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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 in intestinal epithelial cell immunometabolism and mucosal immunity. C. rodentium injects 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 disease and speculate on the role of T3SS effector networks in host adaption.

The gut microbiota plays a crucial role in susceptibility to enteric pathogens, including Citrobacter rodentium, a model extracellular mouse pathogen that colonizes the colonic mucosa. C. rodentium infection outcomes vary between mouse strains, with C57BL/6 and C3H/HeN mice clearing and succumbing to the infection, respectively. Kanamycin (Kan) treatment at the peak of C57BL/6 mouse infection with Kan-resistant C. rodentium resulted in relocalization of the pathogen from the colonic mucosa and cecum to solely the cecal luminal contents; cessation of the Kan treatment resulted in rapid clearance of the pathogen. We now show that in C3H/HeN mice, following Kan-induced displacement of C. rodentium to the cecum, the pathogen stably colonizes the cecal lumens of 65% of the mice in the absence of continued antibiotic treatment, a phenomenon that we term antibiotic-induced bacterial commensalization (AIBC). AIBC C. rodentium was well tolerated by the host, which showed few signs of inflammation; passaged AIBC C. rodentium robustly infected naive C3H/HeN mice, suggesting that the AIBC state is transient and did not select for genetically avirulent C. rodentium mutants. Following withdrawal of antibiotic treatment, 35% of C3H/HeN mice were able to prevent C. rodentium commensalization in the gut lumen. These mice presented a bloom of a commensal species, Citrobacter amalonaticus, which inhibited the growth of C. rodentium in vitro in a contact-dependent manner and the luminal growth of AIBC C. rodentium in vivo. Overall, our data suggest that commensal species can confer colonization resistance to closely related pathogenic species. IMPORTANCE Gut bacterial infections involve three-way interactions between virulence factors, the host immune responses, and the microbiome. While the microbiome erects colonization resistance barriers, pathogens employ virulence factors to overcome them. Treating mice infected with kanamycin-resistant Citrobacter rodentium with kanamycin caused displacement of the pathogen from the colonic mucosa to the cecal lumen. Following withdrawal of the kanamycin treatment, 65% of the mice were persistently colonized by C. rodentium, which seemed to downregulate virulence factor expression. In this model of luminal gut colonization, 35% of mice were refractory to stable C. rodentium colonization, suggesting that their microbiotas were able to confer colonization resistance. We identify a commensal bacterium of the Citrobacter genus, C. amalonaticus, which inhibits C. rodentium growth in vitro and in vivo. These results show that the line separating commensal and pathogenic lifestyles is thin and multifactorial and that commensals may play a major role in combating enteric infection.

