Proteome analysis of Ehrlichia chaffeensis containing phagosome membranes revealed the presence of numerous bacterial and host proteins.

Tick-transmitted Ehrlichia chaffeensis, the causative agent for human monocytic ehrlichiosis, resides and multiplies within a host cell phagosome. Infection progression of E. chaffeensis includes internalization into a host cell by host cell membrane fusion events following engulfment leading to the formation of E. chaffeensis containing vacuole (ECV). Revealing the molecular composition of ECV is important in understanding the host cellular processes, evasion of host defense pathways and in defining host-pathogen interactions. ECVs purified from infected host cells were analyzed to define both host and bacterial proteomes associated with the phagosome membranes. About 160 bacterial proteins and 2,683 host proteins were identified in the ECV membranes. The host proteins included predominantly known phagosome proteins involved in phagocytic trafficking, fusion of vesicles, protein transport, Ras signaling pathway and pathogenic infection. Many highly expressed proteins were similar to the previously documented proteins of phagosome vacuole membranes containing other obligate pathogenic bacteria. The finding of many bacterial membrane proteins is novel; they included multiple outer membrane proteins, such as the p28-Omps, the 120 kDa protein, preprotein translocases, lipoproteins, metal binding proteins, and chaperonins, although the presence of ankyrin repeat proteins, several Type I and IV secretion system proteins is anticipated. This study demonstrates that ECV membrane is extensively modified by the pathogen. This study represents the first and the most comprehensive description of ECV membrane proteome. The identity of many host and Ehrlichia proteins in the ECV membrane will be a valuable to define pathogenic mechanisms critical for the replication of the pathogen within macrophages.

Protein and DNA Biosynthesis Demonstrated in Host Cell-Free Phagosomes Containing Anaplasma phagocytophilum or Ehrlichia chaffeensis in Axenic Media.

Rickettsiae belong to the Anaplasmataceae family, which includes mostly tick-transmitted pathogens causing human, canine, and ruminant diseases. Biochemical characterization of the pathogens remains a major challenge because of their obligate parasitism. We investigated the use of an axenic medium for growth of two important pathogens-Anaplasma phagocytophilum and Ehrlichia chaffeensis-in host cell-free phagosomes. We recently reported that the axenic medium promotes protein and DNA biosynthesis in host cell-free replicating form of E. chaffeensis, although the bacterial replication is limited. We now tested the hypothesis that growth on axenic medium can be improved if host cell-free rickettsia-containing phagosomes are used. Purification of phagosomes from A. phagocytophilum- and E. chaffeensis-infected host cells was accomplished by density gradient centrifugation combined with magnet-assisted cell sorting. Protein and DNA synthesis was observed for both organisms in cell-free phagosomes with glucose-6-phosphate and/or ATP. The levels of protein and DNA synthesis were the highest for a medium pH of 7. The data demonstrate bacterial DNA and protein synthesis for the first time in host cell-free phagosomes for two rickettsial pathogens. The host cell support-free axenic growth of obligate pathogenic rickettsiae will be critical in advancing research goals in many important tick-borne diseases impacting human and animal health.

Proteome Analysis Revealed Changes in Protein Expression Patterns Caused by Mutations in Ehrlichia chaffeensis.

The tick-borne rickettsial pathogen, Ehrlichia chaffeensis, causes monocytic ehrlichiosis in people and other vertebrate hosts. Mutational analysis in E. chaffeensis genome aids in better understanding of its infection and persistence in host cells and in the development of attenuated vaccines. Our recent RNA deep sequencing study revealed that three genomic mutations caused global changes in the gene expression patterns, which in turn affect the ability of pathogen's survival in a host and the host's ability to induce protection against the pathogen. In this follow-up study, we document the impact of mutations on the pathogen's global protein expression and the influence of protein abundance on a mutant's attenuation and protection of vertebrate host against infection. iTRAQ labeling and mass spectrometry analysis of E. chaffeensis wildtype and mutants identified 564 proteins covering about 63% of the genome. Mutation in ECH_0379 gene encoding for an antiporter protein, causing attenuated growth in vertebrate hosts, led to overexpression of p28 outer membrane proteins, molecular chaperons, and metabolic enzymes, while a mutation downstream to the ECH_0490 gene that caused minimal impact on the pathogen's in vivo growth resulted in major changes in the expression of outer membrane proteins, transcriptional regulators and T4SS proteins. ECH_0660 gene mutation, causing the pathogen's rapid clearance and offering protection against wild type infection challenge in a vertebrate host, had a minimal impact on proteome similar to our prior observations from transcriptome analysis. While the global proteome data revealed fewer translated proteins compared to the transcripts identified from RNA deep sequencing analysis, there is a great deal of correlation noted between the global proteome and transcriptome analysis. Further, global proteome analysis, including the assessment of 2D resolved total and immunoproteomes revealed greater variations in the highly immunogenic p28-Omp proteins.

Impact of Three Different Mutations in Ehrlichia chaffeensis in Altering the Global Gene Expression Patterns.

The rickettsial pathogen Ehrlichia chaffeensis causes a tick-borne disease, human monocytic ehrlichiosis. Mutations within certain genomic locations of the pathogen aid in understanding the pathogenesis and in developing attenuated vaccines. Our previous studies demonstrated that mutations in different genomic sites in E. chaffeensis caused variable impacts on their growth and attenuation in vertebrate and tick hosts. Here, we assessed the effect of three mutations on transcriptional changes using RNA deep-sequencing technology. RNA sequencing aided in detecting 66-80% of the transcripts of wildtype and mutant E. chaffeensis. Mutation in an antiporter gene (ECH_0379) causing attenuated growth in vertebrate hosts resulted in the down regulation of many transcribed genes. Similarly, a mutation downstream to the ECH_0490 coding sequence resulted in minimal impact on the pathogen's in vivo growth, but caused major changes in its transcriptome. This mutation caused enhanced expression of several host stress response genes. Even though the ECH_0660 gene mutation caused the pathogen's rapid clearance in vertebrate hosts and aids in generating a protective response, there was minimal impact on the transcriptome. The transcriptomic data offer novel insights about the impact of mutations on global gene expression and how they may contribute to the pathogen's resistance and/or clearance from the host.