Download Chemical genetics discloses the importance of heme

Chemical genetics discloses the importance of
heme and glucose metabolism in Chlamydia
trachomatis pathogenesis
Patrik Engström
Department of Molecular Biology
Labratory of Molecular Infection Medecine Sweden (MIMS)
Umeå Center for Microbial research (UCMR)
Umeå University, Sweden, 2013
Cover: Inside the eukaryotic host cell; a fluorescent chemical compound
(green) binds to Chlamydia trachomatis membrane (red). DNA (blue).
Cover by: Patrik Engström
Copyright © Patrik Engström
ISBN: 978-91-7459-673-1
Electronic version avaliable at http://diva-portal.org/
Printed by: Print & Media
Umeå, Sweden 2013
Till farmor
Table of Contents
Table of Contents
Sammanfattning på svenska
Abstract
Abbreviations and terms
Papers included in this thesis
Introduction
Chlamydiae infections and treatment
The life cycle of chlamydiae
Adhesion and invasion of the host cell
A life inside the chlamydial inclusion
Regulation of the EB-to-RB transition
Subversion of host cell vesicles and organelles
Genomic recombination
Inclusion expansion
Evasion of innate immunity and prevention of host cell apoptosis
Preparing for the next round of infection
Generation of infectious EB progeny
Exit and spreading
Acquisition of carbon and energy sources from the host
Glucose metabolism of the eukaryotic host cell
Carbon and energy metabolism of C. trachomatis
ATP acquisition from the host cell
Generation of ATP
Glucose uptake and metabolism
Tricarboxylic acid (TCA) cycle
Heme metabolism
Heme biosynthesis
Bacterial infections and bone resorption
Aims of this thesis
Key findings and relevance
Additional data
Main conclusions
Approaches to study bacterial pathogenesis
Chemical genetics and chemical biology
Target identification
Acknowledgements
References
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Sammanfattning på svenska
Vilka processer i klamydia är viktiga för att denna bakterie ska bli kompetent att
infektera nya celler? Jag har i denna avhandling identifierat heme- och
glukosmetabolismen som viktiga för att klamydia ska kunna infektera nya celler och
människor.
Klamydia har ett komplicerat sätt att producera nya infektiösa klamydiabakterier.
Första steget är att en klamydia infekterar sin värdcell. Den infektiösa
klamydiabakterien kan inte föröka sig utan måste först övergå till en icke-infektiös
bakterieform. Övergången sker inuti våra humana celler och det möjliggör att en
klamydia blir flera hundra nya bakterier. Förökningen sker genom att klamydia
använder näringsämnen (t.ex. ATP och glukos) som finns i våra humana celler. Efter
ungefär en dag inuti våra celler börjar de hundratals icke-infektiösa bakterierna att
sakta övergå till den infektiösa formen som då är kompetenta att infektera nya celler.
Med andra ord så har en infektiös klamydia producerat hundratals infektiösa
bakterier genom att använda resurser som finns inuti våra egna humana celler. Vad
inducerar den icke-infektiösa formen att övergå till den infektiösa formen? Vilka
molekyler hos klamydia är viktiga för denna övergång? Jag har i denna avhandling
identifierat klamydias heme- och glukosmetabolism som viktiga för omvandlingen till
den infektiösa klamydiabakterien. Jag har gjort dessa upptäckter genom att använda
kemiska föreningar som specifikt slår ut heme- och glukosmetabolismen hos
klamydia. Både heme- och glukosmetabolismen är kopplade till energiproduktion
vilket tyder på en gemensam koppling mellan dessa processer och övergången till den
infektiösa formen. I samband med dessa upptäckter har jag också utvecklat flera
metoder att isolera klamydiastammar med bara en genförändring i sitt genom. Genom
att sedan testa hur dessa stammar växer i olika näringsförhållanden har jag kunnat
visa att klamydia kan använda glukos som den huvudsakliga energi- och kolkällan när
det finns lite näringsämnen i värdcellen.
Jag har också i detta avhandlingsarbete utvecklat metoder för att undersöka hur nya
antibiotika-liknande läkemedel påverkar klamydia. Med hjälp av dessa metoder har
jag identifierat en ny klass av kemiska föreningar (t.ex. förening ksk120) som slår ut
glukosmetabolismen hos klamydia. Förhoppningen är att dessa antibiotika-liknande
läkemedel kommer att kunna användas som behandling av framtida
klamydiainfektioner. I ett sådant scenario finns det en rad fördelar med dessa nya
läkemedel jämfört med de antibiotika som används idag, t.ex. kommer inte våra goda
bakterier att påverkas på samma sätt vilket gör att vi blir motståndskraftigare mot
sekundära infektioner. En annan möjlig fördel med dessa antibiotika-liknande
läkemedel är att immunresponsen mot klamydia blir starkare vilket medför att en
efterföljande klamydiainfektion kommer att tas hand av vårt immunförsvar.
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Abstract
Chlamydiae are important human bacterial pathogens with an intracellular life cycle
that consists of two distinct bacterial forms, an infectious form (EB) that infects the
eukaryotic host cell, and a non-infectious form (RB) that allows intracellular
proliferation. To be successful, chlamydiae need to alternate between EB and RB to
generate infectious EB’s which are competent to infect new host cells.
Chemical genetics is an attractive approach to study bacterial pathogenesis; in
principal this approach relies on an inhibitory compound that specifically inhibits a
protein of interest. An obstacle in using this approach is target identification, however
whole genome sequencing (WGS) of spontaneous mutants resistant to novel
inhibitory compounds has significantly extended the utility of chemical genetic
approaches by allowing the identification of their target proteins and/or biological
pathways.
In this thesis, a chemical genetics approach is used, I have found that heme and
glucose metabolism of C. trachomatis is specifically important for the transition from
the RB form to the infectious EB form. Heme and glucose metabolism are both
coupled to energy metabolism, which suggests a common link between the RB-to-EB
transitions. In connection with the above findings I have developed strategies that
enable the isolation of isogenic C. trachomatis mutant strains. These strategies are
based on WGS of spontaneous mutant populations and subsequent genotyping of
clonal strains isolated from these mutant populations. Experiments with the mutant
strains suggest that the uptake of glucose-6-phosphate (G-6-P) regulates the RB-to-EB
transition, representing one of the first examples where genetics has been used to
study C. trachomatis pathogenesis. Additional experiments with the mutant strains
indicate that G-6-P promotes bacterial growth during metabolic stress.
In concert with other findings presented in this thesis, I have fine-tuned methods that
could be employed to reveal how novel inhibitory chemical compounds affect
chlamydiae. In a broader context, I suggest that C. trachomatis could be used as a
model organism to understand how new inhibitory drugs affect other bacterial
pathogens.
In addition, I observed that C. pneumoniae infections resulted in generalized bone
loss in mice and that these mice display a cytokine profile similar to infected bone
cells in vitro. Thus, this study indicates that C. pneumoniae potentially can infect
bone cells in vivo, resulting in bone loss, alternatively, the inflammatory responses
seen in vivo could be the causative factor of the bone loss observed.
4
Abbreviations and terms
G-6-P
Glucose-6-phosphate
UhpC
G-6-P transporter
E-4-P
Erythrose-4-phosphate, transported via UhpC
HemG
Protoporphyrinogen oxidase, involved in heme metabolism
ATP
Adenosine-5’-triphosphate
DNA
Deoxyribonucleic acid
NADH
Nicotinamide adenine dinucleotide
NADPH
Nicotinamide adenine dinucleotide phosphate
T3SS
Type III secretion system, a virulence system
RANKL
Receptor activator of nuclear kappa-B ligand
KSK120
Compound that target Chlamydia´s glucose metabolism
IS-INP0341
Compound that target Chlamydia´s heme metabolism
WGS
Whole Genome Sequencing
SAH
Salicylidene acylhydrazide
Chemical genetics: Chemical compounds that inhibit or regulate protein
function
5
Papers included in this thesis
I.
Engström P, Bailey L, Önskog T, Bergström S, Johansson J. 2010. A
comparative study of RNA and DNA as internal gene expression controls early
in the developmental cycle of Chlamydia pneumoniae. FEMS Immunol
Med Microbiol. 58(2):244-53
II.
Engström P, Nguyen BD, Normark J, Nilsson I, Bastidas RJ, Gylfe Å,
Elofsson M, Fields KA, Valdivia RH, Wolf-Watz H, Bergström S. Mutations in
hemG mediate resistance to Salicylidene acylhydrazides. A novel link between
protoporphyrinogen oxidase (HemG) and Chlamydia trachomatis infectivity.
(Submitted manuscript)
III.
Engström P, Nguyen BD, Krishan S, Silver J, Bastidas RJ, Chorell E,
Normark J, Hultgren S, Wolf-Watz H, Valdivia RH, Almqvist F, Bergström S.
Glucose uptake and metabolism is required for the generation of infectious
Chlamydia trachomatis progeny. (Manuscript)
IV.
Engström P and Bergström S. Role of the glucose-6-phosphate transporter
UhpC in Chlamydia trachomatis growth. (Manuscript)
V.
Bailey L, Engström P, Nordström A, Bergström S, Waldenström A,
Nordström P. 2008. Chlamydia pneumoniae infection results in generalized
bone loss in mice. Microbes Infect. 10(10-11):1175-81
Additional papers not included in the thesis
1.
Wiklund P, Nordström A, Högström, Alfredsson H, Engström P, Gustafsson
T, Franks P, Nordström P. 2012. High impact loading on the skeleton is
associated with a decrease in glucose levels in young men. Clin. Endocrinol
(Oxf). 77(6):823-7
2. Sellsted M, Nyberg A, Rosenbaum E, Engström P, Wickström M, Gullbo J,
Bergström S, Lennart B.-Å, Almqvist F. 2010. Synthesis and Characterization
of a Multi Ring-Fused 2-Pyridone-Based Fluorescent Scaffold. European
Journal of Organic Chemistry. 32:6171-78
6
Introduction
Chlamydiae infections and treatment
Chlamydiae are pathogens that cause infections in humans and animals (Horn
2008). In humans, more than 100 million new chlamydiae infections are
estimated each year (Batteiger 2012). The taxonomy is confusing (Stephens,
Myers et al. 2009) and therefore I will refer to chlamydiae, which in fact
includes all species identified in this phylum, both pathogenic and nonpathogenic (Horn 2008). Chlamydiae are Gram-negative bacteria that only
can grow inside the eukaryotic host cell, thus chlamydiae species are obligate
intracellular bacteria. The most well-known chlamydiae species is Chlamydia
trachomatis (Serovar D-L), which is the most common cause of sexually
transmitted diseases (Miller, Ford et al. 2004). Untreated C. trachomatis
infections can lead to pelvic inflammatory disease (PID) and infertility
(Haggerty, Gottlieb et al. 2010). C. trachomatis (Serovar A-C) cause the eye
disease trachoma and untreated infections might lead to blindness (Burton
and Mabey 2009). Up to 50% of persons worldwide are positive for C.
pneumoniae antibodies by the age of 20 suggesting that most of us acquire
this airborne disease during our lifetime (Grayston 1992). C. psittaci is a
zoonotic bird pathogen that can be transmitted to humans and cause severe
pneumonia that might be mortal (Petrovay and Balla 2008).
Antibiotics such as doxycycline are effective in treating most chlamydial
infections, although resistant strains have been identified (Somani, Bhullar et
al. 2000) and failures in treatment have been described (Batteiger BE 2010
JID). New anti-chlamydial drugs are needed that target virulence factors such
that the host’s normal bacterial flora is left unaffected (Cegelski, Marshall et
al. 2008). Drugs that allow limited bacterial proliferation but do not allow
transition to infectious stages are also needed. Such drugs would induce
robust immune responses that protect against a second infection (Nguyen,
Cunningham et al. 2011, Valdivia 2012).
1
The life cycle of chlamydiae
Chlamydiae have an unusual biphasic life cycle where the bacteria alternate
between the two distinct forms, the EB form (elementary body) and the RB
form (reticulate body). The EB is the infectious form that adheres to and
invades the target host cell by promoting endocytosis. After entry, the EB
transitions to the RB which is the form that proliferates within the
endocytosed vacuole, termed the chlamydial inclusion. Midway in the
infection, triggered by an undefined signal, the transition of RB to infectious
EB progeny occur (Fields and Hackstadt 2002). Rupture of the host cell
releases infectious EBs and new host cells can be infected. Alternatively, intact
inclusions leave the host cell via an extrusion mechanism (Hybiske and
Stephens 2007). During the chlamydiae life cycle many genes are temporally
expressed and expression of these genes is linked to the progression of the life
cycle (Shaw, Dooley et al. 2000, Belland, Zhong et al. 2003, Nicholson,
Olinger et al. 2003, Maurer, Mehlitz et al. 2007, Engström, Bailey et al. 2010,
Rosario and Tan 2012). Upon induction of stress such as nutrition and energy
limitation chlamydiae enters a form were DNA replication continues and
bacteria become enlarged, however, the aberrant RBs are unable to divide
(wyrrick 2010).
