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HOST AND PATHOGEN TRANSCRIPTIONAL PROFILES OF ACUTE Brucella
melitensis INFECTION
A Dissertation
by
CARLOS ALBERTO ROSSETTI
Submitted to the Office of Graduate Studies of
Texas A&M University
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
August 2007
Major Subject: Veterinary Microbiology
HOST AND PATHOGEN TRANSCRIPTIONAL PROFILES OF ACUTE Brucella
melitensis INFECTION
A Dissertation
by
CARLOS ALBERTO ROSSETTI
Submitted to the Office of Graduate Studies of
Texas A&M University
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Approved by:
Chair of Committee, L. Garry Adams
Committee Members, Renée M. Tsolis
Terry L. Thomas
James E. Womack
Head of Department, Gerald Bratton
August 2007
Major Subject: Veterinary Microbiology
iii
ABSTRACT
Host and Pathogen Transcriptional Profiles of Acute Brucella melitensis Infection.
(August 2007)
Carlos Alberto Rossetti, D.V.M., Universidad de Buenos Aires;
M.S., Universidad de Buenos Aires
Chair of Advisory Committee: Dr. L. Garry Adams
The parallel gene expression profiles of Brucella melitensis and the host have not
been elaborated. In this study, I analyze and discuss the transcriptional profiles of B.
melitensis invasive-associated genes, the expression profile of intracellular B. melitensis
and B. melitensis-infected non-phagocytic cells in the first 12 h post-infection (PI), and
the in vivo temporal global transcriptome of both B. melitensis and the infected bovine
host in the first 4 h PI. The initial study found that B. melitensis at late-log phase of
growth were more invasive in non-phagocytic cells than at early-log or stationary growth
phase. Microarray-based studies identified 454 Brucella genes differentially expressed
between the most and the least invasive growth phases. Additionally, B. melitensis
strains with transposon interrupted in loci BMEII0380 (acrA) and BMEI1538
(hypothetical protein) were found to be deficient in internalization compare with the
wild-type strain. A second experiment was designed with the goal of characterizing host
and pathogen transcriptome in parallel. For detecting intracellular Brucella gene
expression, a combined protocol consisting of a linear amplification of sense-stranded
RNA biased to pathogen transcripts to the previously enriched host:pathogen RNA
iv
mixed sample, was developed. RNA samples were hybridized on human and Brucella
cDNA microarrays, which analysis revealed a common down-regulation transcriptional
profile at 4 h PI that was reverse at 12 h PI. The integrity of B. melitensis virB operon
and the expression of host MAPK1 were confirmed as critical for early B. melitensis
intracellular survival and replication in non-phagocytic cells. Finally, a temporal
morphological and molecular characterization of the initial B. melitensis:bovine host
interaction using a calf ileal loop model was performed. B. melitensis was isolated from
intestinal Peyer’s patches as soon as 15 min and from systemic blood after 30 min post-
intra luminal inoculation. Microarray results revealed a common transcriptional profile
in Brucella, but two different transcriptional profiles were identified in the host in the
first 4 h PI. The importance of differentially expressed biological processes, pathways
and individual genes in the initial Brucella pathogenesis is discussed.
v
DEDICATION
To my lovely wife Paola
and my two amazing sons Fernando and Pablo
vi
ACKNOWLEDGMENTS
I am entirely grateful to my advisory committee’s chair, Dr. L. Garry Adams for
his initial trust in me and for his guidance throughout my doctoral studies. I also extend
my acknowledgments to the other members of my Ph.D. Committee, Dr. Renée Tsolis,
Dr. Terry Thomas and Dr. James Womack for their advice and support during the course
of this study.
This research would not be possible without help from many other people. I
thank all members of Dr. Adams’ lab: Dr. Josely Figueiredo, Tamara Gull, Doris Hunter,
Dr. Sangeeta Khare, Jairo Nunes, Roberta Pugh and Tiffany Fausett for their invaluable
technical assistance; Dr. Sara D. Lawhon, for helpful discussions and critical reading;
Alan Patranella for taking care of experimental animals; Linda McCallum for helping
with surgical and anesthetic procedures; Rhonda Friedberg, Dr. Mitchell McGee and Dr.
