Identification of a family of effectors secreted by the type III secretion system that are conserved in pathogenic Chlamydiae.

Chlamydiae are Gram-negative, obligate intracellular pathogens that replicate within a membrane-bounded compartment termed an inclusion. Throughout their development, they actively modify the eukaryotic environment. The type III secretion (TTS) system is the main process by which the bacteria translocate effector proteins into the inclusion membrane and the host cell cytoplasm. Here we describe a family of type III secreted effectors that are present in all pathogenic chlamydiae and absent in the environment-related species. It is defined by a common domain of unknown function, DUF582, that is present in four or five proteins in each Chlamydiaceae species. We show that the amino-terminal extremity of DUF582 proteins functions as a TTS signal. DUF582 proteins from C. trachomatis CT620, CT621, and CT711 are expressed at the middle and late phases of the infectious cycle. Immunolocalization further revealed that CT620 and CT621 are secreted into the host cell cytoplasm, as well as within the lumen of the inclusion, where they do not associate with bacterial markers. Finally, we show that DUF582 proteins are present in nuclei of infected cells, suggesting that members of the DUF582 family of effector proteins may target nuclear cell functions. The expansion of this family of proteins in pathogenic chlamydiae and their conservation among the different species suggest that they play important roles in the infectious cycle.

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.

Exploitation of host lipids by bacteria.

Bacteria that interact with eukaryotic cells have developed a variety of strategies to divert host lipids, or cellular processes driven by lipids, to their benefit. Host lipids serve as building blocks for bacterial membrane formation and as energy source. They promote the formation of specific microdomains, facilitating interactions with the host. Host lipids are also critical players in the entry of bacteria or toxins into cells, and, for bacteria growing inside parasitophorous vacuoles, in building a secure shelter. Bacterial dissemination is often dependent on enzymatic activities targeting host lipids. Finally, on a larger scale, long lasting parasitic association can disturb host lipid metabolism so deeply as to 'reprogram' it, as proposed in the case of Mycobacterium infection.

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.

Multilocus variable-number tandem repeat analysis: a helpful tool for subtyping French Clostridium difficile PCR ribotype 027 isolates.

The objective of this study was to evaluate the usefulness of multilocus variable-number tandem repeat analysis (MLVA) for typing and subtyping of Clostridium difficile. Sixty-eight strains were studied, including strains from PCR ribotypes 027, 078/126, 014/020/077, 017 and 023. The stability of variable-number tandem repeat (VNTR) loci was tested by comparing the MLVA results of two strains subcultured 11 times. After DNA extraction, seven tandem repeat loci (A6, B7, C6, E7, F3, G8, H9) from published MLVA schemes were amplified by PCR and sequenced. The distance between two strains was determined by calculating the summed tandem repeat difference. Genomic diversity was evaluated by using the minimum spanning tree (Bionumerics 5.1 software program; Applied Maths). Among the 68 C. difficile isolates examined, 65 unique MLVA types were identified, suggesting a high discriminatory power. An overall good agreement was observed between MLVA types and PCR ribotypes. The stability of VNTR loci was good. MLVA could separate isolates of the hypervirulent PCR ribotype 027 clone in several clusters; all 027 strains isolated within a hospital were grouped in a specific cluster or were placed very close to each other. Results of MLVA confirmed that strains from PCR ribotypes 078 and 126 were closely related although some were located in different branches of the tree. Similar results were observed for most strains from PCR ribotypes 014, 020 and 077. This highly discriminatory method is time-consuming and expensive, but is a valuable tool for subtyping of C. difficile, especially of 027 strains.