Citrobacter rodentium Relies on Commensals for Colonization of the Colonic Mucosa.

We investigated the role of commensals at the peak of infection with the colonic mouse pathogen Citrobacter rodentium. Bioluminescent and kanamycin (Kan)-resistant C. rodentium persisted avirulently in the cecal lumen of mice continuously treated with Kan. A single Kan treatment was sufficient to displace C. rodentium from the colonic mucosa, a phenomenon not observed following treatment with vancomycin (Van) or metronidazole (Met). Kan, Van, and Met induce distinct dysbiosis, suggesting C. rodentium relies on specific commensals for colonic colonization. Expression of the master virulence regulator ler is induced in germ-free mice, yet C. rodentium is only seen in the cecal lumen. Moreover, in conventional mice, a single Kan treatment was sufficient to displace C. rodentium constitutively expressing Ler from the colonic mucosa. These results show that expression of virulence genes is not sufficient for colonization of the colonic mucosa and that commensals are essential for a physiological infection course

Phytochrome-based fluorophores: sensitive tools for heme detection and in vivo imaging in bacterial pathogens

The acquisition of iron is crucial for bacterial pathogenesis. In this thesis I describe the development and validation of a biosensor for heme in bacteria, an important host source of iron for human pathogens. I demonstrate that the co-expression of a phytochrome-based fluorophore (PBF), which utilises the heme-derived metabolite biliverdin IXα as a chromophore, with a heme oxygenase (HO) in Gram-negative bacteria results in heme-dependent near infra-red fluorescence. Using this PBF+HO reporter, I probe heme metabolism in the opportunistic human pathogen Pseudomonas aeruginosa and for the first time provide direct evidence linking the previously putative heme receptor HxuC to heme metabolism. Further, coupling of the heme biosensor to transposon mutagenesis and Fluorescence-Activated Cell Sorting was used to screen a P. aeruginosa transposon library leading to the identification of several putative novel components of heme metabolism. Finally, I demonstrate the potential of our fluorescent biosensor to be used to study heme metabolism and host-pathogen interactions in vivo using a live adult zebrafish model of infection. The PBF+HO heme biosensor allows the highthroughput, non-invasive study of bacterial heme metabolism in real-time and proves a useful tool for gaining further insight into iron-heme metabolism in bacterial pathogens.Open Acces

Intestinal epithelial cells and the microbiome undergo swift reprogramming at the inception of colonic Citrobacter rodentium infection

We used the mouse attaching and effacing (A/E) pathogen Citrobacter rodentium, which models the human A/E pathogens enteropathogenic Escherichia coli and enterohemorrhagic E. coli (EPEC and EHEC), to temporally resolve intestinal epithelial cell (IEC) responses and changes to the microbiome during in vivo infection. We found the host to be unresponsive during the first 3 days postinfection (DPI), when C. rodentium resides in the caecum. In contrast, at 4 DPI, the day of colonic colonization, despite only sporadic adhesion to the apex of the crypt, we observed robust upregulation of cell cycle and DNA repair processes, which were associated with expansion of the crypt Ki67-positive replicative zone, and downregulation of multiple metabolic processes (including the tricarboxylic acid [TCA] cycle and oxidative phosphorylation). Moreover, we observed dramatic depletion of goblet and deep crypt secretory cells and an atypical regulation of cholesterol homeostasis in IECs during early infection, with simultaneous upregulation of cholesterol biogenesis (e.g., 3-hydroxy-3-methylglutaryl–coenzyme A reductase [Hmgcr]), import (e.g., low-density lipoprotein receptor [Ldlr]), and efflux (e.g., AbcA1). We also detected interleukin 22 (IL-22) responses in IECs (e.g., Reg3γ) on the day of colonic colonization, which occurred concomitantly with a bloom of commensal Enterobacteriaceae on the mucosal surface. These results unravel a new paradigm in host-pathogen-microbiome interactions, showing for the first time that sensing a small number of pathogenic bacteria triggers swift intrinsic changes to the IEC composition and function, in tandem with significant changes to the mucosa-associated microbiome, which parallel innate immune responses

Type III secretion system effector subnetworks elicit distinct host immune responses to infection

