# Membrane proteocomplexome of Campylobacter jejuni using 2-D blue native/SDS-PAGE combined to bioinformatics analysis

Alizée Guérin, Sheiam Sulaeman, Laurent Coquet, Armelle Ménard, Frédérique Barloy-Hubler, Emmanuelle Dé, Odile Tresse

► To cite this version:

Alizée Guérin, Sheiam Sulaeman, Laurent Coquet, Armelle Ménard, Frédérique Barloy-Hubler, et al. Membrane proteocomplexome of Campylobacter jejuni using 2-D blue native/SDS-PAGE combined to bioinformatics analysis. Frontiers in Microbiology, 2020, 11, 10.3389/fmicb.2020.530906. hal-03765916

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Membrane proteocomplexome of Campylobacter jejuni
using 2-D blue native/SDS-PAGE combined to
bioinformatics analysis

Odile Tresse, Alizée Guérin, Sheiam Sulaeman, Laurent Coquet, Armelle

Menard, Frédérique Barloy-Hubler, Emmanuelle Dé

► To cite this version:

Odile Tresse, Alizée Guérin, Sheiam Sulaeman, Laurent Coquet, Armelle Menard, et al.. Membrane proteocomplexome of Campylobacter jejuni using 2-D blue native/SDS-PAGE combined to bioinformatics analysis. Frontiers in Microbiology, 2020, 10.3389/fmicb.2020.530906. hal-02995535

HAL Id: hal-02995535

https://hal.science/hal-02995535

Submitted on 9 Nov 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
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# Membrane proteocomplexome of Campylobacter jejuni using 2-D blue native/SDS-PAGE combined to bioinformatics analysis

Odile Tresse¹*, Alizée Guérin¹, Sheiam Sulaeman¹, Laurent COQUET², ³, Armelle Menard⁴, Frederique Barloy-Hubler⁵, Emmanuelle Dé², ³

¹INRA Centre Angers-Nantes Pays de la Loire, France, ²UMR CNRS 6270 Polymères de laboratoire
Biopolymères Surfaces (PBS), France, ³Plateforme protéomique PISSARO IRIB - ROUEN, France, ⁴INSERM
U1053 Bordeaux Recherche en Oncologie Translationnelle, France, ⁵UMR6290 Institut de Genetique et
Developpement de Rennes (IGDR), France

**Submitted to Journal:**

Frontiers in Microbiology

**Specialty Section:**

Food Microbiology

**Article type:**

Original Research Article

**Manuscript ID:**

530906

**Received on:**

30 Jan 2020

**Revised on:**

12 Oct 2020

**Accepted on:**

14 Oct 2020

**Frontiers website link:**

www.frontiersin.org
**Conflict of interest statement**

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest

**Author contribution statement**

OT conceived the work; SS, LC and AG performed the experiments; AG prepared the manuscript and the figures, OT finished the writing and the figures, OT, ED, LC, FBH and AM revised the manuscript.

**Keywords**

Foodborne pathogen, proteomics, Functional Genomics, complexes, Membrane Proteins, efflux pumps, regulation, Blue native electrophoresis

**Abstract**

Word count: 260

Campylobacter is the leading cause of human bacterial foodborne infections in developed countries. The perception cues from biotic or abiotic environments by the bacteria are often related to bacterial surface and membrane proteins that mediate the cellular response for the adaptation of Campylobacter jejuni to the environment. These proteins function rarely as a unique entity, they are often organized in functional complexes. In C. jejuni, these complexes are not fully identified and some of them remain unknown. To identify putative functional multi-subunit entities at the membrane subproteome level of C. jejuni, a holistic non a priori method was addressed using two-dimensional blue native /Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in strain C. jejuni 81-176. Couples of acrylamide gradient/migration-time, membrane detergent concentration and hand-made strips were optimized to obtain reproducible extraction and separation of intact membrane protein complexes (MCPs). MCPs were subsequently denatured using SDS-PAGE and each spot from each MCP was identified by mass spectrometry. Altogether, 21 MPCs could be detected including multihomooligomeric and multiheteroligomeric complexes distributed in both inner and outer membranes. Function, conservation and regulation of MPCs across C. jejuni strains were inspected by functional and genomic comparison analyses. In this study, relatedness between subunits of two efflux pumps, CmeABC and MacABputC was observed. In addition, a consensus sequence CosR-binding box in promoter regions of MacABputC was identified in C. jejuni but not in C. coli. The MPCs identified in C. jejuni 81-176 membrane are involved in protein folding, molecules trafficking, oxidative phosphorylation, membrane structuration, peptidoglycan biosynthesis, motility and chemotaxis, stress signaling, efflux pumps and virulence.

**Contribution to the field**

On behalf of the authors, I would like to submit our manuscript entitled " Membrane proteocomplexome of Campylobacter jejuni using 2-D blue native/SDS-PAGE combined to bioinformatics analysis" to Frontiers in Microbiology in your research topic. The perception cues from biotic or abiotic environments by the bacteria are often related to bacterial surface and membrane proteins that mediate the cellular response for the adaptation of to the environment. As Campylobacter jejuni is the leading cause of human bacterial foodborne infections and its adaptation mechanisms remains to be elucidated, we aimed at exploring protein machineries of C. jejuni at the membrane level using proteocomplexomic approach. This work results of a fruitful collaboration between experts on proteomics in Campylobacter (Dr Odile Tresse), LC MS/MS protein identification through PISSARO proteomic plateform (Dr Emmanuelle Dé and Dr Laurent Coquet), bioinformatics (Dr Barbloy-Hubler) and protein complexomics (Armelle Ménard). Results indicated the presence of multihomooligomeric and multiheteroligomeric complexes distributed in both inner and outer membranes of C. jejuni. Function, conservation and regulation of complexes across C. jejuni strains were inspected by functional and genomic comparison analyses. Relatedness between subunits of two efflux pumps, CmeABC and MacABputC was observed. A consensus sequence CosR-binding box in promoter regions of MacABputC was also identified in C. jejuni but not in C. coli. Altogether, this work contributes to better understand proteins machineries and their regulations in C. jejuni membrane. All co-authors are in agreement with the content of the manuscript and there is neither financial interest nor conflict of interest to report. We certify that this submission corresponds to an original work that it is not under review in any other journals. Dr Odile Tresse, CR-HAB INRAE

**Funding statement**

Alizée Guérin was financed by INRAE through the program RFI "Food for Tomorrow in region Pays de la Loire and this study was supported by CompCamp project.
**Ethics statements**

**Studies involving animal subjects**

Generated Statement: No animal studies are presented in this manuscript.

**Studies involving human subjects**

Generated Statement: No human studies are presented in this manuscript.

**Inclusion of identifiable human data**

Generated Statement: No potentially identifiable human images or data is presented in this study.

**Data availability statement**

Generated Statement: No datasets were generated or analyzed for this study.

In review
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Membrane proteocomplexome of *Campylobacter jejuni* using 2-D blue native/SDS-PAGE

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combined to bioinformatics analysis

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Alizée Guérin¹, Sheiam Sulaeman¹, Laurent Coquet²,³, Armelle Ménard⁴, Frédérique Barloy-

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Hubler⁵, Emmanuelle Dé² and Odile Tresse¹*

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¹INRAE, 44316 Nantes, France

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²Normandie Université, UNIROUEN, INSA Rouen, CNRS, UMR6270 Laboratoire Polymères

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Biopolymères Surfaces, 76000 Rouen, France,

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³Normandie Université, UNIROUEN, plateforme PISSARO, IRIB, Mont-Saint-Aignan, France

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⁴Univ. Bordeaux, INSERM, UMR1053 Bordeaux Research in Translational Oncology,

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BaRITOn, 33076 Bordeaux, France

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⁵ Univ. Rennes, CNRS, IGDR (Institut de génétique et développement de Rennes) - UMR 6290,

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F35000 Rennes, France

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*Corresponding author. Mailing address: ¹INRAE, CS71627, 44316 Nantes, France. Phone:

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+33 (0)240 675 006 Email: odile.tresse@inrae.fr

