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: 4.16). 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 · 2.62 Impact Factor
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    • "chaffeensis (Lin et al., 2011). Among the several proteins detected, ER was found to share several proteins with other obligate intracellular bacteria (Chao et al. (2004), Hajem et al. (2009), Marques et al. (2008), Ramabu et al. (2010), Renesto et al. (2005), Samoilis et al. (2007), Seo et al. (2008) and Vandahl et al. (2001), Table S2) such as DnaK, PepA and Ssb (detected on 11 bacteria of 12 total analyzed), AtpA/FusA/ClpB/GroS (8/12), Lpd/ClpP (7/12) and SdhA/RpoB/TrpSRpsA/ClpB (6/10). Tme and TsaA were exclusively found in R. parkeri and ER and other were exclusively found in Anaplasmataceae: ArgJ was only found in E. chaffeensis (using the nano-LC–MS/MS method) and ER, XerC in A. phagocytophilum and ER, SurE in the E. chaffeensis, A. phagocytophilum and ER. "
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    ABSTRACT: The Rickettsiales Ehrlichia ruminantium (ER) is the causative agent of heartwater, a fatal tick-borne disease of livestock in sub-Saharan Africa and in the Caribbean, posing strong economical constraints to livestock production. In an attempt to identify the most prominent proteins expressed by this bacterium, especially those encoded by the major antigenic protein 1 (map1) multigene family, a proteome map of ER cultivated in endothelial cells was constructed by using two dimensional gel electrophoresis combined with mass spectrometry. Among the sixty-four spots detected, we could identify only four proteins from the MAP1-family; the other proteins detected were mainly related to energy, amino acid and general metabolism (26%), to protein turnover, chaperones and survival (21%) and to information processes (14%) or classified as hypothetical proteins (23%). Additional studies on MAP1-family protein using immunochemical labeling also revealed that these proteins are differentially expressed along the bacterium life cycle, presenting different structural organization. Interestingly, when infectious elementary bodies (EBs) are released from host cells, MAP1 appears to be organized in SDS and heat-resistant dimers and trimers stabilized by disulfide bridges. Overall, the results presented herein not only reveal the first partial proteome map of ER but provide new insights on the expression ER MAP1-family proteins in host endothelial cells.
    Veterinary Microbiology 12/2011; 156(3-4):305-14. DOI:10.1016/j.vetmic.2011.11.022 · 2.73 Impact Factor
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    • "However, the kinetics of the production of antibody in dogs against 21 E. chaffeensis outer membrane proteins over a period of 462 days suggests that there may not be sequential/differential expression of the proteins during the course of the infection (Zhang et al., 2004a). Recent proteome studies by Ge and Rikihisa (2007) and Seo et al., (2008) found that all but a few p28-Omp proteins were expressed in macrophage cells, while the major expression in tick cells is limited to one p28-Omp protein 14 (Seo et al. 2008). It is clear that a better understanding of when these genes are expressed and how the host deals with them is important to understanding Ehrlichia immunity. "
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    ABSTRACT: Pathogenic bacteria belonging to the family Anaplasmataceae include species of the genera Ehrlichia and Anaplasma. Ehrlichia chaffeensis, first known as the causative agent of human monocytic ehrlichiosis, also infects several vertebrate hosts including white-tailed deer, dogs, coyotes and goats. E. chaffeensis is transmitted from the bite of an infected hard tick, such as Amblyomma americanum. E. chaffeensis and other tick-transmitted pathogens have adapted to both the tick and vertebrate host cell environments. Although E. chaffeensis persists in both vertebrate and tick hosts for long periods of time, little is known about that process. Immunological studies will be valuable in assessing how the pathogen persists in nature in both vertebrate and invertebrate hosts. Understanding the host immune response to the pathogen originating from dual host backgrounds is also important to develop effective methods of diagnosis, control and treatment. In this paper, we provide our perspective of the current understanding of the immune response against E. chaffeensis in relation to other related Anaplasmataceae pathogens.
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