Abstract
It is now apparent that nearly all bacteria species have at least a single glycosylation system, but the direct effect(s) of these protein post translational modifications are unresolved. In this study, we used the general N-linked glycosylation pathway from Campylobacter jejuni to investigate the biophysical roles of protein modification on the CmeABC multidrug efflux pump complex. The study reveals the multifunctional role of N-linked glycans in enhancing protein thermostability, stabilising protein complexes and the promotion of protein-protein interaction. Our findings demonstrate, for the first time, that regardless of glycan diversification among domains of life, N-linked glycans confer a common evolutionary intrinsic role.
Introduction
Glycosylation, the process of covalently attaching a glycan (mono-, oligo-, or poly-saccharide) to an amino acid side chain, is the most prevalent post-translational modification found on proteins, occurring in all domains of life. The attachment of a carbohydrate moiety to certain amino acid side chains in proteins was traditionally thought to be exclusive to eukaryotes and archaea. In the last decade, protein glycosylation, both N- and O-linked, has been increasingly reported in pathogenic bacteria such as well as commensal bacteria1. Functional analysis of the role of N-glycans in eukaryotes shows multiple roles. Intrinsically, glycans confer protein stabilisation through a plethora of mechanisms such as, promoting secondary structure formation, accelerating protein folding, slowing the unfolding rate2, enhancing protein thermostability3 and reducing aggregation4. Extrinsically, N-linked glycosylation modulates protein-protein interactions and protein targeting5. Notably, prokaryotes and eukaryotes share similar biosynthetic pathways of general N-linked glycosylation, indicating a common evolutionary pathway6. However, the role of N-linked glycans remains poorly studied in prokaryotes.
The first characterisation of an N-linked glycosylation in a bacterium was in the enteric pathogen Campylobacter jejuni which now has one of the most studied of all prokaryotic glycosylation pathways7. Genomic and proteomic studies have demonstrated that, PglB is an oligosaccharyltransferase responsible for the covalent attachment of N-linked glycan (GalNAc -α1,4-GalNAc-α1,4-GalNAc-[Glcβ1,3-]GalNAc-α1,4-GalNAc-α1,4-GalNAc- α1,3-Bac-β1; where GalNAc is N- acetylgalactosamine; Glc is glucose; diBacNAc is 2,4- diacetamido −2,4,6- trideoxyglucopyronose) to the asparagine residue in the acceptor sequon D/E-X1-N-X2- S/T where X1 and X2 are any amino acid except proline7. In depth studies on N-linked glycan diversity have revealed that there are 16 different glycans present in Campylobacter genus. Surprisingly, the first two glycans (diBacNAc and HexNAc) were conserved in all species8, 9. Whilst the role of N-linked glycan remains to be elucidated, disruption of the N-linked glycosylation pathway resulted in pleotropic effects such as decreased chicken colonisation10, reduced adherence to intestinal cells11 as well as impaired bacterial competence12. These studies did not ascertain the direct role of N- linked glycans in the C. jejuni glycoproteome. To address this, we sought to investigate the biophysical role of N-linked glycosylation on a representative post translationally modified glycoprotein. We demonstrate a critical role for glycosylation by focusing on a resistance-nodulation-division type (RND) multidrug efflux pump denoted Campylobacter multidrug efflux; CmeABC.
CmeABC is a tripartite molecular assembly of glycoproteins, CmeB; an inner membrane multidrug transport protein, CmeA; a periplasmic fusion protein and CmeC; an outer membrane associated channel. The complex plays a key role in chicken colonization13, as well as being responsible for multidrug resistance (MDR)14. Previously, we demonstrated that disrupting pglB in C. jejuni impaired the efflux activity of CmeABC resulting in significantly higher ethidium bromide accumulation when compared to the wildtype (Abouelhadid S, et al submitted manuscript). The absence of glycosylation on the CmeABC locus within C. jejuni was also shown to reduce resistance to four different antibiotic classes. Here in, we show that the loss of N-linked glycans in CmeABC is the sole reason for this phenotype and not a pleiotropic effect caused by pglB disruption. We also unravel the intrinsic role of N-linked glycans in a) modulating global protein structure b) enhancing glycosylated CmeA; g2CmeA thermostability and c) significantly slowing the unfolding rate of g2CmeA. Finally, we evaluate the extrinsic role of N-linked glycans in the molecular assembly of CmeABC to discern the difference in binding kinetics of CmeA variants to CmeC. The study highlights the multifunctional role of N-linked glycans in enhancing protein thermostability, stabilising protein complexes and the promotion of protein-protein interaction. Here we present a model N-linked glycosylation system with a tractable phenotype to be used in studying glycan evolution, function and diversity. Our findings demonstrate, for the first time, that regardless of glycan diversification among domains of life, N-linked glycans seem to confer a common evolutionary intrinsic role.
