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Abstract Background The NF-κB family of transcription factors and associated signalling pathways are abundant and ubiquitous in human immune responses. Activation of NF-κB transcription factors by viral pathogen-associated molecular patterns, such as viral RNA and DNA, is fundamental to anti-viral innate immune defences and pro-inflammatory cytokine production that steers adaptive immune responses. Diverse non-viral stimuli, such as lipopolysaccharide and cytokines, also activate NF-κB and the same anti-pathogen gene networks. Viruses adapted to human cells often encode multiple proteins targeting the NF-κB pathway to mitigate the anti-viral effects of NF-κB-dependent host immunity. Results In this study we have demonstrated using a variety of assays, in a number of different cell types including primary cells, that plasmid-encoded or virus-delivered simian immunodeficiency virus (SIV) accessory protein Vpx is a broad antagonist of NF-κB signalling active against diverse innate NF-κB agonists. Using targeted Vpx mutagenesis, we showed that this novel Vpx phenotype is independent of known Vpx cofactor DCAF1 and other cellular binding partners, including SAMHD1, STING and the HUSH complex. We found that Vpx co-immunoprecipitated with canonical NF-κB transcription factor p65, but not NF-κB family members p50 or p100, preventing nuclear translocation of p65. We found that broad antagonism of NF-κB activation by Vpx was conserved across distantly related lentiviruses as well as for Vpr from SIV Mona monkey (SIVmon), which has Vpx-like SAMHD1-degradation activity. Conclusions We have discovered a novel mechanism by which lentiviruses antagonise NF-κB activation by targeting p65. These findings extend our knowledge of how lentiviruses manipulate universal regulators of immunity to avoid the anti-viral sequelae of pro-inflammatory gene expression stimulated by both viral and extra-viral agonists. Importantly our findings are also relevant to the gene therapy field where virus-like particle associated Vpx is routinely used to enhance vector transduction through antagonism of SAMHD1, and perhaps also through manipulation of NF-κB. Graphical Abstract

Abstract Background Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide and the main source of infection is contaminated chicken meat. Although this important human pathogen is an obligate microaerophile, it must survive atmospheric oxygen conditions to allow transmission from contaminated chicken meat to humans. It is becoming increasingly evident that formation of biofilm plays a key role in the survival of this organism for extended periods on poultry products. We have recently demonstrated a novel inducible model for the study of adherent C. jejuni biofilm formation under aerobic conditions. By taking advantage of supercoiling mediated gene regulation, incubation of C. jejuni with subinhibitory concentrations of the Gyrase B inhibitor novobiocin was shown to promote the consistent formation of metabolically active adherent biofilm. Results In this study, we implement this model in conjunction with the fluorescent markers: TAMRA (live cells) and SytoX (dead cells, eDNA) to develop a novel systematic high-content imaging approach and describe how it can be implemented to gain quantifiable information about the integrity and extracellular polymeric substance (EPS) composition of adherent C. jejuni biofilm in aerobic conditions. We show that this produces a model with a consistent, homogenous biofilm that can be induced and used to screen a range of inhibitors of biofilm adherence and matrix formation. Conclusions This model allows for the first time a high throughput analysis of C. jejuni biofilms which will be invaluable in enabling researchers to develop mechanisms to disrupt these biofilms and reduce the viability of these bacteria under aerobic conditions.