Citrobacter rodentium, a natural mouse pathogen which colonises the colon of immuno-competent mice, provides a robust model for interrogating host-pathogen-microbiota interactions in vivo. This model has been key to providing new insights into local host responses to enteric infection, including changes inintestinal epithelial cell immuno metabolism and mucosal immunity. C. rodent iuminjects 31 bacterial effectors into epithelial cells via a type III secretion system (T3SS). Recently, these effectors were shown to be able to form multiple intracellular subnetworks which can withstand significant contractions whilst maintaining virulence. Here we highlight recent advances in understanding gut mucosal responses to infection and effector biology, as well as potential uses for artificial intelligence (AI) in understanding infectious diseaseand speculate on the role of T3SS effector networks in host adaption

P-700 photooxidation in state 1 and state 2 in cyanobacteria upon flash illumination with phycobilin- and chlorophyll-absorbed light

We have measured the flash yield of P-700 photooxidation in cells of the cyanobacteria Synechococcus 6301 and Nostoc MAC adapted to light-states 1 and 2. Using excitation at 337 nm, the flash yield of P-700 photooxidation at limiting flash intensity was larger in state 2 in both species, indicating an increased absorption cross-section of PS I in state 2 for light absorbed by both chlorophyll and phycobilin pigments. Using excitation at 532 nm, the flash yield of P-700 photooxidation at limiting intensity was also larger in state 2 in both species, indicating an increased absorption cross-section of PS I for light absorbed specifically by phycocyanin or phycoerythrin. Differences in P-700 re-reduction kinetics between states 1 and 2 were consistently observed following flash excitation at either wavelength. Our results are consistent with a model for redistribution of excitation energy in state 2 that involves decoupling of the phycobilisome from PS II and its functional reassociation with PS I. Photosynthesis; Light harvesting; Photosystem I; State transition; Phycobilisome; Excitation energy distribution; Cyanobacteria; ( Nostoc MAC; Synechococcus 6301

Citrobacter rodentium induces rapid and unique metabolic and inflammatory responses in mice suffering from severe disease

The mouse pathogen Citrobacter rodentium is used to model infections with enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC). Pathogenesis is commonly modelled in mice developing mild disease (e.g., C57BL/6). However, little is known about host responses in mice exhibiting severe colitis (e.g., C3H/HeN), which arguably provide a more clinically relevant model for human paediatric enteric infection. Infection of C3H/HeN mice with C. rodentium results in rapid colonic colonisation, coinciding with induction of key inflammatory signatures and colonic crypt hyperplasia. Infection also induces dramatic changes to bioenergetics in intestinal epithelial cells, with transition from oxidative phosphorylation (OXPHOS) to aerobic glycolysis and higher abundance of SGLT4, LDHA, and MCT4. Concomitantly, mitochondrial proteins involved in the TCA cycle and OXPHOS were in lower abundance. Similar to observations in C57BL/6 mice, we detected simultaneous activation of cholesterol biogenesis, import, and efflux. Distinctly, however, the pattern recognition receptors NLRP3 and ALPK1 were specifically induced in C3H/HeN. Using cell‐based assays revealed that C. rodentium activates the ALPK1/TIFA axis, which is dependent on the ADP‐heptose biosynthesis pathway but independent of the Type III secretion system. This study reveals for the first time the unfolding intestinal epithelial cells' responses during severe infectious colitis, which resemble EPEC human infections

Citrobacter rodentium relies on commensals for colonization of the colonic mucosa

We investigated the role of commensals at the peak of infection with the colonic mouse pathogen Citrobacter rodentium. Bioluminescent and kanamycin (Kan)-resistant C. rodentium persisted avirulently in the cecal lumen of mice continuously treated with Kan. A single Kan treatment was sufficient to displace C. rodentium from the colonic mucosa, a phenomenon not observed following treatment with vancomycin (Van) or metronidazole (Met). Kan, Van, and Met induce distinct dysbiosis, suggesting C. rodentium relies on specific commensals for colonic colonization. Expression of the master virulence regulator ler is induced in germ-free mice, yet C. rodentium is only seen in the cecal lumen. Moreover, in conventional mice, a single Kan treatment was sufficient to displace C. rodentium constitutively expressing Ler from the colonic mucosa. These results show that expression of virulence genes is not sufficient for colonization of the colonic mucosa and that commensals are essential for a physiological infection course