Chlamydiae, as other obligate intracellular bacteria, sequester metabolites
from the host cell cytoplasm and therefore lack coding capacity for multiple
biosynthetic pathways (Stephens, Kalman et al. 1998). Biosynthetic pathways
are likely to exist according to the “use it or lose it” principle, however there
are examples where intracellular bacteria have lost biosynthetic capacity of
pathways that are “in use” (Moran 2002). Lack of genetic tools have hampered
the understanding of the chlamydiae life cycle, however recent advances in
isolation of isogenic C. trachomatis mutant strains (Kari, Goheen et al. 2011,
Nguyen and Valdivia 2012, Sandoz, Eriksen et al. 2012) have the potential to
significantly increase our understanding of chlamydiae pathogenesis. In the
following section I will guide you through the life cycle of chlamydiae, from
the initial interaction between chlamydiae and the host cell to the phase where
hundreds of infectious chlamydiae are released from the host cell now
competent to infect new host cells.
2
Figure 1. (A) The life cycle of chlamydiae. (B) A matured C. trachomatis inclusion
(2 days after infection), photographed with transmission electron microscopy.
3
Adhesion and invasion of the host cell
Chlamydiae are completely dependent on adhesion and invasion of the
mammalian host cell for intracellular propagation and subsequent spreading.
Multiple chlamydial surface exposed factors have been suggested to mediate
adhesion to the host cell, e.g., the major outer membrane protein (MOMP),
OmcB, heat shock protein 60 (GroEL), polymorphic outer membrane proteins
(Pmps) and heparin sulphate like glycosaminoglycans (GAGs) (Su, Watkins et
al. 1990, Zhang and Stephens 1992, Stephens and Lammel 2001, Wehrl,
Brinkmann et al. 2004, Moelleken and Hegemann 2008). GAGs are negatively
charged repeating disaccharides that are found in the extracellular matrix of
mammalian cells. GAGs have been suggested to mediate the initial interaction
between many microbes and the target cell (Rostand and Esko 1997). It has
been suggested that chlamydiae initially interacts with GAGs in a reversible
manner and thereafter binds irreversibly to receptors such as the mannose,
estrogen and cystic fibrosis transmembrane conductance receptors (CFTR).
Thus, current knowledge indicates that chlamydiae employ a two-step
mechanism for adherence to the mammalian host cell (Carabeo and Hackstadt
2001, Davis, Raulston et al. 2002, Puolakkainen, Kuo et al. 2005).
RNAi screening revealed platelet derived growth factor receptor (PDGFR) and
Abelson (Abl) kinase as essential host factors for EB invasion (Elwell 2008).
In addition, Abromatis and Stephens showed that defects of the host cell
protein disulphide isomerase (PDI) reduce attachment and invasion of
chlamydiae. The reduced invasion was coupled to the activity of the isomerase
while adherence was coupled to the presence of the PDI (Abromaitis and
Stephens 2009). In line with that, an observation by Betts-Hampikian and
Fields indicated that disulphide bonding within proteins of the Type III
secretion system (T3SS) are reduced during adhesion, which most likely leads
to increased secretion that promotes invasion (Betts-Hampikian and Fields
2011). T3SS is a secretion system used by many Gram-negative pathogens to,
e.g., stimulate or inhibit polymerization of the host cell cytoskeleton, to avoid
or stimulate uptake via endocytosis (Hueck 1998). In the case of C.
trachomatis, the T3SS effectors Tarp (translocating actin recruiting
phosphoprotein) and CT694 are translocated into the host cell cytoplasm
during invasion or early development for modulation of the actin cytoskeleton,
likely to potentiate invasion and/or establishment of the infection (Clifton,
Fields et al. 2004, Hower, Wolf et al. 2009). Using inhibitory drugs that block
actin polymerization, it has been shown that C. trachomatis invasion is partly
dependent on actin polymerization (Ward and Murray 1984, Prain and Pearce
1989). The fact that invasion is not blocked by these drugs indicates that
additional mechanisms beyond actin polymerization potentiate C.
4
trachomatis invasion (Ward and Murray 1984). All together, to secure
establishment of the infection, chlamydiae uses multiple factors and
mechanisms to adhere to and promote invasion of target host cells.
5
A life inside the chlamydial inclusion
Regulation of the EB-to-RB transition. Chlamydiae is intracellularly
located within the expanding endocytosed vacuole termed the “inclusion”, a
membrane-bound compartment that separates the bacteria from the host cell
cytoplasm (Moulder 1991). After invasion, chlamydiae stimulate the transport
of the inclusion to a peri-Golgi region of the host cell for interception of
vesicles derived from the exocytic pathway. This transport is dependent on the
host cell microtubule and C. trachomatis protein synthesis. In addition, the
nascent chlamydial inclusion shows no lysosomal or endocytic markers, which
suggests that C. trachomatis avoids interaction with this degradation pathway
(Scidmore, Rockey et al. 1996, Clausen, Christiansen et al. 1997, Fields and
Hackstadt 2002). During this phase of the infection the EB-to-RB transition is
initiated and the cross-linked proteins of the outer membrane become reduced
(Hackstadt and Caldwell 1985). The bacteria then become transcriptionally
active due to de-condensation of the chromatin (Shaw, Dooley et al. 2000,
Clifton, Fields et al. 2004).
The dissociation of the eukaryotic histone H1 homolog (Hc1) from the
chromatin of chlamydiae is required for transcriptional activation.
Interestingly, a metabolite within the nonmevalonate methylerythritol 4phosphate (MEP) pathway is important for the dissociation of Hc1
(Grieshaber, Fischer et al. 2004). It is known that the first metabolite of the
MEP pathway is generated by the condensation of pyruvate and
glycerolaldehyde 3-phosphate (GAPD) (Lange, Rujan et al. 2000), two
intermediate metabolites of the glycolysis. Chlamydial enzymes that belong to
the MEP pathway are expressed directly after invasion, suggesting that the
metabolite that binds Hc1 is generated at this initial phase of the infection
(Grieshaber, Fischer et al. 2004). On the other hand, the inner membrane
hexose-phosphate transporter, UhpC, is exclusively detectable in RB (Saka,
Thompson et al. 2011). UhpC facilitates the uptake of glucose-6-phosphate
(Schwoppe, Winkler et al. 2002), a molecule produced in the first step of
glycolysis. Thus, this indicates that pyruvate and GAPD pre-exist in
extracellular EBs, alternatively, they are generated by metabolically active EBs
that use stored glucose sources to feed glycolysis. In line with this idea,
Omsland and co-workers showed that EBs are metabolically active and
consume glucose-6-phosphate (Omsland, Sager et al. 2012). Thus, it appears
that storage of metabolites and/or glucose might be essential for the EB-to-RB
transition.
6
Subversion of host cell vesicles and organelles is required for
bacterial growth. Adjacent to the Golgi apparatus is the chlamydial
inclusion intercepting a subset of lipid-containing vesicles derived from the
exocytic pathway (Hackstadt 2000), while vesicles from the endocytic
pathway are avoided (Scidmore, Fischer et al. 2003). The vesicles from the
exocytic pathway fuse with the inclusion, and sphingolipids as well as
cholesterol are delivered to the bacteria for incorporation into the cell walls of
growing chlamydiae (Hackstadt, Scidmore et al. 1995, Hackstadt, Rockey et al.
1996, Carabeo, Mead et al. 2003). In addition, it has been described that
multivesicular bodies and lipid droplets are translocated into the inclusion for
delivery of building blocks such as lipids (Beatty 2006, Cocchiaro, Kumar et al.
2008).
Current evidence suggests that the specific interaction with host vesicles and
organelles are dependent on chlamydiae secreting proteins that decorate the
cytoplasmic face of the inclusion membrane (Betts, Wolf et al. 2009,
Cocchiaro and Valdivia 2009). The majority of these inclusion membrane
proteins (Inc proteins) are transcribed early in the infection (Shaw, Dooley et
al. 2000, Belland, Zhong et al. 2003) and quite a few of them have been
suggested to be secreted via T3SS (Fields and Hackstadt 2000, Subtil, Parsot
et al. 2001, Fields, Mead et al. 2003). The mechanisms behind the selective
vesicle fusion are dependent on the interaction of Inc proteins with specific
Rab GTPases and SNAREs (Rzomp, Moorhead et al. 2006, Cortes, Rzomp et
al. 2007, Betts, Wolf et al. 2009, Moore, Mead et al. 2011). Rab GTPases are
host cell proteins that are localized to the cytoplasmic face of vesicles with the
role of regulating vesicle budding, motility and fusion (Stenmark and
Olkkonen 2001, Stenmark 2009). SNAREs (soluble N-ethylmaleimidesensitive factor attachment protein receptors) are also localized to the
cytoplasmic face of intracellular membranes but have complementary
functions to Rabs. Specifically, Rabs initiate the interaction with the target
membrane while SNAREs facilitate the fusion event (Stenmark 2009).
Additionally, chlamydial Inc proteins have been suggested to mimic host
SNARE proteins for selective interaction with complementary host cell
SNARE proteins to stimulate vesicle fusion (Delevoye, Nilges et al. 2008). In
summary, chlamydiae have developed unique strategies for selective fusion
with host vesicles and organelles for acquisition of essential building blocks
that are needed for bacterial propagation.
7
Genomic recombination. In addition to fusion of chlamydiae with host
vesicles, it is also known that two C. trachomatis inclusions can fuse with each
other when located in the same cell (Ridderhof and Barnes 1989, Matsumoto,
Bessho et al. 1991, Van Ooij, Homola et al. 1998). During recent years it has
become obvious that related chlamydial strains can exchange genomic DNA by
homologues recombination (Hayes, Yearsley et al. 1994, Gomes, Bruno et al.
2004, Gomes, Nunes et al. 2006, Gomes, Bruno et al. 2007, Jeffrey, Suchland
et al. 2010) representing an evolutionary path for chlamydiae that is likely
potentiated by fusion of inclusions. First it was thought that genomic
recombination only occurred between chlamydiae, however, Dugan and coworkers revealed that C. suis had acquired tetracycline resistance genes from
Helicobacter, suggesting that chlamydiae can acquire and incorporate foreign
DNA (Dugan, Rockey et al. 2004). The mechanisms that facilitate genomic
recombination are unknown but it should be emphasized that this
phenomenon represents an attractive strategy to create isogenic mutant
strains. Recently this strategy was used to “clean up” chemical mutagenized C.
trachomatis strains (Nguyen and Valdivia 2012) and I have also used this
strategy to isolate isogenic mutant strains (Paper II).
Inclusion expansion. Chlamydiae proliferate within the expanding
inclusion which is surrounded by a structural scaffold consisting of F-actin
and the intermediate filaments vimentin and cytokeratins. RhoA, a regulator
of actin (Etienne-Manneville and Hall 2002), is recruited to the inclusion
membrane to coordinate actin polymerization. In addition, intermediate
filaments (IF) are also recruited to enhance the rigidity of the “actin-cage” and
these IFs may be linked to actin (Kumar and Valdivia 2008). IFs are static
structures but it is believed that C. trachomatis proteases such as CPAF
(Dong, Sharma et al. 2004) is processing those for more dynamic IFs
structures that surround the chlamydial inclusion, allowing inclusion
expansion (Kumar and Valdivia 2008).
8
Evasion of innate immunity and prevention of host cell apoptosis.
Chlamydiae have developed many fascinating strategies to avoid innate
immunity and host cellular protection systems such as apoptosis, to ensure
host and bacterial survival (Cocchiaro and Valdivia 2009, Sharma and Rudel
2009). A potential mechanism to avoid host cell apoptosis is the recruitment
of 14-3-3β to the inclusion membrane by IncG (Scidmore and Hackstadt
2001). In a normal scenario, 14-3-3β grips phosphorylated BAD and prevent
recruitment of BAD to the mitochondria where it otherwise would stimulate
apoptosis by release of cytochrome c (Jiang, Du et al. 2006). Phosphorylated
BAD co-localizes with 14-3-3β at the inclusion membrane where it probably
cannot function as a pro-apoptotic component (Verbeke, Welter-Stahl et al.
2006). Moreover, NF-κB proteins are transcriptional factors that regulate
innate and adaptive immunity (Hayden, West et al. 2006, Hayden, West et al.
2006). Modulation of the NF-κB signaling by C. trachomatis appears to be a
mechanism that suppresses host innate immunity. Specifically, it has been
shown that the C. trachomatis Tsp-like protease (CT441) process a subunit of
NF-κB resulting in blocked nuclear translocation which results in a reduced
immune response upon a C. trachomatis infection (Lad, Yang et al. 2007).
9
Preparing for the next round of infection
Generation of infectious EB progeny. In order to be a successful
pathogen, RBs proliferate inside the inclusion, and subsequently, triggered by
an unknown signal, the transition occurs from RBs to infectious EB progeny,
the form that is required for infection of a new host cell (Fields and Hackstadt
2002).
Possible signals that trigger the RB-to-EB transition are the uptake of glucose6-phosphate (Omsland, Sager et al. 2012) and/or glycogen accumulation
(Stephens and Lammel 2001). Glycogen biosynthesis is a secondary metabolic
pathway that is activated when excess sources of carbon such as glucose are
available (Preiss 1984). Chlamydial EBs are spore-like (Wyllie, Ashley et al.
1998), and in the case of another bacterium, Bacillus, it has been suggested
that glycogen has a role in sporulation by providing energy (Slock and Stahly
1974). In line with this idea is the fact that glycogen stores decline as Bacillus
spores are generated (Slock and Stahly 1974), which is similar to what occurs
when C. trachomatis EBs are generated (Chiappino, Dawson et al. 1995).