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33 *Campylobacter* is the leading cause of the human bacterial foodborne infections in the 34 developed countries. The perception cues from biotic or abiotic environments by the bacteria 35 are often related to bacterial surface and membrane proteins that mediate the cellular response 36 for the adaptation of *Campylobacter jejuni* to the environment. These proteins function rarely 37 as a unique entity, they are often organized in functional complexes. In *C. jejuni*, these 38 complexes are not fully identified and some of them remain unknown. To identify putative 39 functional multi-subunit entities at the membrane subproteome level of *C. jejuni*, a holistic 40 non *a priori* method was addressed using two-dimensional blue native/Sodium dodecyl 41 sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in strain *C. jejuni* 81-176. Couples 42 of acrylamide gradient/migration-time, membrane detergent concentration and hand-made 43 strips were optimized to obtain reproducible extraction and separation of intact membrane 44 protein complexes (MCPs). The MCPs were subsequently denatured using SDS-PAGE and 45 each spot from each MCP was identified by mass spectrometry. Altogether, 21 MPCs could 46 be detected including multihomooligomeric and multiheteroligomeric complexes distributed 47 in both inner and outer membranes. The function, the conservation and the regulation of the 48 MPCs across *C. jejuni* strains were inspected by functional and genomic comparison analyses. 49 In this study, relatedness between subunits of two efflux pumps, CmeABC and MacABputC 50 was observed. In addition, a consensus sequence CosR-binding box in promoter regions of 51 MacABputC was present in *C. jejuni* but not in *C. coli*. The MPCs identified in *C. jejuni* 81- 52 176 membrane are involved in protein folding, molecule trafficking, oxidative 53 phosphorylation, membrane structuration, peptidoglycan biosynthesis, motility and 54 chemotaxis, stress signaling, efflux pumps and virulence. 55
56 Keywords

57 Foodborne pathogen, proteomics; functional genomics; complexes; blue native

58 electrophoresis; membrane proteins; efflux pumps; regulation

In review
## 1. Introduction

*Campylobacter* is a Gram-negative spiral-shaped bacterium. It has emerged as the leading cause of foodborne bacterial gastroenteritis in humans (Epps et al., 2013; Kaakoush et al., 2015; EFSA and ECDC, 2018). The number of campylobacteriosis cases has been increasing in Europe since 2005 and has reached an incidence of 65 per 100 000 people with 246 158 confirmed cases in 2018 (EFSA and ECDC, 2018). Most cases were attributed to *C. jejuni*, an invasive microorganism causing gastroenteritis associated with fever and frequent watery bloody diarrhoea, abdominal pains and occasionally nausea (Moore et al., 2005; Silva et al., 2011; Epps et al., 2013). It is also associated with post-infection complications including the immune-mediated neurological disease Guillain-Barré Syndrome (Nachamkin, 2002; Alshekhlee et al., 2008), its variant Miller Fisher Syndrome (Ang et al., 2001) or reactive arthritis (Altekruse et al., 1999). Notably, the infectious dose is considered to be lower than the one for other foodborne pathogens as only 500-800 bacteria trigger human infection (Robinson, 1981; Black et al., 1988; Boyanova et al., 2004; Castano-Rodriguez et al., 2015). *Campylobacter* cost of illness was estimated at 2.4 billion euros per year in Europe (EFSA, 2016). *C. jejuni* infections are mainly associated with consumption of poultry and cross-contamination from poultry products (Hue et al., 2010; Guyard-Nicodeme et al., 2013; Hald et al., 2016). For the first time, the European Commission regulation has amended the regulation (EC) No 2073/2005 in 2017 on the hygiene of foodstuffs as regards *Campylobacter* on broiler carcasses stating a limit of 1000 CFU/g applied from January 2018. This microaerophilic, capnophilic and thermophilic microorganism requires fastidious growth conditions and it growth is rapidly hampered by several environmental stress conditions. Optimal growth is obtained using a modified atmosphere limited in dioxygen and enriched in carbon dioxide, a temperature between 37 °C to 45 °C and a pH between 6.5 and 7.5 (Mace et al., 2015). Nonetheless, *C. jejuni* is able to survive harmful conditions by developing
adaptation mechanisms in response to stress conditions throughout the food chain (Atack and Kelly, 2009; Rodrigues et al., 2016). Living as biofilms is also a phenotypical feature that was demonstrated for *C. jejuni*, indicating multiple surviving ways outside hosts (Turonova et al., 2015).

Proteomic techniques have been applied to *Campylobacter* to better understand how changes in genetic expression, bacterial state, nutrient limitation, food plant processing and environmental conditions could affect *C. jejuni* at the protein level (Tresse, 2017). Natural compartmentalization has facilitated subfraction proteome analyses of *Campylobacter* such as the cytosolic proteome (Kalmokoff et al., 2006; Bieche et al., 2012; Asakura et al., 2016), the membrane proteome (Seal et al., 2007; Cordwell et al., 2008; Scott et al., 2014; Watson et al., 2014), the inner or outer membrane proteome (Sulaeman et al., 2012) and the exoproteome (Kaakoush et al., 2010). In addition, the genomic and computational era have brought exciting and challenging prospects for proteomics like assigning a function to each protein and subsequently its relationship to other proteins in the cell. Functional genomics and protein structural modelling approaches can predict protein-protein interactions (PPIs), which constitutes the theoretical protein interactome of an organism. Predicted interactomes, including potential stable or transient PPIs, are limited to databases content but PPIs already demonstrated to be biologically functional, specific genetic organizations (operons, gene clusters and regulons) or structural features (domains and loops) (Planas-Iglesias et al., 2013; Wetie et al., 2013). Genomic analyses of the main pathogenic species of *Campylobacter*, revealed a lack of some of the well-described organizations into operons or gene clusters in Gram-negative bacteria (Parkhill et al., 2000). For instance, genes involved in the amino-acid biosynthesis are scattered in distinct loci across the genome of *C. jejuni* whereas they are organised into operons in other bacteria. In *H. pylori*, the closest specie relative to *Campylobacter*, the presence of some genetic elements organized into operons,
gene clusters or islands could have contributed to the specialization of this pathogen (Sohn and Lee, 2011; You et al., 2012). In *C. jejuni*, the virulence variation among strains could not be assigned to any specific genetic organization other than point mutations in the virulence-associated genes or indels in individual loci (Bell et al., 2013). A reduced genetic organization has probably participated to the idiosyncrasy of *C. jejuni*.

The alternative method to identify PPIs, which does not result necessary from a specific genetic organization, is to detect complexes of proteins using non-hypothesis driven methods. When these complexes are composed of only protein subunits, the global approach is called proteocomplexomic. This is the case of the two-dimensional (2-D) blue native (BN)/SDS-PAGE which aims at highlighting intact protein complexes using mild non-ionic and non-denaturing detergents (Dresler et al., 2011; Lasserre and Menard, 2012; Wohlbrand et al., 2016). This method consists in separating native protein complexes according to their molecular mass during the first dimension and subsequently in separating protein subunits of each complex in SDS-denaturated conditions in an orthogonal second dimension. It has been applied with success to monitor oligomeric state, stoichiometry and protein subunit composition of protein complexes.

This study aimed at exploring protein machineries of *C. jejuni* at the membrane level. The bacterial membrane as a hydrophobic lipid structure is a suitable *site* for protein complex organization. Numerous well-characterized proteins *embedded* in the membrane are organized into functional units involved in various cellular processes. These membrane protein complexes (MPCs) could be also influenced by the membrane structural integrity and their molecular environment (Sachs and Engelman, 2006). In *didermata* such as *C. jejuni*, MPCs could be either organized throughout both membranes and the *periplasmic* space or specifically in the inner or in the outer membrane. The first objective was to apply and to optimize 2-D BN/SDS-PAGE technique on the *C. jejuni* membrane proteins to obtain
reproducible gels. The second goal was to identify MPCs present in *C. jejuni* during optimal growth. As this analysis was conducted on the membrane compartment it was called membrane proteocomplexomic analysis.

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## 2. Material and methods

2.1. 1. Bacterial cell cultures and sample preparation

The virulent *C. jejuni* strain 81-176 (NC_008787), whose whole genome is available in **Genoscope Platform (MicroScope Vallenet et al., 2017)**, was selected for the experiments. A loopful of frozen 81-176 cells culture, conserved at -80 °C in Brain-Heart Infusion (BHI) broth (Biokar, Beauvais, France) containing 20% sterile glycerol, was cultured on fresh Karmali agar plates (Oxoid, Dardilly, France) (Air Liquid, Paris, France) at 42 °C for 48 h in microaerobic conditions (MAC) generated using gas replacement jars operated by MACSmics gassing system (BioMérieux, France) with a gas blend composed of 5% O₂, 10% CO₂ and 85% N₂ (Air Liquid, Paris, France) and 4 filled/flushed cycles at -50 kPa as described in Mace *et al.*, 2015 (Mace *et al.*, 2015). Cultures were obtained by inoculating 500 mL of BHI broth in a 1-L flask and incubating them for 16 h under MAC at 42 °C in a rotary shaker.