Results
N-linked glycans do not affect CmeABC protein expression nor protect CmeC from native proteolytic degradation
Scrutiny of the C. jejuni NCTC11168 genome revealed the presence of 13 multidrug efflux transporters, which appear conserved in the species15. Genetic and biochemical testing demonstrated that cmeABC is located in an operon and encodes the predominant multidrug efflux pump in C. jejuni. In addition to its central role in extruding structurally non-related compounds such as antimicrobials, bile salts, dyes and heavy metals14, CmeABC has been reported to function interactively with CmeDEF16; a secondary multidrug efflux pump in C. jejuni. Glycoproteomic analysis of C. jejuni demonstrated that the CmeABC complex is multi N-linked glycosylated where CmeA is glycosylated at positions 121DFNR124 and 271DNNNS275, CmeB is glycosylated at position 634DRNVS648 and CmeC is glycosylated at position 47ETNSS51. Notably, CmeE has also been shown to be N-glycosylated17. Previously, we showed that the multidrug efflux pump is impaired in the glycosylation deficient C. jejuni. This resulted in a significant increase in ethidium bromide accumulation more than the parent strain and a reduction in antibiotics resistance (Abouelhadid S, et al submitted manuscript). We hypothesise that this deficiency may be due to assembly destabilisation as a consequence of glycosylation aberration. To address this question, we sought to study the major multidrug efflux pump of C. jejuni, in a glycosylation deficient CmeABC complex. We conducted the experiments in a C. jejuni cmeD::catr background strain in order to avoid the interaction of CmeDEF with CmeABC that might mask the functional role of N-linked glycans. This parent strain was used to construct a C. jejuni WTCmeABC strain and a C. jejuni glycosylation altered strain; g0CmeABC whereby, N->Q in each glycosylation sequon (D/E-X1-N-X2- S/T where X1 and X2 are any amino acids other than proline). Since cmeABC is an operon, we added a 6x His tag at the C-terminal of CmeC to monitor changes in CmeABC expression. We then grew both WTCmeABC and g0CmeABC strains to an OD600 =0.4-0.5, tetracycline was then added to inhibit protein synthesis. CmeC levels in both strains were monitored for 2 hours. Our data show no difference in CmeC expression in WTCmeABC and g0CmeABC strains Fig 1, A. Once inserted in the outer membrane CmeC might be unreachable region to proteases. Therefore, we conclude that N-linked glycans, in this case, do not protect CmeC from native proteolytic degradation. We observed two bands in the WTCmeC (lanes 2, 4, 6, 8) that migrated slower than the band corresponding to non-glycosylated CmeC in g0CmeC (lanes 1, 3, 5, 7). Western blot analysis confirmed that the two bands observed in WTCmeC lanes correspond to two glycospecies for the protein. Fig 1, B. Our bioinformatic analysis of the primary amino acid sequence shows that CmeC has two glycosylation sequons; 30EANYS34 and 47ETNSS51 in C. jejuni NCTC11168. Structurally both of the glycosylation sequons are located in flexible loops18 Fig 6, B.
N-linked glycans affect multidrug efflux pump efficiency
To examine the role of N-linked glycans in CmeABC molecular assembly, we assessed the efficiency of the multidrug efflux pump using an ethidium bromide accumulation assay. Ethidium bromide accumulation was 22% higher in g0CmeABC when compared to WTCmeABC. This difference was consistent at 5, 10 and 15 minutes, indicating an impairment in the extrusion of ethidium bromide from g0CmeABC Fig 1, C. To confirm this finding, E-test antibiotic strips were used to calculate the minimum inhibitory concentration (MIC) of four non-structurally related antibiotics that have different mechanisms of actions. In comparison to WTCmeABC, an increase in antibiotic susceptibility was noticed in g0CmeABC confirming the previous finding Table 1. The results indicate that N-linked glycans play a role in enabling the multidrug efflux pump to work efficiently in the C. jejuni cell.
