeybee
 larvae
 (Apis
 mellifera)
 worldwide.
 The
 etiological
 agent
 of
 AFB
 is
 the
 Gram-­‐
positive,
 spore-­‐forming
 bacterial
 pathogen,
 Paenibacillus
 larvae.
 Treatment
 with
 antibiotics,

specifically
 oxytetracycline,
 has
 led
 to
 the
 development
 of
 antibiotic
 resistance
 in
 P.
 larvae.

Therefore,
 there
 is
 a
 pressing
 need
 for
 an
 alternative
 treatment
 method.
 The
 overall
 goal
 of

this
 project
 was
 to
 test
 naturally
 occurring,
 active
 antimicrobial
 peptides
 (AMPs),
 which
 are

produced
 in
 the
 adult
 honeybee,
 against
 vegetative
 P.
 larvae.
 These
 active
 AMPs
 could

potentially
 be
 used
 as
 a
 prophylactic
 treatment
 to
 prevent
 P.
 larvae
 infection
 of
 honeybee

larvae.
 The
 use
 of
 AMPs
 is
 a
 superior
 approach
 to
 preventing/treating
 AFB
 than
 common

practices,
 such
 as
 antibiotics,
 due
 to
 the
 unlikeliness
 of
 developing
 AMP-­‐resistant
 bacteria

and
 the
 prevention
 of
 burning
 and
 destroying
 hives.
 My
 research
 first
 focuses
 on
 adapting

and
 creating
 two
 in
 vitro
 methods
 in
 order
 to
 test
 the
 antimicrobial
 activity
 of
 honeybee

AMPs
 against
 vegetative
 P.
 larvae.
 Once
 methods
 were
 established,
 a
 total
 of
 five
 AMPs
 were

tested
 against
 P.
 larvae
 either
 individually
 or
 in
 certain
 combinations.
 I
 found
 that
 four
 of

the
 five
 AMPs
 were
 capable
 of
 inhibiting
 P.
 larvae
 growth;
 however,
 only
 two
 AMPs
 met
 my

threshold
 requirements
 and
 were
 deemed
 suitable
 candidates
 for
 AFB
 prophylaxis.

Furthermore,
 based
 on
 statistical
 significance,
 none
 of
 the
 AMP
 combinations
 tested

inhibited
 the
 growth
 of
 P.
 larvae
 more
 than
 the
 single
 active
 AMP.
 Overall,
 my
 research

  iii
demonstrates
 that
 honeybee
 AMPs
 do
 inhibit
 the
 growth
 of
 P.
 larvae
 in
 vitro
 and
 have
 the

potential
 to
 be
 used
 in
 use
 in
 treatments
 and
 /or
 prophylaxis
 strategies
 for
 AFB.

  iv
ACKNOWLEDGEMENTS

  I
 would
 first
 like
 to
 acknowledge
 and
 thank
 my
 mentor,
 Dr.
 Helen
 J.
 Wing.
 Without

her
 unwavering
 support,
 vast
 knowledge,
 and
 persistent
 challenges,
 I
 would
 not
 have

grown
 so
 much
 as
 a
 scientist
 and
 as
 a
 person
 throughout
 my
 degree.
 She
 has
 taught
 me
 the

importance
 of
 using
 a
 critical
 eye
 and
 to
 question
 everything
 I
 do
 before
 I
 attempt
 them.
 I

would
 also
 like
 to
 thank
 Dr.
 Wing
 for
 allowing
 me
 to
 be
 part
 of
 the
 scientific
 writing
 process

and
 teaching
 me
 what
 it
 entails.

  I
 would
 also
 like
 to
 acknowledge
 the
 members
 of
 my
 committee:
 Drs.
 Ernesto
 Abel-­‐
Santos,
 Andrew
 Andres,
 and
 Eduardo
 Robleto.
 They
 have
 been
 an
 incredible
 resource
 to
 me

whenever
 I
 have
 questions
 or
 needed
 help
 with
 an
 experiment
 or
 concept,
 and
 for
 that,
 I

thank
 them.

