Transcriptome dynamics in chemostat culture reveal how Campylobacter jejuni transports glutamine as a nitrogen source for optimal growth and survival

Abstract S2

Presenter: Ashley Griffin (University of Sheffield)

Background: Campylobacter jejuni is a highly prevalent, invasive human pathogen capable of survival and transmission across a variety of hosts and environments. Despite this most C. jejuni strains are unable to metabolise exogenous sugars. Therefore, this asaccharolytic organism is dependent on amino acids and TCA cycle intermediates as its major carbon sources. Amino-acids can also act as nitrogen sources but ammonium cannot be used because the ammonium transporter AmtB is encoded by a pseudogene. Preliminary growth experiments in a minimal media formula confirmed this and revealed that only glutamine significantly stimulates growth on a variety of substrates suggesting its importance as an N-source for C. jejuni growth and survival. However, how glutamine is transported into the cell is unclear, with several candidate transporter genes potentially involved.

Aims and methods: To identify the genes responsible for glutamine transport, metabolism and regulation. The initial approach taken was to perform RNA-sequencing after addition of excess glutamine to steady-state glutamine-limited chemostat cultures and to identify the major up- and downregulated genes. Multiple mutants in candidate transporter genes were then constructed and analysed for their growth and glutamine uptake phenotypes.

Results: RNA-sequencing revealed a variety of possible glutamine transporter candidates including an annotated putative amino-acid transport protein with homology to a sodium:alanine symporter that was highly down-regulated by excess glutamine. Growth analysis of the corresponding mutant on glutamine and treatment with toxic glutamine analogue, -l-glutamylhydrazide (GGH), indicate this gene to be responsible for the majority of glutamine-stimulated growth in culture. Further analysis using radio labelled glutamine in an uptake experiment confirmed this gene to be the dominant glutamine transport protein. No other genes in question resulted in a reduction in glutamine uptake when knocked out. In vivo infection assays also indicate reduced fitness of the mutant.

Conclusion: We have identified a novel glutamine transport protein using RNA-sequencing on chemostat culture samples with and without the addition of glutamine. Despite reports in the literature of other transporters being responsible for glutamine uptake, this transport protein appears to be the sole glutamine transporter in the C. jejuni genome and is crucial for optimal growth and survival in culture. Further work is ongoing with in vivo phenotypes such as fitness for intracellular survival within HeLa cells.

Presenting in Speaking session 4 - Survival and application