Chlamydia trachomatis development requires both host glycolysis and oxidative phosphorylation but has only minor effects on these pathways.

The obligate intracellular bacteria Chlamydia trachomatis obtain all nutrients from the cytoplasm of their epithelial host cells and stimulate glucose uptake by these cells. They even hijack host ATP, exerting a strong metabolic pressure on their host at the peak of the proliferative stage of their developmental cycle. However, it is largely unknown whether infection modulates the metabolism of the host cell. Also, the reliance of the bacteria on host metabolism might change during their progression through their biphasic developmental cycle. Herein, using primary epithelial cells and 2 cell lines of nontumoral origin, we showed that between the 2 main ATP-producing pathways of the host, oxidative phosphorylation (OxPhos) remained stable and glycolysis was slightly increased. Inhibition of either pathway strongly reduced bacterial proliferation, implicating that optimal bacterial growth required both pathways to function at full capacity. While we found C. trachomatis displayed some degree of energetic autonomy in the synthesis of proteins expressed at the onset of infection, functional host glycolysis was necessary for the establishment of early inclusions, whereas OxPhos contributed less. These observations correlated with the relative contributions of the pathways in maintaining ATP levels in epithelial cells, with glycolysis contributing the most. Altogether, this work highlights the dependence of C. trachomatis on both host glycolysis and OxPhos for efficient bacterial replication. However, ATP consumption appears at equilibrium with the normal production capacity of the host and the bacteria, so that no major shift between these pathways is required to meet bacterial needs.

Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis in epithelial cells.

Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme with transamidating activity. We report here that both expression and activity of TG2 are enhanced in mammalian epithelial cells infected with the obligate intracellular bacteria Chlamydia trachomatis. Genetic or pharmacological inhibition of TG2 impairs bacterial development. We show that TG2 increases glucose import by up-regulating the transcription of the glucose transporter genes GLUT-1 and GLUT-3. Furthermore, TG2 activation drives one specific glucose-dependent pathway in the host, i.e., hexosamine biosynthesis. Mechanistically, we identify the glucosamine:fructose-6-phosphate amidotransferase (GFPT) among the substrates of TG2. GFPT modification by TG2 increases its enzymatic activity, resulting in higher levels of UDP-N-acetylglucosamine biosynthesis and protein O-GlcNAcylation. The correlation between TG2 transamidating activity and O-GlcNAcylation is disrupted in infected cells because host hexosamine biosynthesis is being exploited by the bacteria, in particular to assist their division. In conclusion, our work establishes TG2 as a key player in controlling glucose-derived metabolic pathways in mammalian cells, themselves hijacked by C. trachomatis to sustain their own metabolic needs.

The Loss of Expression of a Single Type 3 Effector (CT622) Strongly Reduces Chlamydia trachomatis Infectivity and Growth.

Invasion of epithelial cells by the obligate intracellular bacterium Chlamydia trachomatis results in its enclosure inside a membrane-bound compartment termed an inclusion. The bacterium quickly begins manipulating interactions between host intracellular trafficking and the inclusion interface, diverging from the endocytic pathway and escaping lysosomal fusion. We have identified a previously uncharacterized protein, CT622, unique to the Chlamydiaceae, in the absence of which most bacteria failed to establish a successful infection. CT622 is abundant in the infectious form of the bacteria, in which it associates with CT635, a putative novel chaperone protein. We show that CT622 is translocated into the host cytoplasm via type three secretion throughout the developmental cycle of the bacteria. Two separate domains of roughly equal size have been identified within CT622 and a 1.9 Å crystal structure of the C-terminal domain has been determined. Genetic disruption of ct622 expression resulted in a strong bacterial growth defect, which was due to deficiencies in proliferation and in the generation of infectious bacteria. Our results converge to identify CT622 as a secreted protein that plays multiple and crucial roles in the initiation and support of the C. trachomatis growth cycle. They reveal that genetic disruption of a single effector can deeply affect bacterial fitness.

The DUF582 Proteins of Chlamydia trachomatis Bind to Components of the ESCRT Machinery, Which Is Dispensable for Bacterial Growth In vitro.

