# Campylobacter jejuni Modulates Reactive Oxygen Species Production and NADPH Oxidase 1 Expression in Human Intestinal Epithelial Cells

Authors: Geunhye Hong; Cadi Davies; Zahra Omole; Janie Liaw; Anna D. Grabowska; Barbara Canonico; Nicolae Corcionivoschi; Brendan W. Wren; Nick Dorrell; Abdi Elmi; Ozan Gundogdu
Journal: Cellular Microbiology
 (2023)

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## Abstract

Campylobacter jejuni is the major bacterial cause of foodborne gastroenteritis worldwide. Mechanistically, how this pathogen interacts with intrinsic defence machinery of human intestinal epithelial cells (IECs) remains elusive. To address this, we investigated how C. jejuni counteracts the intracellular and extracellular reactive oxygen species (ROS) in IECs. Our work shows that C. jejuni differentially regulates intracellular and extracellular ROS production in human T84 and Caco-2 cells. C. jejuni downregulates the transcription and translation of nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase (NOX1), a key ROS-generating enzyme in IECs and antioxidant defence genes CAT and SOD1. Furthermore, inhibition of NOX1 by diphenylene iodonium (DPI) and siRNA reduced C. jejuni ability to interact, invade, and intracellularly survive within T84 and Caco-2 cells. Collectively, these findings provide mechanistic insight into how C. jejuni modulates the IEC defence machinery.


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## Body

## 1. Introduction

Microbial pathogens have evolved to possess subversion strategies to alter the functionality of host cells upon infection [1]. These include modulation of host cell functions that involve vesicle trafficking, apoptosis, and immune activation [2–4]. Crucially, these host cell functions are essential for elimination of foreign pathogens. The evolving battle between pathogen and host adds to the complexity of the pathogenesis of infection [1].Campylobacter jejuni is the leading foodborne bacterial cause of human gastroenteritis worldwide [5]. C. jejuni causes watery or bloody diarrhoea, abdominal pain, and fever. C. jejuni infection can also lead to Guillain-Barré syndrome (GBS), a rare but severe postinfectious autoimmune complication of the peripheral nervous system [5–7]. Importantly, campylobacteriosis in low-income countries is associated with child growth impairment and can be fatal in children [8]. Although C. jejuni is a microaerophilic bacterium, its omnipresence in the environment and various hosts is mitigated by regulatory mechanisms against oxidative stress [9]. Upon adhering and invading human intestinal epithelial cells (IECs), C. jejuni manipulates host cytoskeleton regulation to maximise its invasion [10]. Following invasion, C. jejuni resides in cytoplasmic vacuoles named Campylobacter containing vacuoles (CCVs) which can escape the canonical endocytic pathway and avoid fusion with lysosomes [11, 12]. These findings demonstrate that modulation and invasion of host IECs are a prerequisite for human intestinal disease caused by C. jejuni.A vital mechanism used by host cells in response to pathogens is the production of reactive oxygen species (ROS) which are highly reactive molecules, such as oxygen radicals and nonradicals, produced by the partial reduction of oxygen [13]. When phagocytes such as macrophages detect and engulf pathogens using the respiratory burst, ROS are rapidly generated to eradicate the engulfed pathogens through oxidative damage [14]. Interestingly, the level of ROS produced by human IECs is lower in comparison to resident macrophages and blood leukocytes (neutrophils and monocytes); however, ROS in IECs can also exhibit antimicrobial activity by inducing inflammation [14–16]. The precarious nature of ROS production by IECs is demonstrated by exhibiting both deleterious and beneficial host effects; thus, homeostasis of ROS is essential. To counter the damaging effects of ROS, host IECs possess antioxidant components that neutralise ROS, such as catalase, superoxide dismutase, and glutathione peroxidase. Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase; NOX) and mitochondria have central roles as predominant sources of ROS in human IECS [13].NOX is an essential multicomponent enzyme which catalyses production of superoxide (O2−) [17, 18]. In IECs, the most abundant types of NOX are NOX1 and NOX4. Intriguingly, NOX4 is constitutively active, whereas NOX1 is not. The NOX1 complex is composed of NOX1, p22phox, NOX organiser 1 (NOXO1), NOX activator (NOXA1), and small GTPase Rac1. NOX1 is the catalytic subunit of the complex on the plasma membrane, and its activation is dependent on supplementary cytosolic subunits. Following this, p22phox is transported to the plasma membrane promoted by NOX1 expression [17]. Upon activation, NOXO1 binds to both NOXA1 and p22phox targeting NOXA1 to the plasma membrane. In turn, NOXA1 binds to guanosine triphosphate- (GTP-) bound Rac1 and promotes electron flow through flavocytochrome in NOX1 in a GTP-dependent manner. Studies have shown that GTP-bound Rac1 is essential for activity of NOX1 [19, 20]. Electrons travel from NADPH initially to flavin adenine dinucleotide (FAD), then through the NOX heme groups and finally to oxygen, forming O2− [19]. Notably, NOX1-mediated ROS play important roles in IECs including regulation of growth and proliferation, epithelial wound healing, intestinal host defence, and maintenance of bacterial homeostasis in the GI tract [21–23].HowC. jejuni interacts with the inherent defence machinery of human IECs remains unclear. To explore this further, we examined the mechanisms C. jejuni uses to counteract the intracellular and extracellular ROS in IECs. Previous findings demonstrated the upregulation of NOX1 in IECs by enteric pathogens such as Escherichia coli [24], Salmonella enteritidis [25], and Helicobacter pylori [26, 27]. C. jejuni lacks classical stress response regulators such as OxyR and SoxRS that are found in other enteric bacteria [28, 29]. Instead, it possesses unique strategies to counteract oxidative stress such as CosR, PerR, and MarR-type regulators RrpA and RrpB [9, 30, 31]. Given these survival properties of C. jejuni, we hypothesised that C. jejuni may have distinct host cell modulation mechanisms in play. In this study, we show that diverse C. jejuni strains downregulate both intracellular and extracellular ROS production in human IECs by modulating the expression of NOX1. We demonstrate inhibition of NOX1 by diphenylene iodonium (DPI), and siRNA reduced the ability of C. jejuni to interact, invade, and intracellularly survive within T84 and Caco-2 cells. Our results highlight a unique strategy of C. jejuni survival and emphasise the importance of NOX1 in C. jejuni-IEC interactions. This represents a distinctive mechanism that C. jejuni uses to modulate IEC defence machinery.


## 2. Experimental Procedures

### 2.1. Bacterial Strains and Growth Conditions

C. jejuni wild-type strains used in this study are listed in Table S1. For general growth, all C. jejuni strains were grown on Columbia Blood Agar (CBA) plates (Oxoid, UK) supplemented with 7% (v/v) horse blood (TCS Microbiology, UK) and Campylobacter selective supplement Skirrow (Oxoid) at 37 °C under microaerobic conditions (10% CO2, 5% O2, and 85% N2) (Don Whitley Scientific, UK).


### 2.2. Human Intestinal Epithelial Cell Culture

T84 cells (ECACC 88021101) and Caco-2 cells (ECACC 86010202) were obtained from European Collection of Authenticated Cell Cultures (ECACC). T84 and Caco-2 cells were cultured in a 1 : 1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (DMEM/F-12; Thermo Fisher Scientific, USA) with 10% fetal bovine serum (FBS; Labtech, UK), 1% nonessential amino acid (Sigma-Aldrich, USA), and 1% penicillin-streptomycin (Sigma-Aldrich). Both cell lines were cultured at 37°C in a 5% CO2-humidified environment. DMEM/F-12 without penicillin-streptomycin was used for the coculture assays. DMEM/F-12 without phenol red was used for the ROS detection assays.


### 2.3. T84 and Caco-2 Cell Infection Assays

Human IECs were counted using hemocytometer (Thermo Fisher Scientific, USA), and for general infection assays, approximately 105 cells were seeded in 24-well tissue culture plates 7 days prior to initiation of the C. jejuni infection. The plates were incubated at 37°C in a 5% CO2 atmosphere. For Western blotting, approximately 2×105 cells were seeded in 6-well tissue culture plates. Prior to the infection, IECs were washed with phosphate-buffered saline (PBS; Thermo Fisher Scientific) three times and the medium was replaced with DMEM/F-12 without penicillin-streptomycin. C. jejuni strains grown on CBA plates for 24 hours were resuspended in PBS, and bacterial suspension with appropriate OD600 was then incubated with IECs for various time periods giving a multiplicity of infection (MOI) of 200 : 1. In some experiments, T84 and Caco-2 cells were pretreated with 10 μM DPI for 1 hour, washed three times with PBS, and then infected with C. jejuni. As with our previous infection assays [32, 33], we primarily focus on relatively early time points as many of the transcriptional and translational changes that we observe in the crosstalk between C. jejuni and IECs in our in vitro assays are ephemeral in nature, and so our focus is to detect these subtle deviations that lead to mechanistic impact.


