Quorum sensing has an unexpected role in virulence in the model pathogen Citrobacter rodentium

The bacterial mouse pathogen Citrobacter rodentium causes attaching and effacing (AE) lesions in the same manner as pathogenic Escherichia coli, and is an important model for this mode of pathogenesis. Quorum sensing (QS) involves chemical signalling by bacteria to regulate gene expression in response to cell density. E. coli has never been reported to have N-acylhomoserine lactone (AHL) QS, but it does utilize luxS-dependent signalling. We found production of AHL QS signalling molecules by an AE pathogen, C. rodentium. AHL QS is directed by the croIR locus and a croI mutant is affected in its surface attachment, although not in Type III secretion. AHL QS has an important role in virulence in the mouse as, unexpectedly, the QS mutant is hypervirulent; by contrast, we detected no impact of luxS inactivation. Further study of QS in Citrobacter should provide new insights into AE pathogenesis. As the croIR locus might have been horizontally acquired, AHL QS might exist in some strains of pathogenic E. coli.     

Citrobacter rodentium of mice and man

The major classes of enteric bacteria harbour a conserved core genomic structure, common to both commensal and pathogenic strains, that is most likely optimized to a life style involving colonization of the host intestine and transmission via the environment. In pathogenic bacteria this core genome framework is decorated with novel genetic islands that are often associated with adaptive phenotypes such as virulence. This classical genome organization is well illustrated by a group of extracellular enteric pathogens, which includes enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Citrobacter rodentium, all of which use attaching and effacing (A/E) lesion formation as a major mechanism of tissue targeting and infection. Both EHEC and EPEC are poorly pathogenic in mice but infect humans and domestic animals. In contrast, C. rodentium is a natural mouse pathogen that is related to E. coli, hence providing an excellent in vivo model for A/E lesion forming pathogens. C. rodentium also provides a model of infections that are mainly restricted to the lumen of the intestine. The mechanism's by which the immune system deals with such infections has become a topic of great interest in recent years. Here we review the literature of C. rodentium from its emergence in the mid-1960s to the most contemporary reports of colonization, pathogenesis, transmission and immunity.     

Organ specificity, colonization and clearance dynamics in vivo following oral challenges with the murine pathogen Citrobacter rodentium

Citrobacter rodentium belongs to a family of human and animal enteric pathogens that includes the clinically significant enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens use attaching and effacing (A/E) lesions to colonize the host gastrointestinal tract. In this study we have used bioluminescence imaging (BLI) to investigate the organ specificity, dynamics of colonization and clearance of mice by C. rodentium in situ and in real time. The bioluminescent C. rodentium derivative, strain ICC180, expresses the luxCDABE operon from the entemopathogenic nematode symbiont Photorhabdus luminescens and light levels accurately reflect bacterial numbers both in vitro and in vivo. We have demonstrated that primary colonization of the mouse by C. rodentium takes place within the caecum, specifically within the specialized patch of lymphoid tissue known as the caecal patch. Following colonization of the caecum C. rodentium established a colonic infection. Clearance of C. rodentium ICC180 parallels the colonization dynamics, i.e. the caecum was first to be cleared followed by the colon. A bioluminescent eae (encoding the outer membrane adhesin intimin) C. rodentium mutant failed to establish long-term colonization, although low levels of bacteria could be recovered for up to 3 days post challenge from the caecum.     

Western-style diet impedes colonization and clearance of Citrobacter rodentium

Western-style diet (WSD), which is high in fat and low in fiber, lacks nutrients to support gut microbiota. Consequently, WSD reduces microbiota density and promotes microbiota encroachment, potentially influencing colonization resistance, immune system readiness, and thus host defense against pathogenic bacteria. Here we examined the impact of WSD on infection and colitis in response to Citrobacter rodentium. We observed that, relative to mice consuming standard rodent grain-based chow (GBC), feeding WSD starkly altered the dynamics of Citrobacter infection, reducing initial colonization and inflammation but frequently resulting in persistent infection that associated with low-grade inflammation and insulin resistance. WSD’s reduction in initial Citrobacter virulence appeared to reflect that colons of GBC-fed mice contain microbiota metabolites, including short-chain fatty acids, especially acetate, that drive Citrobacter growth and virulence. Citrobacter persistence in WSD-fed mice reflected inability of resident microbiota to out-compete it from the gut lumen, likely reflecting the profound impacts of WSD on microbiota composition. These studies demonstrate potential of altering microbiota and their metabolites by diet to impact the course and consequence of infection following exposure to a gut pathogen.     

Molecular pathogenesis of Citrobacter rodentium and transmissible murine colonic hyperplasia

Here we review the history, clinical significance, pathology and molecular pathogenesis of Citrobacter rodentium, the causative agent of transmissible murine colonic hyperplasia. C. rodentium serves as an important model pathogen for investigating the mechanisms controlling attaching and effacing pathology, epithelial hyperproliferation, and tumor promotion in the distal colon of the mouse.     