Chlamydiae are Gram negative bacteria that develop exclusively inside eukaryotic host cells, within a membrane-bounded compartment. Members of the family Chlamydiaceae, such as Chlamydia trachomatis, are pathogenic species infecting vertebrates. They have a very reduced genome and exploit the capacities of their host for their own development, mainly through the secretion of proteins tailored to interfere with eukaryotic processes, called effector proteins. All Chlamydiaceae possess genes coding for four to five effectors that share a domain of unknown function (DUF582). Here we show that four of these effectors, which represent the conserved set in all Chlamydiaceae, accumulate in the infectious form of C. trachomatis, and are therefore likely involved in an early step of the developmental cycle. The fifth member of the family, CT621, is specific to C. trachomatis, and is secreted during the growth phase. Using a two-hybrid screen in yeast we identified an interaction between the host protein Hrs and the DUF582, which we confirmed by co-immunoprecipitations in co-transfected mammalian cells. Furthermore, we provide biochemical evidence that a second domain of one of the DUF582 proteins, CT619, binds the host protein Tsg101. Hrs and Tsg101 are both implicated in a well conserved machinery of the eukaryotic cell called the ESCRT machinery, which is involved in several cellular processes requiring membrane constriction. Using RNA interference targeting proteins implicated at different stages of ESCRT-driven processes, or inhibition by expression of a dominant negative mutant of VPS4, we demonstrated that this machinery was dispensable for bacterial entry, multiplication and differentiation into infectious progeny, and for uptake of glycogen into the parasitophorous vacuole. In light of these observations we discuss how the DUF582 proteins might target the ESCRT machinery during infection.

Sequestration of host metabolism by an intracellular pathogen.

For intracellular pathogens, residence in a vacuole provides a shelter against cytosolic host defense to the cost of limited access to nutrients. The human pathogen Chlamydia trachomatis grows in a glycogen-rich vacuole. How this large polymer accumulates there is unknown. We reveal that host glycogen stores shift to the vacuole through two pathways: bulk uptake from the cytoplasmic pool, and de novo synthesis. We provide evidence that bacterial glycogen metabolism enzymes are secreted into the vacuole lumen through type 3 secretion. Our data bring strong support to the following scenario: bacteria co-opt the host transporter SLC35D2 to import UDP-glucose into the vacuole, where it serves as substrate for de novo glycogen synthesis, through a remarkable adaptation of the bacterial glycogen synthase. Based on these findings we propose that parasitophorous vacuoles not only offer protection but also provide a microorganism-controlled metabolically active compartment essential for redirecting host resources to the pathogens.

Quantitative monitoring of the Chlamydia trachomatis developmental cycle using GFP-expressing bacteria, microscopy and flow cytometry.

Chlamydiae are obligate intracellular bacteria. These pathogens develop inside host cells through a biphasic cycle alternating between two morphologically distinct forms, the infectious elementary body and the replicative reticulate body. Recently, C. trachomatis strains stably expressing fluorescent proteins were obtained. The fluorochromes are expressed during the intracellular growth of the microbe, allowing bacterial visualization by fluorescence microscopy. Whether they are also present in the infectious form, the elementary body, to a detectable level has not been studied. Here, we show that a C. trachomatis strain transformed with a plasmid expressing the green fluorescent protein (GFP) accumulates sufficient quantities of the probe in elementary bodies for detection by microscopy and flow cytometry. Adhesion of single bacteria was detected. The precise kinetics of bacterial entry were determined by microscopy using automated procedures. We show that during the intracellular replication phase, GFP is a convenient read-out for bacterial growth with several advantages over current methods. In particular, infection rates within a non-homogenous cell population are easily quantified. Finally, in spite of their small size, individual elementary bodies are detected by flow cytometers, allowing for direct enumeration of a bacterial preparation. In conclusion, GFP-expressing chlamydiae are suitable to monitor, in a quantitative manner, progression throughout the developmental cycle. This will facilitate the identification of the developmental steps targeted by anti-chlamydial drugs or host factors.

The chlamydial OTU domain-containing protein ChlaOTU is an early type III secretion effector targeting ubiquitin and NDP52.