### 2.4. DCFDA Measurement of Intracellular Reactive Oxygen Species (ROS)

To analyse the levels of intracellular ROS production in human IECs under experiments conditions, DCFDA Cellular ROS Detection Assay Kit (Abcam, UK) was used according to the manufacturer’s instructions. Briefly, IECs grown in 96-well cell culture plates were washed three times with PBS and incubated withC. jejuni for 3 or 24 hours (MOI 200 : 1). For positive controls, IECs were treated with 500 μM H2O2 for 45 minutes. 45 minutes prior to completion of the infection, 100 μM 2′,7′-dichlorofluorescin diacetate (DCFDA) was added into each well giving a final concentration of 50 μM. After C. jejuni infection, the fluorescence was detected using SpectraMax M3 Multi-Mode Microplate Reader (Molecular Devices, USA) with 485 nm excitation and 535 nm emission.


### 2.5. Measurement of Extracellular H2O2

Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen, USA) was used to measure extracellular H2O2 in culture media after incubation with C. jejuni. Briefly, Amplex® Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) with horseradish peroxidase (HRP) reacts with H2O2 in a 1 : 1 stoichiometry producing a fluorescent product called resorufin. After incubation with C. jejuni for 3 or 24 hours, 100 μl of culture media was transferred to a 96-well plate and 100 μl of reaction mixture containing 50 μM Amplex® Red reagent was added followed by incubation for 10 minutes at 37°C in a 5% CO2-humidified environment. Using SpectraMax M3 Multi-Mode Microplate Reader, fluorescence was measured at 530 nm excitation and 590 nm emission.


### 2.6. Real-Time Quantitative Polymerase Chain Reaction (qRT-PCR) Analysis

For qRT-PCR, RNA was isolated from infected and uninfected IECs using PureLink™ RNA Mini Kit (Thermo Fisher Scientific) and contaminating DNA was removed using TURBO DNA-free kit (Ambion, USA) according to the manufacturer’s instructions. Concentration and purity of RNA samples were determined in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). 400 ng of RNA per sample were first denatured at 65°C for 5 minutes and snap cooled on ice. Complementary DNA (cDNA) was generated with random hexamers and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). Each reaction had 10μl of SYBR Green PCR Master Mix (Applied Biosystems, USA), 1 μl of primer (20 pmol), 1 μl of cDNA, and 10 μl of HyClone™ water (Thermo Fisher Scientific). The sequence of each primer is described in Table S2. All reactions were run in triplicate on an ABI-PRISM 7500 instrument (Applied Biosystems). Relative expression changes were calculated using the comparative CT cycle (2-ΔΔCT) method [34]. Expression levels of all target genes were normalised to gapdh expression determined in the same sample as an internal control. A minimum of three biological replicates were always analysed, each with three technical replicates.


### 2.7. Semiquantitative Reverse Transcription (RT-PCR) Analysis

Each PCR reaction had 50μl of FasTaq PCR master mix (Qiagen, Netherlands), 2 μl of primer (0.4 nmol) described in Table S2, and 1.5 μl of cDNA. For PCR reactions, Tetrad-2 Peltier thermal cycler (Bio-Rad, UK) was used. One cycle of PCR programme performs 95°C for 15 seconds after 2 minutes in the first cycle, annealing at 50°C for 20 seconds, and extension at 72°C for 30 seconds. Total 36 cycles were repeated. The PCR products were loaded on the 1% agarose gel, and the gel was running for 1 hour at 120 V. The gel was imaged using G:BOX Chime XRQ (Syngene, USA). Quantification of relative mRNA level was performed using ImageJ software [35].


### 2.8. SDS-PAGE and Western Blot Analysis

After infection, IECs were washed three times with PBS and lysed with cold RIPA lysis and extraction buffer (Thermo Fisher Scientific) with cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Roche, Switzerland) and cleared by centrifugation (4°C,13,000×g, 20 min). Protein concentration was determined using Pierce™ Bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Afterward, samples were diluted to a desired concentration in HyClone™ water and 4× Laemmli sample buffer (Sigma-Aldrich) and incubated for 5 minutes at 95°C. 30 μg of protein samples was separated using 4-12% NuPAGE™ Bis-Tris gel in 1× NuPAGE™ MES buffer or MOPS buffer (Thermo Fisher Scientific). Proteins were transferred from the gel using the iBlot® 2 transfer stacks (Life Technologies, USA) using the iBlot® Gel Transfer Device (Invitrogen). These stacks were integrated with nitrocellulose transfer membrane. After the transfer, membranes were blocked with 1× PBS containing 2% (w/v) milk. Membranes were then probed with primary antibodies overnight as described previously [32]. The following primary antibodies were used: GAPDH (ab181602, Abcam), NOX1 (ab101027, Abcam), or NOX1 (NBP-31546, Novus Biologicals). Blots were developed using LI-COR infrared secondary antibody (IRDye 800CW Donkey anti-rabbit IgG) and imaged on a LI-COR Odyssey Classic (LI-COR Biosciences, USA). Quantification of relative protein levels normalised to GAPDH expression was performed using ImageJ software [35].


### 2.9. Detection of GTP-Bound Active Rac1

The levels of active GTP-bound Rac1 were measured by using Rac1 G-LISA kit (Cytoskeleton Inc., USA) according to the manufacturer’s instructions. Briefly, before the infection, IECs were incubated with reduced serum (0.1% FBS) for 24 hours. Infected or uninfected human IECs were washed with 1× PBS and lysed using the supplied 1× lysis buffer. Cell lysates were centrifuged for 1 minute at10,000×g at 4°C and adjusted to 1 mg/ml for the further process of the assay. As a positive control, constitutively active Rac1 (RCCA) was provided in the kit. Three biological replicates were conducted in all experiments, along with two technical replicates for each assay.


### 2.10. Inhibition of NOX1 with Diphenyleneiodonium Chloride (DPI)

A stock solution of 3.25 mM DPI (Sigma-Aldrich) in dimethyl sulfoxide (DMSO, Sigma-Aldrich) was prepared and stored at -20°C. For treatment, the DPI stock solution was diluted to 10μM DPI in culture media without antibiotics and then incubated with IECs for 1 hour at 37°C in a 5% CO2 atmosphere. After treatment, IECs were washed with PBS for three times before coincubation with C. jejuni for various time points.


### 2.11. Small Interfering (si) RNA Transfection

On the day of reverse transfection, 500μl of Caco-2 cells (105 cells/ml) was seeded in 24-well plates and treated for 24 hours with 30 pmol siRNA from either NOX1 siRNA (sc-43939; Santa Cruz Biotechnology Inc., USA) or Ambion® Silencer Negative Control #1 siRNA (Invitrogen) for the negative control. For preparation of siRNA transfection reagent complex, 3 μl of 10 μM stock siRNA was diluted with 100 μl of Opti-MEM® Reduced-Serum Medium (Thermo Fisher Scientific) and mixed with 1.5 μl of Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific). After 24 hours transfection, media was replaced with DMEM/F-12 containing 10% FBS. After additional 48 or 72 hours, RNA and protein were extracted to check efficacy of transfection.


### 2.12. Adhesion, Invasion, and Intracellular Survival Assay

Adherence, invasion, and intracellular assays were performed as described previously with minor modifications [32, 33]. T84 and Caco-2 cells seeded in a 24-well plate were washed three times with PBS and treated with 10 μM DPI for 1 hour or transfected with NOX1 siRNA as described above. Then, IECs were inoculated with C. jejuni with OD600 0.2 at a MOI of 200 : 1 and incubated for 3 hours at 37°C in 5% CO2. For the interaction (adhesion and invasion) assay, monolayers were washed three times with PBS to remove unbound extracellular bacteria and then lysed with PBS containing 0.1% (v/v) Triton X-100 (Sigma-Aldrich) for 20 min at room temperature. The cell lysates were diluted and plated on blood agar plates to determine the number of interacting bacteria (CFU/ml).Invasion assays were performed by additional step of treatment of gentamicin (150μg/ml) for 2 hours to kill extracellular bacteria, washed three times with PBS, lysed, and plated as described above. For intracellular survival assays, after infection with C. jejuni for 3 hours, T84 and Caco-2 cells were treated with gentamicin (150 μg/ml) for 2 hours to kill extracellular bacteria followed by further 18 hours incubation with gentamicin (10 μg/ml). Cell lysis and inoculation were performed as described above.