INTRODUCTION Infections with many Gram-negative pathogens, including Escherichia coli, Salmonella, Shigella, and Yersinia, rely on the injection of effectors via type III secretion systems (T3SSs). The effectors hijack cellular processes through multiple mechanisms, including molecular mimicry and diverse enzymatic activities. Although in vitro analyses have shown that individual effectors can exhibit complementary, interdependent, or antagonistic relationships, most in vivo studies have focused on the contribution of single effectors to pathogenesis. Citrobacter rodentium is a natural mouse pathogen that shares infection strategies and virulence factors with the human pathogens enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC). The ability of these pathogens to colonize the gastrointestinal tract is mediated by the injection of effectors via a T3SS. Although C. rodentium infects 31 effectors, the prototype EPEC strain E2348/69 translocates 21 effectors. RATIONALE The aim of this study was to test the hypotheses that, rather than operating individually, the T3SS effectors form robust intracellular networks that can sustain large contractions and that expanded effector repertoires play a role in distinct disease phenotypes and host adaption. RESULTS We tested the effector-network paradigm by infecting mice with >100 C. rodentium effector mutant combinations. First, using machine learning prediction algorithms, we discovered additional effectors, NleN and NleO. We then sequentially deleted effector genes from two distinct starting points to reach sustainable endpoints, which resulted in strains missing 19 unrelated effectors (CR14) or 10 effectors involved in the modulation of innate immune responses in intestinal epithelial cells (IECs) (CRi9). Moreover, we deleted Map and EspF, which target the mitochondria and disrupt tight junctions. Unexpectedly, all strains colonized the colon and activated conserved metabolic and antimicrobial processes in the IECs while eliciting distinct cytokine and immune cell infiltration responses. In particular, although infection with C. rodentium Δmap/ΔespF failed to induce secretion of interleukin-22 (IL-22), CR14 and CRi9 triggered heightened secretion of IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) and of IL-22, interferon-γ (IFN-γ), and IL-17 from colonic explants, respectively. Nonetheless, infection with CR14 or CRi9 induced protective immunity against secondary infections. Although Tir, EspZ, and NleA are essential, other effectors exhibit context-dependent essentiality in vivo. Moreover, C. rodentium expressing the effector repertoire of EPEC E2348/69 failed to efficiently colonize mice. We used curated functional information and our in vivo data to train a machine learning model that predicted values for colonization efficiency of previously uncharacterized mutant combinations. Notably, a mutant with a low predicted value, lacking only nleF, nleG8, nleG1, nleB, and espL, failed to colonize. CONCLUSION Our analysis revealed that T3SS effectors form robust networks, which can sustain substantial contractions while maintaining virulence, and that the composition of the effector network contributes to host adaptation. Alternative effector networks within a single pathogen triggered markedly different immune responses yet induced protective immunity. CR14 did not tolerate any further contraction, which suggests that this network reached its robustness limit with only 12 effectors. As the robustness limits of other effector networks depend on the contraction starting point and the order of the deletions, machine learning models could transform our ability to predict alternative network functions. Together, this study demonstrates the robustness of T3SS effector networks and the ability of IECs to withstand drastic perturbations while maintaining antibacterial functions. The work of T.I.R. and J.S.C. was funded by the CRUK Centre grant with reference no. C309/A25144. N.W. was supported by PhD fellowships from the Manna Center Program for Food Safety and Security at Tel Aviv University and the Edmond J. Safra Center for Bioinformatics at Tel-Aviv University. The work of E. N.-B. was funded by a PhD FPU grant from the Spanish government (FPU2017/04179). A. R.-P. was partially funded by project PID2019-106960GB-I00 (AEI/FEDER, Spain) and by InGEMICS-CM, FSE/FEDER, Comunidad de Madrid Project B2017/BMD-3691. L.A.F. is supported by grant BIO2017-89081-R (AEI/MICIU/FEDER, European Union). G.F. is supported by an MRC program grant (MR/R02671/), a Royal Society grant (IC160080), and a Wellcome Investigator Award (107057/Z/15/Z). Peerreview

The ability to simultaneously and more directly correlate genes with metabolite levels on a global level would provide novel information for many biological platforms yet has thus far been challenging. Here, we describe a method to help address this problem, which we dub “Met-Seq” (metabolite-coupled Tn sequencing)., Heme is an essential metabolite for most life on earth. Bacterial pathogens almost universally require iron to infect a host, often acquiring this nutrient in the form of heme. The Gram-negative pathogen Pseudomonas aeruginosa is no exception, where heme acquisition and metabolism are known to be crucial for both chronic and acute infections. To unveil unknown genes and pathways that could play a role with heme metabolic flux in this pathogen, we devised an omic-based approach we dubbed “Met-Seq,” for metabolite-coupled transposon sequencing. Met-Seq couples a biosensor with fluorescence-activated cell sorting (FACS) and massively parallel sequencing, allowing for direct identification of genes associated with metabolic changes. In this work, we first construct and validate a heme biosensor for use with P. aeruginosa and exploit Met-Seq to identify 188 genes that potentially influence intracellular heme levels. Identified genes largely consisted of metabolic pathways not previously associated with heme, including many secreted virulence effectors, as well as 11 predicted small RNAs (sRNAs) and riboswitches whose functions are not currently understood. We verify that five Met-Seq hits affect intracellular heme levels; a predicted extracytoplasmic function (ECF) factor, a phospholipid acquisition system, heme biosynthesis regulator Dnr, and two predicted antibiotic monooxygenase (ABM) domains of unknown function (PA0709 and PA3390). Finally, we demonstrate that PA0709 and PA3390 are novel heme-binding proteins. Our data suggest that Met-Seq could be extrapolated to other biological systems and metabolites for which there is an available biosensor, and provides a new template for further exploration of iron/heme regulation and metabolism in P. aeruginosa and other pathogens. IMPORTANCE The ability to simultaneously and more directly correlate genes with metabolite levels on a global level would provide novel information for many biological platforms yet has thus far been challenging. Here, we describe a method to help address this problem, which we dub “Met-Seq” (metabolite-coupled Tn sequencing). Met-Seq uses the powerful combination of fluorescent biosensors, fluorescence-activated cell sorting (FACS), and next-generation sequencing (NGS) to rapidly identify genes that influence the levels of specific intracellular metabolites. For proof of concept, we create and test a heme biosensor and then exploit Met-Seq to identify novel genes involved in the regulation of heme in the pathogen Pseudomonas aeruginosa. Met-Seq-generated data were largely comprised of genes which have not previously been reported to influence heme levels in this pathogen, two of which we verify as novel heme-binding proteins. As heme is a required metabolite for host infection in P. aeruginosa and most other pathogens, our studies provide a new list of targets for potential antimicrobial therapies and shed additional light on the balance between infection, heme uptake, and heme biosynthesis.