Chlamydia are obligate intracellular pathogens. Upon contact with the host, they use type III secretion to deliver proteins into the cell, thereby triggering actin-dependent entry and establishing the infection. We observed that Chlamydia caviae elicited a local and transient accumulation of ubiquitinated proteins at the entry sites, which disappeared within 20 min. We investigated the mechanism for the rapid clearance of ubiquitin. We showed that the OTU-like domain containing protein CCA00261, predicted to have deubiquitinase activity, was detected in infectious particles and was a type III secretion effector. This protein is present in several Chlamydia strains, including the human pathogen Chlamydia pneumoniae, and we further designate it as ChlaOTU. We demonstrated that ChlaOTU bound ubiquitin and NDP52, and we mapped these interactions to distinct domains. NDP52 was recruited to Chlamydia entry sites and was dispensable for infection and for bacterial growth. ChlaOTU functioned as a deubiquitinase in vitro. Heterologousexpression of ChlaOTU reduced ubiquitin accumulation at the entry sites, while a catalytic mutant of the deubiquitinase activity had the opposite effect. Altogether, we have identified a novel secreted protein of chlamydiae. ChlaOTU targets both ubiquitin and NDP52 and likely participates in the clearance of ubiquitin at the invasion sites.

Histone methylation by NUE, a novel nuclear effector of the intracellular pathogen Chlamydia trachomatis.

Sequence analysis of the genome of the strict intracellular pathogen Chlamydia trachomatis revealed the presence of a SET domain containing protein, proteins that primarily function as histone methyltransferases. In these studies, we demonstrated secretion of this protein via a type III secretion mechanism. During infection, the protein is translocated to the host cell nucleus and associates with chromatin. We therefore named the protein nuclear effector (NUE). Expression of NUE in mammalian cells by transfection reconstituted nuclear targeting and chromatin association. In vitro methylation assays confirmed NUE is a histone methyltransferase that targets histones H2B, H3 and H4 and itself (automethylation). Mutants deficient in automethylation demonstrated diminished activity towards histones suggesting automethylation functions to enhance enzymatic activity. Thus, NUE is secreted by Chlamydia, translocates to the host cell nucleus and has enzymatic activity towards eukaryotic substrates. This work is the first description of a bacterial effector that directly targets mammalian histones.

SNARE protein mimicry by an intracellular bacterium.

Many intracellular pathogens rely on host cell membrane compartments for their survival. The strategies they have developed to subvert intracellular trafficking are often unknown, and SNARE proteins, which are essential for membrane fusion, are possible targets. The obligate intracellular bacteria Chlamydia replicate within an intracellular vacuole, termed an inclusion. A large family of bacterial proteins is inserted in the inclusion membrane, and the role of these inclusion proteins is mostly unknown. Here we identify SNARE-like motifs in the inclusion protein IncA, which are conserved among most Chlamydia species. We show that IncA can bind directly to several host SNARE proteins. A subset of SNAREs is specifically recruited to the immediate vicinity of the inclusion membrane, and their accumulation is reduced around inclusions that lack IncA, demonstrating that IncA plays a predominant role in SNARE recruitment. However, interaction with the SNARE machinery is probably not restricted to IncA as at least another inclusion protein shows similarities with SNARE motifs and can interact with SNAREs. We modelled IncA's association with host SNAREs. The analysis of intermolecular contacts showed that the IncA SNARE-like motif can make specific interactions with host SNARE motifs similar to those found in a bona fide SNARE complex. Moreover, point mutations in the central layer of IncA SNARE-like motifs resulted in the loss of binding to host SNAREs. Altogether, our data demonstrate for the first time mimicry of the SNARE motif by a bacterium.

A directed screen for chlamydial proteins secreted by a type III mechanism identifies a translocated protein and numerous other new candidates.

Chlamydiae are strict intracellular parasites that induce their internalization upon contact with the host cell and grow inside an intracellular compartment called an inclusion. They possess a type III secretion (TTS) apparatus, which allows for the translocation of specific proteins in the host cell cytosol. In particular, chlamydial proteins of the Inc family are secreted to the inclusion membrane by a TTS mechanism; other TTS substrates are mostly unknown. Using a secretion assay based on the recognition of TTS signals in Shigella flexneri, we searched for TTS signals in the proteins of unknown function, conserved between three different chlamydial species, Chlamydia pneumoniae, C. trachomatis and C. caviae. We identified 24 new candidate proteins which did not belong to the Inc family. Four of these proteins were also secreted as full-length proteins by a TTS mechanism in S. flexneri, indicating that their translocation does not require other chlamydial proteins. One of these proteins was detected in the cytosol of infected cells using specific antibodies, directly demonstrating that it is translocated in the host cell during bacterial proliferation. More generally, this work represents the first directed search for TTS effectors not based on genetic information or sequence similarity. It reveals the abundance of proteins secreted in the host cell by chlamydiae.