Environmental Pseudomonas are a source of Novel Antibiotics that inhibit Cystic fibrosis derived

183 Pages·2017·4.77 MB·English

by  Payel Chatterjee

ENVIRONMENTAL PSEUDOMONAS ARE A SOURCE OF NOVEL ANTIBIOTICS
THAT INHIBIT CYSTIC FIBROSIS DERIVED PATHOGENIC PSEUDOMONAS
AERUGINOSA
Payel Chatterjee
A Dissertation
Submitted to the Graduate College of Bowling Green
State University in partial fulfillment of the
requirements for the degree of
DOCTOR OF PHILOSOPHY
December 2017
Committee:
Hans Wildschutte, Advisor
Kurt S. Panter
Graduate Faculty Representative
Robert McKay
Paul Morris
Scott Rogers
© 2017
Payel Chatterjee
All Rights Reserved
iii
ABSTRACT
Hans Wildschutte, Advisor
The emergence of antimicrobial resistance bacteria has become a major threat to
human society. The rapid spread of resistant pathogens and the associated loss of efficacy of
available drugs needs to be met with the development of antibiotics and alternative treatments.
Pseudomonas aeruginosa is an opportunistic human pathogen evolving resistance to many
currently used antibiotics. Chronic lung infections with the bacterium P. aeruginosa are the
leading cause of morbidity and mortality in cystic fibrosis (CF patients. Escalating this
problem is that pharmaceutical companies have dropped drug development due to low
profitability, thus making the efforts of drug discovery of prime importance. To address this
global health threat research institutes have now stepped forward to aid in discovery of novel
compounds. P. aeruginosa dominates the lungs during chronic infections in CF patients, yet
it’s abundance in non-human habitats such as water and soil is less compared to other diverse
groups of pseudomonads. A trait that could contribute to such decreased abundance is bacterial
competition from other Pseudomonas populations that dominate water and soil habitats. We
hypothesized that environmental Pseudomonas from diverse soil and water habitats produce
secondary metabolites capable of inhibiting the growth of CF derived P. aeruginosa. Here, we
sought to determine if clinical isolates of P. aeruginosa are susceptible to competition by
environmental pseudomonads which may provide a source of inhibitory factors. We have used
a population based study in association with transposon mutagenesis, PCR techniques, whole
genome sequencing and bioinformatic analysis to identify environmental Pseudomonas
iv
biosynthetic gene clusters (BGCs) and characterize antagonistic compounds that are effective
against CF-derived P. aeruginosa. A total of five BGCs have been identified in this study from
environmental Pseudomonas strains S4B6, S3F9 (soil-derived) and LE6C6 (water-derived)
encoding diverse compounds such as bacteriocins, NRPSs, phenazines, and siderophores
involved in antagonistic activity. Extending this analysis, we have also identified
environmental Pseudomonas that inhibit not only CF-derived P. aeruginosa but are effective
against other pathogens including ESKAPE pathogens and carbapenem resistant P.
aeruginosa. Overall, this research serves as a platform for the identification of novel
antibiotics from these environmental isolates.
v
I dedicate this dissertation to my parents for their constant love and support. I am truly
thankful for having you both in my life; to the loving memory of my grandfather, who would
have been proud today and to my grandmother, for always believing in me.
vi
ACKNOWLEDGMENTS
I would like to express my special appreciation and thanks to my advisor Dr.
Hans Wildschutte, for his encouragement and his excellent guidance and mentorship in
completion of my work. His guidance helped me in all the time of research and writing of this
thesis. I could not have imagined having a better advisor and mentor for my Ph.D study. His
advice on both research as well as on my career have been priceless. I am extremely grateful
to the committee members, Dr. Paul Morris, Dr. Robert Michael McKay, Dr. Scott O. Rogers,
and Dr. Kurt S. Panter for their suggestions and help in the completion of this dissertation.
I would like to extend my thanks to my lab members for the stimulating discussions
and for all the fun we have had in the last couple of years. Thank you, Elizabeth Davis for all
your suggestions and help. Thank you, Joe Basalla for your suggestions on my research and
helping me with my graph analysis. Also thanks to both Elizabeth and Joe for being my mutant
hunt and coffee time buddies. I would also like to thank Sarah James, Abigail Joy, Stephen
Norris, Britney Eggly, Mahnur Khan and Emily Verbrugge for helping me with mutant hunts
and other lab duties. Also, a special thank you to my colleague and friend Chandra Sarkar in
the department for giving me insights and suggestion on my research related questions. Many
thanks to Chris Hess, Susan schooner, Steve Queen, Sheila Kratzer and former graduate
secretary DeeDee for all their help, sweet greetings and occasional chit-chats.
Finally, I would like to thank my family for their love and support throughout writing
this thesis and my life in general. Words cannot express how grateful I am to my parents for
all of the sacrifices that you’ve made on my behalf. Your prayer for me was what sustained
me thus far. At the end I would like to give the biggest thank you to my beloved mother who
vii
spent sleepless nights and was always my support in the moments when there was no one to
answer my queries.
viii
TABLE OF CONTENTS
Page
CHAPTER I: INTRODUCTION ........................................................................................... 1
1.1 The evolution of antibiotic resistance .................................................................. 1
1.2 Cystic fibrosis and Pseudomonas aeruginosa ..................................................... 4
