Marjan Ghaem-Maghami

Central role for B lymphocytes and CD4+ T cells in immunity to infection by the attaching and effacing pathogen Citrobacter rodentium.

ABSTRACT Citrobacter rodentium, an attaching-effacing bacterial pathogen, establishes an acute infection of the murine colonic epithelium and induces a mild colitis in immunocompetent mice. This study describes the role of T-cell subsets and B lymphocytes in immunity to C. rodentium. C57Bl/6 mice orally infected with C. rodentium resolved infection within 3 to 4 weeks. Conversely, systemic and colonic tissues of RAG1−/− mice orally infected with C. rodentium contained high and sustained pathogen loads, and in the colon this resulted in a severe colitis. C57Bl/6 mice depleted of CD4+ T cells, but not CD8+ T cells, were highly susceptible to infection and also developed severe colitis. Mice depleted of CD4+ T cells also had diminished immunoglobulin G (IgG) and IgA antibody responses to two C. rodentium virulence-associated determinants, i.e., EspA and intimin, despite having a massively increased pathogen burden. Mice with an intact T-cell compartment, but lacking B cells (μMT mice), were highly susceptible to C. rodentium infection. Systemic immunity, but not mucosal immunity, could be restored by adoptive transfer of convalescent immune sera to infected μMT mice. Adoptive transfer of immune B cells, but not naïve B cells, provided highly variable immunity to recipient μMT mice. The results suggest that B-cell-mediated immune responses are central to resolution of a C. rodentium infection but that the mechanism through which this occurs requires further investigation. These data are relevant to understanding immunity to enteric attaching and effacing bacterial pathogens of humans.

Characterization of Salmonella enterica Derivatives Harboring Defined aroC and Salmonella Pathogenicity Island 2 Type III Secretion System (ssaV) Mutations by Immunization of Healthy Volunteers

ABSTRACT The attenuation and immunogenicity of two novel Salmonella vaccine strains, Salmonella enterica serovar Typhi (Ty2 ΔaroC ΔssaV, designated ZH9) and S. enterica serovar Typhimurium (TML ΔaroC ΔssaV, designated WT05), were evaluated after their oral administration to volunteers as single escalating doses of 107, 108, or 109 CFU. ZH9 was well tolerated, not detected in blood, nor persistently excreted in stool. Six of nine volunteers elicited anti-serovar Typhi lipopolysaccharide (LPS) immunoglobulin A (IgA) antibody-secreting cell (ASC) responses, with three of three vaccinees receiving 108 and two of three receiving 109 CFU which elicited high-titer LPS-specific serum IgG. WT05 was also well tolerated with no diarrhea, although the administration of 108 and 109 CFU resulted in shedding in stools for up to 23 days. Only volunteers immunized with 109 CFU of WT05 mounted detectable serovar Typhimurium LPS-specific ASC responses and serum antibody responses were variable. These data indicate that mutations in type III secretion systems may provide a route to the development of live vaccines in humans and highlight significant differences in the potential use of serovars Typhimurium and Typhi.

Impaired Resistance and Enhanced Pathology During Infection with a Noninvasive, Attaching-Effacing Enteric Bacterial Pathogen, Citrobacter rodentium, in Mice Lacking IL-12 or IFN-γ

Mice infected with Citrobacter rodentium represent an excellent model in which to examine immune defenses against an attaching-effacing enteric bacterial pathogen. Colonic tissue from mice infected with C. rodentium harbors increased transcripts for IL-12 and IFN-γ and displays mucosal pathology compared with uninfected controls. In this study, the role of IL-12 and IFN-γ in host defense and mucosal injury during C. rodentium infection was examined using gene knockout mice. IL-12p40−/− and IFN-γ−/− mice were significantly more susceptible to mucosal and gut-derived systemic C. rodentium infection. In particular, a proportion of IL-12p40−/− mice died during infection. Analysis of the gut mucosa of IL-12p40−/− mice revealed an influx of CD4+ T cells and a local IFN-γ response. Infected IL-12p40−/− and IFN-γ−/− mice also mounted anti-Citrobacter serum and gut-associated IgA responses and strongly expressed inducible NO synthase (iNOS) in mucosal tissue, despite diminished serum nitrite/nitrate levels. However, iNOS does not detectably contribute to host defense against C. rodentium, as iNOS−/− mice were not more susceptible to infection. However, C57BL/6 mice infected with C. rodentium up-regulated expression of the mouse β-defensin (mBD)-1 and mBD-3 in colonic tissue. In contrast, expression of mBD-3, but not mBD-1, was significantly attenuated during infection of IL-12- and IFN-γ-deficient mice, suggesting mBD-3 may contribute to host defense. These studies are among the first to examine mechanisms of host resistance to an attaching-effacing pathogen and show an important role for IL-12 and IFN-γ in limiting bacterial infection of the colonic epithelium.

