Coxiella burnetii: Recent Advances and New Perspectives in Research of the Q Fever Bacterium

412 Pages·2012·7.03 MB·English

by  Karim E. Hechemy (auth.), Rudolf Toman, Robert A. Heinzen, James E. Samuel, Jean-Louis Mege (eds.)

Coxiella burnetii: Recent Advances and New
Perspectives in Research of the Q Fever Bacterium
ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY
Editorial Board:
IRUN R. COHEN, The Weizmann Institute of Science
ABEL LAJTHA, N.S. Kline Institute for Psychiatric Research
JOHN D. LAMBRIS, University of Pennsylvania
RODOLFO PAOLETTI, University of Milan
For further volumes:
http://www.springer.com/series/5584
Rudolf Toman (cid:129) Robert A. Heinzen
James E. Samuel (cid:129) Jean-Louis Mege
Editors
Coxiella burnetii:
Recent Advances and New
Perspectives in Research
of the Q Fever Bacterium
Editors
Rudolf Toman Robert A. Heinzen
Department of Rickettsiology Coxiella Pathogenesis Section
Institute of Virology Rocky Mountain Laboratories
Slovak Academy of Sciences NIH/NIAID
Bratislava, Slovakia Hamilton, MT, USA
James E. Samuel Jean-Louis Mege
Department of Microbial and Molecular Unité de Recherche sur les Maladies
Pathogenesis Infectieuses Transmissibles et
College of Medicine Emergentes
Texas A&M Health Science Center CNRS-IRD UMR 6236
3112 Medical Research and Education Institut Fédératif de Recherche 48
Building Faculté de Médecine
Bryan, TX, USA Université de la Méditerranée
Marseille, France
ISSN 0065-2598
ISBN 978-94-007-4314-4 ISBN 978-94-007-4315-1 (eBook)
DOI 10.1007/978-94-007-4315-1
Springer Dordrecht Heidelberg New York London
Library of Congress Control Number: 2012941642
© Springer Science+Business Media Dordrecht 2012
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Preface

Over 20 years have elapsed since the publication of the seminal two-volume series
entitled Q Fever: The Biology of Coxiella burnetii (edited by J.C. Williams and
H.A. Thompson) and Q Fever: The Disease (edited by T.J. Marrie) that described
the current state of C oxiella burnetii research. The ensuing years have brought the
post-genomic era and accompanying technologies that have catalyzed major advances
in the fi eld, including milestone discoveries of genetic transformation and host
cell-free growth of this former obligate intracellular bacterium. C oxiella has matured
from a niche organism, investigated by a handful of laboratories worldwide, to a
model system to study macrophage parasitism, developmental biology, host-pathogen
interactions, and immune evasion/modulation. Further bolstering interest are recent
bioterrorism concerns that have delegated C oxiella to the list of regulated microbial
agents with potential for illegitimate use, a designation confounded by the organism’s
ubiquitous nature and abundant natural reservoirs. Indeed, illustrating the zoonotic
character of Q fever is a large outbreak (>4,000 cases) associated with intensive dairy
goat farming that is currently subsiding in the southern Netherlands. The outbreak
has highlighted the public health impact and re-emergent nature of Q fever, in
addition to the need for improved surveillance and control methods.
Coxiella is a fascinating example of intracellular parasitism. The organism has
uniquely evolved to thrive in the most inhospitable of cellular compartments—the
phagolysosome. Understanding how C oxiella resist the degradative functions of this
vacuole, and the host cell functions co-opted for successful parasitism, is central to
understanding Q fever pathogenesis. These topics are among the relevant and timely
research areas comprehensively reviewed in this volume. Structure-function
relationships are discussed in chapters on C oxiella proteomics, lipopolysaccharide,
and lipids (bacterial and host). Improved knowledge in these subject areas is guiding
development of enhanced detection schemes for C oxiella in addition to shedding
light on the host immune response to the Q fever pathogen and how it survives
within the host. The cellular microbiology of the pathogen-host relationship is
explored in chapters detailing how C oxiella responds to oxidative stress, manipulates
host cell pro-survival signaling, and directs biogenesis of a replication vacuole.