### 2.13. Cytotoxicity Assay with Trypan Blue Exclusion Methods

After treatment with DPI and gentamicin or transfection with siRNA as previously described, IECs were washed three times with PBS and were detached using trypsin-EDTA (Thermo Fisher Scientific) and resuspended with culture media. 50μl of cell suspension was added into 50 μl of 0.4% trypan blue solution (Thermo Fisher Scientific), and the numbers of viable and dead cells were counted using hemocytometer under a microscope.


### 2.14.Campylobacter jejuni Viability Test with DPI Treatment

T84 cells were treated with 10μM DPI for 1 hour, and the cells were washed three times with PBS. After DPI treatment for 1 hour, C. jejuni strains (OD600 0.2) were coincubated for 1 hour with PBS from the last wash. After incubation, serial dilution was performed and each dilution was spotted on to blood agar plates. The plates were incubated under microaerobic condition at 37°C for 48 hours. CFU of each spot was recorded.


### 2.15. Statistical Analysis and Graphing

At least three biological replicates were performed in all experiments. Each biological replicate was performed in three technical replicates. For statistical analysis and graphing, GraphPad Prism 8 for Windows (GraphPad Software, USA) was used. One samplet-test or unpaired t-test was used to compare two data sets for significance with ∗ indicating p<0.05, ∗∗ indicating p<0.01, ∗∗∗ indicating p<0.001, and ∗∗∗∗ indicating p<0.0001.


## 2.1. Bacterial Strains and Growth Conditions

C. jejuni wild-type strains used in this study are listed in Table S1. For general growth, all C. jejuni strains were grown on Columbia Blood Agar (CBA) plates (Oxoid, UK) supplemented with 7% (v/v) horse blood (TCS Microbiology, UK) and Campylobacter selective supplement Skirrow (Oxoid) at 37 °C under microaerobic conditions (10% CO2, 5% O2, and 85% N2) (Don Whitley Scientific, UK).


## 2.2. Human Intestinal Epithelial Cell Culture

T84 cells (ECACC 88021101) and Caco-2 cells (ECACC 86010202) were obtained from European Collection of Authenticated Cell Cultures (ECACC). T84 and Caco-2 cells were cultured in a 1 : 1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (DMEM/F-12; Thermo Fisher Scientific, USA) with 10% fetal bovine serum (FBS; Labtech, UK), 1% nonessential amino acid (Sigma-Aldrich, USA), and 1% penicillin-streptomycin (Sigma-Aldrich). Both cell lines were cultured at 37°C in a 5% CO2-humidified environment. DMEM/F-12 without penicillin-streptomycin was used for the coculture assays. DMEM/F-12 without phenol red was used for the ROS detection assays.


## 2.3. T84 and Caco-2 Cell Infection Assays

Human IECs were counted using hemocytometer (Thermo Fisher Scientific, USA), and for general infection assays, approximately 105 cells were seeded in 24-well tissue culture plates 7 days prior to initiation of the C. jejuni infection. The plates were incubated at 37°C in a 5% CO2 atmosphere. For Western blotting, approximately 2×105 cells were seeded in 6-well tissue culture plates. Prior to the infection, IECs were washed with phosphate-buffered saline (PBS; Thermo Fisher Scientific) three times and the medium was replaced with DMEM/F-12 without penicillin-streptomycin. C. jejuni strains grown on CBA plates for 24 hours were resuspended in PBS, and bacterial suspension with appropriate OD600 was then incubated with IECs for various time periods giving a multiplicity of infection (MOI) of 200 : 1. In some experiments, T84 and Caco-2 cells were pretreated with 10 μM DPI for 1 hour, washed three times with PBS, and then infected with C. jejuni. As with our previous infection assays [32, 33], we primarily focus on relatively early time points as many of the transcriptional and translational changes that we observe in the crosstalk between C. jejuni and IECs in our in vitro assays are ephemeral in nature, and so our focus is to detect these subtle deviations that lead to mechanistic impact.


## 2.4. DCFDA Measurement of Intracellular Reactive Oxygen Species (ROS)

To analyse the levels of intracellular ROS production in human IECs under experiments conditions, DCFDA Cellular ROS Detection Assay Kit (Abcam, UK) was used according to the manufacturer’s instructions. Briefly, IECs grown in 96-well cell culture plates were washed three times with PBS and incubated withC. jejuni for 3 or 24 hours (MOI 200 : 1). For positive controls, IECs were treated with 500 μM H2O2 for 45 minutes. 45 minutes prior to completion of the infection, 100 μM 2′,7′-dichlorofluorescin diacetate (DCFDA) was added into each well giving a final concentration of 50 μM. After C. jejuni infection, the fluorescence was detected using SpectraMax M3 Multi-Mode Microplate Reader (Molecular Devices, USA) with 485 nm excitation and 535 nm emission.


## 2.5. Measurement of Extracellular H2O2

Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen, USA) was used to measure extracellular H2O2 in culture media after incubation with C. jejuni. Briefly, Amplex® Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) with horseradish peroxidase (HRP) reacts with H2O2 in a 1 : 1 stoichiometry producing a fluorescent product called resorufin. After incubation with C. jejuni for 3 or 24 hours, 100 μl of culture media was transferred to a 96-well plate and 100 μl of reaction mixture containing 50 μM Amplex® Red reagent was added followed by incubation for 10 minutes at 37°C in a 5% CO2-humidified environment. Using SpectraMax M3 Multi-Mode Microplate Reader, fluorescence was measured at 530 nm excitation and 590 nm emission.


## 2.6. Real-Time Quantitative Polymerase Chain Reaction (qRT-PCR) Analysis

For qRT-PCR, RNA was isolated from infected and uninfected IECs using PureLink™ RNA Mini Kit (Thermo Fisher Scientific) and contaminating DNA was removed using TURBO DNA-free kit (Ambion, USA) according to the manufacturer’s instructions. Concentration and purity of RNA samples were determined in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). 400 ng of RNA per sample were first denatured at 65°C for 5 minutes and snap cooled on ice. Complementary DNA (cDNA) was generated with random hexamers and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). Each reaction had 10μl of SYBR Green PCR Master Mix (Applied Biosystems, USA), 1 μl of primer (20 pmol), 1 μl of cDNA, and 10 μl of HyClone™ water (Thermo Fisher Scientific). The sequence of each primer is described in Table S2. All reactions were run in triplicate on an ABI-PRISM 7500 instrument (Applied Biosystems). Relative expression changes were calculated using the comparative CT cycle (2-ΔΔCT) method [34]. Expression levels of all target genes were normalised to gapdh expression determined in the same sample as an internal control. A minimum of three biological replicates were always analysed, each with three technical replicates.


## 2.7. Semiquantitative Reverse Transcription (RT-PCR) Analysis

Each PCR reaction had 50μl of FasTaq PCR master mix (Qiagen, Netherlands), 2 μl of primer (0.4 nmol) described in Table S2, and 1.5 μl of cDNA. For PCR reactions, Tetrad-2 Peltier thermal cycler (Bio-Rad, UK) was used. One cycle of PCR programme performs 95°C for 15 seconds after 2 minutes in the first cycle, annealing at 50°C for 20 seconds, and extension at 72°C for 30 seconds. Total 36 cycles were repeated. The PCR products were loaded on the 1% agarose gel, and the gel was running for 1 hour at 120 V. The gel was imaged using G:BOX Chime XRQ (Syngene, USA). Quantification of relative mRNA level was performed using ImageJ software [35].


