Data_Sheet_1_Proteome Analysis Revealed Changes in Protein Expression Patterns Caused by Mutations in Ehrlichia chaffeensis.PDF

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.

蜱传播的立克次氏体病原体——查菲埃里希体，能够引发人类及其他脊椎动物宿主的单核细胞埃里希体病。对查菲埃里希体基因组中的突变分析有助于更深入地理解其在宿主细胞中的感染和持续存在机制，以及为研制减毒疫苗提供帮助。我们近期进行的RNA深度测序研究揭示，三种基因组突变导致了基因表达模式的整体变化，进而影响了病原体在宿主体内的生存能力以及宿主对病原体的免疫保护能力。在本项后续研究中，我们记录了突变对病原体全局蛋白质表达的影响，以及蛋白质丰度对突变体减毒作用及其对脊椎动物宿主感染保护的效应。通过对查菲埃里希体野生型和突变体的iTRAQ标记及质谱分析，鉴定出564种蛋白质，覆盖了大约63%的基因组。ECH_0379基因突变，该基因编码一种逆向转运蛋白，导致在脊椎动物宿主中生长减弱，引发了p28外膜蛋白、分子伴侣和代谢酶的过度表达；而位于ECH_0490基因下游的突变，该突变对病原体体内生长影响微乎其微，却导致了外膜蛋白、转录调控因子和T4SS蛋白表达的重大变化。ECH_0660基因突变导致病原体迅速清除，并在脊椎动物宿主中提供对野生型感染挑战的保护，与转录组分析的前期观察相似，对蛋白质组的影响最小。尽管全局蛋白质组数据显示与RNA深度测序分析中识别的转录本相比，翻译蛋白质较少，但全局蛋白质组与转录组分析之间存在着显著的关联。此外，包括二维分离的全蛋白质组和免疫蛋白质组评估在内的全局蛋白质组分析，揭示了高度免疫原性的p28-Omp蛋白表达存在更大变异。