These and other host cell processes are likely modulated by a repertoire of secreted
v
vi Preface
proteins with speci fi c effector functions examined in a thorough review of C oxiella
secretory systems and veri fi ed secreted proteins. The biological relevance and
molecular biology of Coxiella ’s bi-phasic developmental cycle is explored. Several
chapters survey immune functions that control or potentially exacerbate C oxiella
infection and delve into correlates of protective immunity elicited by vaccination.
These topics are particularly relevant to development of a safe and effective Q fever
vaccine that does not require pre-vaccination skin testing. C oxiella genetic diversity
is reviewed with the aim of better understanding the importance of strain variation
and pathogenetic potential. Comparative genomics is also the foundation of
chapters discussing diagnostic antigen discovery and molecular typing of Coxiella,
with signi fi cance for development of new clinical, epidemiologic, and forensic
tools. A rather underexplored area with relevance to the evolution of C oxiella as
human pathogen is discussed in a chapter on C oxiella -like endosymbionts of
ticks. The Netherlands Q fever outbreak is described with subject areas including
surveillance, challenges in laboratory diagnostics, post-acute Q fever sequela, risks
for pregnant women, and mitigating veterinary measures. Finally, chapters on host
cell-free (axenic) growth and genetic manipulation of C oxiella illustrate how one
technological advance feeds another. Axenic growth has relieved the considerable
experimental constraints imposed by C oxiella’s previous obligate intracellular
lifestyle and enabled new lines of investigation. The increasing genetic tractability
of Coxiella will inevitably lead to novel insight into intracellular parasitism and
disease pathogenesis.
In Chap. 1 , Hechemy introduces Coxiella as a pathogen and the natural history
of the disease Q fever. Events leading to the co-discovery of Coxiella by Australian
and American researchers are covered. Focused overviews of disease pathogenesis,
epidemiology, diagnosis, treatment, and vaccination provide a prospective of Q fever
as a zoonosis of continuing concern.
In Chap. 2 , van Schaik and Samuel summarize studies that have compared the
variety of isolates of C . burnetii from clinical, animal, and environmental sources,
which phylogenetically separate these isolates into groups of close relationship
using molecular techniques with increasing resolution. At the most precise level of
investigation, whole genome sequence analysis has predicted a variety of poly-
morphic loci that might contribute to pathotype differences established in animal
models of acute disease and consistent with difference in acute and chronic human
infections. The chapter then predicts genes that might mediate differences in
virulence with various mechanisms reviewed in subsequent chapters.
In Chap. 3 , Mertens and Samuel detail predictions based on the sensitivity to
oxidative stress identi fie d in C. burnetii . Reactive oxygen intermediates (ROI)
and reactive nitrogen intermediates (RNI) are the principal host innate defense
mechanisms employed to control infection. A principal strategy of pathogenesis
is to avoid activating ROI and RNI responses, and extracellular growth restricted
to low oxygen environment supports the hypothesis that C oxiella has evolved to
occupy such an environment. Important genome reductions have resulted in reduced
detoxi fi cation mechanisms and DNA repair enzymes for survival in oxidative
environment.
Preface vii
In Chap. 4 , Narasaki and Toman provide a summary of studies on the only
con fi rmed virulence macromolecular complex, lipopolysaccharide, providing a
summary of studies on its composition and structure, biological activities and bio-
synthetic pathways for the unique sugars (virenose and dihydrohydroxystreptose)
found in the O-chain. The differential expression of O-chain and core LPS is the
bases of serodiagnostic antigens (phase I and II), targets for vaccine-induced
protective antibody, and alternate interactions with host cells. While the structural
prediction for the lipid A moiety of LPS appears to support studies that demonstrate
antagonistic activity for TLR4 host receptors, other studies suggest LPS involve-
ment in a variety of cellular responses.