## 2.8. SDS-PAGE and Western Blot Analysis

After infection, IECs were washed three times with PBS and lysed with cold RIPA lysis and extraction buffer (Thermo Fisher Scientific) with cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Roche, Switzerland) and cleared by centrifugation (4°C,13,000×g, 20 min). Protein concentration was determined using Pierce™ Bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Afterward, samples were diluted to a desired concentration in HyClone™ water and 4× Laemmli sample buffer (Sigma-Aldrich) and incubated for 5 minutes at 95°C. 30 μg of protein samples was separated using 4-12% NuPAGE™ Bis-Tris gel in 1× NuPAGE™ MES buffer or MOPS buffer (Thermo Fisher Scientific). Proteins were transferred from the gel using the iBlot® 2 transfer stacks (Life Technologies, USA) using the iBlot® Gel Transfer Device (Invitrogen). These stacks were integrated with nitrocellulose transfer membrane. After the transfer, membranes were blocked with 1× PBS containing 2% (w/v) milk. Membranes were then probed with primary antibodies overnight as described previously [32]. The following primary antibodies were used: GAPDH (ab181602, Abcam), NOX1 (ab101027, Abcam), or NOX1 (NBP-31546, Novus Biologicals). Blots were developed using LI-COR infrared secondary antibody (IRDye 800CW Donkey anti-rabbit IgG) and imaged on a LI-COR Odyssey Classic (LI-COR Biosciences, USA). Quantification of relative protein levels normalised to GAPDH expression was performed using ImageJ software [35].


## 2.9. Detection of GTP-Bound Active Rac1

The levels of active GTP-bound Rac1 were measured by using Rac1 G-LISA kit (Cytoskeleton Inc., USA) according to the manufacturer’s instructions. Briefly, before the infection, IECs were incubated with reduced serum (0.1% FBS) for 24 hours. Infected or uninfected human IECs were washed with 1× PBS and lysed using the supplied 1× lysis buffer. Cell lysates were centrifuged for 1 minute at10,000×g at 4°C and adjusted to 1 mg/ml for the further process of the assay. As a positive control, constitutively active Rac1 (RCCA) was provided in the kit. Three biological replicates were conducted in all experiments, along with two technical replicates for each assay.


## 2.10. Inhibition of NOX1 with Diphenyleneiodonium Chloride (DPI)

A stock solution of 3.25 mM DPI (Sigma-Aldrich) in dimethyl sulfoxide (DMSO, Sigma-Aldrich) was prepared and stored at -20°C. For treatment, the DPI stock solution was diluted to 10μM DPI in culture media without antibiotics and then incubated with IECs for 1 hour at 37°C in a 5% CO2 atmosphere. After treatment, IECs were washed with PBS for three times before coincubation with C. jejuni for various time points.


## 2.11. Small Interfering (si) RNA Transfection

On the day of reverse transfection, 500μl of Caco-2 cells (105 cells/ml) was seeded in 24-well plates and treated for 24 hours with 30 pmol siRNA from either NOX1 siRNA (sc-43939; Santa Cruz Biotechnology Inc., USA) or Ambion® Silencer Negative Control #1 siRNA (Invitrogen) for the negative control. For preparation of siRNA transfection reagent complex, 3 μl of 10 μM stock siRNA was diluted with 100 μl of Opti-MEM® Reduced-Serum Medium (Thermo Fisher Scientific) and mixed with 1.5 μl of Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific). After 24 hours transfection, media was replaced with DMEM/F-12 containing 10% FBS. After additional 48 or 72 hours, RNA and protein were extracted to check efficacy of transfection.


## 2.12. Adhesion, Invasion, and Intracellular Survival Assay

Adherence, invasion, and intracellular assays were performed as described previously with minor modifications [32, 33]. T84 and Caco-2 cells seeded in a 24-well plate were washed three times with PBS and treated with 10 μM DPI for 1 hour or transfected with NOX1 siRNA as described above. Then, IECs were inoculated with C. jejuni with OD600 0.2 at a MOI of 200 : 1 and incubated for 3 hours at 37°C in 5% CO2. For the interaction (adhesion and invasion) assay, monolayers were washed three times with PBS to remove unbound extracellular bacteria and then lysed with PBS containing 0.1% (v/v) Triton X-100 (Sigma-Aldrich) for 20 min at room temperature. The cell lysates were diluted and plated on blood agar plates to determine the number of interacting bacteria (CFU/ml).Invasion assays were performed by additional step of treatment of gentamicin (150μg/ml) for 2 hours to kill extracellular bacteria, washed three times with PBS, lysed, and plated as described above. For intracellular survival assays, after infection with C. jejuni for 3 hours, T84 and Caco-2 cells were treated with gentamicin (150 μg/ml) for 2 hours to kill extracellular bacteria followed by further 18 hours incubation with gentamicin (10 μg/ml). Cell lysis and inoculation were performed as described above.


## 2.13. Cytotoxicity Assay with Trypan Blue Exclusion Methods

After treatment with DPI and gentamicin or transfection with siRNA as previously described, IECs were washed three times with PBS and were detached using trypsin-EDTA (Thermo Fisher Scientific) and resuspended with culture media. 50μl of cell suspension was added into 50 μl of 0.4% trypan blue solution (Thermo Fisher Scientific), and the numbers of viable and dead cells were counted using hemocytometer under a microscope.


## 2.14.Campylobacter jejuni Viability Test with DPI Treatment

T84 cells were treated with 10μM DPI for 1 hour, and the cells were washed three times with PBS. After DPI treatment for 1 hour, C. jejuni strains (OD600 0.2) were coincubated for 1 hour with PBS from the last wash. After incubation, serial dilution was performed and each dilution was spotted on to blood agar plates. The plates were incubated under microaerobic condition at 37°C for 48 hours. CFU of each spot was recorded.


## 2.15. Statistical Analysis and Graphing

At least three biological replicates were performed in all experiments. Each biological replicate was performed in three technical replicates. For statistical analysis and graphing, GraphPad Prism 8 for Windows (GraphPad Software, USA) was used. One samplet-test or unpaired t-test was used to compare two data sets for significance with ∗ indicating p<0.05, ∗∗ indicating p<0.01, ∗∗∗ indicating p<0.001, and ∗∗∗∗ indicating p<0.0001.


## 3. Results

### 3.1.Campylobacter jejuni Modulates Intracellular and Extracellular ROS in T84 and Caco-2 Cells in a Time- and Strain-Dependent Manner

AsC. jejuni possesses distinct physiological characteristics compared to more studied enteric pathogens, we assessed the ability of three distinct C. jejuni strains to modulate intracellular and extracellular ROS in T84 and Caco-2 cells [28]. We observed strain-specific ROS modulation at 3- and 24-hour postinfection (Figure 1). All three C. jejuni strains reduced the levels of intracellular ROS in T84 and Caco-2 cells compared to the uninfected control (Figures 1(a), 1(b), 1(e), and 1(f)). A similar pattern was observed for extracellular ROS where all C. jejuni strains reduced the levels of extracellular ROS in T84 and Caco-2 cells compared to the uninfected control (Figures 1(c), 1(d), 1(g), and 1(h)). A distinct pattern was observed when assessing levels of extracellular ROS for C. jejuni 81-176 strain at a 3-hour postinfection (Figures 1(c) and 1(g)). At this early time point, extracellular ROS is increased in T84 and Caco-2 cells infected with C. jejuni 81-176, although we observed similar reduced levels of ROS at 24 hours. As controls, C. jejuni infection did not affect viability of both T84 and Caco-2 cells (Figure S1) and levels of ROS were unchanged when C. jejuni was resuspended in DMEM in the absence of IECs (Figure S2). Collectively, these results indicate a strain- and time-specific pattern linking the ability of different C. jejuni strains to modulate intracellular and extracellular ROS levels in T84 and Caco-2 cells.Figure 1
Detection of intracellular and extracellular ROS in T84 and Caco-2 cells after infection withC. jejuni 11168H, 81-176, or 488 strains. Intracellular ROS in T84 cells after infection with C. jejuni for (a) 3 hours or (b) 24 hours and extracellular ROS from T84 cells after infection with C. jejuni for (c) 3 hours or (d) 24 hours were measured. Intracellular ROS in Caco-2 cells after infection of C. jejuni for (e) 3 hours or (f) 24 hours and extracellular ROS from Caco-2 cells after infection for (g) 3 hours or (h) 24 hours were measured. For detection of intracellular ROS, DCFDA was used. For detection of extracellular ROS, Amplex® Red reagent with HRP was used. H2O2 was used as a positive control. Experiments were repeated in three biological and three technical replicates. Asterisks denote a statistically significant difference (∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001).
(a)(b)(c)(d)(e)(f)(g)(h)