Stephen A. Johnston from the Western Regional Center of Excellence (WRCE)
Pathogen Expression Core (Center for Innovations in Medicine, A.S.U.) for developing
and printing the B. melitensis cDNA microarrays; Dr. Robin E. Everts and Dr. Harris A.
Lewin, from W.M. Keck Center for Comparative and Functional Genomics, U.I.U.C. for
printing the bovine cDNA microarrays; Dr. Tom A. Ficht for sharing B. melitensis
mutants from his B. melitensis mutant’s bank; Jonathan Lawson, for sharing protocols
before publishing; Helga Bhatkar and Dr. Ross Payne, for guidance on TEM and SEM
observation; and Dr. Cristi Galindo and Dr. Harold “Skip” Garner from U.T.S.W.
vii
Medical School, Dallas, and Dr. Bryan Kamery and Dr. Ken Drake from Seralogix, Inc.,
Austin, for carrying out the microarray analysis.
Financial support for my Ph.D. program was provided by I.N.T.A. (Instituto
Nacional de Tecnología Agropecuaria) – Fulbright Argentina fellowship, and
NIH/NIAID Western Regional Center of Excellence and U.S. Department of Homeland
Security -National Center of Excellence for Foreign Animal and Zoonotic Disease
(FAZD) Defense grants.
viii
TABLE OF CONTENTS
Page
ABSTRACT.............................................................................................................. iii
DEDICATION.......................................................................................................... v
ACKNOWLEDGMENTS......................................................................................... vi
TABLE OF CONTENTS.......................................................................................... viii
LIST OF FIGURES................................................................................................... xi
LIST OF TABLES.................................................................................................... xiii
CHAPTER
I INTRODUCTION............................................................................. 1
General aspects of genus Brucella.............................................. 1
Clinical manifestations of brucellosis......................................... 2
Cell biology of Brucella infection............................................... 3
Brucella virulence factors........................................................... 8
The host response to Brucella infection...................................... 15
Models to study Brucella:host interaction.................................. 18
The goal....................................................................................... 21
II IDENTIFICATION OF Brucella melitensis INVASIVE
CANDIDATE GENES IN NON-PROFESSIONAL
PHAGOCYTIC CELLS BY MICROARRAY ANALYSIS............ 23
Introduction................................................................................. 23
Materials and methods................................................................ 24
Results......................................................................................... 32
Discussion................................................................................... 52
ix
TABLE OF CONTENTS (continued)
CHAPTER Page
III HOST AND Brucella melitensis TEMPORAL GENE
EXPRESSION PROFILES IN AN in vitro MODEL
OF INFECTION................................................................................ 65
Introduction................................................................................. 65
Materials and methods................................................................ 66
Results......................................................................................... 80
Discussion................................................................................... 111
IV TEMPORAL GLOBAL GENE EXPRESSION ANALYSIS
OF THE in vivo INITIAL INTERACTION OF BOTH
Brucella melitensis AND THE BOVINE HOST.............................. 125
Introduction................................................................................. 125
Materials and methods................................................................ 126
Results......................................................................................... 133
Discussion................................................................................... 159
V CONCLUSIONS............................................................................... 170
REFERENCES.......................................................................................................... 173
APPENDIX A........................................................................................................... 213
APPENDIX B........................................................................................................... 226
APPENDIX C........................................................................................................... 231
APPENDIX D........................................................................................................... 235
APPENDIX E............................................................................................................ 239
APPENDIX F............................................................................................................ 252
x
TABLE OF CONTENTS (continued)
Page
APPENDIX G........................................................................................................... 266
APPENDIX H........................................................................................................... 275
APPENDIX I............................................................................................................. 297
APPENDIX J............................................................................................................. 302
VITA......................................................................................................................... 306
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