Chlamydial glycogen enzymes are secreted for de novo glycogen synthesis

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Biphasic developmental cycle

All Chlamydiaceae are obligate intracellular pathogens sharing a biphasic developmental cycle. The gram negative bacteria exist in two different forms, the infectious elementary body (EB) and the replicative reticulate body (RB). They are easily distinguished based on their shapes, the EB being around 0.3 « m and the RB being around 1 « m. The infectious EBs attach and enter the host cell, where they stay in a parasitophorous vacuole, called inclusion, throughout their whole developmental cycle. Four to eight hours post infection (hpi) the EB converts into the RB, which is metabolically highly active and proliferates through binary fission. At around 20 hpi RBs start to convert into EBs in an asynchronous manner. By around 48 hpi most RBs have differentiated to EBs, now prepared to leave the host cell, either through lysis of the host cell or extrusion of the inclusion (Figure 5). A new cycle of infection starts (AbdelRahman and Belland, 2005).

Building the inclusion and survival

Chlamydiae are endocytosed into a membrane-bound vacuole known as the inclusion, which grows throughout the developmental cycle to harbour the increasing number of progeny (Figure 7). Interestingly, the inclusion membrane does not seem to display markers of the endocytic or lysosomal pathway (Scidmore et al., 2003). Bacterial activity is required to inhibit fusion with the lysosomal pathway, as inhibition of bacterial protein synthesis results in degradation of the bacteria in lysosomes (Scidmore et al., 1996). Early gene  expression is first detected within an hour after entry, and chlamydial proteins called Inc proteins are produced, that are inserted into the inclusion membrane. Inc proteins probably play a central role in controlling cellular interactions of the nascent inclusion with the host cytoplasm, but the function of only a handful of them (out of more than 50 Inc proteins) has been investigated. One of them is probably responsible for the interaction with host dynein, resulting in the transport of the inclusion along microtubules to the microtubule organizing centre (MTOC) (Clausen et al., 1997; Grieshaber et al., 2003). While establishing the appropriate intracellular niche, the EB converts to an RB within the first few hpi (Shaw et al., 2000), which will be explained in more detail in chapter 3.2. A different set of genes, the midcycle genes are expressed, controlling bacterial metabolism, replication and interactions with the host (Belland et al., 2003). RBs repeatedly divide by binary fission, yielding up to 1000 bacteria per infected cell (Shaw et al., 2000). At 18 to 24 hpi some RBs start to undergo conversion into EBs in an asynchronous manner, strongly linked to the switch from the transcription of mid-cycle genes to late genes (see chapter 3.2). At around 48 hpi Chlamydia exit the cell through pathogen-driven lysis of the inclusion and the host plasma membrane or through extrusion of the intact inclusion. However, the latter has only been observed in cell culture and not in vivo (Rank et al., 2011).

Host-pathogen interactions

As an obligate intracellular pathogen with a highly reduced genome of around 900 genes C. trachomatis depends strongly on the host cell (Stephens et al., 1998). Interactions with the host are indispensible for the establishment and maintenance of the niche, and for the acquisition of nutrients. They are aimed at subverting the host defence systems. Specialised secretion systems enable the delivery of bacteria-derived exoproteins that play a pivotal role in regulating host-pathogen interactions, thereby contributing to the virulence of the pathogens.

Recruitment and uptake of host organelles and lipids

Consistent with the obligate intracellular lifestyle, Chlamydiae have lost the ability to synthesize many vital metabolites. They rely on the host cell for essential nutrients, such as amino acids, nucleotides and lipids (Stephens et al., 1998). The acquisition of nutrients occurs hence through a tight interaction with the host and its organelles.