### 3.2.Campylobacter jejuni Modulates Intracellular and Extracellular ROS in T84 and Caco-2 Cells via the Downregulation of NOX1 Complex

Given the observed modulation of intracellular and extracellular ROS in T84 and Caco-2 cells, we next explored the mechanism by whichC. jejuni strains orchestrate ROS modulation. We analysed the transcription and translation of NOX1 which is the main source of ROS production in IECs [17, 18]. As shown in Figure 2, NOX1 transcription and translation levels were significantly reduced in both T84 (Figure 2(a)) and Caco-2 cells (Figure 2(b)) infected with C. jejuni when compared to uninfected cells. Notably, at a 24-hour postinfection, mRNA levels of NOX1 in T84 cells are significantly reduced compared with C. jejuni-infected Caco-2 cells. We measured the relative levels of mRNA between T84 and Caco-2 cells and identified T84 cells expressed a higher basal level of NOX1 mRNA compared to Caco-2 cells (Figure 2(c)). As a result of this higher basal level of NOX1 mRNA in T84 cells, we validated our qRT-PCR data using RT-PCR where less expression of NOX1 in C. jejuni-infected T84 cells was observed (Figures 2(d) and 2(e)). Reduction in the translational level of NOX1 in C. jejuni-infected T84 cells was confirmed independently by Western blotting (Figures 2(f) and 2(g)).Figure 2
C. jejuni modulates NOX1 expression in T84 and Caco-2 cells. qRT-PCR showing expression of NOX1 in (a) T84 and (b) Caco-2 cells. (c) RT-PCR showing expression of NOX1 in uninfected T84 and Caco-2 cells. GAPDH was used as an internal control. (d) RT-PCR showing expression of NOX1 in T84 cells infected with C. jejuni for 24 hours and (e) relative mRNA levels as a percentage from RT-PCR data. (f) Western blotting showing NOX1 in T84 cells infected with C. jejuni for 24 hours and (g) relative protein level as a percentage from Western blotting. Asterisks denote a statistically significant difference (∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001).
(a)(b)(c)(d)(e)(f)(g)


### 3.3.Campylobacter jejuni Modulates Activity of Small GTPase Rac1 in T84 and Caco-2 Cells in a Time-Dependent Manner

To gain further insight into the mechanism that leads toC. jejuni modulation of ROS in T84 and Caco-2 cells, we examined the ability of C. jejuni to activate Rac1, a member of the Rho family of small GTPases. Rac1 undergoes cycling between active GTP- and inactive GDP-bound form which switches activation of cellular response upon stimuli [36]. Although GTP-bound Rac1 is implicated in NOX1 activation in several eukaryotic cell lines [19, 20], the contribution of Rac1 in C. jejuni-mediated NOX1 modulation is unknown. As shown in Figure 3, active GTP-bound Rac1 is an integral part of the NOX1 complex. Given that C. jejuni activates Rac1 in human INT 407 cells via Campylobacter invasion antigen D (CiaD) [10, 37] and that Rac1 supports NOX1 activity only in its GTP-bound active form, we examined the abundance of GTP-bound Rac1 to investigate if downregulation of NOX1 is linked to modulation of Rac1 by C. jejuni. Interestingly, C. jejuni 11168H strain induced Rac1 1 and 3 hours after infection in T84 cells (Figure 4(a)). After 24 hours of infection, Rac1 activity was reduced (though not statistically significant; p=0.0714) (Figure 4(a)). Similarly, C. jejuni 11168H induced Rac1 activity after 1 hour of infection in Caco-2 cells (Figure 4(b)). However, this activity was reduced after 3- and 24-hours of infection (Figure 4(b)). These results suggest that the downregulation of NOX1 by C. jejuni is inversely correlated with an increase in Rac1 GTPase activity.Figure 3
Proposed structure of the NOX1 complex consisting of NOX1, p22phox, GTP-bound Rac1, NOXA1, and NOXO1. p22phox and other subcellular subunits are assembled to activate catalytic subunit NOX1 which results in the generation of O2− by oxidising NADPH [17]. Created with http://BioRender.com.Figure 4
C. jejuni modulates activity of small GTPase Rac1 in T84 and Caco-2 cells. (a) T84 and (b) Caco-2 cells were infected with C. jejuni 11168H strain for 1, 3, and 24 hours, and the activation of small GTPase Rac1 in each time point was measured. Constitutively, active Rac1 (RCCA) was used as a positive control. Experiments were repeated in three biological and three technical replicates. Asterisks denote a statistically significant difference (∗p<0.05; ∗∗p<0.001).
(a)(b)


### 3.4.Campylobacter jejuni Modulates Transcription of Antioxidant-Related Genes in T84 and Caco-2 Cells

To gain further insight into the ability ofC. jejuni to modulate intracellular and extracellular ROS in T84 and Caco-2 cells, we sought to understand if C. jejuni modulates the expression of two important antioxidant genes, superoxide dismutase 1 (SOD1) and catalase (CAT). SOD1 decomposes O2- to H2O2, and CAT breaks down H2O2 to H2O and O2 [13]. Intriguingly, as shown in Figures 5(a) and 5(b), there is a significant downregulation of the mRNA levels of CAT and SOD1 at 24 hours of postinfection in T84 cells. A similar pattern was observed when compared with Caco-2 cells where the expression of CAT and SOD1 at 24 hours of postinfection is significantly downregulated (Figures 5(c) and 5(d)). In contrast, the expression of CAT and SOD1 at 3 hours of postinfection is unaffected. These results may indicate C. jejuni-mediated reduction in intracellular and extracellular ROS is independent of modulation of CAT and SOD1.Figure 5
qRT-PCR showing expression of human catalase (CAT) and superoxide dismutase 1 (SOD1) in T84 and Caco-2 cells. (a, b) T84 and (c, d) Caco-2 cells were infected with C. jejuni for 3 or 24 hours, and transcriptional levels of CAT and SOD1 were measured. GAPDH was used as an internal control. Experiments were repeated in three biological and three technical replicates. Asterisks denote a statistically significant difference (∗∗p<0.01; ∗∗∗p<0.001; ∗∗∗∗p<0.0001).
(a)(b)(c)(d)


### 3.5. Chemical Inhibition of NOX1 Activity by DPI ImpairsCampylobacter jejuni Interaction, Invasion, and Intracellular Survival of T84 and Caco-2 Cells in vitro

Having established thatC. jejuni significantly reduced the transcription and translation of NOX1 in T84 and Caco-2 cells in a time-dependent manner, and that Rac1 is not only known as a key component of the NOX1 complex, but also implicated in cell dynamic morphology [19, 20], we hypothesised Rac1-mediated NOX1 might modulate membrane ruffling and cytoskeleton rearrangement which might in turn affect C. jejuni interaction with IECs. Therefore, we investigated the role of NOX1 in C. jejuni interaction, invasion, and intracellular survival in IECs by transiently pretreating T84 and Caco-2 cells with DPI (10 μM) which is known to inhibit activity of flavoenzymes including NOX complex [38]. First, we demonstrated that DPI reduced ROS in T84 and Caco-2 cells (Figure S3). As shown in Figures 6(a), 6(c), and 6(e), pretreatment of T84 cells by DPI significantly reduced the ability of C. jejuni to interact, invade, and survive intracellularly in T84 cells. Similarly, as shown in Figures 6(b), 6(d), and 6(f), C. jejuni infected with DPI-treated Caco-2 cells showed significant reduction in interaction, invasion, and intracellular survival compared to untreated Caco-2 cells. Since our data revealed C. jejuni reduced interaction, invasion, and intracellular survival between the control- and DPI-treated T84 and Caco-2 cells, we next evaluated the viability of C. jejuni, T84, and Caco-2 cells coincubated with DPI. Treatment with DPI did not affect viability of IECs (Figure S4) or C. jejuni (Figure S5). Thus, our observations suggest further inhibition of NOX1 with DPI is detrimental to C. jejuni interaction, invasion, and intracellular survival in IECs.Figure 6
The effect of DPI onC. jejuni interaction, invasion, and intracellular survival. T84 and Caco-2 cells were pretreated with 10 μM of DPI for 1 hour and infected with C. jejuni for 3 hours. (a) T84 and (b) Caco-2 cells were washed with PBS and lysed, and the numbers of interacting bacteria were assessed. (c, d) For invasion assay, after infection with C. jejuni, IECs were incubated with gentamicin (150 μg/ml) for 2 hours to kill extracellular bacteria and then lysed, and the numbers of intracellular bacteria were assessed. (e, f) For intracellular survival assay, 2 hours of gentamicin treatment was followed by further incubation with gentamicin (10 μg/ml) for 18 hours. Then, cells were lysed, and the numbers of intracellular bacteria were assessed. Experiments were repeated in three biological and three technical replicates. Asterisks denote a statistically significant difference (∗p<0.05; ∗∗p<0.01; ∗∗∗∗p<0.0001).
(a)(b)(c)(d)(e)(f)