  I
 would
 also
 like
 to
 acknowledge
 the
 incredible
 intellectual
 and
 emotional
 support

that
 has
 come
 with
 past
 and
 current
 members
 of
 the
 Wing
 lab.
 I
 would
 like
 to
 especially

thank
 Michael
 Picker,
 Natasha
 Weatherspoon-­‐Griffin,
 and
 Monika
 Karney
 who
 have
 offered

me
 honest
 and
 unbiased
 advice
 that
 has
 been
 applied
 to
 my
 research
 throughout
 the
 years.

  I
 would
 also
 like
 to
 thank
 members
 of
 the
 Amy,
 Robleto,
 Hedlund,
 Abel-­‐Santos,
 and

Elekonich
 labs.
 They
 have
 helped
 with
 so
 many
 parts
 of
 my
 research
 that
 I
 cannot
 list
 them

all
 here.

  I
 would
 like
 to
 acknowledge
 the
 funding
 provided
 by
 USDA
 Grant
 NEVR-­‐2010-­‐
03755
 and
 the
 UNLV
 GPSA.

  Finally,
 I
 would
 like
 to
 thank
 my
 family
 ad
 friends
 for
 all
 of
 their
 emotional
 support.

I
 would
 like
 to
 thank
 my
 husband,
 Nick,
 for
 always
 participating
 in
 my
 research
 and
 always

encouraging
 me
 to
 pursue
 my
 passions.

 My
 parents
 have
 always
 encouraged
 me
 to
 look
 at

the
 world
 from
 a
 different
 perspective,
 and
 for
 that,
 I
 will
 be
 forever
 grateful.

  v
DEDICATION

To
 my
 family,

for
 always
 believing
 in
 me
 and

supporting
 me
 in
 any
 venture
 I
 dare
 take.

Thank
 you
 and
 I
 love
 you.

  vi
TABLE
 OF
 CONTENTS

ABSTRACT
 ....................................................................................................................................................................
 iii

ACKNOWLEDGEMENTS
 ..........................................................................................................................................
 v

LIST
 OF
 TABLES
 ........................................................................................................................................................
 ix

LIST
 OF
 FIGURES
 ........................................................................................................................................................
 x

CHAPTER
 1:
 INTRODUCTION
 ...............................................................................................................................
 1

1.1.
 The
 Importance
 of
 Honeybees
 in
 Agriculture
 .................................................................................
 1

1.2.
 American
 Foulbrood
 Disease
 ..................................................................................................................
 2

1.3.
 The
 Organism
 Paenibacillus
 larvae
 .......................................................................................................
 7

1.4.
 Treatment
 Methods
 of
 AFB
 ......................................................................................................................
 8

1.5.
 Honeybee
 Immunity
 ...................................................................................................................................
 9

1.5.1.
 Social
 Immunity

 ...............................................................................................................................
 10

1.5.2.
 Innate
 Immunity

 ..............................................................................................................................
 11

1.5.2.1.
 Antimicrobial
 Peptides
 .........................................................................................................
 13

1.6.
 Overall
 Goal
 of
 the
 Project
 &
 Rationale
 ...........................................................................................
 15

1.7.
 References
 ...................................................................................................................................................
 17

CHAPTER
 2:
 PROTOCOLS
 TO
 TEST
 THE
 ANTIMICROBIAL
 ACTIVITY
 OF
 ANTIMICROBIAL

PEPTIDES
 AGAINST
 THE
 HONEY
 BEE
 PATHOGEN
 PAENIBACILLUS
 LARVAE
 .............................
 23

2.1.
 Forward
 .........................................................................................................................................................
 23

2.2.
 Keywords
 ......................................................................................................................................................
 23

2.3.
 Abstract
 .........................................................................................................................................................
 23

2.4.
 Introduction
 ................................................................................................................................................
 24

2.5.
 Methods

 ........................................................................................................................................................
 25

2.5.1.
 Growth
 of
 Bacterial
 Strains

 .........................................................................................................
 25

2.5.2.
 Zone
 of
 Inhibition
 Assay

 ..............................................................................................................
 26

2.5.3.
 96-­‐well
 Plate
 Assay

 ........................................................................................................................
 26

2.6.
 Results
 ............................................................................................................................................................
 27

2.7.
 Conclusions

 .................................................................................................................................................
 29

2.8.
 Acknowledgements

 .................................................................................................................................
 30

2.9.
 References
 ....................................................................................................................................................
 31