2.2. Membrane protein complex (MPC) extraction

The cells were harvested by centrifugation for 20 min at 4 °C at 6 000 g. The supernatant was discarded and about 3 g of dry pellet was obtained. The cells were washed twice with lysis buffer containing 50 mM Tris, 750 mM 6-amino-n-caproic acid as a zwitterionic salt, with each wash followed by centrifugation at 6 000 g for 20 min at 4 °C. The cells were then resuspended in 5.5 mL of lysis buffer supplemented with 60 µL phenylmethylsulfonyl fluoride (PMSF) and sonicated at 50 kHz for 6 x 30 s with 5 min intervals on ice (Vibracell 72434, Bioblock Scientific, Illkirch, France) as previously described by Bieche *et al*. (Bieche
et al., 2012). The proteins present in the supernatant were then collected and centrifuged twice at 10 000 g for 30 min at 4 °C in order to remove the cellular debris. The whole protein lysate was treated with 0.2 mg/ml DNaseI for 1 h at 25 °C and then ultracentrifuged at 100 000 g for 1 h at 4 °C. The pellet containing membrane complexes was resuspended in 10 mL of lysis buffer with 50 µL PMSF supplemented with the mild detergent Dodecyl-β-D-Maltoside (DDM) (Sigma, France) at concentrations ranging from 1 to 5% (w/v) to maintain the integrity of protein complexes and limiting dissociation or denaturation as previously recommended by Bernarde *et al.* (Bernarde et al., 2010b). After 15 min on ice, each sample solubilized with DDM was directly ultra-centrifuged at 100 000 g for 1 h at 4 °C. The MPC extraction was performed in triplicate from three independent cultures. Aliquots of the supernatant containing the membrane protein complexes were stored at -80 °C. The protein concentration of membrane complexes was determined using the Micro BCA™ Protein Assay Kit (Perbio Science, Brebieres, France) according to the manufacturer protocol.

## 2.3. MPC separation using 2-D BN/SDS-PAGE

### 2.3.1. First dimension in native conditions (BN-PAGE)

The first dimension was performed in a blue native polyacrylamide gel (BN-PAGE) according to Schagger (Schagger and von Jagow, 1991) with the following modifications. The MPC separation using BN-PAGE gels (15 cm x 16 cm x 0.1 cm) was assayed on linear acrylamide gradients: 4-14% (w/v), 4-18% (w/v), 8-18% (w/v) or 10-20% (w/v) using a gradient forming unit and Protean II cell (Biorad, Hercules, CA, USA). Each separating gel was overlaid with a 3% stacking BN-PAGE. Both anode and cathode buffers contained 50 mM Tris and 75 mM Glycine. Only the cathode buffer was supplemented with 0.002% (w/v) Coomassie Blue G250 (Serva Biochemicals, Heidelberg, Germany). The assembly of gels were embedded with anode and cathode buffers and maintained at 4 °C for 3 h before loading the protein sample. A volume of 1-5 µL of sample buffer (500 mM 6-amino-*n*-caproic
acid and 5% Serva blue G) was added to DDM-solubilized membrane protein complex
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samples. Thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate
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dehydrogenase (140 kDa) and BSA (67 kDa) were used as high molecular weight native
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protein marker mixture (GE Healthcare, Buckinghamshire, UK). The migration was run at
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4 °C with 1 W per gel and limited at 150 V and 90 mA during 4 h to 48 h according to the
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assays.
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To check the optimal solubilisation of the protein complexes using DDM, migration through
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BN-PAGE was performed as described above with 3% stacking gel. Samples of 10 or 20 µg
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of protein complexes solubilized in 1, 2 or 5% (w/v) DDM were prepared as described above
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and loaded for each lane of the BN-PAGE. For protein complex analyses, gels were silver
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stained and scanned with a GS-800 densitometer (Bio-Rad) operated with the QuantityOne®
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software (Bio-Rad) at the resolution of 42.3 microns as described previously by
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Sulaeman et al. (Sulaeman et al., 2012). For the protein identification, the gels were loaded
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with 50 µg of protein complexes. Following the 1-D migration, the protein complexes in the
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BN-PAGE were fixed using the kit Bio SafeTM Coomassie G-250 Stain (Biorad) according to
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the manufacturer's instructions.
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2.3.2. Second-dimension SDS-PAGE

The second dimension was performed under denaturing conditions using 10% (w/v)
201
acrylamide SDS-PAGE (15 cm x 16 cm x 0.15 cm). An individual lane was cut off from the
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first dimension BN-PAGE using a glass plate. Gel lane was equilibrated for 5 min in a buffer
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containing 1% (w/v) SDS and 125 mM Tris. Then, the proteins were reduced for 15 min into
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equilibrating buffer supplemented with 50 mM dithiothreitol (DTT) (Sigma, France), and
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subsequently alkylated for 15 min in equilibrating buffer supplemented with 125 mM
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iodoacetamide (Biorad). An ultimate washing step lasting 5 min was performed in the
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equilibrating buffer without supplement. After polymerization of the separating SDS-PAGE and equilibration, the gel lane was laid on a plastic support and introduced between the gel glass plates over the separation gel and embedded with low-melting agarose. Migration was carried out for 4 h at 16 °C at 300 V maximum and 10 mA/gel for the first 45 min and then at 20 mA/gel. After migration, proteins were silver stained and **scanned as described above**.

## 2.4. In-gel trypsin digestion

The silver-stained spots separated by SDS-PAGE were excised manually. At first, the spots were discoloured, then washed and reduced/alkylated using an automated system (MultiProbe II, Perkin Elmer, France) as following: each spot was washed several times in water, once in 25 mM ammonium carbonate and dehydrated with acetonitrile (ACN). After drying the gel pieces, the reduction was achieved by incubation for 1 h with 10 mM DTT at 55 °C. The alkylation was achieved by incubation the samples with 25 mM iodoacetamide for 1 h at room temperature. Finally, the gel spots were washed three times in water for 10 min, again alternating between ammonium carbonate and ACN. The gel pieces were completely dried before trypsin digestion and rehydrated by trypsin addition. The digestion was carried out overnight at 37 °C. The gel fragments were subsequently incubated twice for 15 min in a H₂O/ACN solution and in ACN to allow extraction of peptides from the gel pieces. The peptide extracts were then pooled, dried and dissolved in 10 µL starting buffer for chromatographic elution, consisting of 3% (v/v) ACN and 0.1% (v/v) formic acid in water.

## 2.5. Protein identification by LC MS/MS

The peptides were enriched and separated using a lab-on-a-chip technology (Agilent, Massy, France) and fragmented using an on-line XCT mass spectrometer (Agilent). The fragmentation data were interpreted using the Data Analysis program (version 3.4, Bruker Daltonic, Billerica, MA, USA). For the protein identification, the MS/MS peak lists were
232 extracted, converted into mgf-format files and compared to the *C. jejuni*, strain 81-176 protein
233 database (UniprotKB, CP000538 for the chromosome, CP000549 for plasmid pTet and
234 CP000550 for plasmid pVir) with the MASCOT Daemon search engine (version 2.6.0; Matrix
235 Science, London, UK). The following search parameters were used: trypsin was used as the
236 cutting enzyme, the mass tolerance for monoisotopic peptide window was set to ± 1.0 Da and
237 the MS/MS tolerance window was set to ± 0.5 Da. Two missed cleavages were allowed.

238 Carbamidomethylation, oxidized methionine, acetylation and pyroglutamate in Nt and
239 amidation in Ct were chosen as variable modifications. Generally, the peptides with
240 individual ions scores higher than the score indicated for p<0.05 were selected. The proteins
241 with two or more unique peptides matching the protein sequence were automatically
242 considered as a positive identification. The main raw data are presented in Table S1. Other
243 raw data are available upon request.