Role of inflammasomes in host defense against Citrobacter rodentium infection

Citrobacter rodentium is an enteric bacterial pathogen of the mouse intestinal tract that triggers inflammatory responses resembling those of humans infected with enteropathogenic and enterohemorrhagic Escherichia coli. Inflammasome signaling is emerging as a central regulator of inflammatory and host responses to several pathogens, but the in vivo role of inflammasome signaling in host defense against C. rodentium has not been characterized. Here, we show that mice lacking the inflammasome components Nlrp3, Nlrc4, and caspase-1 were hypersusceptible to C. rodentium-induced gastrointestinal inflammation. This was due to defective interleukin (IL)-1β and IL-18 production given that il-1β(-/-) and il-18(-/-) mice also suffered from increased bacterial burdens and exacerbated histopathology. C. rodentium specifically activated the Nlrp3 inflammasome in in vitro-infected macrophages independently of a functional bacterial type III secretion system. Thus, production of IL-1β and IL-18 downstream of the Nlrp3 and Nlrc4 inflammasomes plays a critical role in host defense against enteric infections caused by C. rodentium.     

Transcriptional and metabolic regulation of EHEC and Citrobacter rodentium pathogenesis

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Cell attachment properties and infectivity of host-adapted and environmentally adapted Citrobacter rodentium

Citrobacter rodentium belongs to a family of extracellular enteric pathogens that include enterohaemorrhagic and enteropathogenic Escherichia coli, which colonises the gastrointestinal mucosa by the attaching and effacing (A/E) mechanism. We previously described the appearance of a 'hyper-infectious' state after passage of C. rodentium through the murine gastrointestinal tract. Here we report that host-adapted C. rodentium is able to efficiently adhere and trigger actin polymerisation on cultured epithelial cells. Consistent with these observations we recorded higher levels of expression of genes carried on the LEE pathogenicity island and type III secretion system effector genes carried on prophages compared with in vitro-grown bacteria; importantly, the level of ler gene expression was unchanged. These phenotypes were lost after shed C. rodentium was adapted to the external environment. Upon exposure of C57Bl/6 mice, environmentally adapted C. rodentium was no longer infectious at the low doses associated with host-adapted bacteria and the bacteria were found to be localised in the caecal patch in a similar way to C. rodentium cultured in laboratory media. Thus, the 'hyper-infectious' host-adapted state, allowing efficient transmission and colonisation of naive hosts, is transient in nature and gradually lost after shedding into the environment.     

A Comprehensive Proteomic Analysis of the Type III Secretome of Citrobacter rodentium

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium belong to the family of attaching and effacing (A/E) bacterial pathogens. They intimately attach to host intestinal epithelial cells, trigger the effacement of intestinal microvilli, and cause diarrheal disease. Central to their pathogenesis is a type III secretion system (T3SS) encoded by a pathogenicity island called the locus of enterocyte effacement (LEE). The T3SS is used to inject both LEE- and non-LEE-encoded effector proteins into the host cell, where these effectors modulate host signaling pathways and immune responses. Identifying the effectors and elucidating their functions are central to understanding the molecular pathogenesis of these pathogens. Here we analyzed the type III secretome of C. rodentium using the highly sensitive and quantitative SILAC (stable isotope labeling with amino acids in cell culture)-based mass spectrometry. This approach not only confirmed nearly all known secreted proteins and effectors previously identified by conventional biochemical and proteomic techniques, but also identified several new secreted proteins. The T3SS-dependent secretion of these new proteins was validated, and five of them were translocated into cultured cells, representing new or additional effectors. Deletion mutants for genes encoding these effectors were generated in C. rodentium and tested in a murine infection model. This study comprehensively characterizes the type III secretome of C. rodentium, expands the repertoire of type III secreted proteins and effectors for the A/E pathogens, and demonstrates the simplicity and sensitivity of using SILAC-based quantitative proteomics as a tool for identifying substrates for protein secretion systems.     

Critical role of T cell-dependent serum antibody, but not the gut-associated lymphoid tissue, for surviving acute mucosal infection with Citrobacter rodentium, an attaching and effacing pathogen

Citrobacter rodentium uses virulence factors similar to the enteropathogenic Escherichia coli to produce attaching and effacing lesions in the distal colon of mice. We used this infection model to determine components of adaptive immunity needed to survive infection. During acute infection, wild-type mice develop breaks across infected epithelial surfaces but resolve infection. Surprisingly, mice markedly deficient in mucosal lymphocyte populations from beta(7) integrin deficiency resolve infection, as do CD8alpha-/- or TCR-delta-/- mice. In contrast, CD4-/- or TCR-beta-/- mice develop polymicrobial sepsis and end-organ damage, and succumb during acute infection, despite epithelial damage similar to wild-type mice. B cell-deficient (MuMT-/-) or B and T cell-deficient (recombinase-activating gene 2-/-) mice develop severe pathology in colon and internal organs, and deteriorate rapidly during acute infection. Surviving mice develop robust Citrobacter-specific serum IgM responses during acute infection, whereas mice that succumb do not. However, CD4-/- mice receiving serum Igs from infected wild-type mice survive and clear the infection. Our data show that survival of apparently self-limited and luminal mucosal infections requires a systemic T cell-dependent Ab response against bacteria that enter through damaged mucosa. These findings have implications for understanding host defense against mucosal infections, including the pathogenesis of these diseases in immunocompromised populations.     

