Vibrio cholerae accessory colonisation factor AcfC: a chemotactic protein with a role in hyperinfectivity.

Vibrio cholerae O1 El Tor is an aquatic Gram-negative bacterium responsible for the current seventh pandemic of the diarrheal disease, cholera. A previous whole-genome analysis on V. cholerae O1 El Tor strains from the 2010 epidemic in Pakistan showed that all strains contained the V. cholerae pathogenicity island-1 and the accessory colonisation gene acfC (VC_0841). Here we show that acfC possess an open reading frame of 770 bp encoding a protein with a predicted size of 28 kDa, which shares high amino acid similarity with two adhesion proteins found in other enteropathogens, including Paa in serotype O45 porcine enteropathogenic Escherichia coli and PEB3 in Campylobacter jejuni. Using a defined acfC deletion mutant, we studied the specific role of AcfC in V. cholerae O1 El Tor environmental survival, colonisation and virulence in two infection model systems (Galleria mellonella and infant rabbits). Our results indicate that AcfC might be a periplasmic sulfate-binding protein that affects chemotaxis towards mucin and bacterial infectivity in the infant rabbit model of cholera. Overall, our findings suggest that AcfC contributes to the chemotactic response of WT V. cholerae and plays an important role in defining the overall distribution of the organism within the intestine

Impact of the Mk VI SkinSuit on skin microbiota of terrestrial volunteers and an International Space Station-bound astronaut.

Microgravity induces physiological deconditioning due to the absence of gravity loading, resulting in bone mineral density loss, atrophy of lower limb skeletal and postural muscles, and lengthening of the spine. SkinSuit is a lightweight compression suit designed to provide head-to-foot (axial) loading to counteract spinal elongation during spaceflight. As synthetic garments may impact negatively on the skin microbiome, we used 16S ribosomal RNA (rRNA) gene amplicon procedures to define bacterial skin communities at sebaceous and moist body sites of five healthy male volunteers undergoing SkinSuit evaluation. Each volunteer displayed a diverse, distinct bacterial population at each skin site. Short (8 h) periods of dry hyper-buoyancy flotation wearing either gym kit or SkinSuit elicited changes in the composition of the skin microbiota at the genus level but had little or no impact on community structure at the phylum level or the richness and diversity of the bacterial population. We also determined the composition of the skin microbiota of an astronaut during pre-flight training, during an 8-day visit to the International Space Station involving two 6-7 h periods of SkinSuit wear, and for 1 month after return. Changes in composition of bacterial skin communities at five body sites were strongly linked to changes in geographical location. A distinct ISS bacterial microbiota signature was found which reversed to a pre-flight profile on return. No changes in microbiome complexity or diversity were noted, with little evidence for colonisation by potentially pathogenic bacteria; we conclude that short periods of SkinSuit wear induce changes to the composition of the skin microbiota but these are unlikely to compromise the healthy skin microbiome

Investigating the interaction between Campylobacter jejuni and intestinal epithelial cells resulting in activation of the unfolded protein response

Problem Statement Campylobacter jejuni adheres, invades and resides within intestinal epithelial cells (IECs). However, the exact process at the cellular level leading to diarrhoeal disease is poorly understood. There have been studies that link intestinal inflammation and the unfolded protein response (UPR), which is a conserved pathway to restore homeostasis in the endoplasmic reticulum. Recent data has shown that C. jejuni activates UPR through IRE1α pathway. Here, we investigated the activation of the UPR through PERK, IRE1α, and ATF6 pathways by C. jejuni. Approach To investigate whether C. jejuni infection induces UPR in IECs, T84 cells were infected with C. jejuni 11168H, 81-176, and 488 wild-type strains with different exposure times. RNA and proteins were isolated from infected cells, and the induction of UPR by C. jejuni wild-type strains was observed using transcription and proteomic methods. Results The activation of all three UPR branches, PERK, IRE1α, and ATF6 was demonstrated by increased levels of transcription of chop, spliced xbp1, and atf6, respectively compared to uninfected control. The upregulation of these genes was confirmed by western blot, which showed increased amount of proteins for CHOP, phosphorylated eIF2α, and spliced XBP1 compared to the control. Conclusions The combined results demonstrate that C. jejuni induces the UPR through all three pathways. This study opens the way forward for improved understanding of the interaction between C. jejuni and IECs via UPR activation leading to intestinal inflammation. This understanding will further benefit the investigation of cellular pathways triggered by C. jejuni which can lead to diarrhoeal disease

Campylobacter jejuni modulates reactive oxygen species production and NADPH oxidase 1 expression in human intestinal epithelial cells

