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Proteomic profiling of the outer membrane fraction of the obligate intracellular bacterial pathogen Ehrlichia ruminantium.

ABSTRACT: The outer membrane proteins (OMPs) of Gram-negative bacteria play a crucial role in virulence and pathogenesis. Identification of these proteins represents an important goal for bacterial proteomics, because it aids in vaccine development. Here, we have developed such an approach for Ehrlichia ruminantium, the obligate intracellular bacterium that causes heartwater. A preliminary whole proteome analysis of elementary bodies, the extracellular infectious form of the bacterium, had been performed previously, but information is limited about OMPs in this organism and about their role in the protective immune response. Identification of OMPs is also essential for understanding Ehrlichia's OM architecture, and how the bacterium interacts with the host cell environment. First, we developed an OMP extraction method using the ionic detergent sarkosyl, which enriched the OM fraction. Second, proteins were separated via one-dimensional electrophoresis, and digested peptides were analyzed via nano-liquid chromatographic separation coupled with mass spectrometry (LC-MALDI-TOF/TOF). Of 46 unique proteins identified in the OM fraction, 18 (39%) were OMPs, including 8 proteins involved in cell structure and biogenesis, 4 in transport/virulence, 1 porin, and 5 proteins of unknown function. These experimental data were compared to the predicted subcellular localization of the entire E. ruminantium proteome, using three different algorithms. This work represents the most complete proteome characterization of the OM fraction in Ehrlichia spp. The study indicates that suitable subcellular fractionation experiments combined with straightforward computational analysis approaches are powerful for determining the predominant subcellular localization of the experimentally observed proteins. We identified proteins potentially involved in E. ruminantium pathogenesis, which are good novel targets for candidate vaccines. Thus, combining bioinformatics and proteomics, we discovered new OMPs for E. ruminantium that are valuable data for those investigating new vaccines against this organism. In summary, we provide both pioneering data and novel insights into the pathogenesis of this obligate intracellular bacterium.

SUBMITTER: Moumene A

PROVIDER: S-EPMC4339577 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Proteomic profiling of the outer membrane fraction of the obligate intracellular bacterial pathogen Ehrlichia ruminantium.

Moumène Amal A Marcelino Isabel I Ventosa Miguel M Gros Olivier O Lefrançois Thierry T Vachiéry Nathalie N Meyer Damien F DF Coelho Ana V AV

PloS one 20150224 2

The outer membrane proteins (OMPs) of Gram-negative bacteria play a crucial role in virulence and pathogenesis. Identification of these proteins represents an important goal for bacterial proteomics, because it aids in vaccine development. Here, we have developed such an approach for Ehrlichia ruminantium, the obligate intracellular bacterium that causes heartwater. A preliminary whole proteome analysis of elementary bodies, the extracellular infectious form of the bacterium, had been performed ...[more]

PMID: 25710494

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Project description:BACKGROUND: Whole genome transcriptomic analysis is a powerful approach to elucidate the molecular mechanisms controlling the pathogenesis of obligate intracellular bacteria. However, the major hurdle resides in the low quantity of prokaryotic mRNAs extracted from host cells. Our model Ehrlichia ruminantium (ER), the causative agent of heartwater, is transmitted by tick Amblyomma variegatum. This bacterium affects wild and domestic ruminants and is present in Sub-Saharan Africa and the Caribbean islands. Because of its strictly intracellular location, which constitutes a limitation for its extensive study, the molecular mechanisms involved in its pathogenicity are still poorly understood. RESULTS: We successfully adapted the SCOTS method (Selective Capture of Transcribed Sequences) on the model Rickettsiales ER to capture mRNAs. Southern Blots and RT-PCR revealed an enrichment of ER's cDNAs and a diminution of ribosomal contaminants after three rounds of capture. qRT-PCR and whole-genome ER microarrays hybridizations demonstrated that SCOTS method introduced only a limited bias on gene expression. Indeed, we confirmed the differential gene expression between poorly and highly expressed genes before and after SCOTS captures. The comparative gene expression obtained from ER microarrays data, on samples before and after SCOTS at 96 hpi was significantly correlated (R2 = 0.7). Moreover, SCOTS method is crucial for microarrays analysis of ER, especially for early time points post-infection. There was low detection of transcripts for untreated samples whereas 24% and 70.7% were revealed for SCOTS samples at 24 and 96 hpi respectively. CONCLUSIONS: We conclude that this SCOTS method has a key importance for the transcriptomic analysis of ER and can be potentially used for other Rickettsiales. This study constitutes the first step for further gene expression analyses that will lead to a better understanding of both ER pathogenicity and the adaptation of obligate intracellular bacteria to their environment.

