Total, Membrane, and Immunogenic Proteomes of Macrophage- and Tick Cell-Derived Ehrlichia chaffeensis Evaluated by Liquid Chromatography-Tandem Mass Spectrometry and MALDI-TOF Methods

Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, 1800 Denison Avenue, Manhattan, KS 66506, USA.
Infection and immunity (Impact Factor: 3.73). 09/2008; 76(11):4823-32. DOI: 10.1128/IAI.00484-08
Source: PubMed

ABSTRACT Ehrlichia chaffeensis, a tick-transmitted rickettsial, is the causative agent of human monocytic ehrlichiosis. To examine protein expression patterns, we analyzed total, membrane, and immunogenic proteomes of E. chaffeensis originating from macrophage and tick cell cultures. Total proteins resolved by one-dimensional gel electrophoresis and subjected to liquid chromatography-electrospray ionization ion trap mass spectrometry allowed identification of 134 and 116 proteins from macrophage- and tick cell-derived E. chaffeensis, respectively. Because a majority of immunogenic proteins remained in the membrane fraction, individually picked total and immunogenic membrane proteins were also surveyed by liquid chromatography-tandem mass spectrometry and matrix-assisted laser desorption ionization-time of flight methods. The analysis aided the identification of 48 additional proteins. In all, 278 genes of the E. chaffeensis genome were verified as functional genes. They included genes for DNA and protein metabolism, energy metabolism and transport, membrane proteins, hypothetical proteins, and many novel proteins of unknown function. The data reported in this study suggest that the membrane of E. chaffeensis is very complex, having many expressed proteins. This study represents the first and the most comprehensive analysis of E. chaffeensis-expressed proteins. This also is the first study confirming the expression of nearly one-fourth of all predicted genes of the E. chaffeensis genome, validating that they are functionally active genes, and demonstrating that classic shotgun proteomic approaches are feasible for tick-transmitted intraphagosomal bacteria. The identity of novel expressed proteins reported in this study, including the large selection of membrane and immunogenic proteins, will be valuable in elucidating pathogenic mechanisms and developing effective prevention and control methods.