Glycogen is not detectable in C. psittaci or C. pneumonaie (Moulder 1991),
which questions the role of glycogen accumulation in the chlamydiae infection
cycle and its potential role in the generation of infectious EB progeny.
However, these chlamydial species have the coding capacity for the enzyme
required for glycogen biosynthesis and Iliffe-lee and McClarty speculate that
glycogen synthesis and degradation is equal in C. psittaci and C. pneumonaie,
and therefore might glycogen accumulation be under the limit of detection in
these chlamydial species (Iliffe-Lee and McClarty 2000). In addition, plasmidcured C. trachomatis strains do not accumulate any detectable glycogen
(Matsumoto, Izutsu et al. 1998). However, expression of the enzymes coupled
to this pathway remains detectable (O'Connell, AbdelRahman et al. 2011), thus
it is possible that a plasmid-cured C. trachomatis strain also has glycogen
synthesis equal to glycogen degradation.
Glucose-6-phosphate (G-6-P) is a molecule with the potential to play a role in
the RB-to-EB transition (Omsland, Sager et al. 2012), which is supported by
Paper III in this thesis. In Paper III, I isolated isogenic mutant strains and the
characterization of these strains revealed that a strain with increased uptake of
G-6-P transitions to infectious EB earlier than wild-type. In contrast, a strain
with reduced uptake of G-6-P transitions later than the wild-type, suggesting
that glucose uptake and metabolism is coupled to the generation of infectious
EB (Paper III). This is further supported by my work with the inhibitory
compound ksk120 that targets glucose metabolism of C. trachomatis (Paper
III). In accordance with my results, Iliffe-lee and McClarty show that the
10
amount of glucose supplemented to culture media is associated with the
generation of infectious EB progeny (Iliffe-Lee and McClarty 2000). However,
caution should be taken when glucose levels are altered due to potentially
indirect effects on the host cell, which is also pointed out by Iliffe-lee and
McClarty.
Interestingly, myriocin, an inhibitor of sphingolipids results in loss of
inclusion membrane integrity and surprisingly increases the generation of
infectious EB progeny early during the infection. Although, later in the
infection the yield of infectious EBs is fewer compared to the control
treatment (Robertson, Gu et al. 2009). These authors speculate about why the
RB-to-EB transition is accelerated early in the infection. The reasons might be
the following: i) physical detachment coupled to inactivation of T3SS, ii) loss
of inclusion membrane integrity that might lead to increased permabilisation
of environmental changes, and iii) lack of sphingomyelin might push the RBto-EB transition because RBs cannot proliferate due to lack of building blocks
(Robertson, Gu et al. 2009). I favor the second hypothesis whereby increased
permeabilization might lead to the flow of metabolites from the host cell
cytoplasm into the inclusion of C. trachomatis.
T3SS is suggested to play an essential role throughout the infection cycle of
chlamydiae (Betts-Hampikian and Fields 2010). These needle-like structures
(T33Ss) may be involved in the interaction between the replicating RBs and
the inclusion membrane (Wilson, Timms et al. 2006, Betts-Hampikian and
Fields 2010). Detachment of RBs from the inclusion membrane coincides with
the RB to EB transition (Abdelrahman and Belland 2005) and it has been
suggested that the detachment results in decreased T3S activity and that this
would be the signal for RB-to-EB transition (Wilson, Timms et al. 2006,
Hoare, Timms et al. 2008). Regardless of the exact function(s) of the T3SS, I
do not think that T3S activity per se has a direct role in the RB-to-EB
transition. Instead, it is likely that the T3SS is involved in acquisition of
metabolites (by coordinating selective fusion with host cell vesicle) required
for the RB-to-EB transition.
In Paper II of this thesis, I present a novel link between protoporphyrinogen
oxidase (HemG) and the RB-to-EB transition. HemG catalyzes the second last
step of heme biosynthesis (Heinemann, Jahn et al. 2008), and heme is known
to be a cofactor of peroxidases, cytochromes, sensor molecules and catalases
(Mayfield, Dehner et al. 2011). Heme function has a prosthetic group that
binds to respiratory complexes (Mayfield, Dehner et al. 2011), thus heme is
required for cellular respiration, and therefore is it possible that respiration
play a role in regulating the RB-to-EB transition in C. trachomatis (Paper II).
11
Recently, Ngyuen and Valdivia showed that the synthesis of
lipooligosaccharide is required for RB-to-EB transition (Nguyen, Cunningham
et al. 2011) and from a chlamydial point of view this finding is, to my
knowledge, the only proof that gives insight into this crucial developmental
step. In concert, it appears that glucose metabolism, heme metabolism and
lipooligosaccharides are important components for the generation of
infectious EB progeny. Additionally, G-6-P might be the authentic signal that
triggers the RB-to-EB transition. Furthermore, G-6-P or downstream
metabolites might fuel EBs during invasion. The latter suggestions have been
proposed by a number of investigators (Stephens, Kalman et al. 1998,
Vandahl, Birkelund et al. 2001, Skipp, Robinson et al. 2005, Saka, Thompson
et al. 2011).
Exit and spreading. To be successful, chlamydiae need to spread and infect
new host cells. Few studies have described this critical phase of the infection,
however Hybiske and Stephens showed that chlamydiae exit the host cell by
two distinct mechanisms—lysis of the host cell or by an extrusion mechanism
that facilitates the release of an intact chlamydial inclusion, a process that is
actin-dependent and in the majority of events leaves the host cell viable
(Hybiske and Stephens 2007). Future progress in dissecting the molecular
mechanism behind this phase of the infection will be interesting to follow, e.g.,
what are the signal(s) that initiate host cell lysis or extrusion, mechanical
stress of the inclusion membrane, nutrient limitations, or T3SS activity?
12
Acquisition of carbon and energy sources from the host
Chlamydiae sequester carbon sources such as glucose-6-phosphate (Iliffe-Lee
and McClarty 2000, Saka, Thompson et al. 2011) and energy in the form of
ATP from the host cell (Stephens, Kalman et al. 1998, Tjaden, Winkler et al.
1999). Thus, chlamydiae are totally dependent on the metabolic status of the
eukaryotic host cell for efficient propagation.
Glucose metabolism of the eukaryotic host cell. Sugars are the main
energy source for all eukaryotic cells. When a person eats, sugar molecules
(glucose, galactose and fructose) accumulate in the intestine and are thereafter
transported into the blood stream (Ferraris 2001) and absorbed by glucose
transporters (GLUTs) that are expressed in all eukaryotic cells for local use or
for further delivery to other cells (Thorens and Mueckler 2010). Because
glucose is the body’s major sugar molecule, I will continue to give a glimpse
into how one molecule of glucose is metabolized.
After entering the eukaryotic cell, glucose is phosphorylated by hexokinase, a
step that requires input of energy in the form of one ATP molecule, which
generates a polar molecule that cannot diffuse out of the cell (Romano and
Conway 1996). Next, G-6-P enters one of the two major metabolic pathways: i)
glycolysis, to generate pyruvate for the tricarboxylic acid (TCA-cycle) or ii) the
pentose phosphate pathway (PPP) to generate ribose-5-phosphate and
nicotinamide adenine dinucleotide phosphate (NADPH). Ribose-5-phosphate
is further metabolized and subsequently used for the synthesis of nucleic acid.
NADPH has multiple roles in a cell, e.g., as a co-factor for the biosynthesis of
amino acids and fatty acids. Pyruvate, the end product of glycolysis, enters the
mitochondria for processes that generate NADH which is, e.g., used as
reducing agent that donates an electron and a proton to respiration complexes
for the generation of ATP (Andersen and Kornbluth 2013). In addition,
glycogen, a polymer of glucose molecules is synthesized when excess G-6-P is
available. In fact, G-6-P is the allosteric activator of the glycogen synthase,
while ATP acts as an inhibitor (Palm, Rohwer et al. 2013). The structure of
glycogen is optimized to store large amounts for glucose that can be used as a
source of glucose upon metabolic starvation (Melendez-Hevia, Waddell et al.
1993). In summary, the end products of the PPP mainly function in
biosynthetic reactions while glycolytic end products generate energy.
13
Figure 2. Summary of the major routes, from which chlamydiae acquires nutrients.
14
Carbon and energy metabolism of C. trachomatis
Both glycolysis and PPP are functional in C. trachomatis (Stephens, Kalman et
al. 1998, Iliffe-Lee and McClarty 1999) suggesting an essential role for these
metabolic pathways in vivo. In the following paragraphs, I have gathered most
of the current knowledge concerning C. trachomatis carbon and energy
metabolism.
ATP acquisition from the host cell. Interestingly, Ojcius and co-worker
revealed that Chlamydia infections increase glucose consumption and
stimulate ATP synthesis with a peak around 24 hours post infection (Ojcius,
Degani et al. 1998). In line with that discovery, C. trachomatis encodes the
antiporter Npt1, which facilitates the import of host cell ATP coupled to the
export of ADP (Stephens, Kalman et al. 1998, Tjaden, Winkler et al. 1999). In
addition, C. trachomatis also encodes another antiporter, Npt2, which
imports ATP and other nucleotides (CTP, GTP and UTP) in a protondependent fashion (Tjaden, Winkler et al. 1999). These membrane proteins
are almost exclusively expressed in the RB form of C. trachomatis, which is in
line with the high demand of energy needed to fuel biosynthetic reactions in
replicating RBs (Saka, Thompson et al. 2011). An interesting finding by Saka
and co-worker was the presence of Npt1 in the inclusion membrane fraction
(Saka, Thompson et al. 2011). If this is not due to contamination, it represents
an intriguing mechanism to transport host cell ATP into the chlamydial
inclusion.
Generation of ATP. C. trachomatis lacks the capacity to synthesize
nucleotides de novo (Tipples and McClarty 1993), however chlamydial V-type
ATPases and respiratory complexes (McClarty 1999) are likely involved in the
generation of ATP from ADP and P i . These components are expressed both in
RBs and EBs but are more abundant in RBs (Saka, Thompson et al. 2011). I
believe that V-type ATPases and chlamydial respiration complexes function
late in the infection (~20 h p.i. and thereafter) to promote the last rounds of
bacterial division. Potentially, these systems are further activated when RBs
are detached from the inclusion membrane to energize the RB-to-EB
transition and to provide formed EBs with a large amount of ATP. Large stores
of ATP are likely required for invasion and subsequently the EB-to-RB
transition, ideas that have already been proposed by others (Saka, Thompson
et al. 2011).
15
Glucose uptake and metabolism. Using a heterologous system it has
been shown that C. pneumoniae UhpC can transport glucose-6-phosphate and
erythrose-4-phosphate (Schwoppe, Winkler et al. 2002). Because C.
trachomatis UhpC has 89% identity to C. pneumoniae UhpC, identical
functions are assumed, which is confirmed by Paper III in this thesis. It is
interesting to note that the analogous protein (UhpC) in E. coli functions as a
sensor that, upon sensing G-6-P, activates the downstream kinase (UhpB) and
transduction component (UhpA) activated for expression of UhpT which is the
protein that facilitates transport of G-6-P (Wright and Kadner 2001).
In general, little is known about the role for glucose metabolism during C.
trachomatis development. It is suggested that C. trachomatis has functional
glycolysis and PPP (Stephens, Kalman et al. 1998, Iliffe-Lee and McClarty
1999) pathways, although recent data indicates that RBs use ATP as a primary
energy source while G-6-P (i.e., glycolysis and PPP) might fuel EBs or RBs that
are transitioning to EBs (Saka, Thompson et al. 2011, Omsland, Sager et al.
2012). Saka and co-workers found that proteins required for glucose
metabolism is predominately detectable in EBs while UhpC is exclusively
detectable in RBs, analogous to the observation by Albrecht and co-workers
(Albrecht, Sharma et al. 2010) that supports a hypothesis where RB sequesters
G-6-P and EBs metabolizes it (Saka, Thompson et al. 2011). The RB proteome
analyzed in this paper was collected from RBs at 18 h p.i., and the findings
presented in this paper may not apply to RBs in other stages of infection,
which is also pointed out by the authors (Saka, Thompson et al. 2011).
Noteworthy, the cell culture media used in this study contains a high level of
glucose. The high level of glucose will result in a host cell with high metabolic
activity, leading to results that might be biased for the use and uptake of host
cell derived ATP instead of the generation of ATP via C. trachomatis glycolysis
and respiration. A proteomic analysis using different conditions (i.e., low and
high glucose) would therefore be interesting in with the aim to investigate if
chlamydial RBs can change its metabolism due to metabolic variations. Cell
culture media with high glucose is often used in experiments investigating C.
trachomatis metabolism (Ojcius, Degani et al. 1998, Nicholson, Chiu et al.
2004, Saka, Thompson et al. 2011), however one should be aware that normal
levels of glucose are in vivo roughly between 0.5-1g per liter while “high
glucose cell culture media” contains 5 g per liter (Dulbecco and Freeman
1959). Cell culture media used in my papers contains 2 g per litre (Moore and
Glick 1967).
In summary, glucose-6-phosphate might be important for the RB-to-EB
transition and subsequently to fuel EBs during invasion or early development.
These hypotheses are supported by data presented in Paper III of this thesis.
16
In addition, glucose metabolism of C. trachomatis L2 seems to play a role in
replicating RBs, at least in the late phase of the infection. In the case of two
other C. trachomatis serovars (serovar A and D), glucose metabolism has a
minor role in replication of RBs (Paper III). Therefore, caution should be
taken when conclusions are made regarding general roles for certain
metabolic pathways in C. trachomatis. In line with this observation, Thomson
and co-worker suggest that there are metabolic differences between C.
trachomatis L2 and C. trachomatis serovar A and D (Thomson, Holden et al.
