Exploring small proteins in the foodborne pathogen Campylobacter jejuni

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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.