Generation of fully glycosylated CmeA in glycocompetent E. coli
Previous studies showed that UDP-N-acetylglucosamine—undecaprenyl-phosphate N- acetylglucosaminephosphotransferase; WecA, could interfere with the biosynthesis of heterologous expression of polysaccharides built on the undecaprenyl-phosphate lipid anchor-rendering it built on incorrect glycan at the reducing end. To circumvent this problem and ascertain that g2CmeA is glycosylated with the native C. jejuni N-linked glycan we used glycocompetent E. coli SDB119. The heterologous expression of an acceptor protein with protein glycosylation locus (pgl) usually yielded a mix population of glycosylated and non-glycosylated protein variant, indicating a suboptimal glycosylation process20, 21. We observed that pglB expression from pACYC(pgl) is insufficient to achieve optimal glycosylation (data not shown). To overcome this bottleneck, we sought boosting PglB expression by introducing pGXVN114 to E. coli SDB1 expressing CmeA and N-linked glycan biosynthetic pathway. Fig 2 Lane 1 shows the optimal glycosylation of constitutively expressed CmeA and N-linked glycosylation pathway along with IPTG inducible PglB from pGXVN114 backbone.
Glycosylation modulates protein global structure
In eukaryotes the enzymatic attachment of N-linked glycans to amide group of asparagine in the glycosylation consensus sequon, is correlated with changes in the biophysical properties of the protein such as thermostability3, aggregation, function4 and structure22. Glycans can confer interactions not only at the glycosylation site but also other regions in the protein. Serving as bulky hydrophilic groups, glycans can also favour certain conformational modifications that stabilise protein structure22. Bioinformatic studies suggested that glycans force the polypeptide chain to adopt a more extended conformation through restricting residual structures formation in the unfolded state23, 24. To investigate the role that N-linked glycans play in modulating the biophysical properties of CmeABC, we used circular dichroism (CD) spectroscopy. This allowed us to monitor the secondary structure as well as the conformational changes upon thermal denaturation of CmeA variants. Far-UV spectra for both g0CmeA and g2CmeA in 10 mM sodium phosphate, 75 mM sodium chloride, 10% glycerol buffer (pH 8) were collected at 20 °C. The spectrum exhibited helical structure signature with two negative minima at 208 and 222 nm. It also showed a positive maximum at 196 nm suggesting the presence of beta-sheets structure Fig 3 Superimposed CD spectra exhibit g0CmeA spectrum, shown in black, was slightly red shifted towards the β sheet. The CD spectra of both proteins were then analysed by BESTSEL25 for secondary structure content. Table 2 shows the secondary structure content of g0CmeA and g2CmeA at room temperature. Our results show that N-linked glycans confer subtle changes in protein global conformation.
Glycans enhances protein thermostability
It has been established that N-linked glycans play a role in stabilising glycoproteins thermodynamically in eukarotes3, 22. The intrinsic role of N-linked glycans in stabilising CmeA was investigated through analysing the CD spectra recorded for g0CmeA and g2CmeA at elevated temperatures. The multi–wavelength melting profiles monitored at 260- 195 nm were recorded during the heating of g0CmeA and g2CmeA from 6°C to 94°C at 1°C per minute rate with a 2°C step size. Isodichroic points were observed in the far-UV CD spectra at Fig 4, A and B supports more than two-state nature of the unfolding transition. Derivative of CD spectra were used to calculate the melting temperature (Tm) of both g0CmeA and g2CmeA Suppl 1. The loss of CD spectra was observed upon incremental rises in temperature, melting curves measured for g0CmeA and g2CmeA show that both proteins have three transitional phases at Tm1 46.1°C ± 0.2, Tm2 53.5°C ± 0.4 and Tm3 56.7°C ± 0.6 for g0CmeA and Tm1 43.8°C ± 0.3, Tm2 49.3°C ± 0.2 and Tm3 62.8°C ± 0.2 for g2CmeA. No light scattering was observed in the UV spectra indicating turbidity presented at high temperature. This shift in final melting temperature suggests that glycans stabilise g2CmeA at elevated temperature Fig 4, C.
To confirm the previous findings, we examined conformational folding reversibility and unfolding rate for both g0CmeA and g2CmeA. The assay is based on three successive cycles whereby CmeA variants were cooled at 20°C, heated up to the corresponding Tm for 5 minutes and then cooled again at 20°C. To assess conformational folding reversibility, CD spectra that were recorded at 20°C, before and after increasing the temperature to the corresponding Tm were compared. CD spectra of CmeA variants were superimposable before and after the first two cycles of heating (Tm1 and Tm2) but not after heating at Tm3, indicating conformational changes Fig 4, D and E. Unfolding rate was evaluated according to changes in CD spectra with respect to time at Tm3. A significant reduction in CD spectra was observed when g0CmeA and g2CmeA were heated at their corresponding Tm3. The unfolding of CmeA was achieved in 5 minutes at its Tm3. Notably, the CD spectra recorded for g2CmeA at its corresponding Tm3 kept changing for 30 minutes indicating a slower unfolding rate. This result along with the above data highly suggest that N-linked glycans play a pivotal intrinsic role in protein thermodynamic stabilisation.
