IMIB - Institute for Molecular Infection Biology

Exploring small proteins in the foodborne pathogen Campylobacter jejuni

Defining the entire gene complement of a bacterial pathogen is a key step for understanding how it survives and causes disease. Deep sequencing technologies have revolutionized genome analysis and revealed an unexpected complexity in bacterial chromosomes. For example, ribosome profiling (Ribo-seq), which is based on deep sequencing of ribosome-protected fragments, allows for a global translatome analysis and has revealed a wealth of novel open reading frames (ORFs) encoding potential micro-proteins (<50 amino acids) in diverse organisms. Small proteins are encoded by a small ORF owing their own start and stop codons and are actively translated. Therefore, small proteins are clearly distinguished from peptides which arise from proteolytic cleavage of a much larger precursor protein such as leader peptides or from peptides which are synthesized by a ribosome-independent mechanism. The few examples of characterized bacterial small proteins are involved in diverse physiological processes, including modulation of virulence and antibiotic resistance. It is suggested, that most of them are membrane-associated and modulate enzymatic activity of larger complexes.

The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the leading cause of bacterial, food-borne gastroenteritis. However, since its annotated genome lacks homologs of key virulence factors used by other enteric pathogens, little is known about how it causes disease. A 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 my PhD project, I aim to define the C. jejuni small proteome under survival and infection-relevant conditions using Ribo-seq, and/or proteomics and to functionally characterize selected small proteins. A first Ribo-seq analysis of C. jejuni grown in rich broth in our lab already identified several potentially translated small ORFs, including one encoding a 48 aa-long protein, for which we have validated translation using a GFP reporter. This small protein is highly conserved in Campylobacter species and its amino acid sequence suggests it is basic, amphipathic, and might associate with the cell envelope. Moreover, it appears to be regulated post-transcriptionally by a flagellar co-regulated sRNA, and is transcriptionally induced under iron limitation. During a Tn-seq screen of C. jejuni infections of human Caco-2 cells, we identified a second translated small protein candidate, which is novel factor required for C. jejuni motility. Upon validation of selected small proteins, I aim to investigate their subcellular localization and to identify their biochemical interaction partners using co-immunoprecipitation in order to get insights into their roles and mechanisms in C. jejuni physiology. Furthermore, Gradient-Sequencing (Grad-Seq), a global approach to characterize the protein and RNA interactome, will be used to identify, validate and characterize small proteins in C. jejuni. Phenotypic analyses of mutant strains under different growth conditions, as well as during infection of host cells, will also reveal roles for selected small proteins in virulence-associated phenotypes. Overall, this project will fill in gaps in the genome map of C. jejuni and may reveal novel insights into the functions of small proteins in other bacterial pathogens.