2008).
Tricarboxylic acid (TCA) cycle. It has been suggested by Iliffe-Lee and
McClarty that C. trachomatis can use carbon sources other than glucose to
propagate, including the substrates for gluconeogenesis—glutamate, malate,
oxaloacetate and α-ketoglutarate—however the yield of infectious EB progeny
is strongly reduced in the absence of glucose (Iliffe-Lee and McClarty 2000).
The authors also proposed that Chlamydia is unable to regulate expression of
genes (semi-quantitative) involved in carbon metabolism, which was
corroborated by another group. In this paper the carbon sources were
exchanged for six hours (Nicholson, Chiu et al. 2004), I believe that six hours
is too short to significantly affect the host cell and C. trachomatis metabolism.
In both discussed papers C. trachomatis serovar L2 was used. As mentioned
above there are likely metabolic differences between C. trachomatis serovars,
more specifically, serovar L2 appears to have lost its fumarase hydratase
(fumC) and succinate dehydrogenase (sdhC) activity, suggesting that the TCA
cycle is not completely functional in the L2 serovar. In contrast, fumC and
sdhC are intact in serovar A and D (Thomson, Holden et al. 2008). I believe
that it would be interesting to revisit the findings presented by, e.g., Iliffe-Lee
and McClarty.
17
Heme metabolism
The many roles for heme in prokaryotes and eukaryotes are extensive. Heme
is a prosthetic group of, e.g., peroxidases, sensor molecules, catalases and
cytochromes. In prokaryotes, the most abundant protein that consists of heme
are cytochromes, which are essential for respiration (Panek and O'Brian
2002). More specific, Heme functions in processes that mediate redox
reactions (Mayfield, Dehner et al. 2011), in which electrons are transferred
between donors and acceptor molecules (Herrmann and Dick 2012).
Heme biosynthesis is a pathway that appears to consist of eight steps in
chlamydiae. The first precursor in heme synthesis is δ-aminolevulinic acid
(ALA), the source of carbon and nitrogen for biosynthesis of heme
(Heinemann, Jahn et al. 2008). Information from the chlamydiae genome
suggests that ALA is synthesized from glutamate; this route (a.k.a., the C 5 pathway) requires NADPH as a reductant (Panek and O'Brian 2002), a
product of the pentose phosphate pathway (Kruger and von Schaewen 2003).
The next step is the condensation of two ALA molecules to porphobilinogen, a
reaction that is catalyzed by porphobilinogen synthase (hemB). Four
porphobilinogen molecules are thereafter linked together by porphobilinogen
deaminase (hemC) to hydroxymethylbilane. Next is uroporphyrinogen III
generated by the uroporphyrigon synthase (hemD), which is a cyclic
intermediate that subsequently is decarboxylated by uroporphyrinogen
decarboxylase (hemE), generating the product coproporhyrinogen III. The
coproporhyrinogen oxigenase (hemN or hemF) is thereafter responsible for an
additional decarboxylation of the heme precursor molecule which generates
protoporhyrinogen IX. The next step is the oxidation of protoporhyrinogen IX
to protohyrin IX, a reaction performed by protoporhyrinogen oxidase (hemG
or hemY). Noteworthy is the fact that HemG is oxygen-independent while
HemY is oxygen-dependent. The last reaction in the biosynthesis of heme is
the insertion of an iron molecule into the protohyrin IX, a reaction that is
catalyzed by ferrochelatase (hemH) (Heinemann, Jahn et al. 2008).
18
Bacterial infections and bone resorption
Osteoporosis is a disease that is associated with generalized bone loss
(Rachner, Khosla et al. 2011). Bone remodeling is a normal process, which
under normal circumstances is kept in balance. This balance involves
osteoclasts—which are monocyte-macrophage-like cells—that facilitate bone
resorption, and osteoblasts—cells that are responsible for the bone formation
(Lacey, Timms et al. 1998). The molecular mechanisms that regulate bone
remodeling are well-defined and characterized by increased expression of
receptor activator nuclear NF-κB ligand (RANKL) on the surface of osteoblast
cells, and its receptor, RANK, on osteoclasts. Osteoprotegerin (OPG) also
influences this balance by functioning as a soluble decoy receptor for RANKL
(Simonet, Lacey et al. 1997, Caidahl, Ueland et al. 2010). Additional players
that influence the balance between bone resorption and bone formation are
cytokines such as interleukin-6 and tumor necrosis factor alpha (TNF-α)
(Lacey, Timms et al. 1998).
Clinical studies have revealed that bacterial infections affect bone remodeling
(Henderson and Nair 2003) and in vitro experiments have shown that
intracellular bacterial pathogens such as Salmonella induce apoptosis in
osteoblasts, suggesting a direct link between osteoporosis and bacterial
infections (Alexander, Bento et al. 2001). Recently, Rizzo and co-workers
showed that C. pneumoniae can infect osteoblast-like cells with increased
levels of, e.g., IL-6, indicating a link to bone diseases (Rizzo, Di Domenico et
al. 2011). In line with this, a recent clinical study found an association between
C. pneumoniae DNA in bone tissue of osteoporotic patients, suggesting that C.
pneumoniae can cause osteoporosis (Di Pietro, Schiavoni et al. 2012). In fact,
this idea had already been suggested in Paper V presented in this thesis
(Bailey, Engström et al. 2008). In this paper, I found that C. pneumoniae can
infect an osteoblast cell line in vitro with a similar cytokine profile as that of
infection in vivo.
19
Aims of this thesis
I.
II.
Analysis of Chlamydia pathogenesis using chemical genetics
Investigate if C. pneumoniae causes generalized bone loss in mice
20
Key findings and relevance
Paper I. A comparative study of RNA and DNA as internal gene
expression controls early in the developmental cycle of Chlamydia
pneumoniae. To understand clinically relevant antibacterial compounds and
their effects on gene expression, appropriate internal controls are required. In
this paper, I compared the advantages and disadvantages of various internal
expression controls during the early phase of C. pneumoniae development.
Relevance of Paper I. In this study, instead of rRNA or mRNA which is
normally used, I identified bacterial DNA as the most accurate internal gene
expression control, because it is stable, abundant and correlates with bacterial
numbers. When using DNA as an internal control, we found that the
inhibitory drug INP0010 inhibited the expression of all mRNAs tested,
suggesting a developmental delay. Thus, DNA is preferred as an internal gene
expression control when chlamydiae development is affected.
21
Paper II. Mutations in hemG mediate resistance to salicylidene
acylhydrazides: A novel link between protoporphyrinogen oxidase
(HemG) and Chlamydia trachomatis infectivity. Small inhibitory
compounds represent an alternative approach to studying molecular
mechanisms of chlamydial pathogenesis. Salicylidene acylhydrazide (SAHs)
compounds used in this paper were identified as inhibitors of Type III
secretion (T3SS) of Yersinia and other Gram-negative bacteria, however the
target is not known. In addition, these compounds have iron-chelating
capacity, which brings into question their role as specific T3S inhibitors. In
agreement with other research groups, I found that excess iron suppresses the
growth inhibitory effect by the SAHs on C. trachomatis. However, the RB-toEB transition was strongly inhibited, indicating that INP0341 has an effect
that is beyond iron chelation. To unravel the mode of action, I selected and
isolated spontaneous INP0341-resistant strains. By combining whole genome
sequencing (WGS) and genotyping, mutations in hemG were identified to
mediate the resistant phenotype. Thus, INP0341 affects C. trachomatis
HemG, which is known to function in the second-to-last step of heme
biosynthesis.
Relevance of Paper II. In this paper, I identified Protoporphyrinogen
oxidase (HemG) as a regulator of RB-to-EB transition in C. trachomatis.
Heme is essential for respiratory complexes; therefore, in accordance with
others, it is tempting to speculate that a general effect by these compounds on
bacterial T3SS is via the energy metabolism.
22
Paper III. Glucose uptake and metabolism is required for the
generation of infectious Chlamydia trachomatis progeny. Inhibitory
compounds from the chemical class of ring-fused 2-pyrridones affect virulence
properties of Escherichia coli. By using a phenotypic screen, I identified the 2pyrridone KSK120 as a potent compound that inhibits replication and blocks
the generation of infectious C. trachomatis progeny. By independent mutant
selections, I identified eight spontaneous point-mutations in KSK120-resistant
populations. Three of these mutations were found in uhpC, encoding the
membrane glucose-6-phosphate transporter (UhpC), one mutation in pgi,
encoding the glucose-6-phosphate isomerase (Pgi). In fact, glycogen
accumulation was abolished by KSK120 treatment, suggesting that the
compound targets the glucose metabolism of C. trachomatis. Furthermore, an
active fluorescent analogue compound showed punctate distribution in the
membrane of C. trachomatis (see cover picture), which in concert with the
acquired mutations strongly indicates that UhpC is the molecular target of
ksk120. Characterization of isogenic uhpC mutant strains revealed that uptake
of glucose-6-phosphate via UhpC coordinates the generation of infectious C.
trachomatis progeny.
Relevance of Paper III. This paper discloses the importance of glucose
metabolism and UhpC activity for the generation of infectious C. trachomatis
progeny. Furthermore, I have in this paper further fine-tuned the approach
presented in Paper II that allows the isolation of isogenic C. trachomatis
mutants. In this paper, I took advantage of the fact that different mutant
populations have different mutational linkages. Together with approaches
described in paper II there is now handful of different approaches that can be
used to isolate isogenic C. trachomatis strains, which can be tested in assays of
interest, for identification of novel phenotypes.
23
Paper IV. Role of the glucose-6-phosphate transporter UhpC in
Chlamydia trachomatis growth. In this paper we used mutant strains
isolated in our laboratory to investigate whether there is a link between uhpC
and C. trachomatis growth. The gene uhpC encodes for the membrane
glucose-6-phosphate transporter, UhpC. When I measured the generation of
infectious EBs, mutant uhpC strains responded differently to metabolic stress.
More specifically, a strain with an UhpCA394T substitution appears to have
decreased uptake of G-6-P while a strain with UhpCM315I, L429I substitutions has
increased G-6-P uptake (paper III). In this study, under normal conditions,
the mutant uhpC strains grow similar to the wild-type strains suggesting that
alterations in G-6-P uptake do not affect the growth of these strains in
favorable in vitro conditions. Furthermore, I monitored C. trachomatis
growth after 2-DG treatment, a glucose analogue that inhibits host cell
hexokinase leading to metabolic stress including ATP and G-6-P starvation.
After 2-DG treatment, I found that the strain with increased uptake of G-6-P
grows better than our wild-type while the strain with reduced G-6-P uptake
exhibited little growth.
Relevance of paper IV. This study reveals a link between UhpC and C.
trachomatis growth during nutrient-limiting conditions, strongly indicating
that G-6-P can promote bacterial growth. The current hypothesis in the field is
that the glycolysis and the pentose phosphate pathway are mainly active in
EBs. The data presented in this paper indicate that these pathways are used in
RBs to promote growth under nutrient-limiting conditions. Thus, this might
open up new interesting questions regarding C. trachomatis metabolism and
how different experimental conditions affect the metabolism of this
bacterium.
24
Paper V. Chlamydia pneumoniae infection results in generalized
bone loss in mice. Associations between some human diseases and
bacterial infections are described in the literature. In this paper, we
investigated if there is an association between C. pneumoniae infections and
bone loss in mice. Results obtained in this study reveal that C. pneumoniae
infection results in generalized bone loss in mice. We also show that C.
pneumoniae can infect an osteoblast cell line in vitro with a similar cytokine
profile as that of infection in vivo.
Relevance of Paper V. This study indicates that C. pneumonaie potentially
can infect bone cells in vivo, resulting in the increased bone loss observed.
Another more likely explanation is that the inflammatory responses seen in
vivo are the causative factor. This study further indicates that bacterial
infection might have a role in osteoporosis (disease of bone).
25
Additional data
Introduction. It has been shown that INP0341 and other compounds from
the chemical class salicylidene acylhydrazides (SAHs) affect T3SS of gramnegative bacteria (Duncan, Linington et al. 2012), including chlamydiae
(Muschiol, Bailey et al. 2006, Wolf, Betts et al. 2006, Bailey, Gylfe et al. 2007).
The target has been elusive however in paper II of this thesis I show that the
SAH INP0341 target HemG in C. trachomatis. I further show that INP0341
does not inhibit secretion of IncA or CPAF. IncA, is a T3S effector known to be
secreted to the chlamydial inclusion membrane during mid-development
(Subtil, Parsot et al. 2001) and CPAF is a mid-late effector secreted via the
general secretion pathway to the host cell cytoplasm (Jorgensen, Bednar et al.
2011). Because INP0341 inhibit the RB-to-EB transition by affecting HemG, it
was of interest to further investigate a potential effect on T3SS. I therefore
investigated the localization of the late T3S effector CT621, an effector that is
secreted to the cytoplasm of the host cell (Hobolt-Pedersen, Christiansen et al.
2009, Muschiol, Boncompain et al.). It is important to have in mind that
CT621 is expressed late in the infection while IncA is expressed in the mid part
of the infection.