Critical Role for Tumor Necrosis Factor Alpha in Controlling the Number of Lumenal Pathogenic Bacteria and Immunopathology in Infectious Colitis

ABSTRACT Infection of mice with the intestinal bacterial pathogenCitrobacter rodentium results in colonic mucosal hyperplasia and a local Th1 inflammatory response similar to that seen in mouse models of inflammatory bowel disease. In these latter models, and in patients with Crohns disease, neutralization of tumor necrosis factor alpha (TNF-α) is of therapeutic benefit. Since there is no information on the role of TNF-α in either immunity to noninvasive bacterial pathogens or on the role of TNF-α in the immunopathology of infectious colitis, we investigated C. rodentiuminfection in TNFRp55−/− mice. In TNFRp55−/−mice, there were higher colonic bacterial burdens, but the organisms were cleared at the same rate as C57BL/6 mice, showing that TNF-α is not needed for protective antibacterial immunity. The most striking feature of infection in TNFRp55−/−mice, however, was the markedly enhanced pathology, with increased mucosal weight and thickness, increased T-cell infiltrate, and a markedly greater mucosal Th1 response. Interleukin-12 p40 transcripts were markedly elevated in C. rodentium-infected TNFRp55−/− mice, and this was associated with enhanced mucosal STAT4 phosphorylation. TNF-α is not obligatory for protective immunity to C. rodentium in mice; however, it appears to play some role in downregulating mucosal pathology and Th1 immune responses.

Intimin-specific immune responses prevent bacterial colonization by the attaching-effacing pathogen Citrobacter rodentium.

ABSTRACT The formation of attaching and effacing (A/E) lesions on gut enterocytes is central to the pathogenesis of enterohemorrhagic (EHEC)Escherichia coli, enteropathogenic E. coli (EPEC), and the rodent pathogen Citrobacter rodentium. Genes encoding A/E lesion formation map to a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Here we show that the LEE-encoded proteins EspA, EspB, Tir, and intimin are the targets of long-lived humoral immune responses in C. rodentium-infected mice. Mice infected with C. rodentium developed robust acquired immunity and were resistant to reinfection with wild-type C. rodentium or a C. rodentium derivative, DBS255(pCVD438), which expressed intimin derived from EPEC strain E2348/69. The receptor-binding domain of intimin polypeptides is located within the carboxy-terminal 280 amino acids (Int280). Mucosal and systemic vaccination regimens using enterotoxin-based adjuvants were employed to elicit immune responses to recombinant Int280α from EPEC strain E2348/69. Mice vaccinated subcutaneously with Int280α, in the absence of adjuvant, were significantly more resistant to oral challenge with DBS255(pCVD438) but not with wild-type C. rodentium. This type-specific immunity could not be overcome by employing an exposed, highly conserved domain of intimin (Int388–667) as a vaccine. These results show that anti-intimin immune responses can modulate the outcome of a C. rodentium infection and support the use of intimin as a component of a type-specific EPEC or EHEC vaccine.

Tyrosine residues at the immunoglobulin-C-type lectin inter-domain boundary of intimin are not involved in Tir-binding but implicated in colonisation of the host

Intimin is an outer membrane adhesion molecule involved in bacterial adhesion to intestinal epithelium by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli and Citrobacter rodentium. Intimin binds to the translocated intimin receptor, Tir, which is delivered to the plasma membrane of the host cell by a type III protein translocation system. Intimin is also implicated in binding to a host cell-encoded intimin receptor (Hir). The receptor-binding activity of intimin resides within the carboxy terminus 280 amino acids (Int280) of the polypeptide. Structural analysis of this region revealed two immunoglobulin-like domains, the second of which forms a number of contacts with the distal C-type lectin-like module. Specific orientation differences at this inter-domain boundary, which consists of several tyrosine residues, were detected between the crystal and solution structures. In this study, we determined the influence of site-directed mutagenesis of each of four tyrosine residues on intimin-Tir interactions and on intimin-mediated intimate attachment. The mutant intimins were also studied using a variety of in vitro and in vivo infection models. The results show that three of the four Tyr, although not essential for A/E lesion formation in vitro, are required for efficient colonisation of the mouse host following oral challenge.