Glycans modulate molecular assembly and protein-protein interaction
Unlike eukaryotes, there is no evidence that N-linked glycans modulate protein-protein interaction or complex assembly in prokaryotes. To explore the potential role of C. jejuni general N-linked glycans in modulating the interaction of glycoproteins with their cognate partners, surface plasmon resonance (SPR) was used. SPR was previously used to investigate the interactions in orthologous membrane fusion proteins (MFP) with TolC from of E. coli26. Quantitative analysis of the binding curves showed multiple reaction events. The model used to determine binding kinetics indicated the presence of two populations of MFP proteins. The two populations exhibited different binding kinetics, notably, fast and slow dissociation rates that contributed to weak and strong interactions, respectively26. We employed a CM5 chip with g0CmeA and g2CmeA immobilised through amine coupling, CmeC was then injected over CmeA variants surfaces in different concentrations. In our model, g0CmeA and g2CmeA exhibited multiple events interaction with CmeC. These interaction events can be attributed to a fast and a slow association and dissociation rates. Quantitative analysis of the sensogram yielded excellent results for slow interactions however, fast interactions could not be fitted in a model to generate accurate binding kinetics. At pH 7.4, both CmeA variants exhibited similar dissociation rate constants (koff) of 9e−4 s−1 for g0CmeA and 7.5e−4 s−1 for g2CmeA Fig 5, A and B. Interestingly, difference in association rate constant (kon) was observed, g0CmeA kon = 5e4 (M−1s−1) whilst g2CmeA kon =1.5e5 (M−1s−1). This difference in the kon rate indicates that g2CmeA possess more binding pockets that allows slow yet high affinity interactions with CmeC compared to g0CmeA. The equilibrium dissociation rate constants (KD) derived from the binding kinetics analysis were 1.7e−8 (M) and 5e− 9 (M) and g0CmeA and g2CmeA, respectively.
To investigate the effect of pH on modulating binding kinetics, we observed CmeA- CmeC interactions at pH=6.0 Fig 5, C and D. At this pH CmeA-CmeC interactions were more avid and with a greater number of sites bound. Similar to binding curves observed at pH 7.4, g2CmeA showed a favourable slow association and dissociation binding curves than g0CmeA. The number of sites for slow interaction were greater for g2CmeA contributing to a modestly higher affinity for interaction with CmeC compared to a weaker affinity for CmeC exhibited by g0CmeA. To confirm that variations in binding kinetics were not due to differences in structural orientation between g0CmeA and g2CmeA, both proteins were immobilised on NTA chip using C-terminal 6xHis tag, CmeC was then passed in different concentrations. Binding kinetics indicated similar kon and koff for both CmeA variants, although fewer sites were available Suppl 2. Interestingly, g2CmeA bound more CmeC than g0CmeA, confirming the data seen with amine coupling. These results show a complex binding pattern between CmeA variants and CmeC. They also suggest an extrinsic role for N- linked glycans, exhibited in the variation in binding kinetics between g0CmeA and g2CmeA, where the glycosylated form of CmeA showed a greater proportion of higher affinity interaction sites than its non-glycosylated counterpart.
Discussion
Whilst the role of glycosylation in eukaryotes has been thoroughly explored, a similar depth of investigation is lacking in prokaryotes. The presence of different glycosylation systems in prokaryotes has been regularly reported2, 7, 11–13. Some have described their effect in virulence, adhesion and motility by creating genetic knock outs of the glycosylation machinery10–12, 32, 33. Nonetheless, these reports have not provided in-depth studies into the direct role that N-linked glycans exert on protein function. Glycoproteomic studies revealed diBacNAc to be conserved across Campylobacter species. Notably, diBacNAc was also found to be at the reducing end of O-linked glycans in Neisseria gonorrhoeae, indicating a parallel evolution between N-linked and O-linked glycosylation systems in bacteria8, 9, 34. The role of N- linked glycans in stabilising major multidrug efflux pump in C. jejuni has been shown to contribute in efficiently extruding antimicrobials and ethidium bromide. Disrupting glycosylation in CmeABC resulted in higher accumulation of ethidium bromide and lowering antibiotic MIC in C. jejuni. These differences in activity are not due to the low abundance of CmeABC complex in g0CmeABC strain Fig 1, A. A protein synthesis arrest assay showed that loss of glycosylation did not promote CmeC proteolytic degradation. This is in agreement with the previous finding that CmeC protein abundance were equal in C. jejuni and C. jejuni pglB::aphA (Abouelhadid S, et al paper submitted). This result suggests that N-linked glycans might be modulating molecular assembly. Studies on truncated N-linked glycans will reveal the role of each glycostructure, it will also help to understand the role of the conserved first two glycans between different Campylobacter strains.