244

245 2.6. Western blotting

246 The western blots of 2-D BN/SDS PAGE were performed according to Sulaeman *et al*.,
247 (2012) (Sulaeman et al., 2012). Briefly, prior to transfer, the 2-D SDS gels were cut into two
248 horizontal sections and each section was soaked for 15 min in transfer buffer. Then, the
249 proteins of each gel section were transferred to a nitrocellulose membrane by electrophoresis
250 using Mini Trans Blot (Bio-Rad). The transferred proteins were then probed with a 1/2000
251 dilution of antibody anti-PorA or antibody anti-CadF. The immunoreactive proteins were
252 detected using a 1/2000 dilution goat-anti-rabbit alkaline phosphatase antibody (Anti-Rabbit
253 IgG, F(avb)2 fragment-Alkaline Phosphatase, Sigma, Saint Quentin Fallavier, France),
254 followed by BCIP/NBT staining (Biorad). Gels were scanned using the GS-800 Imaging
255 densitometer (BioRad).
256 2.7. Bioinformatic analyses

257 2.7.1. *In silico* determination of complex function

258 The presence and organization of genes encoded protein subunits of identified complexes in
259 81-176 and other *Campylobacter* complete genomes was explored using the **Platform**
260 **MicroScope described in** Vallenet et al., 2017. The biological function of these proteins and
261 complexes were inferred using KEGG and Microcyc (Kanehisa et al., 2016). The
262 conformation analyses of the proteins alone or in a complex were performed using UniProt
263 (Zommiti et al., 2016), RCSB (Stephen et al., 2018), RCSB PDB (Young et al., 2018), Swiss-
264 model (Biasini et al., 2014) and OPM (Lomize et al., 2012). The functional links between
265 partners of complex was explored using STRING (Szklarczyk et al., 2017). If the complex
266 protein subunits were not identified in *Campylobacter*, homologous genes were searched
267 using conventional gene alignment tools (BlatN).

268 2.7.2 Phylogenetic tree of efflux pumps

269 The protein sequences of CmeA, CmeB, CmeC and MacA, MacB, putMacC (for putative
270 MacC) on fourteen complete genomes of *C. jejuni* strains and six complete genomes of *C. coli*
271 strains were recovered (Table S1). MAFFT alignments (reference PMID:23329690) and Fast
272 Tree (reference PMID:19377059) phylogenetic trees were performed using Geneious R9 and
273 visualised using FigTree V1.4.3 software (http://tree.bio.ed.ac.uk/software/figtree/). The
274 proteins structures predictions were determined using Philus transmembrane prediction server
275 (reference, PMID:18989393) (www.yeastrc.org/philius/pages/philius/runPhilius.jsp).

276 2.7.3 Distribution of efflux pumps CmeABC and MacABputC in bacteria

277 BlastP analyses were performed for each protein sequence of the components of these efflux
278 pumps in *C. jejuni* 81-176 against domain bacteria in RefSeq (NCBI Reference Sequence
279 Database) protein database. The general parameters applied for similarity validation were
280

Max target sequences at 20000, automatically adjust parameters for short input sequences,
expect threshold at 10, word size at 6 and max matches in a query range at 0. Scoring
parameters were obtained from the blosum62 matrix, gap costs with existence at 11 and
extension at one with a conditional compositional score matrix adjustment.

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2.7.4 Identification of CosR DNA-Binding Box

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The sequence logo of CosR-binding box previously defined by Turonova et al. (Turonova et al., 2017) was used to check the presence of cosR-binding box in the promoter regions of the operons encoding the efflux pumps *cmeABC* and *macABputC* among the complete genomes of *C. jejuni* and *C. coli* (Table S1). For that, a sequence length of maximum 120 pb in the intergenic regions upstream to *cmeA* and *macA* was recovered. Based on these sequences, a new sequence logo of CosR DNA-binding box with sequences was drawn using WebLogo platform (http://weblogo.threeplusone.com (Crooks et al., 2004)).

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3. Results

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3.1. Optimization of protein complex solubilization and separation

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The BN-PAGE was applied first to check the solubilization efficiency of the extracted MPCs.
In order to approach a useful detergent concentration for protein complex solubilization for
BN-PAGE, Dodecyl-β-D-Maltoside (DDM) has turned out to be suitable (Schägger, 2002).
The solubilisation of the membrane was tested in a concentration of 1, 2 or 5% of DDM. The
loading quantities of 10 µg or 20 µg of MPCs on BN-PAGE were found appropriate to
determine the reliable concentration of DDM detergent for *C. jejuni* (Fig. 1). In native
conditions, MPCs migrate according to their mass and their form in space (steric hindrance).
The bands detected in BN-PAGE in the range of 20 kDa to 670 kDa indicate that MPCs were
stable after membrane solubilization and during the separation. All tested concentrations of
304 DDM (1, 2 and 5%) seem to be suitable to solubilize MPCs from *C. jejuni*. However, over the
305 three independent extractions, less reproducibility was obtained with 5% DDM for the lower
306 molecular mass complexes (not shown). Consequently, only 2% DDM were selected to
307 explore MPCs of *C. jejuni* 81-176. In addition, the better separation of MPCs was obtained by
308 extending the migration time until 40 h (Fig. S1). Acrylamide gradients of BN-PAGE were
309 also optimized by testing different couples of lower concentrations (ranging from 4% to 10%)
310 and higher concentration (ranging from 14% to 20%). Even though some protein complexes
311 were more distinct on some gradients, the linear gradient which gives more detectable
312 complexes was 4% to 18% of acrylamide. In general, a lack of reproducibility of the 2-D gels
313 and vertical smearing altering the resolution of MPCs subunits on SDS-PAGE were
314 frequently reported. To circumvent these biases, many gels were run in previous studies until
315 obtaining at least three similar replicates. In the present study, obtaining gel repeatability in a
316 consecutive manner was the first goal. This goal was reached by decreasing vertical smearing,
317 adjusting the thickness of the gels, fixing the supports and isolating the homemade strip from
318 glass using a five-step protocol (Fig. S1). All these optimization steps resulted in performing
319 consecutive reproducible gels. The reproducibility was validated once three consecutive
320 profiles could be aligned with the same number of detected spots. The results of optimisation
321 steps during the first and second dimensions are presented in figure S2.

322 3.2. Analysis of 2D-BN/SDS-PAGE data

323 Using the criteria mentioned by Reisinger and Eichacker (Reisinger and Eichacker, 2006), all
324 protein spots that are located vertically below each other with a similar shape on the 2-D were
325 considered as subunits of one MPC. Consequently, the complexes were numbered from the
326 left side to the right side of the 2-D gel and the detected subunits for each complex with a
327 second number starting from the top of the gel (Fig. 2). The spots, which were located side by
328 side in a horizontal row, could potentially be an identical subunit in protein complexes of
different molecular masses. If we assume that the molecular mass of a protein complex is increased during assembly of its structural subunits, the analysis of the protein pattern allows to determine the stepwise subunit assembly. The lower towards the higher molecular mass should correspond to complexes located from the right side of the gel towards its left side

(Reisinger and Eichacker, 2008). When a complex was composed of subunits from the same gene product, they were called multihomooligomeric complexes and when they were composed of different subunits, they were named multiheterooligomeric complexes as described before (Bernarde et al., 2010b). Some complexes could not be detected due to a relatively low abundance in the membrane, solubilisation parameters or separation parameters. In addition, depending on the solubilisation and separation parameters more than one protein complex could run at the same molecular mass during BN-PAGE. This was observed more frequently for smaller protein complexes. In this case, the spot identification helps to separate different complexes when the biological function was previously described.

The solubilisation, the migration parameters, the interaction between subunits and the subunit organization in or associated with the membrane could also result in partial identification of complexes.

The identification of the spots was achieved by nanoLC MS/MS and validated using Mascot score (Table 1, Table S1). The western blots using polyclonal antibodies anti-PorA and anti-CadF were used to target specific outer membrane proteins (Fig. S3). Three spots of PorA and one spot of CadF could be identified using Western-blot confirming the identification performed by LC MS/MS. In addition, the LC MS/MS identified two supplementary spots of PorA indicating that it has a probable higher sensitivity than Western-blot according to the protein abundance. As expected, PorA, also named major outer membrane protein (MOMP) is among the predominant proteins. It was previously reported that PorA could account for 45% of the total visible membrane proteins of *C. jejuni* (Cordwell et al., 2008).
354
355

3.3. Protein complex identification by Two-dimensional (2-D) blue native (BN)/SDS-PAGE

356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374

Overall, 55 spots were submitted to LC-MS/MS analysis (Fig. 2 and Table 1). Among them, nine isolated spots and all spots of complex 7 could not be identified, **although attempts using different MS technologies were performed. No contamination with exogenous protein, like keratin, was detected. The spectrograms seem to show noise-to-signal trouble shootings.** Two proteins identified with a low scoring (Mascot score < 30) were discarded from the analysis.

