Abstract
Campylobacter jejuni is a major gastrointestinal pathogen generally acquired via consumption of poorly prepared poultry. N-linked protein glycosylation encoded by the pgl gene cluster targets >80 membrane proteins and is required for both nonsymptomatic chicken colonization and full human virulence. Despite this, the biological functions of N-glycosylation remain unknown. We examined the effects of pgl gene deletion on the C. jejuni proteome using label-based liquid chromatography/tandem mass spectrometry (LC-MS/MS) and validation using data independent acquisition (DIA-SWATH-MS). We quantified 1359 proteins corresponding to ∼84% of the C. jejuni NCTC 11168 genome, and 1080 of these were validated by DIA-SWATH-MS. Deletion of the pglB oligosaccharyltransferase (ΔpglB) resulted in a significant change in abundance of 185 proteins, 137 of which were restored to their wild-type levels by reintroduction of pglB (Δaaz.batpglB::ΔpglB). Deletion of pglB was associated with significantly reduced abundances of pgl targets and increased stress-related proteins, including ClpB, GroEL, GroES, GrpE and DnaK. pglB mutants demonstrated reduced survival following temperature (4 °C and 46 °C) and osmotic (150 mm NaCl) shock and altered biofilm phenotypes compared with wild-type C. jejuni Targeted metabolomics established that pgl negative C. jejuni switched from aspartate (Asp) to proline (Pro) uptake and accumulated intracellular succinate related to proteome changes including elevated PutP/PutA (proline transport and utilization), and reduced DctA/DcuB (aspartate import and succinate export, respectively). ΔpglB chemotaxis to some substrates (Asp, glutamate, succinate and α-ketoglutarate) was reduced and associated with altered abundance of transducer-like (Tlp) proteins. Glycosylation negative C. jejuni were depleted of all respiration-associated proteins that allow the use of alternative electron acceptors under low oxygen. We demonstrate for the first time that N-glycosylation is required for a specific enzyme activity (Nap nitrate reductase) that is associated with reduced abundance of the NapAB glycoproteins. These data indicate a multifactorial role for N-glycosylation in C. jejuni physiology.
Highlights
Campylobacter jejuni is a major gastrointestinal pathogen generally acquired via consumption of poorly prepared poultry
Deletion of pglB resulted in loss of the JlpA glycoform, which was restored by complementation (Fig. 1A)
Quantitative PCR further confirmed that expression of pglB was lost in ⌬pglB and restored levels were not significantly different to those observed for WT (Fig. 1B)
Summary
N-linked protein glycosylation (Pgl) in Campylobacter jejuni is required for chicken colonization and human virulence, yet its biological role remains unknown. pgl gene deletion resulted in a significant rearrangement of the C. jejuni proteome that leads to alterations in crucial phenotypes including stress response, nutrient uptake, electron transport and chemotaxis, and is essential for full activity of the Nap nitrate reductase. N-linked protein glycosylation (Pgl) in Campylobacter jejuni is required for chicken colonization and human virulence, yet its biological role remains unknown. C. jejuni can respire at very low oxygen availabilities by using alternative electron acceptors such as nitrate, nitrite, fumarate, trimethylamine-N-oxide (TMAO) or dimethyl sulfoxide (DMSO) [20], for which several reductases have been identified (Nap, nitrate; Nrf, nitrite; Frd, fumarate; and Cj0264c/Cj0265c, TMAO/DMSO) Both electron donors, including NADH and formate dehydrogenases [21], and electron acceptors are required for optimum host colonization [22]. The N-linked glycosylation system is encoded by the 10 gene pgl cluster, which is responsible for the biosynthesis and attachment of a heptasaccharide glycan (GalNAc-␣1,4-GalNAc␣1,4-[Glc1,3]-GalNAc-␣1,4-GalNAc-␣1,4-GalNAc-␣1,3-Bac1; Bac is bacillosamine [2,4-diacetamido-2,4,6 trideoxyglucopyranose]) to proteins at the consensus sequon Asp/ Glu-X-Asn-X-Ser/Thr (X Pro), where Asn is the attachment site [25]. Our group and others have shown that the Pgl system can target more than 130 sequons from ϳ80 membrane-associated proteins [25, 29, 33–35], including periplasmic proteins, lipoproteins, inner membrane proteins and at least one pro-
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