Bioinformatic studies investigating protein structural changes exerted by glycans has been inconclusive. These studies rely heavily on the in-silico analysis of protein structure entries in the protein database bank. Whilst modern advances in crystallographic techniques pave the way for more structural studies, obtaining glycoprotein structure is still challenging and remains poorly represented in the protein database bank. Xin et al reported that protein glycosylation causes significant yet unexpectedly subtle changes in both local and global protein structure (up to 7%) 23. However, Hui Sun lee et al concluded that N-glycosylation causes non-significant changes in protein structure but increases protein stability likely due it a role played in reduction of protein dynamics24. Experimentally, our initial CD study of CmeA variants showed that both have the same conformational fold, however they confer subtle structural differences to the protein Fig 3, B. A small shift has been observed in the percentage of alpha helices and beta sheets between g0CmeA and g2CmeA, 1.3 ±0.2. It is still unclear whether the structural variations are due to local stabilisation resulting from the glycosidic bond between the asparagine side chain in the glycosylation site and N-linked glycans, or global structural rearrangement due to the interaction of the glycan with other distant regions in the protein backbone. A structural elucidation of CmeA in its glycosylated and non-glycosylated might provide insights on the extent of the importance of conformational changes.
It has been suggested that N-linked glycans might enhance protein thermostability. The glycoprotein PEB3 from C. jejuni was used to test the stabilisation effect of N-linked glycans. Average melting temperature of PEB3 (K135E) variants were analysed using SYPRO orange thermoflour. Interestingly, the Tm of glycosylated PEB3 was shown to be 4.7°C higher than its non-glycosylated counterpart; PEB335. This comes with an agreement with CD thermal melts of g0CmeA and g2CmeA. CD thermal melts showed that whilst both of the CmeA variants have the same apparent unfolding behaviour, Tm of g2CmeA was 6.4°C ±0.5 higher than that of g0CmeA. The three transitional phases of both variants showed that g2CmeA seems to be responding to a rise in temperature via conformational rearrangements at 2.4°C ±0.1 lower than g0CmeA Fig 4, A. CD spectra recorded after cooling showed that the structural rearrangements were reversible and the protein could fold again, suggesting that protein fold/unfolding Tm1 and Tm2 are reversible for both of the CmeA variants. Remarkably, the unfolding behaviour of g2CmeA at Tm3 was different to g0CmeA at its correspondent Tm3 in the conformational reversibility assay. Time taken to unfold g2CmeA was at least 5 times more than g0CmeA, thus indicating a role played by N-linked glycans in conferring greater resistance to unfolding Fig 4, C and D. We postulate that N-linked glycans stabilise g2CmeA through a reduction in the unfolding rate in g2CmeA, this finding agrees with the observation that eukaryotic N- linked glycans stabilise the hCD2ad through slowing the unfolding rate of the protein 50- fold when compared to its non-glycosylated counterpart2.
