Unexpected differential metabolic responses of Campylobacter jejuni to the abundant presence of glutamate and fucose

Abstract

Introduction: Campylobacter jejuni is the leading cause of foodborne bacterial enteritis in humans, and yet little is known in regard to how genetic diversity and metabolic capabilities among isolates affect their metabolic phenotype and pathogenicity.

Objectives: For instance, the C. jejuni 11168 strain can utilize both L-fucose and L-glutamate as a carbon source, which provides the strain with a competitive advantage in some environments and in this study we set out to assess the metabolic response of C. jejuni 11168 to the presence of L-fucose and L-glutamate in the growth medium.

Methods: To achieve this, untargeted hydrophilic liquid chromatography coupled to mass spectrometry was used to obtain metabolite profiles of supernatant extracts obtained at three different time points up to 24 h.

Results: This study identified both the depletion and the production and subsequent release of a multitude of expected and unexpected metabolites during the growth of C. jejuni 11168 under three different conditions. A large set of standards allowed identification of a number of metabolites. Further mass spectrometry fragmentation analysis allowed the additional annotation of substrate-specific metabolites. The results show that C. jejuni 11168 upon L-fucose addition indeed produces degradation products of the fucose pathway. Furthermore, methionine was faster depleted from the medium, consistent with previously-observed methionine auxotrophy.

Conclusions: Moreover, a multitude of not previously annotated metabolites in C. jejuni were found to be increased specifically upon L-fucose addition. These metabolites may well play a role in the pathogenicity of this C. jejuni strain.

Keywords: Campylobacter jejuni; HILIC chromatography; Mass spectrometry fragmentation; Metabolomics; Sulphur metabolism.

Fig. 1

Amino acid analysis using IDEOM identifications to determine Log² fold changes (mean of triplicate measurements) of amino acid peaks of 24 h compared with 0 h samples for medium only (Med), medium supplemented with fucose (Fuc), and medium supplemented with glutamic acid (Glu). It can be seen that several amino acids are preferably depleted from the medium. Further inspection revealed that aspartic acid, glutamine, methionine, proline, and serine are all completely depleted from the medium within 4 h. Glutamate was depleted under all conditions; in the glutamate-supplemented condition the Log² fold change is less pronounced due to the glutamate surplus abundance

Fig. 2

Upon growth in MEMα medium Campylobacter jejuni excreted a unexpectedly high number of metabolites into the medium. Plotted are the Log² fold changes of selected metabolites based on 24/4 h non-supplemented MEMα comparison—for all the three conditions Med; non-supplemented MEMα (left), Fuc; fucose-supplemented MEMα (middle), Glu; glutamate-supplemented MEMα (right). It can be observed that most of the metabolite changes occurring upon growth in MEMα medium are similar across the two treatments and control and form a ‘base metabolic response’; however there are some exceptions. Some metabolites were detected in both negative and positive mode; hence their double presence here

Fig. 3

Most-differential metabolomic changes upon glutamate supplementation of selected metabolites based on 24/4 h fucose-supplemented and 24 h fucose-supplemented/24 h medium only comparisons. It can be observed that a wide range of acylated amino acids is excreted into the medium. Some metabolites were detected in both negative and positive ionization mode; hence their double presence here

Fig. 4

Most-differential metabolomic changes upon fucose supplementation of selected metabolites based on 24/4 h fucose-supplemented and 24 h fucose-supplemented/24 h medium only comparisons with metabolites a released into and b depleted from the medium. c Breakdown products of fucose were also found to be excreted in the medium by Campylobacter, thereby supporting the proposal of Stahl et al. that Campylobacter jejuni possesses a Xanthomonas campestris-like fucose utilisation pathway (Stahl et al. 2011). d Molecular networking provided additional evidence for metabolites detected only upon fucose supplementation of the medium. A number of annotated metabolites were thiazole-containing (the heteroatomic ring depicted). Arrows point towards examples of detected structures

Fig. 5

Schematic overview of study: one bacterial strain under three conditions revealed pleiotropic metabolic effects upon growth whereas two different supplementations result in specific metabolic characteristics