The development of innovative high-throughput genomics and metabolomics technologies has considerably expanded our understanding of the commensal microorganisms residing within the human body, collectively termed the microbiota. In recent years, the microbiota has been reported to have important roles in multiple aspects of human health, pathology and host-pathogen interactions. One function of commensals that has attracted particular interest is their role in protection against pathogens and pathobionts, a concept known as colonization resistance. However, pathogens are also able to sense and exploit the microbiota during infection. Therefore, obtaining a holistic understanding of colonization resistance mechanisms is essential for the development of microbiome-based and microbiome-targeting therapies for humans and animals. Achieving this is dependent on utilizing physiologically relevant animal models. In this Perspective, we discuss the colonization resistance functions of the gut microbiota and sieve through the advantages and limitations of murine models commonly used to study such mechanisms within the context of enteric bacterial infection.

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., The outcome of Citrobacter rodentium infection is dependent on the genetic background of the host, with C57BL/6 and C3H/HeN mice developing self‐limiting and fatal colitis, respectively. In this paper, Carson et al have demonstrated accelerated colonisation and onset of conserved infection signatures in C3H/HeN mice. Uniquely to C3H/HeN mice,the expression of the pattern recognition receptors NLRP3 and ALPK1, as well as lactate fermentation associated proteins, LDHA and MCT4, is induced in infected intestinal epithelial cells.

Citrobacter rodentium is an extracellular enteric mouse-specific pathogen used to model infections with human pathogenic Escherichia coli and inflammatory bowel disease. C. rodentium injects type III secretion system effectors into intestinal epithelial cells (IECs) to target inflammatory, metabolic and cell survival pathways and establish infection. While the host responds to infection by activating innate and adaptive immune signalling, required for clearance, the IECs respond by rapidly shifting bioenergetics to aerobic glycolysis, which leads to oxygenation of the epithelium, an instant expansion of mucosal-associated commensal Enterobacteriaceae and a decline of obligate anaerobes. Moreover, infected IECs reprogramme intracellular metabolic pathways, characterized by simultaneous activation of cholesterol biogenesis, import and efflux, leading to increased serum and faecal cholesterol levels. In this Review we summarize recent advances highlighting the intimate relationship between C. rodentium pathogenesis, metabolism and the gut microbiota. Citrobacter rodentium, an extracellular mouse-specific enteric pathogen, provides a robust model for the study of physiological host–pathogen–microbiota interactions. In this Review, Frankel and colleagues highlight how the C. rodentium model has advanced our understanding of enteric infections and inflammatory bowel disease, in particular changes to host metabolism and inflammation.

Summary: 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. : A/E pathogens intimately adhere to the gut mucosa. Mullineaux-Sanders et al. demonstrate that inducing specific dysbiosis at the peak of murine infection with Citrobacter rodentium prevents mucosal colonization. This occurs via a mechanism independent of virulence gene expression modulation, indicating that enteric pathogens may rely on commensals for effective infection. Keywords: C. rodentium, enteric infection, antibiotics, microbiota, colonization resistance, AMR