### 3.6. NOX1 Silencing by siRNA ImpairsCampylobacter jejuni Interaction, Invasion, and Intracellular Survival in Caco-2 Cells in vitro

As DPI is a pan-NOX inhibitor, we silenced NOX1 expression in Caco-2 cells by delivering specific small interfering RNA (siRNA) into cultured Caco-2 cells. We used siRNA sequence which targets regions of NOX1 for silencing. As a negative control, we used a nontargeting scrambled RNA sequence which is not complementary to theNOX1 mRNA. As shown in Figures 7(a) and 7(b), transcriptional and translational levels of NOX1 were significantly decreased in cells treated with NOX1 siRNA, relative to that in mock-treated Caco-2 controls. We further confirmed reduced activity of NOX1 by demonstrating significant reduction in extracellular ROS (Figure 7(c)). We showed that NOX1 siRNA transfection did not affect viability of Caco-2 cells (Figure S6). Based on these results, we further investigated interaction, invasion, and intracellular survival of C. jejuni within Caco-2 cells (Figures 7(d)–7(f)). Our result showed significant decrease in C. jejuni interaction, invasion, and intracellular survival when compared to nontransfected controls. This result highlights a correlation between reduced NOX1 expressions with a reduction in C. jejuni infection. Taken together, our results demonstrate that NOX1 is a critical host factor for C. jejuni interaction, invasion, and intracellular survival.Figure 7
The effect of NOX1 silencing onC. jejuni interaction, invasion, and intracellular survival. Caco-2 cells were transfected with NOX1 siRNA or scrambled siRNA (Scr siRNA). (a) qRT-PCR showing expression of NOX1 after siRNA transfection. (b) Western blotting showing expression of NOX1 after 72 hours of siRNA transfection. (c) Detection of extracellular ROS from Caco-2 cells after 72 hours of siRNA transfection followed by coincubation of C. jejuni for 3 hours. (d) After 72 hours of siRNA transfection followed by C. jejuni infection for 3 hours, Caco-2 cells were washed with PBS and lysed and the numbers of interacting bacteria were assessed or (e) for invasion assay, the cells were incubated with gentamicin (150 μg/ml) for 2 hours to kill extracellular bacteria and then lysed, and the numbers of intracellular bacteria were assessed. (f) For intracellular survival assay, 2 hours of gentamicin treatment was followed by further incubation with gentamicin (10 μg/ml) for 18 hours. Then, the cells were lysed, and the number of intracellular bacteria was determined. Experiments were repeated in three biological and three technical replicates. Asterisks denote a statistically significant difference (∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001; ∗∗∗∗p<0.0001).
(a)(b)(c)(d)(e)(f)


## 3.1.Campylobacter jejuni Modulates Intracellular and Extracellular ROS in T84 and Caco-2 Cells in a Time- and Strain-Dependent Manner

AsC. jejuni possesses distinct physiological characteristics compared to more studied enteric pathogens, we assessed the ability of three distinct C. jejuni strains to modulate intracellular and extracellular ROS in T84 and Caco-2 cells [28]. We observed strain-specific ROS modulation at 3- and 24-hour postinfection (Figure 1). All three C. jejuni strains reduced the levels of intracellular ROS in T84 and Caco-2 cells compared to the uninfected control (Figures 1(a), 1(b), 1(e), and 1(f)). A similar pattern was observed for extracellular ROS where all C. jejuni strains reduced the levels of extracellular ROS in T84 and Caco-2 cells compared to the uninfected control (Figures 1(c), 1(d), 1(g), and 1(h)). A distinct pattern was observed when assessing levels of extracellular ROS for C. jejuni 81-176 strain at a 3-hour postinfection (Figures 1(c) and 1(g)). At this early time point, extracellular ROS is increased in T84 and Caco-2 cells infected with C. jejuni 81-176, although we observed similar reduced levels of ROS at 24 hours. As controls, C. jejuni infection did not affect viability of both T84 and Caco-2 cells (Figure S1) and levels of ROS were unchanged when C. jejuni was resuspended in DMEM in the absence of IECs (Figure S2). Collectively, these results indicate a strain- and time-specific pattern linking the ability of different C. jejuni strains to modulate intracellular and extracellular ROS levels in T84 and Caco-2 cells.Figure 1
Detection of intracellular and extracellular ROS in T84 and Caco-2 cells after infection withC. jejuni 11168H, 81-176, or 488 strains. Intracellular ROS in T84 cells after infection with C. jejuni for (a) 3 hours or (b) 24 hours and extracellular ROS from T84 cells after infection with C. jejuni for (c) 3 hours or (d) 24 hours were measured. Intracellular ROS in Caco-2 cells after infection of C. jejuni for (e) 3 hours or (f) 24 hours and extracellular ROS from Caco-2 cells after infection for (g) 3 hours or (h) 24 hours were measured. For detection of intracellular ROS, DCFDA was used. For detection of extracellular ROS, Amplex® Red reagent with HRP was used. H2O2 was used as a positive control. Experiments were repeated in three biological and three technical replicates. Asterisks denote a statistically significant difference (∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001).
(a)(b)(c)(d)(e)(f)(g)(h)


## 3.2.Campylobacter jejuni Modulates Intracellular and Extracellular ROS in T84 and Caco-2 Cells via the Downregulation of NOX1 Complex

Given the observed modulation of intracellular and extracellular ROS in T84 and Caco-2 cells, we next explored the mechanism by whichC. jejuni strains orchestrate ROS modulation. We analysed the transcription and translation of NOX1 which is the main source of ROS production in IECs [17, 18]. As shown in Figure 2, NOX1 transcription and translation levels were significantly reduced in both T84 (Figure 2(a)) and Caco-2 cells (Figure 2(b)) infected with C. jejuni when compared to uninfected cells. Notably, at a 24-hour postinfection, mRNA levels of NOX1 in T84 cells are significantly reduced compared with C. jejuni-infected Caco-2 cells. We measured the relative levels of mRNA between T84 and Caco-2 cells and identified T84 cells expressed a higher basal level of NOX1 mRNA compared to Caco-2 cells (Figure 2(c)). As a result of this higher basal level of NOX1 mRNA in T84 cells, we validated our qRT-PCR data using RT-PCR where less expression of NOX1 in C. jejuni-infected T84 cells was observed (Figures 2(d) and 2(e)). Reduction in the translational level of NOX1 in C. jejuni-infected T84 cells was confirmed independently by Western blotting (Figures 2(f) and 2(g)).Figure 2
C. jejuni modulates NOX1 expression in T84 and Caco-2 cells. qRT-PCR showing expression of NOX1 in (a) T84 and (b) Caco-2 cells. (c) RT-PCR showing expression of NOX1 in uninfected T84 and Caco-2 cells. GAPDH was used as an internal control. (d) RT-PCR showing expression of NOX1 in T84 cells infected with C. jejuni for 24 hours and (e) relative mRNA levels as a percentage from RT-PCR data. (f) Western blotting showing NOX1 in T84 cells infected with C. jejuni for 24 hours and (g) relative protein level as a percentage from Western blotting. Asterisks denote a statistically significant difference (∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001).
(a)(b)(c)(d)(e)(f)(g)


