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Bacteria have acquired multiple systems to expose proteins on their surface, release them in the extracellular environment or even inject them into a neighboring cell. Protein secretion has a high adaptive value and secreted proteins are implicated in many functions, which are often essential for bacterial fitness. Several secreted proteins or secretion machineries have been extensively studied as potential drug targets. It is therefore important to identify the secretion substrates, to understand how they are specifically recognized by the secretion machineries, and how transport through these machineries occurs. The purpose of this review is to provide an overview of the biochemical, genetic and imaging tools that have been developed to evaluate protein secretion in a qualitative or quantitative manner. After a brief overview of the different tools available, we will illustrate their advantages and limitations through a discussion of some of the current open questions related to protein secretion. We will start with the question of the identification of secreted proteins, which for many bacteria remains a critical initial step toward a better understanding of their interactions with the environment. We will then illustrate our toolbox by reporting how these tools have been applied to better understand how substrates are recognized by their cognate machinery, and how secretion proceeds. Finally, we will highlight recent approaches that aim at investigating secretion in real time, and in complex environments such as a tissue or an organism.

Upon transfer to the inclusion membrane, ceramide might be converted to SM by host SMS, which is enriched around the inclusion, and incorporated by the bacteria. Because the catalytic site of SMS is in the lumenal site of the Golgi apparatus, it would imply that the enzyme traffics to the inclusion membrane to convert ceramide, by a mechanism that remains to be determined. [...]Elwell et al. also provide data showing that depletion of the brefeldin A target GBF1 reproduces the effect of the drug on Chlamydia infection, implicating GBF1 in the vesicular route for SM acquisition [8].

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. Chlamydia trachomatis is the most common sexually transmitted bacteria that causes disease. Infections often do not produce any obvious symptoms, but can lead to infertility or other severe problems if left untreated. This microbe is also the leading cause of blindness by an infectious agent.The bacteria grow in the human body by infecting host cells. Inside these cells, the bacteria are found inside compartments known as inclusions, which protect them from the host’s defense responses and enable them to create a comfortable environment for themselves. However, this comes at a cost because the bacteria lose immediate access to the nutrients in the rest of the host cell. Thus, C.trachomatis has developed ways to import these nutrients into inclusions, and, more generally, to take the control of its interactions with the host cell. The inclusions built up by C. trachomatis contain a high amount of glycogen, a carbohydrate that generally acts as an energy storage molecule. Although this observation was made many decades ago, the molecular mechanism by which such a large molecule accumulates in the inclusion has not been clarified. Gehre et al. have now used a variety of cell biology techniques to address this question. The experiments show that there are two different pathways through which glycogen accumulates within the inclusion. Some glycogen is transported in bulk from the interior of the host cell into the inclusion. However, the bacteria also make new glycogen in the inclusion from a building block molecule called UDP-glucose. To do this, the bacteria recruit a host transport molecule to the membrane that surrounds the inclusion. This transport molecule brings UDP-glucose into the inclusion, where an enzyme called glycogen synthase – which is released by the bacteria – uses the UDP-glucose to make glycogen. The C. trachomatis glycogen synthase is unusual because most other bacteria can only make glycogen from another type of glucose. By using both pathways, C. trachomatis is able to trap most of the glycogen stores of the infected cell within the inclusion so that they are inaccessible to the host but ready for the bacteria to use. Previous work has shown that C. trachomatis is much better at accumulating glycogen than other Chlamydia bacteria are. Therefore, a future challenge will be to find out exactly how this helps C. trachomatis survive inside human cells.

Background Chlamydiae are obligate intracellular bacteria that multiply in a vacuolar compartment, the inclusion. Several chlamydial proteins containing a bilobal hydrophobic domain are translocated by a type III secretion (TTS) mechanism into the inclusion membrane. They form the family of Inc proteins, which is specific to this phylum. Based on their localization, Inc proteins likely play important roles in the interactions between the microbe and the host. In this paper we sought to identify and analyze, using bioinformatics tools, all putative Inc proteins in published chlamydial genomes, including an environmental species. Results Inc proteins contain at least one bilobal hydrophobic domain made of two transmembrane helices separated by a loop of less than 30 amino acids. Using bioinformatics tools we identified 537 putative Inc proteins across seven chlamydial proteomes. The amino-terminal segment of the putative Inc proteins was recognized as a functional TTS signal in 90% of the C. trachomatis and C. pneumoniae sequences tested, validating the data obtained in silico . We identified a macro domain in several putative Inc proteins, and observed that Inc proteins are enriched in segments predicted to form coiled coils. A surprisingly large proportion of the putative Inc proteins are not constitutively translocated to the inclusion membrane in culture conditions. Conclusions The Inc proteins represent 7 to 10% of each proteome and show a great degree of sequence diversity between species. The abundance of segments with a high probability for coiled coil conformation in Inc proteins support the hypothesis that they interact with host proteins. While the large majority of Inc proteins possess a functional TTS signal, less than half may be constitutively translocated to the inclusion surface in some species. This suggests the novel finding that translocation of Inc proteins may be regulated by as-yet undetermined mechanisms.