Results. HeLa cells were infected with C. trachomatis and treated as
described in figure 1. At 42 and 32 hour post infection (h p.i.), infected cells
were fixed and stained for subsequent analysis with confocal laser microscopy.
In DMSO-treated infections I observed that the majority of CT621 was
localized to the bacteria that were in close proximity to the inclusion
membrane while other bacteria in the center of the inclusion showed only
little detectable staining. This rim-like localization was also observed in
infections treated with DFO and iron sulfate. Addition of excess iron to
INP0341 treated infections did not result in the rim-like localization of CT621.
Instead the protein was found in the majority of bacteria, including bacteria
located in the center of the inclusions (Figure 1). To explore if the altered
CT621 distribution was caused by bacterial detachment we investigated the
localization at 32 h p.i., a time point before INP0341-induced redistribution.
As in the later time point, CT621 localization was altered by INP0341 and the
signal was visualized in the majority of the bacteria in each inclusion while the
controls showed a rim-like localization (data not shown). Previously it has
been shown by independent groups that CT621 is secreted into the host cell
cytoplasm (Hobolt-Pedersen, Christiansen et al. 2009, Muschiol, Boncompain
et al. 2011). In our model system we could not detect CT621 outside the
inclusion however data presented in figure 1 indicate that INP0341 affects the
distribution of CT621.
26
Figure 1. (A and B) HeLa cells infected with C. trachomatis were treated with DMSO,
INP0341 or DFO in the presence or absence of FeSO 4 . At 42 h p.i. infected cells were fixed with
4 % PFA and incubation with an anti-CT621 antibody (gift from Sandra Muschiol). DAPI was
used to detect host and bacterial DNA (blue). The photographs were produced using Nikon
Eclipse C1 confocal laser scanning microscopy. (B) CT621 intensity per inclusion, quantified
using the EZ-C1 software (Nikon).
Disscussion. The fact that INP0341 affect distribution of a late T3Ssubstrate (Figure 1) whereas secretion of a mid T3S-substrate is unperturbed
(Paper II) indicates that INP0341 specifically affects the secretion-mechanism
of late substrates. It has been suggested that the Chlamydia T3SS might have
a few interchangeable components including the 4 inner membrane
components CdsV, CdsN, CdsL and CdsJ which are classified as “flagellar” or
“non-flagellar” (Betts-Hampikian and Fields 2010, Wu, Lei et al. 2011).
Interestingly, Belland et al., show that the “flagellar” T3SS genes are
transcribed earlier than the “non-flagellar” T3SS genes (Belland, Zhong et al.
2003). Potentially, the corresponding “non-flagellar” component of the
ATPase CdsN might be dependent on respiration including HemG activity
(INP0341 targets HemG, which is needed to synthesize heme, a prosthetic
group of respiratory complexes), on the other hand CdsN might use ATP
acquired from the host cell. In line with that, it has recently been suggested
that RBs uses host derived ATP while EBs uses G-6-P as primarily energy
sources (Saka, Thompson et al. 2011, Omsland, Sager et al. 2012). Thus, the
central metabolism together with respiration may be essential for the
maturation of infectious EBs. Therefore, inhibition of HemG by INP0341
might limit the ATP pool needed to support secretion of late T3S-substrates
27
that occur in bacteria that initiate the RB-to-EB transition. In contrast,
secretion of mid T3S-substrates does likely rely on ATP acquired from the host
cell. This represents an intriguing but speculative explanation to why INP0341
have a specific effect on late T3S while mid T3S is unperturbed.
In summary, my data indicate a novel link between HemG and late T3SS in C.
trachomatis however investigation of additional effectors are warranted. It
has been shown that T3SS requires proton motive force (PMF) to secret
effector proteins in Yersinia (Wilharm, Lehmann et al. 2004). Consistently,
the flagella, a complex of proteins with high similarity to the T3SS also require
PMF to function (Minamino and Namba 2008). Therefore it is tempting to
speculate that the SAHs target HemG or structural and functional related
proteins in other bacterial species. Thus, I postulate that the effect on T3SS by
the SAHs is via bacterial respiration.
28
Main conclusions
•
DNA is the most accurate internal expression control when C.
pneumoniae development is affected.
•
Protoporphyrinogen oxidase (HemG) is a regulator of RB-to-EB
transition in C. trachomatis.
•
Glucose metabolism and UhpC activity coordinate the generation
of infectious C. trachomatis EB progeny.
•
UhpC has a role in C. trachomatis growth during nutrientlimiting conditions.
•
C. pneumoniae infection results in generalized bone loss in mice.
29
Future directions
Approaches to study bacterial pathogenesis. Classical genetic
approaches have definitely been the most important tool to understand
bacterial pathogenesis and will continue to be for many years to come.
Commonly, these approaches include gene deletions (“knockouts”) and/or
bacterial strains that overexpress the gene of interest. Using such approaches
essential genes are difficult to manipulate, however, there are elegant
examples of both essential and non-essential genes, where point mutations are
introduced to alter the function of the gene product. This approach is timeconsuming and it often includes a selection-marker which potentially has
pleiotropic effects.
It is hard to see why “knockouts” are still used as a foundation to study
bacterial pathogenesis. It is evident that such manipulation might create a new
organism with new properties that are beyond the deleted gene. This
possibility remains for bacteria that contain a point mutation in a gene of
interest; however, the probability for pleiotropic effects will significantly
decline. I believe that caution should be exercised when organisms with gene
deletions are studied in situ.
I believe it’s time to go in new directions as new technologies allow us to do so.
Whole genome sequencing (WGS) has revealed some novel discoveries during
recent years, however, in most of these studies there is a great deal of data
with little understanding of the molecular mechanisms behind it. I think WGS
can be used as a foundation to answer important questions regarding bacterial
pathogenesis, e.g. by generating bacterial mutant strains with novel
phenotypes that are tested in assays of interest. Strains can be generated via
chemical mutagenesis (Kari, Goheen et al. 2011, Nguyen and Valdivia 2012)
and/or by applying selective pressure(s) for the isolation of spontaneous
mutants. Ideally, those strains are isogenic with only one point-mutation in
the whole genome. In that context I believe that bacteria with low genetic drift
will function as eminent model organisms, including e.g. Chlamydia,
Rickettsia and Coxiella, three important human pathogens that are obligate
intracellular bacterial parasites. We know very little about these intracellular
pathogens and the molecular mechanisms behind their critical biological
events. These bacteria grow naturally inside eukaryotic host cells and are
therefore relevant model organisms to study intracellular microbial
pathogenesis and host-pathogen interactions. Experiments with these bacteria
can easily be performed in an in vitro setting where eukaryotic cell lines
function as their natural host. Furthermore, (and important to keep in mind)
experiments with intracellular bacteria will shed light on molecular
mechanisms from a eukaryotic cell-biology perspective.
30
It has become evident during recent years that chemical genetics is an
attractive approach to study how pathogens invade and neutralize their hosts.
Chemical genetics is an approach were specific inhibitory compounds are
applied to a biological system to determine the function of a gene product of
interest (Puri and Bogyo 2009).
By combing sophisticated genetic approaches and chemical genetics I believe
that we will identify novel phenotypes with increased in vivo relevance.
Chemical genetics and chemical biology. Chemical biology was used
before most of us were born including my supervisors. By a coincidence in
1928, Alexander Flemming discovered penicillin as an antibacterial substance
produced by the fungi Penicillium rubens (Flemming 1929). In 1940 Gardner
investigated the effect of penicillin on bacteria and found that this compound
altered the morphology of bacteria, including increasing bacterial size
(Gardner 1940). In 1949, Park and Johnson showed evidence that altered
morphology by penicillin treatment is due to increased amount of organic
phosphates inside the bacterium (Park and Johnson 1949), representing one
of the first examples where a chemical biology approach was used to study
bacterial physiology. Later, in 1957 penicillin was identified as an inhibitor of
peptidoglycans (Park and Strominger 1957), however, the target molecules
were not identified until a few years later.
Chemical genetics was born when the target of aminoglycoside antibiotics,
fluoroquinolones and rifampicin was revealed. Rifampicin targets a subunit of
RNA-polymerase (Ezekiel and Hutchins 1968) and has after this discovery
been used to study bacterial transcription. Quinolones targets the DNA gyrase
which is involved in DNA replication (Tocchini-Valentini, Marino et al. 1968)
and aminoglycosides target the ribosome and subsequently these antibiotics
have been used as chemical probes to study the function of these essential
gene products (Stanley and Hung 2009). These are just a few examples of
when chemical genetics and chemical biology have been used to study
bacterial pathogenesis. It is good to remind ourselves that some of these
antibiotics are still frequently used as chemical tools in research laboratories.
There are two different chemical genetics strategies that can be employed to
identify novel inhibitory compounds, forward chemical genetics or a reversed
chemical genetic approach. In forward chemical genetic screens, also known
as phenotypic screens, compounds are added to the biological system of
interest with the goal to find novel phenotypes, or phenotypes of interest.
There are a few advantages of using this approach, for examples there are no
assumptions made regarding the target or downstream pathway and
compounds with an effect in the whole cell systems that are preselected. One
obstacle with a forward chemical genetic approach is identifying the target or
31
the mode of action. This obstacle is more prominent for compounds that affect
virulence properties but not bacterial growth.
Reversed chemical genetic screens start with a protein of interest and
compounds are screened for modulation of the protein function. This
approach has been successful, however, when applying identified compounds
to a whole cell system the specificity might be lost, another alternative is that
the compound is not cell permeable or cell toxic (Puri and Bogyo 2009).
Personally, I prefer a forward chemical genetics screen were a compound that
causes a phenotypic change of interest is identified. Subsequently, the target
molecule(s) can be identified by approaches that are described below. This is
an unbiased way of doing research and the identified compounds are
preselected for in situ effects.
Target identification is a difficult task and has been a limiting factor in the
use of chemical genetics. Again, two approaches are in use, either a genetic or
a biochemical approach. Historically, the genetic approach has been extremely
fruitful. In fact, the targets of antibiotics and the majority of novel antibacterial compounds have been revealed by genetic approaches (Stanley and
Hung 2009, Stanley, Grant et al. 2012). In most of these cases resistant
mutant strains have been selected, either via spontaneous resistance or
chemical mutagenesis. Subsequently, the genetic lesions can be identified
revealing the target molecule and/or mode of action. Currently, with the use of
WGS this is an easy task in most bacterial pathogens although one should be
aware that selection of resistant bacterial strains requires a selective pressure.
So what is the selective pressure? According to my dictionary the definition of
selective pressure is; “the effect of any feature of the environment in
natural selection” (Lawrence 2005). I like the words “any feature”, I think that
selecting resistant mutants is not only applicable to compounds that cause
growth defects or kill the bacteria. Here I think fantasy is the limiting factor.
An example of such a strategy was recently published; in a paper where they
selected Salmonella mutants resistant to inhibitory compounds. These
compounds inhibit T3SS and swimming in Salmonella, thus these authors
employed an approach were they picked the best Salmonella swimmers in the
presence of compound. Using WGS the authors identified point-mutations in
genes that are involved in energy metabolism indicating that these compounds
affect ATP generation in Salmonella (Martinez-Argudo, Veenendaal et al.
2013).
Biochemical approaches have also been successful. In principle these
approaches are based on the target being fished out by an inhibitory
compound that is conjugated to a column. Protein lysates are applied to the
column and the proteins that remain in the column represent target
32
candidates. Examples where affinity-based approaches have been successful to
identify the target are found in the cell biology field, examples are the
calcineurin inhibitors CsA and F506 (Liu, Farmer et al. 1991). In general, the
biochemical approaches used so far have been relative unsuccessful (Stanley
and Hung 2009). This is due to obvious reasons, first, false positive hits are
frequently captured, second it is very likely that the inhibitory interaction
between the compound and the target requires the whole cell environment
including e.g. pH, membrane integration, metals etc. Therefore, such a
strategy might be impossible in certain cases. Interestingly, techniques have
been developed to distinguish false positive hits from true targets (Ong,
Schenone et al. 2009). In addition, photoreactive compounds which after
exposure to light generate radical intermediates covalently binding to its
target protein, represent a promising strategy to identify targets in a whole cell
system. In summary, biochemical approaches have been relatively
unsuccessful however the development and use of the new techniques
described will likely be fruitful.
Concluding remarks. So far mutant selections have been the most
successful strategy to identify targets of novel anti-bacterial compounds.
Together with genetic approaches, I believe that the new biochemical
techniques will likely revolutionize the field of chemical genetics by
accelerating the target identification process.
33
Acknowledgements
I am extremely grateful to my supervisors, Hans Wolf-Watz and Sven
Bergström, for this opportunity and their professional and personal support.
Thank you so much! I have enjoyed every day!
I am confused about which of you is my main supervisor but maybe that’s a
part of your strategy? Wait a minute, maybe that has been my strategy?
Thanks to my co-supervisor Åsa Gylfe for your valuable support during early
years. To Fredrik Almqvist for your never ending enthusiasm and useful
chemical compounds, I will miss your great ideas and chemical drawings.
Raphael Valdivia, for a great time in your lab and it is an understatement to
say that I learned a lot, also of course for brilliant suggestions coupled to our
projects and practical executions including genome sequencing performed by
professional people in your lab. Ken Fields, for your humane and curiositydriven way of being a scientist, it was a pleasure to be in your great lab and it
was the best two months of my life. If any of those mentioned above (not Sven
or Hans) in the future wants a collaborator or a co-worker, I will be the first
person in line.