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

Biochemical characterization and identification of ferulenol and embelin as potent inhibitors of malate:quinone oxidoreductase from Campylobacter jejuni

Campylobacter jejuni infection poses a serious global threat to public health. The increasing incidence and antibiotic resistance of this bacterial infection have necessitated the adoption of various strategies to curb this trend, primarily through developing new drugs with new mechanisms of action. The enzyme malate:quinone oxidoreductase (MQO) has been shown to be essential for the survival of several bacteria and parasites. MQO is a peripheral membrane protein that catalyses the oxidation of malate to oxaloacetate, a crucial step in the tricarboxylic acid cycle. In addition, MQO is involved in the reduction of the quinone pool in the electron transport chain and thus contributes to cellular bioenergetics. The enzyme is an attractive drug target as it is not conserved in mammals. As a preliminary step in assessing the potential application of MQO from C. jejuni (CjMQO) as a new drug target, we purified active recombinant CjMQO and conducted, for the first time, biochemical analyses of MQO from a pathogenic bacterium. Our study showed that ferulenol, a submicromolar mitochondrial MQO inhibitor, and embelin are nanomolar inhibitors of CjMQO. We showed that both inhibitors are mixed-type inhibitors versus malate and noncompetitive versus quinone, suggesting the existence of a third binding site to accommodate these inhibitors; indeed, such a trait appears to be conserved between mitochondrial and bacterial MQOs. Interestingly, ferulenol and embelin also inhibit the in vitro growth of C. jejuni, supporting the hypothesis that MQO is essential for C. jejuni survival and is therefore an important drug target

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

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

Biochemical characterization and identification of ferulenol and embelin as potent inhibitors of malate:quinone oxidoreductase from Campylobacter jejuni

Campylobacter jejuni infection poses a serious global threat to public health. The increasing incidence and antibiotic resistance of this bacterial infection have necessitated the adoption of various strategies to curb this trend, primarily through developing new drugs with new mechanisms of action. The enzyme malate:quinone oxidoreductase (MQO) has been shown to be essential for the survival of several bacteria and parasites. MQO is a peripheral membrane protein that catalyses the oxidation of malate to oxaloacetate, a crucial step in the tricarboxylic acid cycle. In addition, MQO is involved in the reduction of the quinone pool in the electron transport chain and thus contributes to cellular bioenergetics. The enzyme is an attractive drug target as it is not conserved in mammals. As a preliminary step in assessing the potential application of MQO from C. jejuni (CjMQO) as a new drug target, we purified active recombinant CjMQO and conducted, for the first time, biochemical analyses of MQO from a pathogenic bacterium. Our study showed that ferulenol, a submicromolar mitochondrial MQO inhibitor, and embelin are nanomolar inhibitors of CjMQO. We showed that both inhibitors are mixed-type inhibitors versus malate and noncompetitive versus quinone, suggesting the existence of a third binding site to accommodate these inhibitors; indeed, such a trait appears to be conserved between mitochondrial and bacterial MQOs. Interestingly, ferulenol and embelin also inhibit the in vitro growth of C. jejuni, supporting the hypothesis that MQO is essential for C. jejuni survival and is therefore an important drug target

Biochemical characterization and identification of ferulenol and embelin as potent inhibitors of malate:quinone oxidoreductase from Campylobacter jejuni

Campylobacter jejuni infection poses a serious global threat to public health. The increasing incidence and antibiotic resistance of this bacterial infection have necessitated the adoption of various strategies to curb this trend, primarily through developing new drugs with new mechanisms of action. The enzyme malate:quinone oxidoreductase (MQO) has been shown to be essential for the survival of several bacteria and parasites. MQO is a peripheral membrane protein that catalyses the oxidation of malate to oxaloacetate, a crucial step in the tricarboxylic acid cycle. In addition, MQO is involved in the reduction of the quinone pool in the electron transport chain and thus contributes to cellular bioenergetics. The enzyme is an attractive drug target as it is not conserved in mammals. As a preliminary step in assessing the potential application of MQO from C. jejuni (CjMQO) as a new drug target, we purified active recombinant CjMQO and conducted, for the first time, biochemical analyses of MQO from a pathogenic bacterium. Our study showed that ferulenol, a submicromolar mitochondrial MQO inhibitor, and embelin are nanomolar inhibitors of CjMQO. We showed that both inhibitors are mixed-type inhibitors versus malate and noncompetitive versus quinone, suggesting the existence of a third binding site to accommodate these inhibitors; indeed, such a trait appears to be conserved between mitochondrial and bacterial MQOs. Interestingly, ferulenol and embelin also inhibit the in vitro growth of C. jejuni, supporting the hypothesis that MQO is essential for C. jejuni survival and is therefore an important drug target