Owing to the lack of subcellular compartments, the extrinsic role of prokaryotic N- linked glycans in protein-protein interaction has been not fully appreciated. Despite the scarcity of glycoproteomic data, few molecular assemblies have been reported to have at least one of its component to be glycosylated 17, 31. We demonstrate a potential extrinsic role of N- linked glycans in CmeA interaction with CmeC. In an orthologous multidrug efflux pump; AcrAB-TolC, AcrA showed the presence of two populations, of the same protein, interacting with different kinetics to TolC. The two populations contributed to a fast weak interaction and slow strong interaction Fig 5, A and B. The complexity of these interactions is exaggerated in C. jejuni due to the presence of N-linked glycans, that could modulate interaction of CmeA with CmeC. Quantitative analysis for the interaction kinetics of CmeA variants with CmeC showed that, N-linked glycans increase the binding affinity to CmeC by 3.4-fold. That was clearly demonstrated in the difference in KD between CmeA variants at pH= 7.4. The difference in binding affinity was confirmed when CmeA variants were immobilised with the same orientation on Ni chip. Recently, a pseudoatomic structure provided a detailed picture of interaction between AcrA and TolC. This elaborated the adaptor bridging-binding model that involved an intermesh cogwheel-like binding between AcrA and TolC36. The conserved binding motif Val-Gly-Leu/Thr (VGL) is located at the tip region of the coiled coil α-hairpin of the protein, serving as a site of interaction with the RXXXLXXXXXXS (RLS) motif of AcrA36. In light of this study, our computational analysis showed that CmeC from Campylobacter spp does contain a truncated VGL motif, denoted VGA, whilst we found the RLS motif to be conserved in among C. jejuni, C. lari, C. coli and C. fetus Fig 6, A. To understand whether N-linked glycans modulate protein-protein interaction we analysed the proximity of glycosylation sites to VGA and RLS binding site in both CmeC and CmeA, respectively. The glycosylation sites in CmeC were shown to be distant from the proposed binding site Fig 6, C and probably closer to the transmembrane domain of the protein. Remarkably, we found that one of the glycan modified asparagine (123N) is at the X−1 position to RLS motif and is conserved in C. jejuni and C. coli, but not in C. lari and C. fetus Fig 6, A and C. This strongly suggests that the localisation of N-linked glycan adjacent to RLS might be affecting either the local site conformation and/or promote a stronger interaction with the VGA motif in CmeC; resulting in the interaction kinetics differences between g2CmeA and g0CmeA with CmeC observed by SPR in this study. In eukaryotes, it is established that N-linked glycans at different glycosylation sites in the same protein could play different roles. The roles of these N-linked glycans can be categorised into; (a) promoting protein folding, (b) modulating protein trafficking and localisation and (c) effecting protein functionality.
This study provides the first detailed analysis of the role of bacterial N-linked glycan. The role of bacterial general N-linked glycans has been difficult to elucidate. This led to a notion that bacterial general N-linked glycans do not play any role in protein folding or function39. This notion was based on previous inconclusive results on the role of bacterial general N-linked glycans in modulating proteins function37–40. Our work refutes this widely held notion and demonstrates that bacterial N-linked glycans do not only play a role in slowing protein unfolding process and enhancing its thermostability but also it modulates protein interaction with its cognate partner. It also demonstrates a conserved role of general N- linked glycans previously seen in eukaryotes. This also suggests a common evolutionary role that led to the emergence of N-linked protein post translation modification, in expanding the functionality of proteome repertoire across all domains of life. Our proposed model can be used to interrogate prokaryotic general. glycosylation systems Our proposed model can be used to interrogate prokaryotic general glycosylation systems and help in the development of novel antimicrobials.
Materials and methods
Bacterial strains and growth conditions
Campylobacter jejuni 1116810 and its derivatives; C. jejuni cmeD::cat, C. jejuni cmeD::cat wtcmeABC and C. jejuni cmeD::cat g0cmeABC were used in this study. C. jejuni 11168H was grown on Columbia based agar or Muller Hinton based agar supplemented with 5% horse blood according to manufacturer’s instructions. Strains were grown at 37°C in a variable atmospheric incubator (VAIN) cabinet (Don Whitely, UK) maintaining microaerophilic conditions of: 85% Nitrogen, 5% Oxygen and 10% carbon dioxide. All of the cloning experiments were done in Escherichia coli DH10Beta (New England Biolabs, USA). E. coli DH10B was used in expression of CmeA and cloning and expression of CmeC whilst gCmeA was expressed in E. coli SBD1. E. coli strains were grown on either Luria-Bertani Broth or Luria-Bertani Agar and antibiotics were added when necessary.