In Chap. 5 , Zhang, Zhang and Samuel explore the literature for an understanding
of which components of the host immune response are essential for protective
immunity engendered after infection or as a result of vaccine-induced immunity.
One novel discovery by the authors identi fi ed an important role for antibody in
protection, which is distinct from traditional models of protective immunity against
an intracellular pathogen but has become recently appreciated for a variety of other
bacterium models. The target of antibody-speci fi c immunity appears to include
the O antigen of LPS and may provide a component of next generation subunit
vaccines. But equally well documented are the principal mechanisms of clearance
in infection and vaccine-induced immunity as T-cell-mediated effectors elicited by
activating cytokines, especially interferon-g .
In Chap. 6 , Ihnatko, Shaw and Toman critically evaluate the rapidly expanding
fi eld of C oxiella proteomics. Sub-cellular and whole cell proteomic studies
have pro vided new knowledge of Coxiella developmental biology, physiology,
secreted proteins, and strain variation associated with different virulence potential.
Immunoproteomics, wherein whole proteome microarrays are high-throughput
screened for seroreactive proteins, are revealing candidate antigens with potential
use in a new generation of Q fever diagnostic and vaccine antigens based on recom-
binant protein. The chapter also details how proteome studies dovetail with genomics
to reveal conserved and unique C oxiella biomarkers useful as detection tools.
In Chap. 7 , Hussain and Voth describe the adept ability of C oxiella to manipulate
host cell pro-survival signaling and describe pathogen reliance on various cellular
kinases for proper development of the parasitophorous vacuole (PV). Akt and
Erk1/2 are two pro-survival kinases actively modulated by Coxiella to promote host
viability. Interplay between autophagic and apoptotic pathways is also observed
during C oxiella infection, as exempli fi ed by the interaction of autophagy-related
Beclin-1 and anti-apoptotic Bcl-2 proteins. The general anti-apoptotic activity of
Coxiella is viewed as a virulence strategy to sustain the host for the duration the
pathogen’s lengthy infectious cycle.
In Chap. 8 , Ghigo, Colombo and Heinzen examine the biogenesis and biological
character of the C oxiella PV. The PV is the intracellular microenvironment of
Coxiella from which it interfaces with host cells. With the exception of human
macrophages/monocytes, studies agree that the PV completely matures through the
endolysosomal pathway to acquire characteristic of a phagolysosome. C oxiella
has the uncanny ability to resist the degradative functions of the vacuole and to
viii Preface
exploit its acidic pH for metabolic activation. This chapter presents opposing
traf fi cking models for virulent phase I and avirulent phase II Coxiella in human
macrophages/monocytes and discusses the different behavior of phase variants in
primary murine macrophages. Cumulative evidence indicates that the PV is a spe-
cialized compartment that is actively modi fi ed by the pathogen, with interactions
extending to engagement with autophagic and early secretory pathways.
In Chap. 9 , McDonough, Newton and Roy critically review C oxiella secretion
systems. The ability of Coxiella to transport proteins with effector functions across
its membranes is considered critical for conditioning of the PV lumen and manipu-
lating host cell processes that bene fi t the pathogen. C oxiella encodes components of
the Sec-dependent secretion pathway, and an export system used for type IV pilus
assembly, that are predicted to deliver proteins to the PV lumen. On the other hand,
a Coxiella type IVB secretion system that is functionally analogous to the L egionella
pneumophila Dot/Icm secretion system has recently been veri fi ed to translocate
proteins to the host cell cytoplasm where their speci fi c activities manipulate a
variety of host cell processes, including pro-survival signaling and PV biogenesis.
In Chap. 1 0 , Gilk examines the minimally studied but critically important topic
of lipid metabolism (pathogen and host) and C oxiella infection. C oxiella requires
lipids for both normal bacterial functions and formation of its PV membrane.
