Abstract
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.
Highlights
Ehrlichia chaffeensis is the etiological agent for human monocytic ehrlichiosis
The pellet of cell-free Ehrlichia were resuspended in phosphate buffered saline (PBS), centrifuged at 15,000x g for 15 min and the final pellet was used for protein extraction
Our recent transcriptome assessment of wildtype and three mutant strains of E. chaffeensis aided in gaining considerable understanding of how mutations at certain genomic sites impact global gene expression changes, while other having minimal impact
Summary
Ehrlichia chaffeensis is the etiological agent for human monocytic ehrlichiosis. It is a tick-borne pathogen causing infections in dogs and other vertebrate hosts from Amblyomma americanum ticks (Dumler et al, 1993; Walker and Dumler, 1996; Davidson et al, 2001; Paddock and Childs, 2003). Pathogenesis-associated E. chaffeensis proteins are possibly highly active in a host microenvironment and their differential expression in response to host cell defense is known to occur (Kuriakose et al, 2011). Progress has been made toward identifying proteins crucial for E. chaffeensis survival in a host cell environment (Singu et al, 2006; Seo et al, 2008; Lin et al, 2011). Identifying proteins involved in pathogenesis and virulence, and documenting their differential expression may aid in the discovery of new protein targets valuable for therapeutic interventions and vaccine development against HME and in other related pathogenic rickettsial diseases
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