Inactivation of cmeD and generation of C. jejuni cmeD::cat, C. jejuni cmeD::cat wtcmeABC and C. jejuni cmeD::cat g0cmeABC
The nucleotide sequence of cmeD gene was commercially synthesized (Clonetech, USA) to also carry a chloramphenicol resistant gene; cat was inserted in the middle of cmeD to disrupt the gene. The DNA was then released by restriction digestion with EcoRV and cloned in pJET1.2 -following manufacturer’s instructions- to give pATN. Cloning of cmeABC-aphA was achieved by the following; cmeABC locus was amplified by primer FWDCmeA and primerREVCmeC with Phusion polymerase (New England biolabs, UK) using C. jejuni 11168H genomic DNA as a template, 6Xhis tag was added at the C-terminus of the CmeC to track its expression. The PCR amplicon was cloned in pJET1.2 following the manufacture’s instructions to give pMH3 that was then cut by BamHI to introduce the kanamycin resistant gene aphA, to be used as an antibiotic selection marker after homologous recombination in C. jejuni 11168H to give pMHT. To add homologous recombination arms for cmeABC- aphA, pMH3 was cut by SaCII to ligate cj0364 at the 3’ end of aphA to give pMHTF. For g0cmeABC-aphA, each asparagine in the non-canonical glycosylation sequon (D/E-X1-N-X2- S/T where X1 and X2 are any amino acid except proline) was altered to glutamine in-silico and nucleotide sequence of g0cmeABC was synthesized by (Clonetech, USA) DNA was then treated as above to generate pATKH.
To generate C. jejuni cmeD::cat, electroporation of pATN into C. jejuni 11168H was carried out as previously described10. The transformants were selected on CBA plates supplemented with 10 µg/ml chloramphenicol and the double cross over event was confirmed by PCR, this strain was then used as parent strain to generate other mutants. Plasmids pMHT and pATK were electroporated into C. jejuni cmeD::cat to generate C. jejuni cmeD::cat cmeC::cmeC-aphA and C. jejuni cmeD::cat cmeABC::cmeABC-(N->Q)-aphA, respectively. Transformants were selected on CBA plates supplemented with 10 µg/ml chloramphenicol and 30 µg/ml kanamycin and the double cross over event was confirmed by PCR.
Antibiotic sensitivity test (E-test)
C. jejuni 11168H were grown in suspension in Mueller-Hinton broth equivalent to 1.0 MacFarland’s standard and 100 µl aliquots were spread plated on dry Mueller-Hinton agar plates supplemented with 5 % Sheep blood (Oxoid, UK), the plates were left for 5-10 minutes to dry before the antibiotic strip (Oxoid, UK) was added. Plates were incubated at 37°C overnight. The minimum inhibitory concentration (MIC) was read directly from the strip at the oint where the zone of inhibition of bacterial growth intersected with the antibiotic concentration on the strip.
Ethidium bromide accumulation assay
Bacterial cells were grown to mid log phase (OD 600 0.4-0.5). Cells were harvested, washed and resuspended in 0.1M sodium phosphate buffer pH 7 (previously incubated in the VAIN) to OD 600 0.2. Cells were then incubated in the VAIN for 15 mins at 37°C o before a 100µl aliquot was withdrawn to indicate time zero. Ethidium bromide (Sigma, UK) was added to final concentration 2 µg/ml and fluorescence was measured at 530 nm excitation and 600 nm emission using a plate reader (Molecular Devices M3 plate reader, USA).
Expression of CmeA and gCmeA
Protein expression was carried out in E. coli strains unless stated otherwise. CmeA and CmeC were expressed in E. coli DH10B carrying pMH5 plasmid and pAT3, respectively, whilst gCmeA was expressed in E. coli SDB1 carrying pGVXN114, pWA2 and pACYC(pgl). Initiating cultures were grown overnight in LB broth supplemented with appropriate antibiotics at 37 °C under shaking condition. The following day, 10 ml of culture was withdrawn from the shake flask to inoculate 400 ml LB broth supplemented with appropriate antibiotics. To achieve optimal glycosylation of CmeA, PglB was expressed from pGVXN114 by the addition of 0.5 mM ITPG at OD 600 0.5-0.6. Cultures were incubated at 37°C for 24 hours with shaking. Cultures were centrifuged and cell pellets washed with binding buffer (300 mM NaCl, 50 mM NaH2PO4 with 25mM imidazole) and passed twice through a high pressure cell homogeniser (Stanstead works, UK). Cell debris was removed by centrifugation at 20,000 xg for 45 minutes. Supernatant was collected and incubated with 0.2 ml Ni-NTA for 1 hour at 4 °C then washed with 50 ml binding buffer and eluted four times in 0.5 ml elution buffer (300 mM NaCl, 50 mM NaH2PO4 with 250mM imidazole).