The remaining spots could be grouped into 20 complexes according to their location in the gel, identification and biological functions when available. Overall, 39 proteins predicted as membrane proteins or membrane associated proteins were identified indicating the efficiency of the extraction of the protein complexes from *C. jejuni* 81-176. These complexes were grouped according to biological functions of KEGG classification (Table 1). Four complexes are involved in oxidative phosphorylation, two in the respiration process, seven in molecules trafficking, three in protein biosynthesis and folding, one in motility and three with unknown functions in the membrane. Altogether, 6 multiheterooligomeric and fourteen multihomooligomeric complexes were detected (Table 1). Certain identified complexes were already described in *C. jejuni* such as efflux pump CmeABC (Gibreel et al., 2007) validating this technique to identify MPC in this bacteria. However, novel complexes are presented, such as complexes 2 and 8 comprised of FrdABC and FdhAB, respectively. These complexes were already described in *Helicobacter pylori* Bernarde *et al*. (Bernarde et al., 2010a) and *Eubacterium acidaminophilum* (Graentzdoerffer et al., 2003), respectively.

375

3.4. *In silico* analysis of efflux pumps CmeABC and MacAB

376
377

Among the 20 identified complexes, subunits belonging to two efflux pumps were detected in complex 6 with CmeA, CmeB and CmeC and complex 20 with MacB (Fig. 2 and Table 1).
The subunits of these two pumps were further investigated in this study. CmeABC is a
multidrug efflux system in *Campylobacter* working as an RND efflux pump (Lin et al.,
2002; Akiba et al., 2006; Grinnage-Pulley and Zhang, 2015). It contributes to the resistance
acquisition of *Campylobacter* to various antimicrobials including macrolides and
fluoroquinolones (Yan et al., 2006; Gibreel et al., 2007; Jeon and Zhang, 2009). This efflux
pump has also an important role in the resistance to bile (Lin et al., 2003). It includes the inner
membrane drug transporter CmeB, the periplasmic membrane fusion protein CmeA and the
outer membrane channel CmeC. These proteins can be glycosylated at various sites (Scott et
al., 2011) which could explain two CmeB proteins identified with a different molecular
weight (Fig. 2). The bioinformatics analysis confirmed the organization in operon of the two
efflux pumps amongst both *C. jejuni* and *C. coli*. For the other efflux pump, the subunit
MacB, previously identified to be associated with MacA, was detected in the
multihomooligomeric complex 20 which belongs to the efflux pump specific to macrolides
(Yum et al., 2009; Bogomolnaya et al., 2013). (Yum et al., 2009; Bogomolnaya et al., 2013).
Using Platform MicroScope, STRING, blastp and blastn, both DNA and protein sequences of
these efflux pump partners were analysed across *C. jejuni* and *C. coli*. Genes *cmeA* and *macA*
are homologous and *cmeC* is homologous to a gene encoding a putative outer membrane
protein (CJJ81176_0637) located downstream to *macB*. This putative outer membrane protein
contains the same functional domain TolC as the one described in CmeC suggesting that this
putative protein is probably the third partner of MacAB efflux pump. Further experimental
assays will be required to validate the biological function of this putative protein for
macrolide efflux pump operation. The phylogenetic and functional analyses confirm the
similarities between proteins sequences of subunits of these two efflux pumps across
*Campylobacter* strains (Fig. 4; Table S1). CmeA and MacA belong to the HlyD superfamily
showing a structure composed of the signal peptide and the non-cytoplasmic domain. The
403 phylogenetic analysis of the putative outer membrane protein CJJ81176_0637 confirmed it

404 similarly to CmeC which likely is the third subunit of the efflux pump MacAB (Fig. 4).

405 Considering this data, this putative outer membrane protein was named putative MacC

406 (putMacC) and the system MacABputC. In contrast to the similarly between sequences and

407 structural functions of CmeC and putMacC in one hand and CmeA and MacA in the other

408 hand, differences observed between CmeB and MacB is probably at the origin of the

409 restriction of MacB to macrolides efflux.

410 3.5. Analysis of the potential regulation of MacABC

411 The efflux pump CmeABC was previously shown to be regulated by CmeR and CosR (Lin et

412 al., 2005; Hwang et al., 2012; Grinnage-Pulley et al., 2016) while no regulation was identified

413 for MacABC. Using the CosR binding box sequence (5'-wdnnhdwnwhwwTTwnhhTTd- 3')

414 previously described by Turonova *et al*. (Turonova et al., 2017), *in silico* analysis revealed the

415 presence of a CosR-like DNA-binding box upstream to *macA* in *C. jejuni* 81-176. Screening

416 for the presence of the CosR binding box in the promoter region of *macA* in the complete

417 genomes of *C. jejuni* and *C. coli*, this consensus sequence was found in *C. jejuni* but not in

418 *C. coli*. All the binding box DNA sequences of CosR in the *cmeA* promoter region of both

419 *C. jejuni* and *C. coli* strains and the *macA* promoter region of *C. jejuni* strains were compared

420 so as to propose a consensus sequence logo refined for CosR-binding box (Fig. 5).

421 3.6. Distribution of gene subunit encoding CmeABC and MacABputC across Bacteria

422 domain

423 Analyses of genes encoding proteins belonging to these two efflux pumps using across

424 Bacteria domain Blastp analysis indicates that they are mainly observed in proteobacteria

425 (Fig. 6, Table S1). CmeA, CmeB and CmeC were mainly found in the delta/epsilon

426 proteobacteria groups while MacABputC also highly more represented in beta and gamma
proteobacteria. Proteins of MacABputC were also found in the fusobacteriaceae family belonging to the fusobacterial phylum (Fig. 6, Table S1). The presence of this third subunit in outer membrane (putMaC and CmeC) is probably crucial for the functionality of these complexes.

431

432 **4. Discussion**

Beyond the protein mapping at the organism or a biological compartment scale using holistic approaches, identifying functional multi-subunit entities at the proteome level is a real challenge. Many cellular processes are carried out by sophisticated multi-subunit protein machineries, *i.e.* different protein complexes maintained by stable protein interactions. These functional entities could be defined as protein complexes composed of a minimal biologically structure of assembled protein subunits necessary for a specific cellular process (Reisinger and Eichacker, 2007). Membrane proteocomplexome of *C. jejuni* 81-176 cultivated in optimal growth conditions was explored using 2-D BN/SDS PAGE. The prerequisite goal was to obtain reproducible profiles after optimizing and **stabilising** homemade strips. The objective was reached when three consecutive gels with resolved spots could be performed. Twenty-one MPCs were found with this method in *C. jejuni* 81-176 (Fig. 3). We found **incomplete** complexes suggesting that subunits were probably lost during MPC extraction or unidentified by LC MS/MS.

446 4.1. Oxidative phosphorylation

Oxidative phosphorylation is the metabolic pathway in which bacteria use enzymatic complexes to re-oxidize cofactors and produce ATP. It ensures the electron transfer between electron donors and the final electron acceptor which is oxygen for aerobic and microaerobic bacteria such as *C. jejuni*. The redox reactions are carried out by a series of four protein complexes (I, II, III, IV) located in the inner membrane. Membrane proteocomplexomic
452 profiling revealed the presence of complexes involved in oxidative phosphorylation.