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Available from: Roman Reddy Ganta, Aug 21, 2014
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    • "July 2014 | Volume 4 | Article 86 | 4 encodes for superoxide dismutase) infecting mammalian cells as compared with infected vector cells (Kuriakose et al., 2011) and for A. phagocytophilum (APH_0795 encodes for antioxidant AhpC/Tsa family) (Nelson et al., 2008). Superoxide dismutase was also detected in the global proteome of E. chaffeensis (Seo et al., 2008). In E. ruminantium proteome, TsaA and ElbB proteins, both involved in cell redox homeostasis, were detected. "
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    ABSTRACT: This paper examines how "Omics" approaches improve our understanding of Anaplasmataceae pathogenesis, through a global and integrative strategy to identify genes and proteins involved in biochemical pathways key for pathogen-host-vector interactions. The Anaplasmataceae family comprises obligate intracellular bacteria mainly transmitted by arthropods. These bacteria are responsible for major human and animal endemic and emerging infectious diseases with important economic and public health impacts. In order to improve disease control strategies, it is essential to better understand their pathogenesis. Our work focused on four Anaplasmataceae, which cause important animal, human and zoonotic diseases: Anaplasma marginale, A. phagocytophilum, Ehrlichia chaffeensis, and E. ruminantium. Wolbachia spp. an endosymbiont of arthropods was also included in this review as a model of a non-pathogenic Anaplasmataceae. A gap analysis on "Omics" approaches on Anaplasmataceae was performed, which highlighted a lack of studies on the genes and proteins involved in the infection of hosts and vectors. Furthermore, most of the studies have been done on the pathogen itself, mainly on infectious free-living forms and rarely on intracellular forms. In order to perform a transcriptomic analysis of the intracellular stage of development, researchers developed methods to enrich bacterial transcripts from infected cells. These methods are described in this paper. Bacterial genes encoding outer membrane proteins, post-translational modifications, eukaryotic repeated motif proteins, proteins involved in osmotic and oxidative stress and hypothetical proteins have been identified to play a key role in Anaplasmataceae pathogenesis. Further investigations on the function of these outer membrane proteins and hypothetical proteins will be essential to confirm their role in the pathogenesis. Our work underlines the need for further studies in this domain and on host and vector responses to infection.
    Frontiers in Cellular and Infection Microbiology 07/2014; 4:86. DOI:10.3389/fcimb.2014.00086 · 3.72 Impact Factor
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    • "We recently presented the first evidence that clpB gene expression increases during replication stage of E. chaffeensis [54]. Similarly, we and others reported global macrophage and tick cell-specific protein and gene expression differences [13,14]; the differentially expressed proteins included various outer membrane proteins [11,13,55,56]. "
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    ABSTRACT: Bacterial gene transcription is initiated by RNA polymerase containing a sigma factor. To understand gene regulation in Ehrlichia chaffeensis, an important tick-transmitted rickettsiae responsible for human monocytic ehrlichiosis, we initiated studies evaluating the transcriptional machinery of several genes of this organism. We mapped the transcription start sites of 10 genes and evaluated promoters of five genes (groE, dnaK, hup, p28-Omp14 and p28-Omp19 genes). We report here that the RNA polymerase binding elements of E. chaffeensis gene promoters are highly homologous for its only two transcription regulators, sigma 32 and sigma 70, and that gene expression is accomplished by either of the transcription regulators. RNA analysis revealed that although transcripts for both sigma 32 and sigma 70 are upregulated during the early replicative stage, their expression patterns remained similar for the entire replication cycle. We further present evidence demonstrating that the organism's -35 motifs are essential to transcription initiations. The data suggest that E. chaffeensis gene regulation has evolved to support the organism's growth, possibly to facilitate its intraphagosomal growth. Considering the limited availability of genetic tools, this study offers a novel alternative in defining gene regulation in E. chaffeensis and other related intracellular pathogens.
    PLoS ONE 11/2013; 8(11):e81780. DOI:10.1371/journal.pone.0081780 · 3.23 Impact Factor
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    • "We have presented two novel findings; cell debris in phagosomes of a subset of infected macrophages, which possibly represent degradation of the organisms, and the localization of E. chaffeensis organisms within the nucleus of a subset of infected host cells. The morphological differences in infected tick cells and macrophages parallel to our prior observations that E. chaffeensis organisms express unique host cell specific proteins [13]–[16]. The morphological differences in the pathogen’s progression in infected macrophages and tick cells are further evidence that the pathogen employs unique host-cell specific strategies. "
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    ABSTRACT: Ehrlichia chaffeensis is an emerging tick-borne rickettsial pathogen responsible for human monocytic ehrlichiosis. Despite the induction of an active host immune response, the pathogen has evolved to persist in its vertebrate and tick hosts. Understanding how the organism progresses in tick and vertebrate host cells is critical in identifying effective strategies to block the pathogen transmission. Our recent molecular and proteomic studies revealed differences in numerous expressed proteins of the organism during its growth in different host environments. Transmission electron microscopy analysis was performed to assess morphological changes in the bacterium within macrophages and tick cells. The stages of pathogen progression observed included the attachment of the organism to the host cells, its engulfment and replication within a morulae by binary fission and release of the organisms from infected host cells by complete host cell lysis or by exocytosis. E. chaffeensis grown in tick cells was highly pleomorphic and appears to replicate by both binary fission and filamentous type cell divisions. The presence of Ehrlichia-like inclusions was also observed within the nucleus of both macrophages and tick cells. This observation was confirmed by confocal microscopy and immunoblot analysis. Morphological differences in the pathogen's progression, replication, and processing within macrophages and tick cells provide further evidence that E. chaffeensis employs unique host-cell specific strategies in support of adaptation to vertebrate and tick cell environments.
    PLoS ONE 05/2012; 7(5):e36749. DOI:10.1371/journal.pone.0036749 · 3.23 Impact Factor
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