1.3 Multi-drug resistant Pseudomonas aeruginosa ................................................... 8
CHAPTER II: ENVIRONMENTAL PSEUDOMONADS INHIBIT CYSTIC FIBROSIS
PATIENT-DERIVED PSEUDOMONAS AERUGINOSA .................................................. 13
2.1 Results .......................................................................................................... 13
2.1.1 Population-level diversity of wild pseudomonads ................................ 13
2.1.2 Environmental pseudomonads exhibit antagonistic activity................. 14
2.1.3 Environmental pseudomonads inhibit pathogenic P. aeruginosa......... 15
2.1.4 Identification of a locus involved in antagonistic activity .................... 16
2.1.5 Multilocus Sequence Analysis (MLSA) ............................................... 18
2.2 Discussion ............................................................................................................ 20
CHAPTER III: IDENTIFICATION OF ENVIRONMENTAL PSEUDOMONAS
BIOSYNTHETIC GENE CLUSTERS (BGCs) THAT INHIBIT PATHOGENS ................ 25
3.1 Results .......................................................................................................... 25
3.1.1 Identifying strains for conjugation and transposon mutagenesis .......... 25
3.1.2 Mutant hunt results with env-Ps ........................................................... 28
3.1.2.1 Mutant screening with S06B 330 (S4B6) .............................. 28
3.1.2.2 Mutant screening with S11A 273 (S3A11) ............................ 30
3.1.2.3 Mutant screening with S09F 262 (S3F9) ............................... 31
ix
3.1.2.4 Mutant screening with10B 22 (LE5B10) ............................... 32
3.1.2.5 Mutant screening with11C 131 (LE6C11) ............................. 32
3.1.3 Identification of the mutated gene ........................................................ 32
3.1.4 Identification of disrupted biosynthetic gene cluster (BGC)
in Pseudomonas mutants............................................................................... 34
3.1.4.1 Alignment of Pseudomonas mutants to wildtype genome ..... 34
3.1.4.2 Wildtype genome annotation ................................................. 35
3.1.4.3 Identification of BGCs ........................................................... 36
3.1.4.4 Validation of BGCs using antiSMASH ................................. 38
3.1.4.5 Species Characterization of the Pseudomonas strains ........... 39
3.1.4.6 Comparison of Biosynthetic gene clusters ............................. 40
3.1.5 Growth of soil- and water-derived strains in liquid media ................... 41
3.2 Discussion ............................................................................................................ 42
CHAPTER IV: ENVIRONMENTAL PSEUDOMONADS INHIBIT MULTI-DRUG
RESISTANT PATHOGENS ................................................................................................. 48
4.1 Results .......................................................................................................... 48
4.1.1 Env-Ps inhibit CF-derived non-P. aeruginosa isolates (CF-NPs) ........ 48
4.1.2 Env-Ps inhibit other human pathogens (OHPs) .................................... 49
4.1.3 Determining multi-drug resistance (MDR) of CF-Ps ........................... 51
4.1.4 Env-Ps inhibit MDR P. aeruginosa ...................................................... 51
4.2 Discussion ............................................................................................................ 55
CHAPTER V: MATERIALS AND METHODS .................................................................. 60
5.1 Bacterial strain isolation and culture conditions .................................................. 60
x
5.2 Gene sequencing and phylogenetic analysis ........................................................ 62
5.3 Antagonistic activity ............................................................................................ 64
5.3.1 Spot assay used to verify antagonistic activity ..................................... 65
5.4 General description of Transposon (Tn) mutagenesis ......................................... 65
5.4.1 Tri-parental Mating Filter Method ........................................................ 66
5.4.2 Tri-parental Mating Spotting Method ................................................... 66
5.5 Optimization of Transposon (Tn) mutagenesis .................................................... 67
5.6 Optimization of mutant screening ........................................................................ 68
5.7 Upscale Mutant Hunt and Verification of Mutants.............................................. 69
5.7.1 Transposon mutagenesis and mutant screening
with strain LE6C6 (06C 126) ......................................................................... 70
5.7.2 Transposon mutagenesis and mutant screening
with strain S4B6 (S06B 330) ......................................................................... 71
5.7.3 Transposon mutagenesis and mutant screening
with strain S3A11 (S11A 273) ....................................................................... 72
5.7.4 Transposon mutagenesis and mutant screening
with strain S3F9 (S09F 262) .......................................................................... 72
5.7.5 Transposon mutagenesis and mutant screening
with strain LE5B10 (10B 22) ......................................................................... 73
5.7.6 Transposon mutagenesis and mutant screening
with strain LE6C11 (11C 131) ....................................................................... 73
5.8 Mutant DNA extraction and PCR to identify transposon insertion ..................... 74
5.8.1 Linker-Mediated PCR ........................................................................... 75