453 Complex 19 corresponds to the NADH ubiquinone oxidoreductase, the first complex of the
454 oxidative phosphorylation chain. This complex catalyzes the transfer of two electrons from
455 NADH to quinone with the translocation of four protons across the inner membrane: NADH +

456 H⁺ + Q + 4H⁺<sub>in</sub> → NAD⁺ + QH₂ + 4H⁺<sub>out</sub> (Baranova et al., 2007; Weerakoon and Olson,

457 2008; Efremov and Sazanov, 2011; Baradaran et al., 2013). Genomic analysis revealed
458 fourteen genes *nuo* organized in operon in *C. jejuni* and *C. coli* genomes with a highly

459 conserved synteny. NuoFEGDCBI are involved in the hydrophilic domain of the NADH

460 dehydrogenase complex while NuoAHJKLMN is localized into the membrane (Baradaran et

461 al., 2013). The partners of this complex NuoC, NuoD and NuoG detected from our MPC

462 fingerprintings are predicted to be localized on the basal part of the hydrophilic domain, close

463 to the inner membrane. In *E. coli*, NuoC and NuoD are fused and NuoG is close to them
464 (Baranova et al., 2007; Baradaran et al., 2013). If these subunits are similarly organized in

465 *C. jejuni*, this would indicate that 2% DMM MPC extraction, detection and subunit
466 identification mainly selected this part of NADH dehydrogenase complex. The complex 2

467 corresponds to complex II of the oxidative phosphorylation chain. The fumarate reductase
468 complex is generally composed of FrdA, FrdB and FrdC (Weingarten et al., 2009; Guccione et

469 al., 2010; Jardim-Messeder et al., 2017). All three subunits were detected and identified on all
470 the proteocomplexic fingerprintings performed in the present study. In *C. jejuni*, this inner

471 membrane system is bifunctional being able to catalyze both succinate oxidation and fumarate
472 reduction (Guccione et al., 2010; Hofreuter, 2014). The succinate oxidation is favored under
473 microaerobic conditions while the fumarate reduction is operated under oxygen-limited

474 conditions. All three genes are close located on the genome of both *C. jejuni* and *C. coli*. FrdA

475 is the fumarate reductase flavoprotein, FrdB is the iron-sulfur subunit and FrdC is the

476 cytochrome B-556 subunit. This fumarate reductase complex FrdABC was also described in
H. pylori (Pyndiah et al., 2007). A fourth partner, FrdD is described in E. coli (Rothery et al., 2005). However, the bioinformatic analyses did not reveal any homologous gene to FrdD in C. jejuni and C. coli genomes. This would indicate that complex 2 does exist and might be functional in C. jejuni and H. pylori without FrdD subunit. Any subunit of the ubiquinol-cytochrome c reductase, complex III of the oxidative phosphorylation, was not detected in this study. However, predicted functional partners of this proton pump were detected in C. jejuni genome: petA encoding the iron sulfur subunit, petB encoding the cytochrome b subunit and petC encoding the cytochrome c1 subunit which indicates the absence of this complex in optimal growth conditions or DMM limitations to extract all MPCs. Partners, function and pathways of cytochrome c oxidase (complex IV) in Campylobacter remain elusive. It is encoded by ccoNOPQ. The protein CcoO (Complex 9) was detected in our study. It corresponds to the subunit II of cytochrome c oxidase complex with a high affinity for O₂ (Cosseau and Batut, 2004; Hofreuter, 2014). In complex V, the ATP synthase is usually composed of nine subunits, AtpABCDEFFGH often identified as subunit α, A, ε, β, C, B, B', γ and δ (Rastogi and Girvin, 1999; Altendorf et al., 2000; Cingolani and Duncan, 2011; Okuno et al., 2011). The bioinformatics analyses revealed that genes atpF, atpF', atpH, atpA, atpG, atpD and atpC are close located in C. jejuni and C. coli genomes while genes atpB and atpE are present in different loci. The proteocomplexomic analysis identified AptD (also called subunit β in complex 11). The membrane-bound ATP synthase is a key energy carrier in bacteria using the energy of an electrochemical ion gradient and the synthesis of ATP from ADP with inorganic phosphate (Rastogi and Girvin, 1999; Altendorf et al., 2000; Cingolani and Duncan, 2011; Okuno et al., 2011).

## 4.2 Respiration

The complex formate deshydrogenase (complex 8) contributes to the respiration process by producing CO₂ from formate oxidation (Hofreuter, 2014). This complex is composed of two
502 FdhA and FdhB, two subunits detected on complexomic fingerprintings. As an asaccharolytic
503 microorganism, carbon supply in *Campylobacter* is ensured from amino and organic acids and
504 formate is one of the preferred substrate when hosted in poultry. The second detected complex
505 involved the respiration process is hydrogenase complex HydAB complex 12. Two HydB
506 with different weights (around 75 kDa and 130 kDa) were identified in complex 12 indicating
507 the possible presence of a dimeric form of HydB (spot 12.1) in *C. jejuni* membrane. For these
508 two complexes, single conserved copies of the encoding genes were observed in all analyzed
509 genomes except for *C. jejuni* 4031 where a second copy of FdhA was found. Two other
510 subunits for each complex (FdhC/FdhD and HydC/HydD) were previously identified in
511 *C. jejuni* (Andreesen and Makdessi, 2008; Smart et al., 2009; Weerakoon et al., 2009; Pryjma et
512 al., 2012; Shaw et al., 2012; Hofreuter, 2014). These two other partners interact with the main
513 ones only under environmental stress conditions. The genes encoding these environment
514 dependent conditions are present on *C. jejuni* and *C. coli* genomes. As MPCs of *C. jejuni* 81-
515 176 were explored under optimal growth in our study, it is not a surprise not having detected
516 them.

517 4.3. Biosynthesis and folding of proteins

518 Several proteins are biosynthesized and folded by different complexes localized in membrane.

519 <ins>Different membrane protein complexes were extracted and identified in this study.</ins>

520 The **chaperonin** GroEL (complex 0) is a cylindrical complex with two stacked heptameric
521 rings with ATPase activity that binds non-native substrate protein (SP). GroEL is associated
522 with cofactor GroES and form a nano-cage where SP can be folded up (Klancnik et al.,
523 2006;Chi et al., 2015;Haldar et al., 2015;Motojima and Yoshida, 2015;Hayer-Hartl et al.,
524 2016). As its cofactor GroES has a too small molecular weight (10 kDa), it could not be
525 detected on our profiling fingerprinting. Another complex (complex 4) playing a role in
526 protein folding was detected. DsbB-DsbA is a disulfide bond generation system operating in
the oxidative pathway (Inaba et al., 2006; Inaba and Ito, 2008; Ito and Inaba, 2008; Grabowska et al., 2011; Sperling et al., 2013). In this study, only DsbB subunit was identified although two dsbA genes with 51% homology were present in C. jejuni 81-176 genome. DsbB localized in inner-membrane interacts with the periplasmic dithiol oxidase DsbA (Inaba et al., 2006; Inaba and Ito, 2008).

### 4.4. Molecules trafficking in membrane

Several membrane protein complexes identified in this study have a function in molecule trafficking through the C. jejuni 81-176 membrane. These complexes play an important role in adaptation and virulence capabilities. For instance, the major *Campylobacter* porin PorA was extracted and identified in the complexes 3 and 13. This porin corresponding to MOMP, involved in the adaptation of *Campylobacter* to host environments (De et al., 2000; Zhang et al., 2000; Clark et al., 2007; Ferrara et al., 2016), is a multihomooligomeric porin with three PorA subunits (Zhang et al., 2000; Ferrara et al., 2016). This porin can be identified in three conformational forms including the folded monomer (35 kDa), the denatured monomer (40 to 48 kDa), and the native trimer (120 to 140 kDa) (Huyer et al., 1986; Zhang et al., 2000). In our gel, two complexes corresponding to native trimer MOMP at two different weights were identified. The complex 13 was estimated between 120-140 kDa and after subunit separation proteins corresponding to the folded monomer at 35 kDa and the denatured monomer between 40 and 48 kDa could be detected as previously described by Zhang *et al.* 2000 (Zhang *et al.*, 2000). In the complex 3, the spot with apparent *weight* between 350 kDa and 400 kDa could correspond to the fusion of two trimeric MOMP5. The associated forms of MOMP in the complex 3 with a folded monomer, denatured monomer and a truncated form at 16 kDa was not previously described.

### 4.5. Antibiotic efflux pumps
In this work, units of complexes corresponding to two efflux pumps CmeABC and MacAB were detected (complex 6 and 20). These two efflux pumps were known to play a role in antimicrobials resistance. The multidrug efflux pump CmeABC was more studied compared to the macrolide efflux pump MacABputC in *Campylobacter* (Lin et al., 2002;Lin et al., 2003;Akiba et al., 2006;Yan et al., 2006;Gibreel et al., 2007;Jeon and Zhang, 2009;Grinnage-Pulley and Zhang, 2015). In our study, the genetic and proteomic similarities between the constitutive proteins of these efflux pumps were highlighted by bioinformatics analyses. We were able to identify a potential third partner of the macrolide efflux pump, putMacC with a functional domain TolC similar to CmeC. Subunits composing these two efflux pumps were mainly found in the proteobacteria phylum. Furthermore, the presence of CosR-binding box of the upstream sequences of *macA* was found in *C. jejuni* strains. However, this CosR-binding box could not be detected upstream *macA* in *C. coli* strains. Further biological analyses are required to explore the potential rule of CosR to regulate expression of transcripts of this macrolide efflux pump and to state its presumptive species specificity.