During my first year in a research laboratory I had the pleasure to work
together with three interesting scientists who gave me valuable insight into the
life in a lab. Thanks to Leslie Bailey who boosted my self-confidence with
great leadership and friendship. Thanks to Jörgen Johansson and Ulrich
von Pawel-Rammingen for being excellent scientists with endless spirit.
Without Ingela Nilsson this thesis would not have been materialized. Thank
you for all the help and support! “Jojje” Normark, Susanne, Silver,
Matilda, Malin, M Ottosson, “nöten” Spoerry, Elofsson, Thomas Ö,
P Nordström, Chorell and “robocop” Bastidas for contributions that
made this thesis possible. Bidong, thanks for all fun, help and contributions
during the years! Syam, it has been a pleasure to collaborate with you! I also
want to thank present and former lab members of “my” labs for great
moments and help. Especially, I want to thank “Heffrey” Barker and Jenni
at Duke University for their hospitality, at Miami University people such as
Kate Wolf and Sara Bartra for taking care of a young Swede, in Umeå
Mari B, Marie A, Maria N, Mari K, Michaela, “brorsan”, Tobbe,
“Korv” and Reine for fun in the lab, now I want my shoes back!
Thanks to Sandra Muschiol for gifts of reagents including some Chlamydia.
34
Thanks to Micke Sellin, “biffen” Lindell, Marios, Mukunda and J
Gripenland for delightful discussions and great friendship, to Jonny
Stoneman for being the best superintendent at our department, to PIs such
as Åke, Tomas, Nelson and Markus for a positive and supportive attitude.
Tack till “Large” and “Dr Snuggles” for great friendship.
Thanks to my parents and grandparents who supported curiosity from a
young age, to my brother Magnus for great moments and nice chats that
always boosted my motivation; I wish we could spend more time together.
Astrid, we made it!
35
References
Abdelrahman, Y. M. and R. J. Belland (2005). "The chlamydial
developmental cycle." FEMS Microbiol Rev 29(5): 949-959.
Abromaitis, S. and R. S. Stephens (2009). "Attachment and Entry of
Chlamydia Have Distinct Requirements for Host Protein Disulfide
Isomerase." Plos Pathogens 5(4).
Albrecht, M., C. M. Sharma, R. Reinhardt, J. Vogel and T. Rudel (2010).
"Deep sequencing-based discovery of the Chlamydia trachomatis
transcriptome." Nucleic Acids Res 38(3): 868-877.
Alexander, E. H., J. L. Bento, F. M. Hughes, Jr., I. Marriott, M. C. Hudson
and K. L. Bost (2001). "Staphylococcus aureus and Salmonella enterica
serovar Dublin induce tumor necrosis factor-related apoptosis-inducing
ligand expression by normal mouse and human osteoblasts." Infect Immun
69(3): 1581-1586.
Andersen, J. L. and S. Kornbluth (2013). "The tangled circuitry of
metabolism and apoptosis." Mol Cell 49(3): 399-410.
Bailey, L., P. Engström, A. Nordström, S. Bergström, A. Waldenström and P.
Nordström (2008). "Chlamydia pneumoniae infection results in generalized
bone loss in mice." Microbes Infect 10(10-11): 1175-1181.
Bailey, L., Å. Gylfe, C. Sundin, S. Muschiol, M. Elofsson, P. Nordström, B.
Henriques-Normark, R. Lugert, A. Waldenström, H. Wolf-Watz and S.
Bergström (2007). "Small molecule inhibitors of type III secretion in
Yersinia block the Chlamydia pneumoniae infection cycle." FEBS Lett
581(4): 587-595.
Batteiger, B. (2012). "Chlamydia infection and epidemiology." Chlamydiales;
Intracellular pathogens I, Tan, M and Bavoil, P (ed.). Washington, DC:
American Society for Microbiology,pp. 1-26.
Beatty, W. L. (2006). "Trafficking from CD63-positive late endocytic
multivesicular bodies is essential for intracellular development of Chlamydia
trachomatis." Journal of Cell Science 119(2): 350-359.
Belland, R. J., G. Zhong, D. D. Crane, D. Hogan, D. Sturdevant, J. Sharma,
W. L. Beatty and H. D. Caldwell (2003). "Genomic transcriptional profiling
of the developmental cycle of Chlamydia trachomatis." Proc Natl Acad Sci U
S A 100(14): 8478-8483.
36
Betts-Hampikian, H. J. and K. A. Fields (2010). "The Chlamydial Type III
Secretion Mechanism: Revealing Cracks in a Tough Nut." Front Microbiol 1:
114.
Betts-Hampikian, H. J. and K. A. Fields (2011). "Disulfide Bonding within
Components of the Chlamydia Type III Secretion Apparatus Correlates with
Development." Journal of Bacteriology 193(24): 6950-6959.
Betts, H. J., K. Wolf and K. A. Fields (2009). "Effector protein modulation of
host cells: examples in the Chlamydia spp. arsenal." Current Opinion in
Microbiology 12(1): 81-87.
Burton, M. J. and D. C. W. Mabey (2009). "The Global Burden of Trachoma:
A Review." Plos Neglected Tropical Diseases 3(10).
Caidahl, K., T. Ueland and P. Aukrust (2010). "Osteoprotegerin: a biomarker
with many faces." Arterioscler Thromb Vasc Biol 30(9): 1684-1686.
Carabeo, R. A. and T. Hackstadt (2001). "Isolation and characterization of a
mutant Chinese hamster ovary cell line that is resistant to Chlamydia
trachomatis infection at a novel step in the attachment process." Infection
and Immunity 69(9): 5899-5904.
Carabeo, R. A., D. J. Mead and T. Hackstadt (2003). "Golgi-dependent
transport of cholesterol to the Chlamydia trachomatis inclusion."
Proceedings of the National Academy of Sciences of the United States of
America 100(11): 6771-6776.
Cegelski, L., G. R. Marshall, G. R. Eldridge and S. J. Hultgren (2008). "The
biology and future prospects of antivirulence therapies." Nat Rev Microbiol
6(1): 17-27.
Chiappino, M. L., C. Dawson, J. Schachter and B. A. Nichols (1995).
"Cytochemical-Localization of Glycogen in Chlamydia-Trachomatis
Inclusions." Journal of Bacteriology 177(18): 5358-5363.
Clausen, J. D., G. Christiansen, H. U. Holst and S. Birkelund (1997).
"Chlamydia trachomatis utilizes the host cell microtubule network during
early events of infection." Molecular Microbiology 25(3): 441-449.
Clifton, D. R., K. A. Fields, S. S. Grieshaber, C. A. Dooley, E. R. Fischer, D. J.
Mead, R. A. Carabeo and T. Hackstadt (2004). "A chlamydial type III
translocated protein is tyrosine-phosphorylated at the site of entry and
associated with recruitment of actin." Proceedings of the National Academy
of Sciences of the United States of America 101(27): 10166-10171.
37
Cocchiaro, J. L., Y. Kumar, E. R. Fischer, T. Hackstadt and R. H. Valdivia
(2008). "Cytoplasmic lipid droplets are translocated into the lumen of the
Chlamydia trachomatis parasitophorous vacuole." Proceedings of the
National Academy of Sciences of the United States of America 105(27):
9379-9384.
Cocchiaro, J. L. and R. H. Valdivia (2009). "New insights into Chlamydia
intracellular survival mechanisms." Cellular Microbiology 11(11): 1571-1578.
Cortes, C., M. A. Rzomp, A. Tvinnereim, M. A. Scidmore and B. Wizel
(2007). "Chlamydia pneumoniae inclusion membrane protein cpn0585
interacts with multiple rab GTPases." Infection and Immunity 75(12): 55865596.
Davis, C. H., J. E. Raulston and P. B. Wyrick (2002). "Protein disulfide
isomerase, a component of the estrogen receptor complex, is associated with
Chlamydia trachomatis serovar E attached to human endometrial epithelial
cells." Infection and Immunity 70(7): 3413-3418.
Delevoye, C., M. Nilges, P. Dehoux, F. Paumet, S. Perrinet, A. Dautry-Varsat
and A. Subtil (2008). "SNARE protein mimicry by an intracellular
bacterium." Plos Pathogens 4(3).
Di Pietro, M., G. Schiavoni, V. Sessa, F. Pallotta, G. Costanzo and R. Sessa
(2012). "Chlamydia pneumoniae and osteoporosis-associated bone loss: a
new risk factor?" Osteoporos Int.
Dong, F., J. Sharma, Y. M. Xiao, Y. M. Zhong and G. M. Zhong (2004).
"Intramolecular dimerization is required for the chlamydia-secreted protease
CPAF to degrade host transcriptional factors." Infection and Immunity
72(7): 3869-3875.
Dugan, J., D. D. Rockey, L. Jones and A. A. Andersen (2004). "Tetracycline
resistance in Chlamydia suis mediated by genomic islands inserted into the
chlaravdial inv-like gene." Antimicrobial Agents and Chemotherapy 48(10):
3989-3995.
Dulbecco, R. and G. Freeman (1959). "Plaque production by the polyoma
virus." Virology 8(3): 396-397.
Duncan, M. C., R. G. Linington and V. Auerbuch (2012). "Chemical
inhibitors of the type three secretion system: disarming bacterial pathogens."
Antimicrob Agents Chemother 56(11): 5433-5441.
38
Engström, P., L. Bailey, T. Önskog, S. Bergström and J. Johansson (2010).
"A comparative study of RNA and DNA as internal gene expression controls
early in the developmental cycle of Chlamydia pneumoniae." FEMS
Immunol Med Microbiol 58(2): 244-253.
Etienne-Manneville, S. and A. Hall (2002). "Rho GTPases in cell biology."
Nature 420(6916): 629-635.
Ezekiel, D. H. and J. E. Hutchins (1968). "Mutations affecting RNA
polymerase associated with rifampicin resistance in Escherichia coli."
Nature 220(5164): 276-277.
Ferraris, R. P. (2001). "Dietary and developmental regulation of intestinal
sugar transport." Biochem J 360(Pt 2): 265-276.
Fields, K. A. and T. Hackstadt (2000). "Evidence for the secretion of
Chlamydia trachomatis CopN by a type III secretion mechanism."
Molecular Microbiology 38(5): 1048-1060.
Fields, K. A. and T. Hackstadt (2002). "The chlamydial inclusion: escape
from the endocytic pathway." Annu Rev Cell Dev Biol 18: 221-245.
Fields, K. A., D. J. Mead, C. A. Dooley and T. Hackstadt (2003). "Chlamydia
trachomatis type III secretion: evidence for a functional apparatus during
early-cycle development." Molecular Microbiology 48(3): 671-683.
Flemming, P. (1929). "The Medical Aspects of the Mediaeval Monastery in
England." Proc R Soc Med 22(6): 771-782.
Gardner, A. D. (1940). "An enzyme from bacteria able to destroy penicillin."
Nature, letters to the editors.
Gomes, J. P., W. J. Bruno, M. J. Borrego and D. Dean (2004).
"Recombination in the genome of Chlamydia trachomatis involving the
polymorphic membrane protein C gene relative to ompA and evidence for
horizontal gene transfer." Journal of Bacteriology 186(13): 4295-4306.
Gomes, J. P., W. J. Bruno, A. Nunes, N. Santos, C. Florindo, M. J. Borrego
and D. Dean (2007). "Evolution of Chlamydia trachomatis diversity occurs
by widespread interstrain recombination involving hotspots." Genome
Research 17(1): 50-60.
Gomes, J. P., A. Nunes, W. J. Bruno, M. J. Borrego, C. Florindo and D. Dean
(2006). "Polymorphisms in the nine polymorphic membrane proteins of
Chlamydia trachomatis across all serovars: Evidence for serovar Da
39
recombination and correlation with tissue tropism." Journal of Bacteriology
188(1): 275-286.
Grayston, J. T. (1992). "Infections Caused by Chlamydia-Pneumoniae Strain
Twar." Clinical Infectious Diseases 15(5): 757-763.
Grieshaber, N. A., E. R. Fischer, D. J. Mead, C. A. Dooley and T. Hackstadt
(2004). "Chlamydial histone-DNA interactions are disrupted by a metabolite
in the methylerythritol phosphate pathway of isoprenoid biosynthesis."
Proceedings of the National Academy of Sciences of the United States of
America 101(19): 7451-7456.
Hackstadt, T. (2000). "Redirection of host vesicle trafficking pathways by
intracellular parasites." Traffic 1(2): 93-99.
Hackstadt, T. and H. D. Caldwell (1985). "Effect of proteolytic cleavage of
surface-exposed proteins on infectivity of Chlamydia trachomatis." Infect
Immun 48(2): 546-551.
Hackstadt, T., D. D. Rockey, R. A. Heinzen and M. A. Scidmore (1996).
"Chlamydia trachomatis interrupts an exocytic pathway to acquire
endogenously synthesized sphingomyelin in transit from the Golgi apparatus
to the plasma membrane." Embo Journal 15(5): 964-977.
Hackstadt, T., M. A. Scidmore and D. D. Rockey (1995). "Lipid-Metabolism
in Chlamydia Trachomatis-Infected Cells - Directed Trafficking of GolgiDerived Sphingolipids to the Chlamydial Inclusion." Proceedings of the
National Academy of Sciences of the United States of America 92(11): 48774881.