Recruitment of host lipids

C. trachomatis encodes for the genes required for lipid synthesis, but still acquires preferentially host-derived lipids. Its lipid composition, which consists out of sphingolipids (SLs), glycerophospholipids (such as phosphatidylethanolamines or phosphatidylcholines) and cholesterol consequently resembles strongly to the one of the host (Wylie et al., 1997).
The SL precursor, ceramide, is synthesized within the host in the ER, processed in the Golgi apparatus and accumulates at the plasma membrane (Perry and Ridgway, 2005; Tan and Bavoil, 2012). Inhibition of SL synthesis leads to loss of inclusion membrane integrity and a subsequent disruption of the normal inclusion development, accompanied by premature RBto- EB differentiation and early release of EBs (Robertson et al., 2009). Hackstadt could show that fluorescently labelled ceramide analogues trafficked in live cells to the inclusion in a manner that resembles Golgi apparatus-to-plasma membrane vesicular transport (Hackstadt et al., 1995). On the other hand, plasma membrane SLs are not directed towards the inclusion, concordant with the segregation of the inclusion from the endocytic traffic (Hackstadt et al., 1996). Cholesterol, a lipid not normally identified in prokaryotes, can be found in EBs and in the inclusion membrane. Carabeo and colleagues demonstrated that this cholesterol stems from two different sources: de novo synthesised by the host cell, or derived from the extracellular medium via the low-density lipoproteins (LDL) pathway. It was also suggested that the host cell cholesterol might be co-transported to the inclusion with SL in redirected Golgi-vesicles, as acquisition of both is sensitive to addition of Brefeldin A causing the interruption of normal Golgi apparatus vesicular traffic (Carabeo et al., 2003). In addition to this pathway Chlamydia also acquires host ceramides through a non-vesicular pathway. The host’s interorganelle lipid transfer highly relies on membrane contact sites between organelles, notably with the ER (Levine and Loewen, 2006). The bulk of ceramide ER-to-Golgi transport is coordinated through the ceramide transfer protein (CERT), which directly transfers the precursor from one organelle to the other at the membrane contact sites. Early ultrastructural studies had already reported ER tubules closely apposed to C. trachomatis inclusions and another study revealed the enrichment of certain ER proteins on the inclusion membrane (Majeed et al., 1999; Peterson and de la Maza, 1988). CERT is equally localised to and enriched at the inclusion membrane. These structures resemble to membrane contact sites between the ER and the inclusion membrane (Derre et al., 2011; Dumoux et al., 2012). An attractive candidate for the recruitment of CERT to the inclusion membrane is the inclusion membrane protein IncD (CT115), as it colocalises in patches with CERT on the membrane and interacts with its Pleckstrin homology (PH) domain (Agaisse and Derre, 2014; Derre et al., 2011). Additionally, C. caviae lacks IncD and is unable to recruit CERT. Intriguingly, CERT depletion causes reduced inclusion size and less infectious progeny, a different phenotype to what is observed for Brefeldin A treatment leading to a decrease in Golgi-toinclusion traffic. It has consequently been hypothesised that the ceramides obtained via these two different pathways are subsequently used in different  ways by Chlamydia (Elwell et al., 2011).

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Table of contents :

TABLE OF CONTENTS
TABLE OF FIGURES
ABBREVIATIONS
ABSTRACT
RESUME (UNE PAGE)
RESUME (VERSION LONGUE)
INTRODUCTION
1. Chlamydiae
1.1 Phylogeny
2. Human pathogens
2.1 C. trachomatis
2.1.1 Trachoma, disease and history
2.1.2 Urogenital tract infections
2.1.3 Lymphogranuloma venereum
2.1.4 Diagnosis and treatment
2.2 C. pneumoniae
3. Biphasic developmental cycle
3.1 Entry to Exit
3.1.1 Attachment and Entry
3.1.2 Building the inclusion and survival
3.2 Characteristics of EBs and RBs
3.2.1 Ultrastructure of the chlamydial surface
3.2.2 Transcriptional regulation
3.3 Persistence
4. Host-pathogen interactions
4.1 Chlamydial secretions systems
4.2 Recruitment and uptake of host organelles and lipids
4.2.1 Recruitment of host lipids
4.2.2 Interaction with host organelles
4.3 Interface of interaction: the inclusion membrane
4.4 The host defence and chlamydial immune evasion
5. Chlamydial metabolism
5.1 Energy parasite hypothesis
5.2 Metabolic pathways in the post-genomic era
5.2.1 Substrate level phosphorylation
5.2.2 Oxidative phosphorylation
5.2.3 Other metabolic pathways
5.3 Glycogen
5.3.1 Chlamydial glycogen metabolism
5.3.2 Eukaryotic glycogen metabolism
MATERIAL AND METHODS
Cells and bacteria
Electron microscopy and periodic acid-thiocarbohydrazide-silver proteinate reaction
(PATAg)
Reinfection assay and flow cytometry
Quantitative Reverse Transcription PCR and Reverse Transcription PCR
Transfection
Construction of recombinant plasmids
Immunofluorescence and PAS staining
Quantification of glycogen with CellProfiler
Western Blot and antibodies
Zymogram
Heterologous secretion assay in Shigella flexneri
Transformation of C. trachomatis L2
Glucose uptake assay
RESULTS
1. Glycogen detection in Chlamydia trachomatis inclusion
2. Glucose is essential for Chlamydia trachomatis infection
3. Intraluminal glycogen is not derived from bacterial lysis
4. Kinetics of glycogen accumulation
5. Mechanisms of glycogen accumulation in inclusion lumen 
5.1 Hypotheses
5.2 Vesicular import of host glycogen
5.2.1 Part of luminal glycogen is translocated in bulk from the host cytoplasm
5.2.2 Import of host glycogen and glycogen enzymes is autophagosome independent
5.3 Import of host glucose derivative
5.3.1 UDP-glucose is the host sugar transported into the inclusion lumen
5.3.2 UDP-glucose is a substrate for chlamydial GlgA
5.3.3 Identification of UDP-Glc transporter at inclusion membrane
6. Chlamydial glycogen enzymes are secreted for de novo glycogen synthesis
6.1 Heterologous test of secretion in Shigella flexneri
6.2 GlgX is present in the inclusion lumen
6.3 Overexpression of glycogen enzymes in C. trachomatis
6.4 Ectopically expressed GlgA compensates for the plasmid-less deficiency in glycogen accumulation
7. Chlamydia import Glc6P, but not Glc1P nor Glc
DISCUSSION
BIBLIOGRAPHY
ANNEXE

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