Exploring small proteins in the foodborne pathogen Campylobacter jejuni

确定细菌病原体的整个基因互补对于了解它如何生存和引起疾病至关重要。深度测序技术彻底改变了基因组测序，并揭示了细菌基因组和转录组中意想不到的复杂性。基于核糖体保护片段的深度测序，最近建立的用于全局翻译组分析的核糖体分析技术（Ribo-seq）揭示了多种生物体中编码潜在微蛋白（< 50个氨基酸）的大量新颖开放阅读框架（ORF）。这类细胞大分子仍然是研究不足。细菌中少量的特征性微蛋白参与多种生理过程，包括毒力和抗生素抗性的调节。食源性病原体空肠弯曲杆菌是目前世界范围内细菌性胃肠炎的主要原因。然而，由于其注释的基因组缺乏其他肠道病原体使用的关键毒力因子的同源物，因此对其如何引起疾病知之甚少。我们比较了多种C.空肠菌株揭示了保守的和菌株特异性的转录产物，包括许多新的转录物，并表明关于其表达的基因组如何有助于毒力还有很多东西要了解。在这个项目中，我们的目标是定义C。空肠微蛋白质组和功能性表征所选微蛋白质。在不同生长条件下使用Ribo-seq结合我们的转录组图谱和比较基因组学进行翻译组分析将鉴定表达的C。空肠micro-ORF。将使用质谱和表位标记进一步验证翻译。我们的第一个C.在肉汤培养物中生长的空肠已经鉴定了几种可能翻译的微蛋白，包括Cj 0878（48个密码子），我们已经使用GFP报告子验证了其翻译。Cj 0878在弯曲杆菌中高度保守，其氨基酸序列表明其是碱性的，两亲的，并且可能与细胞被膜结合。Cj 0878似乎也被鞭毛共调节的sRNA转录后调节，并且在铁限制下被转录诱导。亚细胞定位研究以及表型分析，三维肠组织模型中的感染研究，以及野生型和Cj 0878突变株的基因表达分析将用于研究其在C.空肠生理学为了鉴定Cj 0878的潜在生化和遗传相互作用伙伴，我们将分别采用免疫共沉淀和转座子测序。我们还旨在开发一种选择性捕获细菌核糖体的方案，用于感染样本中的Ribo-seq研究。总的来说，该项目将填补C基因组图谱的空白。空肠。此外，它可能揭示新的模式的微蛋白活性，这可能被用作新的目标，在不同的病原体的抗菌策略。

Defining the entire gene complement of a bacterial pathogen is essential to understand how it survives and causes disease. Deep sequencing technologies have revolutionized genome sequencing and revealed an unexpected complexity in bacterial genomes and transcriptomes. The recently established ribosome profiling technique (Ribo-seq) for global translatome analysis, based on deep sequencing of ribosome-protected fragments, is revealing a wealth of novel open reading frames (ORFs) encoding potential micro-proteins (< 50 amino acids) in diverse organisms. This class of cellular macromolecules is still understudied. The small number of characterized micro-proteins in bacteria is involved in diverse physiological processes, including modulation of virulence and antibiotic resistance. The food-borne pathogen Campylobacter jejuni is currently the leading cause of bacterial gastroenteritis worldwide. However, since its annotated genome lacks homologues of key virulence factors used by other enteric pathogens, little is known about how it causes disease. Our comparative RNA-seq-based transcriptome analysis of multiple C. jejuni strains revealed conserved and strain-specific transcriptional output, including many novel transcripts, and suggests much remains to be learned about how its expressed genome contributes to virulence. In this project we aim to define the C. jejuni micro-proteome and functionally characterize selected micro-proteins. Translatome analysis under different growth conditions using Ribo-seq combined with our transcriptome maps and comparative genomics will identify expressed C. jejuni micro-ORFs. Translation will be further validated using mass spectrometry and epitope tagging. Our first Ribo-seq of C. jejuni grown in broth culture has already identified several potentially translated micro-proteins, including Cj0878 (48 codons), for which we have validated translation using a GFP reporter. Cj0878 is highly conserved in Campylobacter and its amino acid sequence suggests it is basic, amphipathic, and might associate with the cell envelope. Cj0878 also appears to be regulated post-transcriptionally by a flagellar co-regulated sRNA, and is transcriptionally induced under iron limitation. Subcellular localization studies as well as phenotypic analyses, infection studies in 3D intestinal tissue models, and gene expression analyses of wild-type and Cj0878 mutant strains will be used to study its role and mechanisms in C. jejuni physiology. To identify potential biochemical and genetic interaction partners of Cj0878, we will employ co-immunoprecipitation and transposon-sequencing, respectively. We also aim to develop a protocol for selective capture of bacterial ribosomes for Ribo-seq studies in infection samples. Overall, this project will fill in gaps in the genome map of C. jejuni. Moreover, it might reveal novel paradigms of micro-protein activity, which could be used as novel targets for antimicrobial strategies in diverse pathogens.