Haggerty, C. L., S. L. Gottlieb, B. D. Taylor, N. Low, F. Xu and R. B. Ness
(2010). "Risk of sequelae after Chlamydia trachomatis genital infection in
women." J Infect Dis 201 Suppl 2: S134-155.
Hayden, M. S., A. P. West and S. Ghosh (2006). "NF-kappaB and the
immune response." Oncogene 25(51): 6758-6780.
Hayden, M. S., A. P. West and S. Ghosh (2006). "SnapShot: NF-kappaB
signaling pathways." Cell 127(6): 1286-1287.
Hayes, L. J., P. Yearsley, J. D. Treharne, R. A. Ballard, G. H. Fehler and M. E.
Ward (1994). "Evidence for Naturally-Occurring Recombination in the Gene
Encoding the Major Outer-Membrane Protein of LymphogranulomaVenereum Isolates of Chlamydia-Trachomatis." Infection and Immunity
62(12): 5659-5663.
40
Heinemann, I. U., M. Jahn and D. Jahn (2008). "The biochemistry of heme
biosynthesis." Arch Biochem Biophys 474(2): 238-251.
Henderson, B. and S. P. Nair (2003). "Hard labour: bacterial infection of the
skeleton." Trends Microbiol 11(12): 570-577.
Herrmann, J. M. and T. P. Dick (2012). "Redox Biology on the rise." Biol
Chem 393(9): 999-1004.
Hoare, A., P. Timms, P. M. Bavoil and D. P. Wilson (2008). "Spatial
constraints within the chlamydial host cell inclusion predict interrupted
development and persistence." BMC Microbiol 8: 5.
Hobolt-Pedersen, A. S., G. Christiansen, E. Timmerman, K. Gevaert and S.
Birkelund (2009). "Identification of Chlamydia trachomatis CT621, a
protein delivered through the type III secretion system to the host cell
cytoplasm and nucleus." FEMS Immunol Med Microbiol 57(1): 46-58.
Horn, M. (2008). "Chlamydiae as symbionts in eukaryotes." Annu Rev
Microbiol 62: 113-131.
Hower, S., K. Wolf and K. A. Fields (2009). "Evidence that CT694 is a novel
Chlamydia trachomatis T3S substrate capable of functioning during
invasion or early cycle development." Molecular Microbiology 72(6): 14231437.
Hueck, C. J. (1998). "Type III protein secretion systems in bacterial
pathogens of animals and plants." Microbiology and Molecular Biology
Reviews 62(2): 379-+.
Hybiske, K. and R. S. Stephens (2007). "Mechanisms of host cell exit by the
intracellular bacterium Chlamydia." Proc Natl Acad Sci U S A 104(27):
11430-11435.
Iliffe-Lee, E. R. and G. McClarty (1999). "Glucose metabolism in Chlamydia
trachomatis: the 'energy parasite' hypothesis revisited." Mol Microbiol
33(1): 177-187.
Iliffe-Lee, E. R. and G. McClarty (2000). "Regulation of carbon metabolism
in Chlamydia trachomatis." Molecular Microbiology 38(1): 20-30.
Jeffrey, B. M., R. J. Suchland, K. L. Quinn, J. R. Davidson, W. E. Stamm and
D. D. Rockey (2010). "Genome Sequencing of Recent Clinical Chlamydia
trachomatis Strains Identifies Loci Associated with Tissue Tropism and
41
Regions of Apparent Recombination." Infection and Immunity 78(6): 25442553.
Jiang, P., W. J. Du, K. Heese and M. Wu (2006). "The bad guy cooperates
with good cop p53: Bad is transcriptionally up-regulated by p53 and forms a
bad/p53 complex at the mitochondria to induce apoptosis." Molecular and
Cellular Biology 26(23): 9071-9082.
Jorgensen, I., M. M. Bednar, V. Amin, B. K. Davis, J. P. Ting, D. G.
McCafferty and R. H. Valdivia (2011). "The Chlamydia protease CPAF
regulates host and bacterial proteins to maintain pathogen vacuole integrity
and promote virulence." Cell Host Microbe 10(1): 21-32.
Kari, L., M. M. Goheen, L. B. Randall, L. D. Taylor, J. H. Carlson, W. M.
Whitmire, D. Virok, K. Rajaram, V. Endresz, G. McClarty, D. E. Nelson and
H. D. Caldwell (2011). "Generation of targeted Chlamydia trachomatis null
mutants." Proc Natl Acad Sci U S A 108(17): 7189-7193.
Kruger, N. J. and A. von Schaewen (2003). "The oxidative pentose
phosphate pathway: structure and organisation." Curr Opin Plant Biol 6(3):
236-246.
Kumar, Y. and R. H. Valdivia (2008). "Actin and intermediate filaments
stabilize the Chlamydia trachomatis vacuole by forming dynamic structural
scaffolds." Cell Host & Microbe 4(2): 159-169.
Lacey, D. L., E. Timms, H. L. Tan, M. J. Kelley, C. R. Dunstan, T. Burgess, R.
Elliott, A. Colombero, G. Elliott, S. Scully, H. Hsu, J. Sullivan, N. Hawkins, E.
Davy, C. Capparelli, A. Eli, Y. X. Qian, S. Kaufman, I. Sarosi, V. Shalhoub, G.
Senaldi, J. Guo, J. Delaney and W. J. Boyle (1998). "Osteoprotegerin ligand
is a cytokine that regulates osteoclast differentiation and activation." Cell
93(2): 165-176.
Lad, S. P., G. Yang, D. A. Scott, G. Wang, P. Nair, J. Mathison, V. S. Reddy
and E. Li (2007). "Chlamydial CT441 is a PDZ domain-containing tailspecific protease that interferes with the NF-kappaB pathway of immune
response." J Bacteriol 189(18): 6619-6625.
Lange, B. M., T. Rujan, W. Martin and R. Croteau (2000). "Isoprenoid
biosynthesis: The evolution of two ancient and distinct pathways across
genomes." Proceedings of the National Academy of Sciences of the United
States of America 97(24): 13172-13177.
Lawrence, E. (2005). "Henderson´s dictionary of biology."
edition: 598.
42
thirteenth
Liu, J., J. D. Farmer, Jr., W. S. Lane, J. Friedman, I. Weissman and S. L.
Schreiber (1991). "Calcineurin is a common target of cyclophilin-cyclosporin
A and FKBP-FK506 complexes." Cell 66(4): 807-815.
Martinez-Argudo, I., A. K. Veenendaal, X. Liu, A. D. Roehrich, M. C.
Ronessen, G. Franzoni, K. N. van Rietschoten, Y. V. Morimoto, Y. SaijoHamano, M. B. Avison, D. J. Studholme, K. Namba, T. Minamino and A. J.
Blocker (2013). "Isolation of Salmonella mutants resistant to the inhibitory
effect of Salicylidene acylhydrazides on flagella-mediated motility." PLoS
One 8(1): e52179.
Matsumoto, A., H. Bessho, K. Uehira and T. Suda (1991). "Morphological
studies of the association of mitochondria with chlamydial inclusions and
the fusion of chlamydial inclusions." J Electron Microsc (Tokyo) 40(5): 356363.
Matsumoto, A., H. Izutsu, N. Miyashita and M. Ohuchi (1998). "Plaque
formation by and plaque cloning of Chlamydia trachomatis biovar
trachoma." Journal of Clinical Microbiology 36(10): 3013-3019.
Maurer, A. P., A. Mehlitz, H. J. Mollenkopf and T. F. Meyer (2007). "Gene
expression profiles of Chlamydophila pneumoniae during the developmental
cycle and iron depletion-mediated persistence." PLoS Pathog 3(6): e83.
Mayfield, J. A., C. A. Dehner and J. L. DuBois (2011). "Recent advances in
bacterial heme protein biochemistry." Curr Opin Chem Biol 15(2): 260-266.
McClarty, G. (1999). "Chlamydial metabolism as inferred from the complete
genome sequence." In Chlamydia: Intracellular Biology, Pathogenesis and
Immunity. Stephens, R.S. (ed.). Washington, DC: American Society for
Microbiology, pp. 69–98.
Melendez-Hevia, E., T. G. Waddell and E. D. Shelton (1993). "Optimization
of molecular design in the evolution of metabolism: the glycogen molecule."
Biochem J 295 ( Pt 2): 477-483.
Miller, W. C., C. A. Ford, M. Morris, M. S. Handcock, J. L. Schmitz, M. M.
Hobbs, M. S. Cohen, K. M. Harris and J. R. Udry (2004). "Prevalence of
chlamydial and gonococcal infections among young adults in the United
States." Jama-Journal of the American Medical Association 291(18): 22292236.
Minamino, T. and K. Namba (2008). "Distinct roles of the FliI ATPase and
proton motive force in bacterial flagellar protein export." Nature 451(7177):
485-488.
43
Moelleken, K. and J. H. Hegemann (2008). "The Chlamydia outer
membrane protein OmcB is required for adhesion and exhibits biovarspecific differences in glycosaminoglycan binding." Molecular Microbiology
67(2): 403-419.
Moore, E. R., D. J. Mead, C. A. Dooley, J. Sager and T. Hackstadt (2011).
"The trans-Golgi SNARE syntaxin 6 is recruited to the chlamydial inclusion
membrane." Microbiology-Sgm 157: 830-838.
Moore, G. E. and J. L. Glick (1967). "Perspective of human cell culture." Surg
Clin North Am 47(6): 1315-1324.
Moran, N. A. (2002). "Microbial minimalism: genome reduction in bacterial
pathogens." Cell 108(5): 583-586.
Moulder, J. W. (1991). "Interaction of Chlamydiae and Host-Cells Invitro."
Microbiological Reviews 55(1): 143-190.
Muschiol, S., L. Bailey, Å. Gylfe, C. Sundin, K. Hultenby, S. Bergström, M.
Elofsson, H. Wolf-Watz, S. Normark and B. Henriques-Normark (2006). "A
small-molecule inhibitor of type III secretion inhibits different stages of the
infectious cycle of Chlamydia trachomatis." Proc Natl Acad Sci U S A
103(39): 14566-14571.
Muschiol, S., G. Boncompain, F. Vromman, P. Dehoux, S. Normark, B.
Henriques-Normark and A. Subtil (2011). "Identification of a family of
effectors secreted by the type III secretion system that are conserved in
pathogenic Chlamydiae." Infect Immun 79(2): 571-580.
Nguyen, B. D., D. Cunningham, X. Liang, X. Chen, E. J. Toone, C. R. Raetz,
P. Zhou and R. H. Valdivia (2011). "Lipooligosaccharide is required for the
generation of infectious elementary bodies in Chlamydia trachomatis." Proc
Natl Acad Sci U S A 108(25): 10284-10289.
Nguyen, B. D. and R. H. Valdivia (2012). "Virulence determinants in the
obligate intracellular pathogen Chlamydia trachomatis revealed by forward
genetic approaches." Proceedings of the National Academy of Sciences of the
United States of America 109(4): 1263-1268.
Nicholson, T. L., K. Chiu and R. S. Stephens (2004). "Chlamydia
trachomatis lacks an adaptive response to changes in carbon source
availability." Infect Immun 72(7): 4286-4289.
44
Nicholson, T. L., L. Olinger, K. Chong, G. Schoolnik and R. S. Stephens
(2003). "Global stage-specific gene regulation during the developmental
cycle of Chlamydia trachomatis." J Bacteriol 185(10): 3179-3189.
O'Connell, C. M., Y. M. AbdelRahman, E. Green, H. K. Darville, K. Saira, B.
Smith, T. Darville, A. M. Scurlock, C. R. Meyer and R. J. Belland (2011).
"Toll-Like Receptor 2 Activation by Chlamydia trachomatis Is Plasmid
Dependent, and Plasmid-Responsive Chromosomal Loci Are Coordinately
Regulated in Response to Glucose Limitation by C-trachomatis but Not by Cmuridarum." Infection and Immunity 79(3): 1044-1056.
Ojcius, D. M., H. Degani, J. Mispelter and A. Dautry-Varsat (1998).
"Enhancement of ATP levels and glucose metabolism during an infection by
Chlamydia. NMR studies of living cells." J Biol Chem 273(12): 7052-7058.
Omsland, A., J. Sager, V. Nair, D. E. Sturdevant and T. Hackstadt (2012).
"Developmental stage-specific metabolic and transcriptional activity of
Chlamydia trachomatis in an axenic medium." Proc Natl Acad Sci U S A
109(48): 19781-19785.
Ong, S. E., M. Schenone, A. A. Margolin, X. Li, K. Do, M. K. Doud, D. R.
Mani, L. Kuai, X. Wang, J. L. Wood, N. J. Tolliday, A. N. Koehler, L. A.
Marcaurelle, T. R. Golub, R. J. Gould, S. L. Schreiber and S. A. Carr (2009).
"Identifying the proteins to which small-molecule probes and drugs bind in
cells." Proc Natl Acad Sci U S A 106(12): 4617-4622.
Palm, D. C., J. M. Rohwer and J. H. Hofmeyr (2013). "Regulation of glycogen
synthase from mammalian skeletal muscle--a unifying view of allosteric and
covalent regulation." FEBS J 280(1): 2-27.
Panek, H. and M. R. O'Brian (2002). "A whole genome view of prokaryotic
haem biosynthesis." Microbiology 148(Pt 8): 2273-2282.
Park, J. T. and M. J. Johnson (1949). "A Submicrodetermination of Glucose."
