The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.     

Cryptococcus Strains with Different Pathogenic Potentials Have Diverse Protein Secretomes

Secreted proteins are the frontline between the host and pathogen. In mammalian hosts, secreted proteins enable invasive infection and can modulate the host immune response. Cryptococcosis, caused by pathogenic Cryptococcus species, begins when inhaled infectious propagules establish to produce pulmonary infection, which, if not resolved, can disseminate to the central nervous system to cause meningoencephalitis. Strains of Cryptococcus species differ in their capacity to cause disease, and the mechanisms underlying this are not well understood. To investigate the role of secreted proteins in disease, we determined the secretome for three genome strains of Cryptococcus species, including a hypovirulent and a hypervirulent strain of C. gattii and a virulent strain of C. neoformans. Sixty-seven unique proteins were identified, with different numbers and types of proteins secreted by each strain. The secretomes of the virulent strains were largely limited to proteolytic and hydrolytic enzymes, while the hypovirulent strain had a diverse secretome, including non-conventionally secreted canonical cytosolic and immunogenic proteins that have been implicated in virulence. The hypovirulent strain cannot establish pulmonary infection in a mouse model, but strains of this genotype have caused human meningitis. To directly test brain infection, we used intracranial inoculation and found that the hypovirulent strain was substantially more invasive than its hypervirulent counterpart. We suggest that immunogenic proteins secreted by this strain invoke a host response that limits pulmonary infection but that there can be invasive growth and damage if infection reaches the brain. Given their known role in virulence, it is possible that non-conventionally secreted proteins mediate this process.     

Genome-Wide Analysis of the Pho Regulon in a pstCA Mutant of Citrobacter rodentium

The phosphate-specific transport operon, pstSCAB-phoU, of Gram-negative bacteria is an essential part of the Pho regulon. Its key roles are to encode a high-affinity inorganic phosphate transport system and to prevent activation of PhoB in phosphate-rich environments. In general, mutations in pstSCAB-phoU lead to the constitutive expression of the Pho regulon. Previously, we constructed a pstCA deletion mutant of Citrobacter rodentium and found it to be attenuated for virulence in mice, its natural host. This attenuation was dependent on PhoB or PhoB-regulated gene(s) because a phoB mutation restored virulence for mice to the pstCA mutant. To investigate how downstream genes may contribute to the virulence of C. rodentium, we used microarray analysis to investigate global gene expression of C. rodentium strain ICC169 and its isogenic pstCA mutant when grown in phosphate-rich medium. Overall 323 genes of the pstCA mutant were differentially expressed by at least 1.5-fold compared to the wild-type C. rodentium. Of these 145 were up-regulated and 178 were down-regulated. Differentially expressed genes included some involved in phosphate homoeostasis, cellular metabolism and protein metabolism. A large number of genes involved in stress responses and of unknown function were also differentially expressed, as were some virulence-associated genes. Up-regulated virulence-associated genes in the pstCA mutant included that for DegP, a serine protease, which appeared to be directly regulated by PhoB. Down-regulated genes included those for the production of the urease, flagella, NleG8 (a type III-secreted protein) and the tad focus (which encodes type IVb pili in Yersinia enterocolitica). Infection studies using C57/BL6 mice showed that DegP and NleG8 play a role in bacterial virulence. Overall, our study provides evidence that Pho is a global regulator of gene expression in C. rodentium and indicates the presence of at least two previously unrecognized virulence determinants of C. rodentium, namely, DegP and NleG8.     

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.     

The bacterial virulence factor NleA undergoes host-mediated O-linked glycosylation

Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are gastrointestinal pathogens responsible for severe diarrheal illness. EHEC and EPEC form “attaching and effacing” lesions during colonization and, upon adherence, inject proteins directly into host intestinal cells via the type III secretion system (T3SS). Injected bacterial proteins have a variety of functions but generally alter host cell biology to favor survival and/or replication of the pathogen. Non-LEE-encoded effector A (NleA) is a T3SS-injected effector of EHEC, EPEC, and the related mouse pathogen Citrobacter rodentium. Studies in mouse models indicate that NleA has an important role in bacterial virulence. However, the mechanism by which NleA contributes to disease remains unknown. We have determined that the following translocation into host cells, a serine and threonine-rich region of NleA is modified by host-mediated mucin-type O-linked glycosylation. Surprisingly, this region was not present in several clinical EHEC isolates. When expressed in C. rodentium, a non-modifiable variant of NleA was indistinguishable from wildtype NleA in an acute mortality model but conferred a modest increase in persistence over the course of infection in mixed infections in C57BL/6J mice. This is the first known example of a bacterial effector being modified by host-mediated O-linked glycosylation. Our data also suggests that this modification may confer a selective disadvantage to the bacteria during in vivo infection.     

