Exploring small proteins in the foodborne pathogen Campylobacter jejuni

确定细菌病原体的整个基因组合对于了解它是如何生存和致病至关重要的。深度测序技术已经彻底改变了基因组测序，并揭示了细菌基因组和转录体中意想不到的复杂性。最近建立的用于全球翻译组分析的核糖体图谱技术(Ribo-seq)，基于核糖体保护片段的深度测序，揭示了不同生物体中编码潜在微蛋白(&lt；50个氨基酸)的丰富的新的开放阅读框架(ORF)。这类细胞大分子仍未得到充分研究。细菌中的少量特征微蛋白参与多种生理过程，包括毒力和抗生素耐药性的调节。食源性空肠弯曲杆菌目前是全球细菌性胃肠炎的主要原因。然而，由于它的注释基因组缺乏其他肠道病原体使用的关键毒力因子的同源基因，人们对它是如何致病的知之甚少。我们对多个空肠弯曲菌菌株进行的基于RNA-seq的比较转录组分析揭示了保守的和菌株特异性的转录输出，包括许多新的转录本，这表明关于其表达的基因组如何对毒力起作用仍有许多需要了解的地方。在这个项目中，我们的目标是定义空肠弯曲菌微型蛋白质组，并对选定的微型蛋白质进行功能鉴定。利用RIBO-SEQ分析不同生长条件下的翻译组，结合我们的转录组图谱和比较基因组学，可以鉴定出表达的空肠弯曲菌微ORF。翻译将使用质谱学和表位标记进一步验证。我们的第一个在肉汤培养中生长的空肠弯曲菌的Ribo-Seq已经确定了几个潜在的翻译微蛋白，包括Cj0878(48个密码子)，我们已经使用GFP记者验证了它的翻译。Cj0878在弯曲杆菌中高度保守，其氨基酸序列表明它是碱性的，具有两亲性，可能与细胞膜有关。Cj0878似乎也受鞭毛共调控的sRNA转录后调控，并在铁限制下转录诱导。亚细胞定位研究以及表型分析、3D肠道组织模型中的感染研究以及野生型和Cj0878突变株的基因表达分析将用于研究其在空肠弯曲菌生理中的作用和机制。为了确定Cj0878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，用于在感染样本中进行核糖核酸序列研究。总体而言，该项目将填补空肠弯曲菌基因组图谱的空白。此外，它可能揭示微蛋白活性的新范例，可以作为不同病原体抗菌策略的新靶点。

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.