## 3.3.Campylobacter jejuni Modulates Activity of Small GTPase Rac1 in T84 and Caco-2 Cells in a Time-Dependent Manner

To gain further insight into the mechanism that leads toC. jejuni modulation of ROS in T84 and Caco-2 cells, we examined the ability of C. jejuni to activate Rac1, a member of the Rho family of small GTPases. Rac1 undergoes cycling between active GTP- and inactive GDP-bound form which switches activation of cellular response upon stimuli [36]. Although GTP-bound Rac1 is implicated in NOX1 activation in several eukaryotic cell lines [19, 20], the contribution of Rac1 in C. jejuni-mediated NOX1 modulation is unknown. As shown in Figure 3, active GTP-bound Rac1 is an integral part of the NOX1 complex. Given that C. jejuni activates Rac1 in human INT 407 cells via Campylobacter invasion antigen D (CiaD) [10, 37] and that Rac1 supports NOX1 activity only in its GTP-bound active form, we examined the abundance of GTP-bound Rac1 to investigate if downregulation of NOX1 is linked to modulation of Rac1 by C. jejuni. Interestingly, C. jejuni 11168H strain induced Rac1 1 and 3 hours after infection in T84 cells (Figure 4(a)). After 24 hours of infection, Rac1 activity was reduced (though not statistically significant; p=0.0714) (Figure 4(a)). Similarly, C. jejuni 11168H induced Rac1 activity after 1 hour of infection in Caco-2 cells (Figure 4(b)). However, this activity was reduced after 3- and 24-hours of infection (Figure 4(b)). These results suggest that the downregulation of NOX1 by C. jejuni is inversely correlated with an increase in Rac1 GTPase activity.Figure 3
Proposed structure of the NOX1 complex consisting of NOX1, p22phox, GTP-bound Rac1, NOXA1, and NOXO1. p22phox and other subcellular subunits are assembled to activate catalytic subunit NOX1 which results in the generation of O2− by oxidising NADPH [17]. Created with http://BioRender.com.Figure 4
C. jejuni modulates activity of small GTPase Rac1 in T84 and Caco-2 cells. (a) T84 and (b) Caco-2 cells were infected with C. jejuni 11168H strain for 1, 3, and 24 hours, and the activation of small GTPase Rac1 in each time point was measured. Constitutively, active Rac1 (RCCA) was used as a positive control. Experiments were repeated in three biological and three technical replicates. Asterisks denote a statistically significant difference (∗p<0.05; ∗∗p<0.001).
(a)(b)


## 3.4.Campylobacter jejuni Modulates Transcription of Antioxidant-Related Genes in T84 and Caco-2 Cells

To gain further insight into the ability ofC. jejuni to modulate intracellular and extracellular ROS in T84 and Caco-2 cells, we sought to understand if C. jejuni modulates the expression of two important antioxidant genes, superoxide dismutase 1 (SOD1) and catalase (CAT). SOD1 decomposes O2- to H2O2, and CAT breaks down H2O2 to H2O and O2 [13]. Intriguingly, as shown in Figures 5(a) and 5(b), there is a significant downregulation of the mRNA levels of CAT and SOD1 at 24 hours of postinfection in T84 cells. A similar pattern was observed when compared with Caco-2 cells where the expression of CAT and SOD1 at 24 hours of postinfection is significantly downregulated (Figures 5(c) and 5(d)). In contrast, the expression of CAT and SOD1 at 3 hours of postinfection is unaffected. These results may indicate C. jejuni-mediated reduction in intracellular and extracellular ROS is independent of modulation of CAT and SOD1.Figure 5
qRT-PCR showing expression of human catalase (CAT) and superoxide dismutase 1 (SOD1) in T84 and Caco-2 cells. (a, b) T84 and (c, d) Caco-2 cells were infected with C. jejuni for 3 or 24 hours, and transcriptional levels of CAT and SOD1 were measured. GAPDH was used as an internal control. Experiments were repeated in three biological and three technical replicates. Asterisks denote a statistically significant difference (∗∗p<0.01; ∗∗∗p<0.001; ∗∗∗∗p<0.0001).
(a)(b)(c)(d)


## 3.5. Chemical Inhibition of NOX1 Activity by DPI ImpairsCampylobacter jejuni Interaction, Invasion, and Intracellular Survival of T84 and Caco-2 Cells in vitro

Having established thatC. jejuni significantly reduced the transcription and translation of NOX1 in T84 and Caco-2 cells in a time-dependent manner, and that Rac1 is not only known as a key component of the NOX1 complex, but also implicated in cell dynamic morphology [19, 20], we hypothesised Rac1-mediated NOX1 might modulate membrane ruffling and cytoskeleton rearrangement which might in turn affect C. jejuni interaction with IECs. Therefore, we investigated the role of NOX1 in C. jejuni interaction, invasion, and intracellular survival in IECs by transiently pretreating T84 and Caco-2 cells with DPI (10 μM) which is known to inhibit activity of flavoenzymes including NOX complex [38]. First, we demonstrated that DPI reduced ROS in T84 and Caco-2 cells (Figure S3). As shown in Figures 6(a), 6(c), and 6(e), pretreatment of T84 cells by DPI significantly reduced the ability of C. jejuni to interact, invade, and survive intracellularly in T84 cells. Similarly, as shown in Figures 6(b), 6(d), and 6(f), C. jejuni infected with DPI-treated Caco-2 cells showed significant reduction in interaction, invasion, and intracellular survival compared to untreated Caco-2 cells. Since our data revealed C. jejuni reduced interaction, invasion, and intracellular survival between the control- and DPI-treated T84 and Caco-2 cells, we next evaluated the viability of C. jejuni, T84, and Caco-2 cells coincubated with DPI. Treatment with DPI did not affect viability of IECs (Figure S4) or C. jejuni (Figure S5). Thus, our observations suggest further inhibition of NOX1 with DPI is detrimental to C. jejuni interaction, invasion, and intracellular survival in IECs.Figure 6
The effect of DPI onC. jejuni interaction, invasion, and intracellular survival. T84 and Caco-2 cells were pretreated with 10 μM of DPI for 1 hour and infected with C. jejuni for 3 hours. (a) T84 and (b) Caco-2 cells were washed with PBS and lysed, and the numbers of interacting bacteria were assessed. (c, d) For invasion assay, after infection with C. jejuni, IECs were incubated with gentamicin (150 μg/ml) for 2 hours to kill extracellular bacteria and then lysed, and the numbers of intracellular bacteria were assessed. (e, f) For intracellular survival assay, 2 hours of gentamicin treatment was followed by further incubation with gentamicin (10 μg/ml) for 18 hours. Then, cells were lysed, and the numbers of intracellular bacteria were assessed. Experiments were repeated in three biological and three technical replicates. Asterisks denote a statistically significant difference (∗p<0.05; ∗∗p<0.01; ∗∗∗∗p<0.0001).
(a)(b)(c)(d)(e)(f)


## 3.6. NOX1 Silencing by siRNA ImpairsCampylobacter jejuni Interaction, Invasion, and Intracellular Survival in Caco-2 Cells in vitro

As DPI is a pan-NOX inhibitor, we silenced NOX1 expression in Caco-2 cells by delivering specific small interfering RNA (siRNA) into cultured Caco-2 cells. We used siRNA sequence which targets regions of NOX1 for silencing. As a negative control, we used a nontargeting scrambled RNA sequence which is not complementary to theNOX1 mRNA. As shown in Figures 7(a) and 7(b), transcriptional and translational levels of NOX1 were significantly decreased in cells treated with NOX1 siRNA, relative to that in mock-treated Caco-2 controls. We further confirmed reduced activity of NOX1 by demonstrating significant reduction in extracellular ROS (Figure 7(c)). We showed that NOX1 siRNA transfection did not affect viability of Caco-2 cells (Figure S6). Based on these results, we further investigated interaction, invasion, and intracellular survival of C. jejuni within Caco-2 cells (Figures 7(d)–7(f)). Our result showed significant decrease in C. jejuni interaction, invasion, and intracellular survival when compared to nontransfected controls. This result highlights a correlation between reduced NOX1 expressions with a reduction in C. jejuni infection. Taken together, our results demonstrate that NOX1 is a critical host factor for C. jejuni interaction, invasion, and intracellular survival.Figure 7
The effect of NOX1 silencing onC. jejuni interaction, invasion, and intracellular survival. Caco-2 cells were transfected with NOX1 siRNA or scrambled siRNA (Scr siRNA). (a) qRT-PCR showing expression of NOX1 after siRNA transfection. (b) Western blotting showing expression of NOX1 after 72 hours of siRNA transfection. (c) Detection of extracellular ROS from Caco-2 cells after 72 hours of siRNA transfection followed by coincubation of C. jejuni for 3 hours. (d) After 72 hours of siRNA transfection followed by C. jejuni infection for 3 hours, Caco-2 cells were washed with PBS and lysed and the numbers of interacting bacteria were assessed or (e) for invasion assay, the cells were incubated with gentamicin (150 μg/ml) for 2 hours to kill extracellular bacteria and then lysed, and the numbers of intracellular bacteria were assessed. (f) For intracellular survival assay, 2 hours of gentamicin treatment was followed by further incubation with gentamicin (10 μg/ml) for 18 hours. Then, the cells were lysed, and the number of intracellular bacteria was determined. Experiments were repeated in three biological and three technical replicates. Asterisks denote a statistically significant difference (∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001; ∗∗∗∗p<0.0001).
(a)(b)(c)(d)(e)(f)