Journal of Biological Chemistry 181(1): 149-151.
Park, J. T. and J. L. Strominger (1957). "Mode of Action of Penicillin Biochemical Basis for the Mechanism of Action of Penicillin and for Its
Selective Toxicity." Science 125(3238): 99-101.
Petrovay, F. and E. Balla (2008). "Two fatal cases of psittacosis caused by
Chlamydophila psittaci." J Med Microbiol 57(Pt 10): 1296-1298.
Prain, C. J. and J. H. Pearce (1989). "Ultrastructural Studies on the
Intracellular Fate of Chlamydia-Psittaci (Strain Guinea-Pig Inclusion
45
Conjunctivitis) and Chlamydia-Trachomatis (Strain Lymphogranuloma
Venereum 434) - Modulation of Intracellular Events and Relationship with
Endocytic Mechanism." Journal of General Microbiology 135: 2107-2123.
Preiss, J. (1984). "Bacterial glycogen synthesis and its regulation." Annu Rev
Microbiol 38: 419-458.
Puolakkainen, M., C. C. Kuo and L. A. Campbell (2005). "Chlamydia
pneumoniae uses the mannose 6-phosphate/insulin-like growth factor 2
receptor for infection of endothelial cells." Infection and Immunity 73(8):
4620-4625.
Puri, A. W. and M. Bogyo (2009). "Using small molecules to dissect
mechanisms of microbial pathogenesis." ACS Chem Biol 4(8): 603-616.
Rachner, T. D., S. Khosla and L. C. Hofbauer (2011). "Osteoporosis: now and
the future." Lancet 377(9773): 1276-1287.
Ridderhof, J. C. and R. C. Barnes (1989). "Fusion of Inclusions Following
Superinfection of Hela-Cells by 2 Serovars of Chlamydia-Trachomatis."
Infection and Immunity 57(10): 3189-3193.
Rizzo, A., M. Di Domenico, C. R. Carratelli, N. Mazzola and R. Paolillo
(2011). "Induction of proinflammatory cytokines in human osteoblastic cells
by Chlamydia pneumoniae." Cytokine 56(2): 450-457.
Robertson, D. K., L. Gu, R. K. Rowe and W. L. Beatty (2009). "Inclusion
Biogenesis and Reactivation of Persistent Chlamydia trachomatis Requires
Host Cell Sphingolipid Biosynthesis." Plos Pathogens 5(11).
Romano, A. H. and T. Conway (1996). "Evolution of carbohydrate metabolic
pathways." Res Microbiol 147(6-7): 448-455.
Rosario, C. J. and M. Tan (2012). "The early gene product EUO is a
transcriptional repressor that selectively regulates promoters of Chlamydia
late genes." Mol Microbiol 84(6): 1097-1107.
Rostand, K. S. and J. D. Esko (1997). "Microbial adherence to and invasion
through proteoglycans." Infection and Immunity 65(1): 1-8.
Rzomp, K. A., A. R. Moorhead and M. A. Scidmore (2006). "The GTPase
Rab4 interacts with Chlamydia trachomatis inclusion membrane protein
CT229." Infection and Immunity 74(9): 5362-5373.
46
Saka, H. A., J. W. Thompson, Y. S. Chen, Y. Kumar, L. G. Dubois, M. A.
Moseley and R. H. Valdivia (2011). "Quantitative proteomics reveals
metabolic and pathogenic properties of Chlamydia trachomatis
developmental forms." Mol Microbiol 82(5): 1185-1203.
Sandoz, K. M., S. G. Eriksen, B. M. Jeffrey, R. J. Suchland, T. E. Putman, D.
E. Hruby, R. Jordan and D. D. Rockey (2012). "Resistance to a novel
antichlamydial compound is mediated through mutations in Chlamydia
trachomatis secY." Antimicrob Agents Chemother 56(8): 4296-4302.
Schwoppe, C., H. H. Winkler and H. E. Neuhaus (2002). "Properties of the
glucose-6-phosphate transporter from Chlamydia pneumoniae (HPTcp) and
the glucose-6-phosphate sensor from Escherichia coli (UhpC)." J Bacteriol
184(8): 2108-2115.
Scidmore, M. A., E. R. Fischer and T. Hackstadt (2003). "Restricted fusion of
Chlamydia trachomatis vesicles with endocytic compartments during the
initial stages of infection." Infection and Immunity 71(2): 973-984.
Scidmore, M. A. and T. Hackstadt (2001). "Mammalian 14-3-3 beta
associates with the Chlamydia trachomatis inclusion membrane via its
interaction with IncG." Molecular Microbiology 39(6): 1638-1650.
Scidmore, M. A., D. D. Rockey, E. R. Fischer, R. A. Heinzen and T. Hackstadt
(1996). "Vesicular interactions of the Chlamydia trachomatis inclusion are
determined by Chlamydial early protein synthesis rather than route of
entry." Infection and Immunity 64(12): 5366-5372.
Sharma, M. and T. Rudel (2009). "Apoptosis resistance in Chlamydiainfected cells: a fate worse than death?" Fems Immunology and Medical
Microbiology 55(2): 154-161.
Shaw, E. I., C. A. Dooley, E. R. Fischer, M. A. Scidmore, K. A. Fields and T.
Hackstadt (2000). "Three temporal classes of gene expression during the
Chlamydia trachomatis developmental cycle." Molecular Microbiology
37(4): 913-925.
Simonet, W. S., D. L. Lacey, C. R. Dunstan, M. Kelley, M. S. Chang, R. Luthy,
H. Q. Nguyen, S. Wooden, L. Bennett, T. Boone, G. Shimamoto, M. DeRose,
R. Elliott, A. Colombero, H. L. Tan, G. Trail, J. Sullivan, E. Davy, N. Bucay, L.
Renshaw-Gegg, T. M. Hughes, D. Hill, W. Pattison, P. Campbell, S. Sander,
G. Van, J. Tarpley, P. Derby, R. Lee and W. J. Boyle (1997).
"Osteoprotegerin: a novel secreted protein involved in the regulation of bone
density." Cell 89(2): 309-319.
47
Skipp, P., J. Robinson, C. D. O'Connor and I. N. Clarke (2005). "Shotgun
proteomic analysis of Chlamydia trachomatis." Proteomics 5(6): 1558-1573.
Slock, J. A. and D. P. Stahly (1974). "Polysaccharide That May Serve as a
Carbon and Energy-Storage Compound for Sporulation in Bacillus-Cereus."
Journal of Bacteriology 120(1): 399-406.
Somani, J., V. B. Bhullar, K. A. Workowski, C. E. Farshy and C. M. Black
(2000). "Multiple drug-resistant Chlamydia trachomatis associated with
clinical treatment failure." J Infect Dis 181(4): 1421-1427.
Stanley, S. A., S. S. Grant, T. Kawate, N. Iwase, M. Shimizu, C. Wivagg, M.
Silvis, E. Kazyanskaya, J. Aquadro, A. Golas, M. Fitzgerald, H. Dai, L. Zhang
and D. T. Hung (2012). "Identification of novel inhibitors of M. tuberculosis
growth using whole cell based high-throughput screening." ACS Chem Biol
7(8): 1377-1384.
Stanley, S. A. and D. T. Hung (2009). "Chemical tools for dissecting bacterial
physiology and virulence." Biochemistry 48(37): 8776-8786.
Stenmark, H. (2009). "Rab GTPases as coordinators of vesicle traffic."
Nature Reviews Molecular Cell Biology 10(8): 513-525.
Stenmark, H. and V. M. Olkkonen (2001). "The Rab GTPase family."
Genome Biology 2(5).
Stephens, R. S., S. Kalman, C. Lammel, J. Fan, R. Marathe, L. Aravind, W.
Mitchell, L. Olinger, R. L. Tatusov, Q. X. Zhao, E. V. Koonin and R. W. Davis
(1998). "Genome sequence of an obligate intracellular pathogen of humans:
Chlamydia trachomatis." Science 282(5389): 754-759.
Stephens, R. S. and C. J. Lammel (2001). "Chlamydia outer membrane
protein discovery using genomics." Current Opinion in Microbiology 4(1):
16-20.
Stephens, R. S., G. Myers, M. Eppinger and P. M. Bavoil (2009). "Divergence
without difference: phylogenetics and taxonomy of Chlamydia resolved."
Fems Immunology and Medical Microbiology 55(2): 115-119.
Su, H., N. G. Watkins, Y. X. Zhang and H. D. Caldwell (1990). "ChlamydiaTrachomatis Host-Cell Interactions - Role of the Chlamydial Major OuterMembrane Protein as an Adhesin." Infection and Immunity 58(4): 10171025.
48
Subtil, A., C. Parsot and A. Dautry-Varsat (2001). "Secretion of predicted Inc
proteins of Chlamydia pneumoniae by a heterologous type III machinery."
Molecular Microbiology 39(3): 792-800.
Thomson, N. R., M. T. Holden, C. Carder, N. Lennard, S. J. Lockey, P.
Marsh, P. Skipp, C. D. O'Connor, I. Goodhead, H. Norbertzcak, B. Harris, D.
Ormond, R. Rance, M. A. Quail, J. Parkhill, R. S. Stephens and I. N. Clarke
(2008). "Chlamydia trachomatis: genome sequence analysis of
lymphogranuloma venereum isolates." Genome Res 18(1): 161-171.
Thorens, B. and M. Mueckler (2010). "Glucose transporters in the 21st
Century." Am J Physiol Endocrinol Metab 298(2): E141-145.
Tipples, G. and G. McClarty (1993). "The obligate intracellular bacterium
Chlamydia trachomatis is auxotrophic for three of the four ribonucleoside
triphosphates." Mol Microbiol 8(6): 1105-1114.
Tjaden, J., H. H. Winkler, C. Schwoppe, M. Van Der Laan, T. Mohlmann and
H. E. Neuhaus (1999). "Two nucleotide transport proteins in Chlamydia
trachomatis, one for net nucleoside triphosphate uptake and the other for
transport of energy." J Bacteriol 181(4): 1196-1202.
Tocchini-Valentini, G. P., P. Marino and A. J. Colvill (1968). "Mutant of E.
coli containing an altered DNA-dependent RNA polymerase." Nature
220(5164): 275-276.
Valdivia, R. H. (2012). "Thinking outside the box: new strategies for
antichlamydial control." Future Microbiol 7(4): 427-429.
Van Ooij, C., E. Homola, E. Kincaid and J. Engel (1998). "Fusion of
Chlamydia trachomatis-containing inclusions is inhibited at low
temperatures and requires bacterial protein synthesis." Infection and
Immunity 66(11): 5364-5371.
Vandahl, B. B., S. Birkelund, H. Demol, B. Hoorelbeke, G. Christiansen, J.
Vandekerckhove and K. Gevaert (2001). "Proteome analysis of the
Chlamydia pneumoniae elementary body." Electrophoresis 22(6): 12041223.
Ward, M. E. and A. Murray (1984). "Control Mechanisms Governing the
Infectivity of Chlamydia-Trachomatis for Hela-Cells - Mechanisms of
Endocytosis." Journal of General Microbiology 130(Jul): 1765-1780.
Wehrl, W., V. Brinkmann, P. R. Jungblut, T. F. Meyer and A. J. Szczepek
(2004). "From the inside out - processing of the Chlamydial autotransporter
49
PmpD and its role in bacterial adhesion and activation of human host cells."
Molecular Microbiology 51(2): 319-334.
Verbeke, P., L. Welter-Stahl, S. Ying, J. Hansen, G. Hacker, T. Darville and
D. M. Ojcius (2006). "Recruitment of BAD by the Chlamydia trachomatis
vacuole correlates with host-cell survival." Plos Pathogens 2(5): 408-417.
Wilharm, G., V. Lehmann, K. Krauss, B. Lehnert, S. Richter, K. Ruckdeschel,
J. Heesemann and K. Trulzsch (2004). "Yersinia enterocolitica type III
secretion depends on the proton motive force but not on the flagellar motor
components MotA and MotB." Infect Immun 72(7): 4004-4009.
Wilson, D. P., P. Timms, D. L. McElwain and P. M. Bavoil (2006). "Type III
secretion, contact-dependent model for the intracellular development of
chlamydia." Bull Math Biol 68(1): 161-178.
Wolf, K., H. J. Betts, B. Chellas-Gery, S. Hower, C. N. Linton and K. A. Fields
(2006). "Treatment of Chlamydia trachomatis with a small molecule
inhibitor of the Yersinia type III secretion system disrupts progression of the
chlamydial developmental cycle." Mol Microbiol 61(6): 1543-1555.
Wright, J. S., 3rd and R. J. Kadner (2001). "The phosphoryl transfer domain
of UhpB interacts with the response regulator UhpA." J Bacteriol 183(10):
3149-3159.
Wu, X., L. Lei, S. Gong, D. Chen, R. Flores and G. Zhong (2011). "The
chlamydial periplasmic stress response serine protease cHtrA is secreted
into host cell cytosol." BMC Microbiol 11: 87.
Wyllie, S., R. H. Ashley, D. Longbottom and A. J. Herring (1998). "The major
outer membrane protein of Chlamydia psittaci functions as a porin-like ion
channel." Infect Immun 66(11): 5202-5207.
Zhang, J. P. and R. S. Stephens (1992). "Mechanism of C. trachomatis
Attachment to Eukaryotic Host-Cells." Cell 69(5): 861-869.
50