Project description:Both prokaryotic and eukaryotic organisms take deterministic decisions to reprogram cell fates. A macroscopic manifestation of such an event is the remodelling of the cellsâ morphology and it typically is governed at the molecular scale by massive reorganization of the cellular transcriptome. With the regulation at the level of transcription initiation representing the most common form for such developmental reprogramming, cells typically rely on one or several master regulators to coordinate the the activity of hundreds of genes simultaneously by directly binding to their promoters. Despite the apparent simplicity of prokaryotes and their reduced genome size compared to that of their eukaryotic counterparts, free-living bacteria typically encode hundreds of transcription factors (TFs) in their genomes that could act as master TFs. By contrast, obligate intracellular bacteria such as Chlamydiae have a drastically reduced genome due to their intimate association with the host and thus a smaller number of TF genes. The genomes of members of the Chlamydiaceae family, which include the well-known bacterial pathogens Chlamydia trachomatis and Chlamydia pneumoniae, are only 1-1.2 Mbp. By contrast, members of the environmental Chlamydiae Waddlia chondrophila and Parachlamydia acanthamoebae have a 2-fold and 3-fold larger genome, respectively, likely allowing for an expansion of the host range while still retaining their host dependence and parasitic life style. Moreover, they all exhibit a characteristic chlamydial developmental cycle via two functionally specialized morphotypes, the infectious non-dividing elementary bodies (EBs) and the non-infectious dividing reticulate bodies (RBs). This developmental cycle is usually divided in three stages: the early stage during which EBs enter host cells and differentiate into RBs; the mid-stage where RBs proliferate inside a vacuole called inclusion and the late stage where RBs differentiate back into EBs and are released after exocytosis or cell lysis. Chlamydial genes are thus classified into three different temporal classes (early, mid and late expressed genes), likely reflecting the need of these transcripts in each of the three developmental stages. W. chondrophila, an emerging pathogen implicated in abortion in bovine and miscarriage in humans, encodes less than 20 TFs, 10 of which are conserved among the Chlamydiae. In light of this low TF multiplicity in the chlamydial pan-genome along with the common developmental cycle and parasitic life style, we aimed to define the regulatory pan-genome of each these conserved TFs to identify the elusive chlamydial master regulator and to characterize underling specificity for its target promoters using chromatin-immunoprecipitation followed by deep-sequencing (ChIP-Seq) of chlamydial cells growing inside the host. The immunochemistry of ChIP-Seq has the advantage of minimizing the contaminating nucleic acids compared to chlamydial transcriptome studies, it has the added benefit of providing the first unambiguous glimpse into the regulatory landscape of a bacterium inside host, offering a solid framework in understanding the stochastic and/or deterministic switches that bacteria rely on during infections. Examination of the regulatory network of an intracellular pathogen

Project description:Gram-negative bacteria constitutively release outer membrane vesicles (OMVs) during cell growth that play significant roles in bacterial survival, virulence and pathogenesis. In this study, comprehensive proteomic analysis of OMVs from a human gastrointestinal pathogen Campylobacter jejuni NCTC11168 was performed using high-resolution mass spectrometry. The OMVs of C. jejuni NCTC11168 were isolated from culture supernatants then characterized using electron microscopy and dynamic light scattering revealing spherical OMVs of an average diameter of 50nm. We then identified 134 vesicular proteins using high-resolution LTQ-Orbitrap mass spectrometry. Subsequent functional analysis of the genes revealed the relationships of the vesicular proteins. Furthermore, known N-glycoproteins were identified from the list of the vesicular proteome, implying the potential role of the OMVs as a delivery means for biologically relevant bacterial glycoproteins. These results enabled us to elucidate the overall proteome profile of pathogenic bacterium C. jejuni and to speculate on the function of OMVs in bacterial infections and communication.Biological significanceThis work demonstrates the importance of understanding vesicular proteomes from a human pathogen Campylobacter jejuni. From the secreted outer membrane vesicles (OMVs) of C. jejuni NCTC11168, we found a variety of virulence factors and essential proteins for bacterial survival. Bioinformatics analysis of these proteins predicted functional enrichment and localization. The most highly enriched were redox enzymes, which are considered to be essential for survival in oxygen-limiting environments and are predicted to be on the twin-arginine translocation (Tat) pathway suggesting a role for this pathway in the biogenesis of OMVs. This study additionally implicates a biological role for N-linked glycoproteins in OMVs. These approaches allow for a better understanding of the physiology of this important human pathogen.

Dataset Information

Proteomic profiling of the outer membrane fraction of the obligate intracellular bacterial pathogen Ehrlichia ruminantium.

Publications

Proteomic profiling of the outer membrane fraction of the obligate intracellular bacterial pathogen Ehrlichia ruminantium.

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