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


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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    • "An alternative approach of dissecting rickettsial virulence is to examine the presence of differentially produced rickettsial proteins during infection. While proteomic and immunoreactive profiles have been described for several rickettsial species (Chao et al., 2004, 2007; Hajem et al., 2009; Ogawa et al., 2007; Pornwiroon et al., 2009; Renesto et al., 2005; Sears et al., 2012; Tucker et al., 2011) and closely related tick-borne bacterial pathogens such as Anaplasma (Lopez et al., 2005) and Ehrlichia (Seo et al., 2008), little has been done with comparative antigenic analysis of different rickettsial species. In this study, a comparative immunoproteomic approach using sera from R. parkeri-infected patients was employed for the identification and distinction of several immunoreactive proteins from R. parkeri and R. amblyommii. "
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    ABSTRACT: Rickettsia parkeri is an Amblyomma-associated, spotted fever group Rickettsia species that causes an eschar-associated, febrile illness in multiple countries throughout the Western Hemisphere. Many other rickettsial species of known or uncertain pathogenicity have been detected in Amblyomma spp. ticks in the Americas, including Rickettsia amblyommii, "Candidatus Rickettsia andeanae" and Rickettsia rickettsii. In this study, we utilized an immunoproteomic approach to compare antigenic profiles of low-passage isolates of R. parkeri and R. amblyommii with serum specimens from patients with PCR- and culture-confirmed infections with R. parkeri. Five immunoreactive proteins of R. amblyommii and nine immunoreactive proteins of R. parkeri were identified by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry. Four of these, including the outer membrane protein (Omp) A, OmpB, translation initiation factor IF-2, and cell division protein FtsZ, were antigens common to both rickettsiae. Serum specimens from patients with R. parkeri rickettsiosis reacted specifically with cysteinyl-tRNA synthetase, DNA-directed RNA polymerase subunit alpha, putative sigma (54) modulation protein, chaperonin GroEL, and elongation factor Tu of R. parkeri which have been reported as virulence factors in other bacterial species. Unique antigens identified in this study may be useful for further development of the better serological assays for diagnosing infection caused by R. parkeri. Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.
    Ticks and Tick-borne Diseases 07/2015; 6(6). DOI:10.1016/j.ttbdis.2015.07.012 · 2.72 Impact Factor
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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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