The organism acquires lipids through d e novo synthesis, and potentially through
subversion of host cell pools. Phospholipids comprising the C oxiella cell envelope
contain a high percentage of branched fatty acids, a modi fi cation that may serve
a protective role. The PV membrane is sterol-rich and C oxiella encodes two
“orphan” eukaryotic-like sterol reductases predicted to catalyze late reduction steps
in cholesterol biosynthesis. Indeed, at least one reductase is enzymatically active,
leading to the hypothesis that Coxiella modi fi es host cell sterols. Other lipid
modifying enzymes of C oxiella include phospholipases of unknown function.
Elucidation of how Coxiella exploits host cell lipids will improve our understanding
of the interplay between pathogen and host.
In Chap. 11 , Omsland discusses the milestone advance of C oxiella host cell-free
(axenic) growth. Coxiella ’s prior obligate intracellular lifestyle presented severe
experimental hurdles to gaining an improved understanding of the pathogen lifestyle.
This chapter summarizes how transcription microarrays, metabolic pathway
reconstruction, and metabolite typing aided development of acidi fi ed citrate cysteine
medium (ACCM), a medium that supports robust growth of C oxiella in liquid
culture as well as colony formation in semi-solid agarose plates. Technical issues
related to ACCM culture are discussed as are utilization of the media in genetic
transformation and viability assays. The technique is currently facilitating new lines
of investigation and dramatically aiding development of new C oxiella genetic tools.
ACCM culture methods should also prove useful in a clinical setting.
In Chap. 12 , Minnick and Raghavan provide an overview of the developmental
biology of C oxiella . Pronounced environmental stability is a hallmark of C oxiella
that promotes ef fi cient aerosol transmission. C oxiella undergoes a biphasic devel-
opmental cycle that generates morphologically distinct small cell variants (SCV)
and large cell variants (LCV). The transcriptional and translational properties of
Preface ix
SCV and LCV, and ultrastructural observations of intracellular C oxiella , support a
model whereby non-replicating SCV differentiate into replicating LCV. Based on
SCV structure (i.e., condensed chromatin and multi-layered cell envelope) and
mechanical disruption experiments, the SCV is considered the extracellular survival
form of Coxiella that initiates most natural infections. Interestingly, SCV and LCV
appear equally infectious for cultured cells. Many interesting questions related to
the molecular biology of C oxiella differentiation remain unanswered . For example,
do two-component regulatory systems drive stringent response physiology that
triggers LCV to SCV transition during stationary phase? A better understanding
of the pathogenic roles of Coxiella developmental forms in the natural history of
Q fever is an important research goal.
In Chap. 1 3 , Beare provides a brief overview of successes in genetic transforma-
tion of obligate intracellular bacteria and discusses the current state of Coxiella
genetic manipulation including practical aspects of genetic transformation protocols.
The rescue of C oxiella from its host cell eliminated several technical obstacles
associated with host cell-based transformation systems, such as dif fi culties in cloning
and transformant expansion/characterization. Axenic culture has allowed the rapid
expansion of the C oxiella genetics toolbox to currently include m ariner -based
Himar1 transposon systems for random mutagenesis, a Tn7 system for site-speci fi c
single-copy, chromosomal gene integration and i n cis complementation, and multiple
permutations of RSF1010 o ri -based shuttle vectors that allow multi-copy i n trans
complementation and heterologous gene expression. A reliable method of targeted
gene inactivation remains a current, but not insurmountable, challenge. The ability
to ful fi ll molecule Koch’s postulates for putative virulence genes will shepherd a
new era of C oxiella virulence factor discovery.
