Combined Experimental Attenuation and Pan-genomic Analysis Allow for Inference of Novel ...

161 Pages·2014·21.67 MB·English

UC San Diego
UC San Diego Electronic Theses and Dissertations
Title
Combined Experimental Attenuation and Pan-genomic Analysis Allow for Inference of Novel
Virulence Genes in Leptospira interrogans
Permalink
https://escholarship.org/uc/item/65w0638z
Author
Lehmann, Jason Steven
Publication Date
2014

Peer reviewed|Thesis/dissertation
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University of California
UNIVERSITY OF CALIFORNIA, SAN DIEGO

Combined Experimental Attenuation and Pan-genomic Analysis
Allow for Inference of Novel Virulence Genes in Leptospira interrogans

A dissertation submitted in partial satisfaction of
the requirements for the degree of Doctor of Philosophy
in

Biomedical Sciences

by
Jason Steven Lehmann

Committee in Charge:
Professor Victor Nizet, Chair
Professor Bruce Hamilton
Professor Dwayne Stupack
Professor Emily Troemel
Professor Joseph Vinetz

2014
Copyright
Jason S. Lehmann, 2014
All rights reserved.
The Dissertation of Jason Steven Lehmann is approved, and is acceptable
in quality and form for publication on microfilm and electronically:

______________________________________________________________

______________________________________________________________

______________________________________________________________

______________________________________________________________

______________________________________________________________
Chair

University of California, San Diego
2014

iii
Dedication

To my Grandfather, Dr. Quentin Lehmann.
His support and encouragement
have been constant longer than my memory,
and without which none of this would have been conceivable.

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Table of Contents

Signature Page……………………………………....…………..……….............. iii
Dedication Page………………………………………..…………….....….....….. iv
Table of Contents……………………………….………………...............….……v
List of Figures and Table……………………….…..……….......................….. xi
Acknowledgments…………………………………………………..............….. xiv
Vita………………………………………………………….......………………...... xv
Abstract of the Dissertation……………....………………........................... xviii
Chapter 1
Introduction: Leptospira and Leptospirosis…………………………….…… 1
1.1 Introduction...................................................................................... 2
1.2 Historical Perspectives of Leptospirosis………………...…………... 3
1.3 Leptospira Biology……………………………………………………... 4
1.4 Transmission Cycle……………………………………...……....…..... 6
1.5 Human Epidemiology………………………………………………….. 7
1.6 Disease Pathogenesis and Manifestations……………………....…. 8
1.7 Virulence Factors……………………………………………………... 11
1.8 Genetic Tools for Leptospira…………………………………......…. 13
1.9 Pathogenomics Overview………………....………...…………….… 15

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Chapter 2
Pathogenomic Inference of Virulence-Associated
Genes in Leptospira interrogans…………...…………….......…….….......….17
2.1 Introduction………………………....………..........……….…………..18
2.2 Methods………………………....………………….............................20
Ethics Statement……………………………...........................…..20
Bacterial Strain Maintenance and Attenuation of
L. interrogans serovar Lai strain 56601…………….............…...20
Genome Assembly of Virulent P1 and Attenuated
P19 L. interrogans serovar Lai strain 56601 and
Non-synonymous SNV (nsSNV) Detection................................21
RT-qPCR In Vivo Gene Expression Analysis.............................22
Domain Architecture Analysis of LA_4008
and Other Related AGC Proteins……………………..................23
Leptospira Concentrated Culture Supernatant (CCS)……....….23
CCS cAMP Elevating Activity.....................................................24
CCS Immunodepletion Studies..................................................24
Phylogenetic Analysis of PF07598
Paralogous Protein Family…………………..………….......…….25
Pan-genomic Analysis of Attenuated
Genes and PF07598 Orthologs……………....…………......…...25
Statistics…………………………………...…………....................27

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2.3 Results…………………………………................................................…....28
Pathogenomic Identification of Protein Coding Genes
in Leptospira interrogans serovar Lai and Patterns of
Tissue-specific Up-regulation In Vivo…………...…….....……....28
Identification of a Putative Leptospiral Protein
with Host cAMP Elevating Activity…....…………….....………....29
Identification of a Paralogous Gene Family Shared by
Pathogenic Leptospira, Bartonella bacilliformis, and
Bartonella australis with Profound, Tissue-specific
Up-regulation In Vivo in an Acute Leptospirosis
Infection Model in Hamsters……….......………………........…....31
Other Pathogenomically-identified Putative
Virulence Genes in Leptospira Species……………......……......33
2.4 Discussion………………………………………………………...........35
2.5 Acknowledgements…..……....…....….....…………….....………......41

Chapter 3
Recombinant LA_4008 Adenylate Cyclase Elevates
Monocyte cAMP-levels In-Vitro…........…....…………...……….......…….......54
3.1 Introduction…………………………………………………....……......55
3.2 Methods………………………………………………….........……......55
Gene Synthesis..........................................................................55
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E. coli Transformation................................................................56
Recombinant Protein Expression...............................................56
Recombinant Protein Purification...............................................57
Gel Electrophoresis and Western Blotting.................................57
Cyclic AMP Elevation Assay......................................................58
3.3 Results………………………...………………………........................59
Heterologous Expression of LA_4008 in E. coli…..........……....59
Recombinant LA_4008 Adenylate Cyclase
Elevates Monocyte cAMP Levels In-vitro……….....………….....60
3.4 Discussion…………………………………...………………......…......60
3.5 Acknowledgements……......…....……………………...............….....61

Chapter 4
Dynamic Deep Sequencing During Attenuation
Shows Selection on Leptospira Regulatory Proteins…....…...……...…....67
4.1 Introduction.....................................................................................68
4.2 Methods..........................................................................................71
Ethics Statement........................................................................71
Attenuation of L. interrogans serovar Lai strain 56601..............71
Genomic Library Preparation and Assembly.............................73
COG Functional Category Analysis of
nsSNV Containing Genes..........................................................75
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Pan Leptospira Genus Analysis of
Study Identified Genes...............................................................75
Amino Acid Residue Conservation Analysis of
Study Identified nsSNV Positions...............................................76
Identification of Potential ncRNAs in the
L. interrogans serovar Lai 56601 Genome.................................77
4.3 Results………………………………………………………….…….....79
Culture Passage-based Attenuation of
Leptospira interrogans serovar Lai strain 56601…....................79
Identification of SNVs Within the Attenuated P8 L. interrogans
serovar Lai Isolate Genome with Differing Allele Frequencies
Compared to the Virulent P1 Parent Isolate………………..……80
Analysis of nsSNVs with Allelic Frequencies that Increased
During Attenuation…………………………………………….....…81
Pan-Leptospira Genomic Analysis of Amino Acid Residue
Conservation at nsSNV Positions in Homologs of
Attenuation-identified Genes………………………………….......83
Intergenic SNV Analysis and Novel Non-coding
RNA Prediction…………………………………………………......87
4.4 Discussion………………………………………………………….......89
4.5 Conclusions……………………………………………………...……..97
4.6 Acknowledgements……………………………………………..……..98

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