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.     

Sec24 interaction is essential for localization and virulence-associated function of the bacterial effector protein NleA

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) are food-borne pathogens that cause severe diarrhoeal disease in humans. Citrobacter rodentium is a related mouse pathogen that serves as a small animal model for EPEC and EHEC infections. EPEC, EHEC and C. rodentium translocate bacterial virulence proteins directly into host cells via a type III secretion system (T3SS). Non-LEE-encoded effector A (NleA) is a T3SS effector that is common to EPEC, EHEC and C. rodentium and is required for bacterial virulence. NleA localizes to the host cell secretory pathway and inhibits vesicle trafficking by interacting with the Sec24 subunit of mammalian coatamer protein II complex (COPII). Mammalian cells express four paralogues of Sec24 (Sec24A-D), which mediate selection of cargo proteins for transport and possess distinct, but overlapping cargo specificities. Here, we show that NleA binds Sec24A-D with two distinct mechanisms. An NleA protein variant with greatly diminished interaction with all Sec24 paralogues does not properly localize, does not inhibit COPII-mediated vesicle budding, and does not confer virulence in the mouse infection model. Together, this work provides strong evidence that the interaction and inhibition of COPII by NleA is an important aspect of EPEC- and EHEC-mediated disease.     

Emergence of a 'hyperinfectious' bacterial state after passage of Citrobacter rodentium through the host gastrointestinal tract

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 exploit attaching and effacing (A/E) lesions to colonize the host gastrointestinal tract. However, both EHEC and EPEC are poorly pathogenic in mice. In contrast, C. rodentium, which is genetically highly related to E. coli, relies on A/E lesion formation as an essential step in both colonization and infection of the murine mucosa, providing an excellent in vivo model. In this study we have used bioluminescence imaging (BLI) to investigate the organ specificity and dynamics of colonization of mice by LB-grown and mouse-passaged C. rodentium in situ and in real time. We have demonstrated the appearance of a 'hyperinfectious' state after passage of C. rodentium through the murine gastrointestinal tract. The 'hyperinfectious' state was found to dramatically reduce the dose required to infect secondary individuals, and also influenced the tissue distribution of colonizing bacteria, removing the requirement for primary colonization of the caecal patch. In addition, the 'hyperinfectious' phenotype was found to be transient with one overnight passage in rich medium sufficient to return C. rodentium to 'culture' infectivity.     

A C-terminal class I PDZ binding motif of EspI/NleA modulates the virulence of attaching and effacing Escherichia coli and Citrobacter rodentium

Enteropathogenic Escherichia coli induces characteristic attaching–effacing (A/E) lesions on the intestinal mucosa during infection. The locus of enterocyte effacement is essential for A/E lesion formation and encodes a type III secretion system that translocates multiple effector proteins into the host cell. Following translocation, EspI/NleA localizes to the Golgi. Using the yeast two-hybrid system (Y2HS) and PSD-95/Disk-large/ZO-1 (PDZ)-domain protein array overlays, we identified 15 putative host-interacting partners of EspI. All but two of the target proteins contained PDZ domains. Examination of the EspI amino acid sequence revealed a C-terminal consensus class I PDZ binding motif. Deletion of the last 7 amino acids of EspI to generate EspI_ΔC7 abrogated the Y2HS interaction between EspI and 5 of the 6 putative host cell target proteins tested. Deletion of the EspI PDZ binding motif also resulted in delayed trafficking of EspI to the Golgi. Using a mouse model of infection, we showed that Citrobacter rodentium expressing truncated EspI_ΔC7 was attenuated when in competition with C. rodentium expressing full-length EspI. Overall, these results suggested that EspI may modulate the virulence of A/E pathogens by binding host PDZ-domain proteins.     