## 4.6. New membrane protein complexes

Two complexes (complex 1 and 14) were extracted and their subunits could be identified. The subunits of the complex 1, IlvC a ketol acid reductoisomerase and Ftn a nonheme iron-containing ferritin were detected on the same horizontal line. IlvC is involved in L-isoleucine and L-valine biosynthesis (Pyndiah et al., 2007;Wu et al., 2016) while Ftn has a role in storing available cytosolic iron and to reduce cellular toxicity under conditions of intermittent or constant iron excess during infection (Wai et al., 1996). There are few information concerning these two proteins in *Campylobacter* and no interactions were reported. Complexes 10 and 14 are made of two isoforms of the methyl accepting chemotaxis protein CJJ_0180. This protein plays a role in chemotaxis and colonization of the gastrointestinal tract (Gonzalez et al., 1998;Zautner et al., 2012;Li et al., 2014;Chandrashekhar et al., 2015). However, weight of
complexes 10 and 14 are different, indicating that these multihomooligomeric complex might
be composed of different forms of the subunit as it was described for MOMP complex.

To conclude, this study is the first 2-D BN/SDS-PAGE method applied to identify membrane proteocomplexome of *Campylobacter jejuni*. Although not all the subunits of functional complexes in the membrane of *C. jejuni* could be detected, functional genomics analyses assisted us in reconstituting probable functional complexes. For instance, we were able to pinpoint a potential third partner in the macrolide efflux pump and raised hypothesis concerning its regulation by CosR. The 2-D BN/SDS-PAGE raised also limitations for studying bacterial proteocomplexes. Assignment of spots to independent membrane protein complexes in low molecular weight areas is less easy. In certain cases, proteins complexes were probably too weakly expressed as compared to others, or absent in optimal conditions. The tune up of DDM concentration, the conformation of complexes and their location, as full or part of the membrane, might contribute to the extraction of entire and stable complexes. Altogether, this study has allowed to better describe the membrane proteocomplexome of *C. jejuni* providing new focus for further studies of protein complexes previously annotated with unknown functions.

Acknowledgments

The authors are grateful to Pr Mégraud (Univ. Bordeaux, INSERM, UMR1053 Bordeaux Research in Translational Oncology, BaRITOn, 33076 Bordeaux) for helping us to develop complexomic analyses. Thank you to Lucile Bougro for her technical assistance. Alizée Guérin was financed by INRAE through the program RFI "Food for Tomorrow in region Pays de la Loire and this study was supported by CompCamp project. This work was presented in
600 part at the 19th International Workshop on Campylobacter, Helicobacter and Related

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941 **Figure Legends**

942 **Fig 1. Separation of membrane protein complexes from C. jejuni strain 81-176 by BN-PAGE.** 10 µg (A) and 20 µg (B) of membrane protein complexes were solubilized with DDM
943
944 at concentrations ranging from 1, 2 and 5% (w/v). Mass marker are indicated in the central
945 lane (MW in kDa).

946 **Fig 2. 2D-BN/SDS-PAGE separation of membrane protein complexes of C. jejuni 81-176.** Acrylamide gradient was 4-18% for BN-PAGE and acrylamide concentration was 10%
947
948 for SDS-PAGE. Mass markers for BN-PAGE and SDS-PAGE are respectively indicated at
949 the top left side of the gel. Proteins identified by LC-MS/MS are indicated by **blue arrows**.
950
Proteins that do not reach identification criteria after LC-MS/MS analysis are indicated with
951 **black arrows**. First numbers correspond to complexes and second numbers to subunits of
952 these complexes.

953 **Fig 3. Membrane protein complexes identified by 2-D BN/SDS-PAGE in C. jejuni 81-176.** Colour of complexes corresponds to their function in cell: Green for molecules
954 trafficking, Red for protein biosynthesis and folding, Blue for respiration, Yellow for motility
955
956 and Orange for the oxidative phosphorylation.

957 **Fig 4. Phylogenetic tree of proteins belonging to efflux pumps CmeABC and MacABputC.** Alignment was performed using MAFFT (GeneiousR9) with functional
958
959 domain and protein structure (Philus) in 14 C. jejuni strains and 6 C. coli strains. Bootstraps
960 percentages were added to internal branches for 1000 replicates. **Strains used in this study are listed in Table S2.**
961

962 **Fig 5. Consensus sequence logo of CosR-binding box of the upstream sequences of genes**
963 with CosR-binding capacity (cmeA and macA). Consensus sequence logo of CosR-binding
964 box defined by Turonova *et al.*, 2017 (A) alignment of upstream sequences of cmeA and
965 macA by Geneious 9.1.8 (B) Consensus sequence logo of CosR-binding box redefined (C).

966 w-A or T; y-C or T; m-A or C and k-G or T.

967 **Fig 6. Distribution of CmeABC and MacABputC subunits in bacteria using Blastp with**

968 **C. jejuni** 81-176 protein sequences of each subunit as queries in RefSeq_protein

969 **database.** The data are based on the hits presented in Table S3.

970 Legend of figures in Supplementary data

971 **Figure S1. Homemade strips in 5 steps to separate the subunits of protein complexes in a reproducing manner.** A- Cut the strip with a clean glass plate following the well where the sample was deposited, equilibrate the gel lane in DTT and iodoacetamide buffers as indicated in material and methods; B- lay the gel strip on the wet plastic support using clean forceps; C- Stick the plastic face of the strip on the glass behind; D- Slide the strip until touching the precast polyacrylamide gel; E- Fill the gap and overlaid the strip with low melting agarose using a sterile syringe.

978 **Figure S2. 2D-BN-SDS-PAGE profiling before (A) and after optimization (B) of protein complexes and their subunit separation.** The first dimension for complex separation was performed on 4 to 18% acrylamide gradient gels with 2% Dodecyl-β-D-Maltoside (DDM).

982 **Figure S3. 2D-BN/SDS-PAGE western blot, using antibody anti-MOMP (A) and antibody anti-CadF (B).** The image in the middle corresponds to the identified proteins complexes before the Western-blot The black arrow indicate the identified proteins with anti-PorA antibody and the white arrow the identify protein with anti-CadF antibody on the 2D-BN/SDS-PAGE.

