Campylobacter jejuni is the leading cause of bacterial foodborne gastroenteritis world wide and represents a major public health concern. Over the past two decades, significant progress in functional genomics, proteomics, enzymatic-based virulence profiling (EBVP), and the cellular biology of C. jejuni have improved our basic understanding of this important pathogen. We review key advances in our understanding of the multitude of emerging virulence factors that influence the outcome of C. jejuni–mediated infections. We highlight, the spatial and temporal dynamics of factors that promote C. jejuni to sense, adapt and survive in multiple hosts. Finally, we propose cohesive research directions to obtain a comprehensive understanding of C. jejuni virulence mechanisms.

Host physical, chemical, and immune responses constitute well-established defences against bacterial invasion. Recent studies have highlighted the critical role of cellular mechanisms, particularly the production of reactive oxygen species (ROS) in antibacterial defence. This review focuses on ROS generation by mammalian intestinal epithelial cells (IECs) and investigates whether ROS production is host-driven to eliminate bacteria or manipulated by bacteria to suppress or exploit ROS for enhanced internalisation. We examine the activation mechanisms of the NADPH oxidase (NOX) enzyme complex and the resulting ROS production in IECs, which, unlike professional phagocytes, lack the ability to engulf bacteria. The downstream effects of NOX-mediated ROS signalling are discussed in detail. Additionally, we explore the dynamic interplay between host and pathogen, with particular attention to how bacterial infection may disrupt or hijack host NOX-mediated ROS responses. The review concludes with key experimental considerations and outlines future directions in this evolving field. Overall, we present ROS as a double-edged sword, an essential antimicrobial effector that is also susceptible to bacterial subversion, highlighting its potential as a target in novel antimicrobial strategies.

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.

Chickens are a key food source for humans yet their microbiome contains bacteria that can be pathogenic to humans, and indeed potentially to chickens themselves. Campylobacter is present within the chicken gut and is the leading cause of bacterial foodborne gastroenteritis within humans worldwide. Infection can lead to secondary sequelae such as Guillain-Barré syndrome and stunted growth in children from low-resource areas. Despite the global health impact and economic burden of Campylobacter , how and when Campylobacter appears within chickens remains unclear. As such, there has been a motivation to decrease the number of Campylobacter within chickens and thus reduce the risk of infection to humans. The lack of day-to-day microbiome data with replicates, relevant metadata, and a lack of natural infection studies have delayed our understanding of the chicken gut microbiome and Campylobacter . Here, we performed a comprehensive day-to-day microbiome analysis of the chicken cecum from day 3 to 35 (12 replicates each day; n=396) combining metadata such as chicken weight and feed conversion rates to investigate what the driving forces are for the microbial changes within the chicken gut over time, and how this relates to Campylobacter appearance within a natural habitat setting. We found a rapidly increasing microbial diversity up to day 12 with variation observed both in terms of genera and abundance, before a stabilisation of the microbial diversity after day 20. In particular, we identified a shift from competitive to environmental drivers of microbial community from days 12 to 20 creating a window of opportunity whereby Campylobacter appears. Campylobacter was identified at day 16 which was one day after the most substantial changes in metabolic profiles observed. In addition, microbial variation over time is most likely influenced by the diet of the chickens whereby significant shifts in OTU abundances and beta dispersion of samples often corresponded with changes in feed. This study is unique in comparison to the most recent studies as neither sampling was sporadic nor Campylobacter was artificially introduced, thus the experiments were performed in a natural setting. We believe that our findings can be useful for future intervention strategies and can help elucidate the mechanism through which Campylobacter within chickens can be reduced.

Campylobacter jejuni is the major bacterial cause of foodborne gastroenteritis worldwide. How this pathogen interacts with the host defence machinery of human intestinal epithelial cells (IECs) and is involved in pathogenesis remains elusive. Bacterial pathogens utilise strategies to gain access to the eukaryotic cell machinery that can involve subversion of biological processes in host. Unfolded protein response (UPR) is a highly conserved host cell stress response to the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and is a conserved evolutionary response against invading pathogens. Several bacterial pathogens can induce the UPR for their own survival and thus design a dual scenario where UPR can both protect and facilitate pathogen evasion. Herein, we investigated whether UPR represents a virulence mechanism exploited by C. jejuni during bacterial invasion in human IECs. Our data show that following C. jejuni infection, we observe consistent upregulation of protein kinase R-like ER kinase (PERK), inositol-requiring enzyme 1α and (IRE1α), with activating transcription factor 6 (ATF6) activation occurring in a strain- and cell line-dependent manner. Chemical induction of UPR by thapsigargin in host cells reduced intracellular survival of C. jejuni while conversely pretreatment with UPR inhibitors increased intracellular survival of C. jejuni and attenuated IL-8 release. Finally, we show using C. jejuni mutants that the capsular polysaccharide and flagella contribute to UPR activation in IECs. Collectively, these findings provide observational insights into UPR activation during infections and how C. jejuni infection leads to UPR activation and inflammation, potentially contributing to downstream C. jejuni-mediated damage.

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.