Cloning and expression of CmeC
To express CmeC in E. coli, cmeC lacking signal peptide sequence was amplified by PCR with CmeCFwd1 and CmeCRev using C. jejuni 11168H genomic DNA as a template. The amplicon was then cut by NheI and SalI and cloned into pEC415 downstream of the DsbA signal peptide sequence to give pCMECDSBA. E. coli carrying pCMECDSBA was grown in LB media supplemented with ampicillin (100 µg/ml) overnight at 37 °C under shaking condition. On the following day, 10 ml were withdrawn from the overnight culture to inoculate 400 ml LB media. Cells were grown to OD 600 0.5-0.6 and 0.2% L-arabinose was added to induce the expression of CmeC. Cultures were incubated at 37 °C for 24 hours with shaking at 180 rpm. Cultures were centrifuged and cell pellets washed with binding buffer (300 mM NaCl, 50 mM NaH2PO4 with 25mM imidazole) and passed twice through cell homogeniser (Stanstead works, UK). Cells debris was removed by centrifugation at 20,000 xg for 45 minutes and then collected and incubated in binding buffer with 2 % DDM for 3 hours at 4 °C. The mixture was then centrifuged at 15,000 xg for 10 minutes. The supernatant was collected, diluted with binding buffer and incubated with 0.2 ml Ni- NTA for 1 hour at 4 °C then washed with 50 ml binding buffer and eluted four times in 0.5 ml elution buffer (300 mM NaCl, 50 mM NaH2PO4 with 250mM imidazole).
CD Spectroscopy
All CD spectra of gCmeA and CmeA were acquired in 0.5mm rectangular cell pathlength using Chirascan spectrometer (Applied Biophysics, UK) equipped with Quantum NorthWest TC125 Peltier unit. Temperature dependent confirmation changes were monitored at wavelength 260-195nm for gCmeA (0.2 mg/ml) and CmeA (0.2 mg/ml) in 10 mM Sodium phosphate, 75 mM Sodium chloride, 10 % glycerol buffer (pH=8.0) during stepwise increase in temperature from 6°C to 94°C. Temperatures were measured directly with a thermocouple probe in the sample solution. Melting temperatures were determined from the derivative CD- Temperature spectra and fitted using a Levenberg–Marquardt algorithm (LMA) on the van’t Hoff isochore. (Global 3, Global Analysis for T-ramp Version 1.2 built 1786, Applied Photophysics Ltd, 2007-2012). For Conformation Reversibility Study, far-UV CD spectra were recorded at 20°C, raised to Tm and re-cooled to 20°C. The temperature at each elevated Tm was kept constant for 5 minutes and the CD spectrum was recorded to assess the rate of protein unfolding process.
Surface Plasmon Resonance
For coupling of CmeA and gCmeA to the CM5 sensor chip, carboxyl groups on the surface were activated by injecting a 1:1 mixture of 0.4M 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide (EDC) and 0.1 M N-hydroxysuccinimide (NHS) for 7 minutes at 5 μl/min. CmeA and gCmeA were diluted to 10-20 µg/ml in 0.1 M acetate pH 5.5 and immobilised at 5 μl/min. Immobilisation was stopped when the required RU was achieved. This was followed by injecting 1M ethanolamine pH 8.5 (7 minutes at 5 μl/min) to inactivate excess reactive groups. To account for non-specific binding, a control flow cell was generated using the same method described minus the protein immobilisation step. For coupling of CmeA and gCmeA to a NTA chip, the chip was cleaned and loaded with NiCl2 (0.5 mM). The flow cells were then activated as above and CmeA and gCmeA (10 ug/ml in HBSP buffer) were loaded into appropriate flow cells until appropriate RU were achieved. Subsequently the flow cells were treated with ethanolamine as above to block remaining activated sites.
Cmec at various concentrations (3 nM- 0.2 µM) was analysed at a constant temperature of 25°C under continuous flow of HBS-PE buffer (10mM HEPES pH 7.4, 3 mM EDTA, 0.005% (w/v) Surfactant P20 (GE Healthcare) at 30μl/min (sufficient to prevent mass transfer effects) at pH 7.4 for 3 minutes association and a dissociation time of 5 minutes. Experiments at pH 6.0 were performed with 10 mM MES pH 6.0, 3 mM EDTA, 0.005% (w/v) Surfactant P20 (GE Healthcare) The surface chip was regenerated by injecting 0.1 M triethanolamine pH 11.5. Data was analysed using the BIAevaluation software version 4.1.1 (Biacore, GE Healthcare, Amersham). Blank flow cell controls were subtracted. The kd was defined between 10s after the end of the sample injection and 300 sec later.
Acknowledgements
We acknowledge the Wellcome Trust grant 102978/Z/13/Z for funding.