CHAPTER
 3:
 CHARACTERIZATION
 OF
 HONEYBEE
 ANTIMICROBIAL
 PEPTIDES
 AGAINST

THE
 HONEYBEE
 PATHOGEN
 PAENIBACILLUS
 LARVAE
 .........................................................................
 32

3.1.
 Introduction
 ................................................................................................................................................
 32

3.2.
 Methods
 .........................................................................................................................................................
 39

3.2.1.
 Source
 and
 Storage
 of
 AMPs
 ........................................................................................................
 39

3.2.2.
 Growth
 Conditions
 of
 Organisms

 ..............................................................................................
 39

3.2.3.
 Zone
 of
 Inhibition
 Assay

 ...............................................................................................................
 40

3.2.4.
 96-­‐well
 Plate
 Assay

 .........................................................................................................................
 40

3.2.5.
 Statistical
 Analysis
 ............................................................................................................................
 41

  vii
3.3.
 Results
 ............................................................................................................................................................
 41

3.3.1.
 In
 vitro
 Testing
 of
 Apidaecin
 for
 Antimicrobial
 Activity
 ..................................................
 42

3.3.2.
 In
 vitro
 Testing
 of
 Abaecin
 for
 Antimicrobial
 Activity
 ......................................................
 45

3.3.3.
 In
 vitro
 Testing
 of
 Hymenoptaecin
 for
 Antimicrobial
 Activity
 ......................................
 48

3.3.4.
 In
 vitro
 Testing
 of
 Defensin
 1
 for
 Antimicrobial
 Activity
 .................................................
 52

3.3.5.
 In
 vitro
 Testing
 of
 Defensin
 2
 for
 Antimicrobial
 Activity
 .................................................
 55

3.3.6.
 In
 vitro
 Testing
 of
 Individual
 AMPs
 for
 Precipitation
 .......................................................
 58

3.3.7.
 96-­‐well
 Plate
 Assays
 for
 AMP
 Combinations
 ........................................................................
 60

3.4.
 Conclusion
 ....................................................................................................................................................
 62

3.5.
 References
 ....................................................................................................................................................
 68

CHAPTER
 4:
 DISCUSSION
 ....................................................................................................................................
 71

4.1.
 Conclusions
 ..................................................................................................................................................
 71

4.1.1.
 Methods
 Development
 ...................................................................................................................
 71

3.2.2.
 Identification
 of
 AMPs
 Active
 Against
 P.
 larvae
 ...................................................................
 75

4.2.
 Future
 Directions
 ......................................................................................................................................
 76

4.3.
 References
 ....................................................................................................................................................
 79

APPENDIX

 .................................................................................................................................................................
 81

CURRICULUM
 VITAE
 .............................................................................................................................................
 93

  viii
LIST
 OF
 TABLES

TABLE
 1.
 Crops
 dependent
 on
 honeybee
 pollination
 and
 attributable
 values
 ...............................
 1

TABLE
 2.
 A
 list
 of
 honeybee
 cationic
 AMPs
 and
 their
 characteristics
 .............................................
 14

TABLE
 3.
 Summary
 of
 honeybee,
 cationic
 AMPs
 and
 their
 characteristics
 ...................................
 38

TABLE
 4.
 Zone
 of
 inhibition
 assay
 data
 for
 apidaecin
 ............................................................................
 42

TABLE
 5.
 Zone
 of
 inhibition
 assay
 data
 for
 abaecin
 .................................................................................
 45

TABLE
 6.
 Zone
 of
 inhibition
 assay
 for
 hymenoptaecin
 ...........................................................................
 48

TABLE
 7.
 Zone
 of
 inhibition
 assay
 for
 defensin
 1
 .....................................................................................
 52

TABLE
 8.
 Zone
 of
 inhibition
 assay
 for
 defensin
 2
 .....................................................................................
 55

TABLE
 9.
 Summary
 of
 actual
 results
 for
 the
 ZoI
 and
 96-­‐well
 plate
 assays
 ....................................
 63

TABLE
 10.
 Summary
 of
 expected
 results
 for
 the
 ZoI
 and
 96-­‐well
 plate
 assays
 ...........................
 65

TABLE
 A1.
 Sequences
 of
 honeybee
 AMPs
 used
 .........................................................................................
 83

  ix