987
<table><caption>988 Table 1. Description of membrane protein complexes identified in *C. jejuni* strain 81-176
989 using two-dimensional BN/SDS-PAGE.</caption><thead><tr><th>Complex function</th><th>complex ID</th><th>Spot ID</th><th>Access No. (NCBI)</th><th>Protein ID</th><th>Gene name</th><th>Mascot score (a)</th><th>NPM/ PC (%) (b)</th><th>pl/MW (kDa) (theoretical)</th></tr></thead><tbody><tr><td rowspan="10">Oxidative phosphorylation</td><td>2</td><td>2.1</td><td>EAQ73056.1</td><td>Fumarate reductase flavoprotein subunit</td><td>frdA</td><td>413</td><td>12/20</td><td>6.36/74</td></tr><tr><td></td><td>2.2</td><td>EAQ73378.1</td><td>Fumarate reductase iron-sulfur subunit</td><td>frdB</td><td>153</td><td>4/22</td><td>5.37/27</td></tr><tr><td></td><td>2.3</td><td>EAQ73136.1</td><td>Fumarate reductase cytochrome b-556 subunit</td><td>frdC</td><td>38</td><td>2*/5</td><td>9.37/30</td></tr><tr><td>9</td><td>9.1</td><td>EAQ72593.1</td><td>Cytochrome c oxidase, cbb3-type, subunit II</td><td>ccoO</td><td>136</td><td>4/23</td><td>5.86/25</td></tr><tr><td>11</td><td>11.1</td><td>EAQ71910.1</td><td>F0F1 ATP synthase subunit beta</td><td>atpD</td><td>102</td><td>2/5</td><td>4.97/51</td></tr><tr><td>19</td><td>19.1</td><td>EAQ72569.1</td><td>NADH dehydrogenase subunit G</td><td>nuoG</td><td>205</td><td>6/9</td><td>5.49/93</td></tr><tr><td></td><td>19.2</td><td>EAQ72908.1</td><td>NADH dehydrogenase subunit D</td><td>nuoD</td><td>121</td><td>3/7</td><td>5.51/47</td></tr><tr><td></td><td>19.3</td><td>EAQ72659.1</td><td>NADH dehydrogenase subunit C</td><td>nuoC</td><td>131</td><td>2/10</td><td>7.77/31</td></tr><tr><td>8</td><td>8.1</td><td>EAQ72956.1</td><td>Formate dehydrogenase, alpha unit, selenocysteine-containing</td><td>fdhA</td><td>401</td><td>12/19</td><td>6.09/83</td></tr><tr><td rowspan="3">Respiration</td><td>12</td><td>8.2</td><td>EAQ72781.1</td><td>Formate dehydrogenase, iron-sulfur subunit</td><td>fdhB</td><td>65</td><td>2*/9</td><td>5.99/24</td></tr><tr><td></td><td>12.1</td><td>EAQ72716.1</td><td>Quinone-reactive Ni/Fe-hydrogenase, large subunit</td><td>hydB</td><td>98</td><td>2/6</td><td>6.26/64</td></tr><tr><td></td><td>12.2</td><td>EAQ72716.1</td><td>Quinone-reactive Ni/Fe-hydrogenase, large subunit</td><td>hydB</td><td>164</td><td>6/14</td><td>6.26/64</td></tr><tr><td rowspan="5">Protein biosynthesis and folding</td><td>0</td><td>0</td><td>EAQ72817.1</td><td>Chaperonin GroEL</td><td>groEL</td><td>698</td><td>16/39</td><td>5.02/58</td></tr><tr><td>4</td><td>4.1</td><td>EAQ71919.1</td><td>DsbB family disulfide bond formation protein</td><td>dsbB</td><td>139</td><td>3/7</td><td>8.57/57</td></tr><tr><td>10</td><td>10.1</td><td>EAQ73315.1</td><td>Penicillin-binding protein 1A</td><td>pbpA</td><td>81</td><td>3/5</td><td>8.35/73</td></tr><tr><td></td><td>10.2</td><td>EAQ73158.1</td><td>Methyl-accepting chemotaxis protein</td><td>CJJ_0180</td><td>101</td><td>3/5</td><td>4.94/73</td></tr><tr><td></td><td>10.3</td><td>EAQ73158.1</td><td>Methyl-accepting chemotaxis protein</td><td>CJJ_0180</td><td>132</td><td>4/6</td><td>4.94/73</td></tr><tr><td rowspan="10">Efflux pumps, virulence and molecules trafficking</td><td>3</td><td>3.1</td><td>EAQ72728.1</td><td>Major outer membrane protein</td><td>porA**</td><td>2004</td><td>14/49</td><td>4.72/46</td></tr><tr><td></td><td>3.2</td><td>EAQ72728.1</td><td>Major outer membrane protein</td><td>porA**</td><td>1989</td><td>14/48</td><td>4.72/46</td></tr><tr><td></td><td>3.3</td><td>EAQ72728.1</td><td>Major outer membrane protein</td><td>porA**</td><td>1133</td><td>8/30</td><td>4.72/46</td></tr><tr><td>5</td><td>5.1</td><td>EAQ73202.1</td><td>Outer membrane protein</td><td>bamA</td><td>70</td><td>2*/4</td><td>5.57/83</td></tr><tr><td></td><td>5.2</td><td>EAQ72997.1</td><td>Conserved hypothetical protein (putative lipoprotein)</td><td>CJJ_0419</td><td>196</td><td>5/17</td><td>8.48/37</td></tr><tr><td>6</td><td>6.1</td><td>EAQ73082.1</td><td>RND efflux system, outer membrane lipoprotein CmeC</td><td>cmeC</td><td>129</td><td>7/10</td><td>5.14/55</td></tr><tr><td></td><td>6.2</td><td>EAQ73146.1</td><td>RND efflux system, inner membrane transporter CmeB</td><td>cmeB</td><td>101</td><td>2/2</td><td>6.48/114</td></tr><tr><td></td><td>6.3</td><td>EAQ73146.1</td><td>RND efflux system, inner membrane transporter CmeB</td><td>cmeB</td><td>121</td><td>2/2</td><td>6.48/114</td></tr><tr><td></td><td>6.4</td><td>EAQ72976.1</td><td>RND efflux system, membrane fusion protein CmeA</td><td>cmeA</td><td>102</td><td>3/10</td><td>8.29/40</td></tr><tr><td>13</td><td>13.1</td><td>EAQ72728.1</td><td>Major outer membrane protein</td><td>porA**</td><td>231</td><td>4/11</td><td>4.72/46</td></tr><tr><td></td><td>13.2</td><td>EAQ72728.1</td><td>Major outer membrane protein</td><td>porA**</td><td>419</td><td>10/30</td><td>4.72/46</td></tr><tr><td>15</td><td>15.1</td><td>EAQ72952.1</td><td>Capsular polysaccharide ABC transporter</td><td>kpsE</td><td>136</td><td>3/8</td><td>6.22/43</td></tr><tr><td></td><td>15.2</td><td>EAQ72738.1</td><td>Outer membrane fibronectin-binding protein</td><td>cadF**</td><td>55</td><td>2/8</td><td>5.89/36</td></tr><tr><td>20</td><td>20.1</td><td>EAQ73027.1</td><td>Macrolide-specific efflux protein macB</td><td>CJJ_0636</td><td>35</td><td>2*/1</td><td>9.25/70</td></tr><tr><td></td><td>18.1</td><td>EAQ72087.1</td><td>CjaA protein</td><td>cjaA</td><td>21</td><td>1/4</td><td>5.69/31</td></tr><tr><td></td><td>18.2</td><td>EAQ72374.1</td><td>CjaC protein</td><td>cjaC</td><td>250</td><td>6/25</td><td>6.48/28</td></tr><tr><td>Mobility</td><td>17</td><td>17.1</td><td>EAQ72823.1</td><td>Flagellar basal body-associated protein FliL</td><td>fliL</td><td>47</td><td>1/15</td><td>4.93/20</td></tr><tr><td rowspan="4">Unknown</td><td>1</td><td>1.1</td><td>EAQ73148.1</td><td>Ketol acid reductoisomerase</td><td>ilvC</td><td>365</td><td>8/27</td><td>6.1/37</td></tr><tr><td></td><td>1.2</td><td>EAQ72988.1</td><td>Nonheme iron-containing ferritin</td><td>ftn</td><td>73</td><td>2/16</td><td>5.34/20</td></tr><tr><td>14</td><td>14.1</td><td>EAQ73158.1</td><td>Methyl-accepting chemotaxis protein</td><td>CJJ_0180</td><td>94</td><td>2/4</td><td>4.94/73</td></tr><tr><td></td><td>14.2</td><td>EAQ73158.1</td><td>Methyl-accepting chemotaxis protein</td><td>CJJ_0180</td><td>111</td><td>4/6</td><td>4.94/73</td></tr><tr><td>Other</td><td>16</td><td>16.1</td><td>EAQ73030.1</td><td>Elongation factor Tu</td><td>tuf</td><td>60</td><td>2/8</td><td>5.11/44</td></tr></tbody></table>

990

991 None of the proteins from the complex no. 7 have been identified.

992 (a) From all identified peptides (b) Peptides with only a significant individual ion score were considered for NPM (Number

993 of Peptide Match) and PC (Protein Coverage). * One peptide with a non-significant score but mainly identified from y and

994 b ions (see MS/MS fragmentation in supplementary data) are included to validate the identification. **proteins verified

995 by Western Blot

996

997
<figure><img src="image_2.png" /><figcaption>Figure 1: JPEG compression of a protein band at 669 kDa.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Figure 2.JPEG</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 3.JPEG</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 4.JPEG</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure 5.JPEG</figcaption></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

1. cmeA_81176_5

2. macA_81176_5

3. cmeA_Bof_5

4. macA_Bof_5

5. cmeA_002425_5

6. macA_002425_5

7. cmeA_002426_5

8. macA_002426_5

9. cmeA_002544_5

10. macA_002544_5

11. cmeA_81116_5

12. macA_81116_5

13. cmeA_IA3902_5

14. macA_IA3902_5

15. cmeA_NCTC11168_5

16. macA_NCTC11168_5

17. cmeA_PT14_5

18. macA_PT14_5

19. cmeA_4031_5

20. macA_4031_5

21. cmeA_M1_5

22. macA_002544_5

23. cmeA_RM1221_5

24. macA_RM1221_5

25. cmeA_S3_5

26. macA_S3_5

27. cmeA_002538_5

28. macA_002538_5
<figure><img src="image_9.png" /><figcaption>Figure 6.JPEG</figcaption></figure>