In Chap. 14 , Capo and Mege provide a wide-ranging view of elements of both
the innate and adaptive immune responses that are engaged in controlling infection
in acute and chronic infections. This chapter also provides an excellent example of
the strength of this book in presenting several of the important controversies associ-
ated with modeling Q fever infection and pathogenesis. The authors provide a model
for innate responses essential for clearance that does not involve IFN-g killing
through generation of ROI or RNI but rather engagement of nutritional restriction
and activation of TNF-mediated apoptosis. The authors also posit an important
role for TLR4, although other studies have shown the lipid A moiety of C . burnetii
LPS does not serve as an agonist for TLR4-mediated activation. A detailed and
important review of the dysregulation of immune suppression noted in chronic
Q fever patients provides a basis for understanding the host component to this
disease. Additionally, the authors provide a summary of recent (some unpublished)
results that may provide a basis for sex-related susceptibility to acute Q fever
symptoms in males.
In Chap. 15 , Amara, Bechah and Mege outline several novel models describing
mechanisms C . burnetii may use to evade the host response, thereby allowing per-
sistence. Adipose and placenta tissues provide unique sites within the host to persist
and C. burnetii may utilize these tissues to avoid host immune surveillance and to
provide a permissive replication environment. The characterization that C . burnetii
x Preface
exists in a non-replicating, viable state in M1 polarized monocytes and replicate
well in M2 polarized macrophage forms the basis for the principle infection model
and con fi rms the general appreciation that infected macrophages do not become
active in vitro. The role of the immunomodulatory cytokine, IL-10, in establishing
the M1/M2 polarization and how dysregulated overexpression is critical for chronic
infection is carefully reviewed. Finally, the role that apoptotic cell engulfment may
play in infection and new data suggesting regulatory T cell involvement in chronic
patient infection provide exciting expansions to the model of persistence.
In Chap. 16 , Hendrix and Chen provide a detailed review of the vaccines and
diagnostic methods that have been applied to Q fever. With this summary of previous
approaches to these related problems, the authors explore current technologies that
are designed to identify novel reagents for both subunit vaccines and diagnostic
methods. The limitations to these approaches require a clear identi fi cation of strain
heterogeneity and studies that summarize this diversity of C. burnetii isolates are
provided. The thrust of this review is how characterization of dominant protein
antigens, through traditional gel separation, mass spectrometric description, and
genome wide expression of the entire proteome, has each contributed to the current
understanding of dominant B and T cell antigens. Accompanying this review is
an extensive table cataloging all proteins identi fi ed in the literature as immuno-
dominant proteins.
In Chap. 1 7 , Van Der Hoek et al. present a summary of the recent Q fever
outbreak in the Netherlands where more than 4,000 human cases were reported
from 2007 to 2010. This important review provides a careful and extensive
analysis of the measures taken to de fi ne and successfully mitigate this seasonal
epidemic. Besides providing a chronological summary of the events between 2007
and 2010, this chapter also provides an interesting analysis of public health policy,
surveillance strategies, zoonotic transmission and selective application of large-scale
animal vaccination. In particular, the ability to survey such a large population of
at-risk and validated infections provides the opportunity to explore the risk factors
associated with pregnancy for infected individuals and various chronic manifestations
including chronic fatigue syndromes. The careful de fi nition and evaluation of
various diagnostic strategies will be important for public health surveillance and
policy worldwide. Finally, the opportunity to administer the human Q fever vaccine,
manufactured and licensed in Australia, to at-risk individuals will provide additional
safety and ef fi cacy data for future licensure consideration in the Netherlands and
possibly other countries.
In Chap. 1 8 , Zhong probes the fascinating fi eld of C oxiella -like endosymbionts.
A diverse collection of Coxiella -like bacteria have recently been found in hard and
soft ticks, as well as in a few vertebrate hosts. In some tick species, colonization
rates approach 100%. C oxiella -like bacteria and virulent C . burnetii are highly
homologous and form a monophyletic clade. This review discusses the techniques
used to study Coxiella -like bacteria and delves into their putative functions.
Improved understanding of the biological signi fi cance of C oxiella -like bacteria will
shed light on the evolution of C. burnetii as a human pathogen.