Long-term Selenium Deficiency Increases the Pathogenicity of a Citrobacter rodentium Infection in Mice

Citrobacter rodentium is a mouse pathogen that causes infectious colitis and shares characteristics with human enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli, including the ability to cause attaching and effacing lesions in the colon and serves as a useful model to study the pathogenicity of these bacteria. In this study, mice were fed a selenium-deficient diet for 5 or 20?weeks and then infected with C. rodentium. Colonization of the colon by C. rodentium was similar in mice fed adequate or selenium-deficient diets, but total bacterial colonization of the spleen was elevated in mice fed selenium-deficient diet for 20?weeks. Infection-induced changes to the colon included inflammatory cell infiltration, gross changes in crypt architecture, and ulceration and denuding of the epithelial layer that were greatest in mice fed a selenium-deficient diet for 20?weeks. Expression of pro-inflammatory genes was significantly higher 12-days post-infection in mice fed the selenium-deficient diet for 20?weeks compared to mice fed a selenium-adequate diet or selenium-deficient diet for 5?weeks. Diarrhea was prevalent in mice fed the selenium-deficient diet for 20?weeks but not 5?weeks, and this was associated with decreased expression of solute carrier family 26a3 and carbonic anhydrase IV, genes involved in ion transport. These results indicated that selenium played an important role in resistance to the pathological effects of a C. rodentium infection, and therefore, selenium status may be important in the expression of human disease caused by common food-borne bacteria.     

Absence of PmrAB-mediated phosphoethanolamine modifications of Citrobacter rodentium lipopolysaccharide affects outer membrane integrity

The PmrAB two-component system of enterobacteria regulates a number of genes whose protein products modify lipopolysaccharide (LPS). The LPS is modified during transport to the bacterial outer membrane (OM). A subset of PmrAB-mediated LPS modifications consists of the addition of phosphoethanolamine (pEtN) to lipid A by PmrC and to the core by CptA. In Salmonella enterica, pEtN modifications have been associated with resistance to polymyxin B and to excess iron. To investigate putative functions of pEtN modifications in Citrobacter rodentium, ΔpmrAB, ΔpmrC, ΔcptA, and ΔpmrC ΔcptA deletion mutants were constructed. Compared to the wild type, most mutant strains were found to be more susceptible to antibiotics that must diffuse across the LPS layer of the OM. All mutant strains also showed increased influx rates of ethidium dye across their OM, suggesting that PmrAB-regulated pEtN modifications affect OM permeability. This was confirmed by increased partitioning of the fluorescent dye 1-N-phenylnaphthylamine (NPN) into the OM phospholipid layer of the mutant strains. In addition, substantial release of periplasmic β-lactamase was observed for the ΔpmrAB and ΔpmrC ΔcptA strains, indicating a loss of OM integrity. This study attributes a new role for PmrAB-mediated pEtN LPS modifications in the maintenance of C. rodentium OM integrity.     

TLR signaling mediated by MyD88 is required for a protective innate immune response by neutrophils to Citrobacter rodentium

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium are classified as attaching and effacing pathogens based on their ability to adhere to intestinal epithelium via actin-filled membranous protrusions (pedestals). Infection of mice with C. rodentium causes breach of the colonic epithelial barrier, a vigorous Th1 inflammatory response, and colitis. Ultimately, an adaptive immune response leads to clearance of the bacteria. Whereas much is known about the adaptive response to C. rodentium, the role of the innate immune response remains unclear. In this study, we demonstrate for the first time that the TLR adaptor MyD88 is essential for survival and optimal immunity following infection. MyD88(-/-) mice suffer from bacteremia, gangrenous mucosal necrosis, severe colitis, and death following infection. Although an adaptive response occurs, MyD88-dependent signaling is necessary for efficient clearance of the pathogen. Based on reciprocal bone marrow transplants in conjunction with assessment of intestinal mucosal pathology, repair, and cytokine production, our findings suggest a model in which TLR signaling in hemopoietic and nonhemopoietic cells mediate three distinct processes: 1) induction of an epithelial repair response that maintains the protective barrier and limits access of bacteria to the lamina propria; 2) production of KC or other chemokines that attract neutrophils and thus facilitate killing of bacteria; and 3) efficient activation of an adaptive response that facilitates Ab-mediated clearance of the infection. Taken together, these experiments provide evidence for a protective role of innate immune signaling in infections caused by attaching and effacing pathogens.