The tumoral origin and extensive passaging of HeLa cells, a most commonly used cervical epithelial cell line, raise concerns on their suitability to study the cell responses to infection. The present study was designed to isolate primary epithelial cells from human ectocervix explants and characterize their susceptibility to C. trachomatis infection. We achieved a high purity of isolation, assessed by the expression of E-cadherin and cytokeratin 14. The infectious progeny in these primary epithelial cells was lower than in HeLa cells. We showed that the difference in culture medium, and the addition of serum in HeLa cultures, accounted for a large part of these differences. However, all things considered the primary ectocervical epithelial cells remained less permissive than HeLa cells to C. trachomatis serovar L2 or D development. Finally, the basal level of transcription of genes coding for pro-inflammatory cytokines was globally higher in primary epithelial cells than in HeLa cells. Transcription of several pro-inflammatory genes was further induced by infection with C. trachomatis serovar L2 or serovar D. In conclusion, primary epithelial cells have a strong capacity to mount an inflammatory response to Chlamydia infection. Our simplified purification protocol from human explants should facilitate future studies to understand the contribution of this response to limiting the spread of the pathogen to the upper female genital tract.

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.

Infection of epithelial cells by Chlamydia trachomatis elicits an innate immune response by these cells. The signaling pathways involved, and their outcomes, are still very poorly understood. In this paper, we described how Chlamydia infection triggered the expression of ISG15, a small molecule normally associated to type I interferon (IFN-I) signaling and control of INF-γ production. ISG15 synthesis by epithelial cells attenuated their immune response to Chlamydia infection. In mice, we observed that ISG15 displayed a marginal role in modulating the production of IFN-γ, a key component of the host immune response to infection, but facilitated bacterial clearance. Overall, our study strengthens the importance of ISG15 not only in the resolution of viral but also of bacterial infection and document its role of “immune brake” in the context of Chlamydia infection.

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.

Under the endosymbiont hypothesis, over a billion years ago a heterotrophic eukaryote entered into a symbiotic relationship with a cyanobacterium (the cyanobiont). This partnership culminated in the plastid that has spread to forms as diverse as plants and diatoms. However, why primary plastid acquisition has not been repeated multiple times remains unclear. Here, we report a possible answer to this question by showing that primary plastid endosymbiosis was likely to have been primed by the secretion in the host cytosol of effector proteins from intracellular Chlamydiales pathogens. We provide evidence suggesting that the cyanobiont might have rescued its afflicted host by feeding photosynthetic carbon into a chlamydia-controlled assimilation pathway.

Chlamydia trachomatis is an obligate intracellular pathogen responsible for loss of eyesight through trachoma and for millions of cases annually of sexually transmitted diseases. The bacteria develop within a membrane-bounded inclusion. They lack enzymes for several biosynthetic pathways, including those to make some phospholipids, and exploit their host to compensate. Three-dimensional fluorescence microscopy demonstrates that small organelles of the host, peroxisomes, are translocated into the Chlamydia inclusion and are found adjacent to the bacteria. In cells deficient for peroxisome biogenesis the bacteria are able to multiply and give rise to infectious progeny, demonstrating that peroxisomes are not essential for bacterial development in vitro. Mass spectrometry-based lipidomics reveal the presence in C. trachomatis of plasmalogens, ether phospholipids whose synthesis begins in peroxisomes and have never been described in aerobic bacteria before. Some of the bacterial plasmalogens are novel structures containing bacteria-specific odd-chain fatty acids; they are not made in uninfected cells nor in peroxisome-deficient cells. Their biosynthesis is thus accomplished by the metabolic collaboration of peroxisomes and bacteria.