One Small Step for a Yeast - Microevolution within Macrophages Renders Candida glabrata Hypervirulent Due to a Single Point Mutation

Candida glabrata is one of the most common causes of candidemia, a life-threatening, systemic fungal infection, and is surpassed in frequency only by Candida albicans. Major factors contributing to the success of this opportunistic pathogen include its ability to readily acquire resistance to antifungals and to colonize and adapt to many different niches in the human body. Here we addressed the flexibility and adaptability of C. glabrata during interaction with macrophages with a serial passage approach. Continuous co-incubation of C. glabrata with a murine macrophage cell line for over six months resulted in a striking alteration in fungal morphology: The growth form changed from typical spherical yeasts to pseudohyphae-like structures – a phenotype which was stable over several generations without any selective pressure. Transmission electron microscopy and FACS analyses showed that the filamentous-like morphology was accompanied by changes in cell wall architecture. This altered growth form permitted faster escape from macrophages and increased damage of macrophages. In addition, the evolved strain (Evo) showed transiently increased virulence in a systemic mouse infection model, which correlated with increased organ-specific fungal burden and inflammatory response (TNFα and IL-6) in the brain. Similarly, the Evo mutant significantly increased TNFα production in the brain on day 2, which is mirrored in macrophages confronted with the Evo mutant, but not with the parental wild type. Whole genome sequencing of the Evo strain, genetic analyses, targeted gene disruption and a reverse microevolution experiment revealed a single nucleotide exchange in the chitin synthase-encoding CHS2 gene as the sole basis for this phenotypic alteration. A targeted CHS2 mutant with the same SNP showed similar phenotypes as the Evo strain under all experimental conditions tested. These results indicate that microevolutionary processes in host-simulative conditions can elicit adaptations of C. glabrata to distinct host niches and even lead to hypervirulent strains.     

R-Spondins Are Expressed by the Intestinal Stroma and are Differentially Regulated during Citrobacter rodentium- and DSS-Induced Colitis in Mice

The R-spondin family of proteins has recently been described as secreted enhancers of β-catenin activation through the canonical Wnt signaling pathway. We previously reported that Rspo2 is a major determinant of susceptibility to Citrobacter rodentium-mediated colitis in mice and recent genome-wide association studies have revealed RSPO3 as a candidate Crohn’s disease-specific inflammatory bowel disease susceptibility gene in humans. However, there is little information on the endogenous expression and cellular source of R-spondins in the colon at steady state and during intestinal inflammation. RNA sequencing and qRT-PCR were used to assess the expression of R-spondins at steady state and in two mouse models of colonic inflammation. The cellular source of R-spondins was assessed in specific colonic cell populations isolated by cell sorting. Data mining from publicly available datasets was used to assess the expression of R-spondins in the human colon. At steady state, colonic expression of R-spondins was found to be exclusive to non-epithelial CD45^- lamina propria cells, and Rspo3/RSPO3 was the most highly expressed R-spondin in both mouse and human colon. R-spondin expression was found to be highly dynamic and differentially regulated during C. rodentium infection and dextran sodium sulfate (DSS) colitis, with notably high levels of Rspo3 expression during DSS colitis, and high levels of Rspo2 expression during C. rodentium infection, specifically in susceptible mice. Our data are consistent with the hypothesis that in the colon, R-spondins are expressed by subepithelial stromal cells, and that Rspo3/RSPO3 is the family member most implicated in colonic homeostasis. The differential regulation of the R-spondins in different models of intestinal inflammation indicate they respond to specific pathogenic and inflammatory signals that differ in the two models and provides further evidence that this family of proteins plays a key role in linking intestinal inflammation and homeostasis.     

Transmission of Hypervirulence Traits via Sexual Reproduction within and between Lineages of the Human Fungal Pathogen Cryptococcus gattii

Since 1999 a lineage of the pathogen Cryptococcus gattii has been infecting humans and other animals in Canada and the Pacific Northwest of the USA. It is now the largest outbreak of a life-threatening fungal infection in a healthy population in recorded history. The high virulence of outbreak strains is closely linked to the ability of the pathogen to undergo rapid mitochondrial tubularisation and proliferation following engulfment by host phagocytes. Most outbreaks spread by geographic expansion across suitable niches, but it is known that genetic re-assortment and hybridisation can also lead to rapid range and host expansion. In the context of C. gattii, however, the likelihood of virulence traits associated with the outbreak lineages spreading to other lineages via genetic exchange is currently unknown. Here we address this question by conducting outgroup crosses between distantly related C. gattii lineages (VGII and VGIII) and ingroup crosses between isolates from the same molecular type (VGII). Systematic phenotypic characterisation shows that virulence traits are transmitted to outgroups infrequently, but readily inherited during ingroup crosses. In addition, we observed higher levels of biparental (as opposed to uniparental) mitochondrial inheritance during VGII ingroup sexual mating in this species and provide evidence for mitochondrial recombination following mating. Taken together, our data suggest that hypervirulence can spread among the C. gattii lineages VGII and VGIII, potentially creating novel hypervirulent genotypes, and that current models of uniparental mitochondrial inheritance in the Cryptococcus genus may not be universal.