## 4. Discussion

Upon infection, host cells induce a range of cellular responses to remove offending pathogens. However, bacterial pathogens often target host organelle(s), signalling pathway(s), or immune responses to evade host defence mechanisms [1]. Disruption of ROS production in host cells by bacterial pathogens has been previously reported [39, 40]. S. typhimurium pathogenicity island-2 encoding Type III Secretion System (T3SS) inhibits ROS production in human macrophages by preventing NOX2 assembly [39, 41]. In addition, Pseudomonas aeruginosa T3SS effector, ExoS, disrupts ROS production in human neutrophils by ADP-ribosylating Ras and inhibiting its activity which is essential for NOX2 assembly [40].We have characterised the ability of distinctC. jejuni strains to modulate intracellular and extracellular ROS from human IECs in vitro. ROS production by human IECs is a major defence mechanism, yet how C. jejuni evades ROS remains unclear. Our work establishes that in contrast to other enteric pathogens, C. jejuni uses a different mechanism involving downregulation of NOX1 expression to modulate ROS in human IECs [24–27]. We examined three different C. jejuni strains using two different human IECs and showed that C. jejuni strains modulate intracellular and extracellular ROS from human IECs via the differential regulation of the transcription and translation of NOX1 which is a major ROS source in IECs [13]. Interestingly, a previous study demonstrated that C. jejuni 81-176 induces extracellular ROS production through NOX1 activation in human ileocecal adenocarcinoma-derived HCT-8 cells [42]. To further understand the implications of C. jejuni transcriptional and translational downregulation of NOX1 in T84 and Caco-2 cells, we revealed similarities with some other enteropathogens and also differences amongst others including the C. jejuni strain 81-176 [24–27]. Enteropathogens such as E. coli, Salmonella spp., and H. pylori upregulate expression of NOX1 and ROS production in infected IECs [24–27]. Our findings confirmed downregulation of ROS production by C. jejuni is strain dependent. In contrast to C. jejuni 11168H and 488 strains, C. jejuni 81-176 induced extracellular ROS in T84 and Caco-2 cells at 3 hours of postinfection. Induction of extracellular ROS by C. jejuni 81-176 at this earlier infection time point was also observed previously [42]. We hypothesise C. jejuni 81-176 might have additional bacterial determinants which may induce host extracellular ROS independent of NOX1 modulation (e.g., the pVir and pTet plasmids which encode putative Type IV Secretion Systems (T4SS)) [43, 44]. We also noted a difference between the ability of C. jejuni strains to regulate expression of NOX1 in T84 and Caco-2 cells. This difference could be due to variations between the two cell lines. Caco-2 cells possess characteristic enterocytes, whereas T84 cells possess characteristic colonocytes throughout differentiation [45]. In addition, previous studies have shown that reduced NOX1 mRNA was present in the ileum than in the colon of healthy patients suggesting there is a gradient in NOX1 expression from the small intestine to large intestine [46]. In our study, the lower expression of NOX1 mRNA detected in Caco-2 cells compared to T84 cells was also observed.As ROS homeostasis in the GI tract is regulated by multiple antioxidant enzymes [13], C. jejuni-mediated modulation of CAT and SOD1 at the transcriptional level was investigated. Our data demonstrated C. jejuni strains did not affect transcriptional levels of CAT and SOD1 in T84 and Caco-2 cells after 3 hours of infection, but they significantly downregulated expression of both genes after 24 hours. To our knowledge, this is the first data on C. jejuni modulation of antioxidant-related genes in human IECs in vitro. Our observations imply C. jejuni might modulate intracellular or extracellular ROS after 3 hours of infection without modulating expression of CAT and SOD1. These results also suggest that there could be additional mechanisms of C. jejuni-mediated reduction of ROS because C. jejuni was able to reduce ROS after 24 hours of infection even though transcription levels of antioxidant-related genes CAT and SOD1 were downregulated. However, we cannot disregard the possibilities that C. jejuni might secrete its own antioxidant-related proteins that may mitigate host cellular ROS and/or C. jejuni might induce expression of other host antioxidant genes such as mitochondrial superoxide dismutase (SOD2), extracellular superoxide dismutase (SOD3), and glutathione peroxidase [13]. A previous study demonstrated Pseudomonas pyocyanin decreased expression of human CAT but not SOD1 in the human A549 alveolar type II epithelial cells [47]. C. jejuni might produce pyocyanin-like metabolites but this is not yet investigated. In addition, downregulation of genes encoding CAT and SOD1 might be a host cellular strategy to produce ROS for clearance of C. jejuni.Upon adhering to host cells,C. jejuni modulates small GTPase Rac1 resulting in actin filament reorganisation to promote invasion. Activation of Rac1 in human embryonic INT 407 cells was observed between 45 minutes and 4 hours after C. jejuni infection [10, 37]. In accordance with previous studies, we demonstrated C. jejuni activates Rac1 at early infection time points. In contrast, a decrease of active Rac1 was detected at the later infection time point. Given the association of the active GTP-bound Rac1 and NOX1 activity, the early activation of Rac1 in IECs suggests that C. jejuni uses an intriguing system which we hypothesise could have temporally nonoverlapping mechanisms. The GTP-bound Rac1 observed in early time points may be linked to the requirement for C. jejuni to establish adhesion/invasion utilising a distinct mechanism in its infection cycle. Although the inactive GDP-bound Rac1 observed at the later time point of 24 hours, this suggests C. jejuni clearly possesses yet-to-be discovered mechanisms that enable differential regulation of NOX1 relative to modulation of Rac1. We also observe the pattern of active GTP-bound Rac1 in Caco-2 cells that is different to T84 cells. Such a difference may be due to the signalling cues between the cells and C. jejuni preference to efficiently interact with individual cells by binding, invading, and intracellularly surviving from distinct states during its infection.The impact of differential regulation of NOX1 onC. jejuni interaction, invasion, and intracellular survival in human IECs remains unclear. Surprisingly, chemical inhibition of NOX1 significantly reduced the ability of C. jejuni to interact, invade, and survive intracellularly in T84 and Caco-2 cells. It is possible that DPI may inadvertently affect local cellular receptors that C. jejuni uses to bind human IECs. Since DPI is not a specific inhibitor of NOX1 [38], we repeated these experiments using siRNA silencing of NOX1 which demonstrated similar findings, suggesting that NOX1 is indirectly necessary for C. jejuni interaction, invasion, and intracellular survival. Previous studies have demonstrated that DPI treatment reduced fibronectin expression in rat renal tubular epithelial cells [48], and a pan-NOX inhibitor APX-115 reduced fibronectin production in mesangial cells [49]. As fibronectin has been demonstrated as a key host receptor that C. jejuni uses to bind and invade human IECs [50], we hypothesise that silencing NOX1 might also affect expression of a key receptor fibronectin as is the case following DPI treatment, and this might be responsible for the reduced interaction and invasion of C. jejuni strains. We hypothesise that C. jejuni fine-tunes the modulation of NOX1 in a cell-specific manner, so that there is no impact on its ability to adhere and invade at early infection time points and then subsequently downregulates NOX1 to obtain the potential benefits of ROS reduction for its enhanced survivability at later infection time points. However, the broader nonspecificity of DPI and siRNA silencing experiments means that there could be alternative mechanisms in play.We have demonstrated thatC. jejuni modulates intracellular and extracellular ROS in human T84 and Caco-2 cells. Our observations link C. jejuni ROS modulation to the transcriptional and translational downregulation of NOX1. These findings also point to a further role of Rac1 in NOX1 modulation and downstream interaction. Based on chemical inhibition and silencing of NOX1 expression and translation, our findings suggest an indirect role of NOX1 for adhesion, invasion, and intracellular survival of C. jejuni. In this context, further understanding C. jejuni determinants that lead to ROS and/or NOX1 modulation in IECs will provide greater insights into how C. jejuni manipulate host defence mechanisms and cause diarrhoeal disease.



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