The outcome of an encounter between pathogen and host is determined both by the virulence potential of the pathogen and the defensive potential of the host. Factors determining bacterial pathogenicity are often proteins acting as virulence factors. As one well-characterized example, Yersinia enterocolitica injects anti-host effector proteins (Yersinia outer proteins, Yops) into professional phagocytes via a type III protein secretion/translocation system (TTSS). Both the TTSS and Yops are encoded by a 70 kb virulence plasmid (pYV) that is common to all pathogenic Yersinia spp. The six established effector proteins interfere with distinct signaling pathways resulting in paralysis of phagocyte function. YopP blocks the nuclear factor-#B (NF#B) and mitogen-activated protein kinase (MAPK) pathways and thus inhibits the production of pro-inflammatory cytokines and adhesion molecules. YopP also induces programmed cell death in macrophages. YopE, YopT and YopO interfere with the actin cytoskeleton dynamics of the host cell through modulating the GTPases Rac and Rho, resulting in disruption of the actin cytoskeleton and impaired phagocytosis. YopH is a tyrosine phosphatase that dephosphorylates focal adhesion complex proteins like FAK and p130Cas. The function of YopM is enigmatic to date.
In contrast to these well-characterized bacterial virulence factors, much less is known about host factors determining resistance or susceptibility against bacterial infections. However, different inbred mouse strains differ remarkably in susceptibility against Yersinia infection, with LD50 doses of resistant strains (e.g. C57BL/6) being 100-1000 fold higher after intravenous (iv) infection compared to susceptible strains (e.g. BALB/c) . This difference in susceptibility seems to be under multigenic control and is not linked to the H-2 locus. An important role in control of systemic Yersinia infection has been attributed to an early induction of IFN-# or interleukin-12 in resistant C57BL/6 mice: Intravenous administration of IFN-# to BALB/c mice induces resistance comparable to that of C57BL/6 mice, and administration of IFN-# specific antibodies renders C57BL/6 mice susceptible. Similarly, systemic administration of IL-12 protects BALB/c mice against Y. enterocolitica challenge. However, how exactly this intricate cytokine network acts to mediate antibacterial resistance remains to be determined.
Based on these studies, it seems plausible that host factors critically influence the outcome of a host – pathogen encounter. The aim of this project is to identify target genes, and later on molecular mechanisms, underlying this host difference in susceptibility to bacterial infections.
Results
Transcriptional responses of immortalized macrophage-like cells to Yersinia infection
Transcriptional responses of J774 murine macrophage-like cells to infection with Yersinia enterocolitica were evaluated with oligonucleotide microarrays interrogating 12488 genes and Expressed Sequence Tags. Virulence plasmid (pYV)-cured Yersiniae induce a transcriptional program resembling a general inflammatory response. pYV-carrying Yersiniae translocating the Yersinia outer proteins (Yops) impact on this transcriptional program in two ways: First, by suppressing this inflammatory response, and, second, by inducing sustained expression of a distinct set of genes with known silencing functions. These tranquilizing patterns of gene expression could be confirmed by real-time RT-PCR, are stable upon reduction of bacterial load, and could also be reproduced in BALB/c derived bone marrow macrophages. Prestimulation of macrophages with IFN-#, but not with IL-4, induces partial resistance against pYV-mediated transcriptional tranquilization. The first effect, suppression of the inflammatory program, is mediated by YopP, whereas no YopH- or YopM-regulated genes could be identified under our stringent statistical criteria. The bacterial protein responsible for the second effect, induction of silencing genes, remains elusive. We suggest that Yersinia enterocolitica might employ two independent mechanisms to inhibit macrophage inflammatory responses on the transcriptional level.
Impact of host resistance factors on the transcriptional response of bone marrow derived macrophages to infection with Yersinia enterocolitica
Extending the results obtained with J774 immortalized macrophage like cells, we next aimed to determine the impact of host resistance factors on Yersinia-elicited gene expression patterns. Bone-marrow derived macrophages were generated from BALB/c and C57BL/6 derived bone marrow and infected with Yersinia strains in vitro. By comparing gene expression patterns in macrophages from resistant (C57BL/6) and susceptible (BALB/c) mice or from susceptible mice rendered resistant by administration of IFN-#, we aimed to identify novel genes associated with resistance to acute bacterial infection. Preliminary results indicate that the impact of host resistance status on the gene expression patterns of bone marrow derived macrophages is limited: only 1.2% and 4.9% of all genes differentially expressed in response to Yersinia infection are altered by IFN-# treatment or host genetic background, respectively. These genes, however, are functionally highly relevant in that they are known to augment and modify the immune response. Moreover, analysis of promoter regions of these genes indicates that transcription factors known to be involved in regulating developmental processes may unexpectedly be involved in regulating gene expression during an immune response.
Transcriptional responses of bone marrow derived dendritic cells to infection with Yersinia
Dendritic cells (DC) are crucial in the initiation of an immune response as they are the most effective antigen-presenting cells. In particular, immature DCs are specialized for uptake and intracellular processing of antigen, while mature DCs are most effective in priming T cell responses. We thus aimed to identify differences in transcriptional responses of DCs to Yersinia infection in resistant (C57BL/6) and susceptible (BALB/c) mice). DCs were differentiated in vitro from bone marrow of C57BL/6 and BALB/c mice, infected with Yersinia strains, and immature DCs (intermediate expression of MHC II) were isolated by high-speed fluorescence activated cell sorting (FACS). Gene expression patterns generated with high-density oligonucleotide arrays revealed that the overall pattern of DC transcriptional responses is similar to that of macrophages described above: an inflammatory program elcited by non virulent Yersiniae is suppressed by action of Yops, and genes with silencing functions are induced. Further analyses focus on the comparison between resistant and susceptible mice.
Yersinia-host cell crosstalk in vivo
To date, the transcriptional changes induced by bacterial infection in vivo are relatively poorly understood, since cell populations isolated from infected animals always contain a significant proportion of uninfected cells. However, Yersinia infection results in localized accumulation of bacteria in the form of abscesses in lymphoid organs. We expect that cells in proximity of these bacteria-loaded foci had most intensive contact with bacteria. Presently, mice are infected with GFP-tagged Yersiniae and the cell populations accumulating at Yersinia-abscesses are characterized by multi-color immunofluorescence stainings. Results indicate a highly regularized architecture around Yersinia abscesses, with granulocytes, T cells, B cells, and dendritic cells being organized in characteristic structures, and a fundamental alteration of the general spleen architecture. Subsequently, we will isolate homogeneous cell populations around focal accumulations of Yersiniae by laser-captured microdissection and perform gene expression profiling with high-density oligonucleotide arrays. This will be one of the first studies investigating transcriptional changes induced by bacterial infections in vivo.
Outlook
Currently, this subproject focuses on screening different cell populations of the innate immune system for differences in gene expression patterns in response to Yersinia infection between resistant and susceptible mouse strains. Future studies will extend the collection of Yersinia-elicited gene expression profiles of innate immune cell populations even more by including e.g. NK cells or mast cells. Finally. cell-based and in vivo assays will aim at identifying the cellular and immunological role of the target genes identified here.
Lit.: 1. Hoffmann, R., T. Seidl and M. Dugas (2002). Profound effect of normalization on detection of differentially expressed genes in oligonucleotide microarray data analysis. Genome Biol 3(7): RESEARCH0033. 2. Hoffmann, R., K. Van Erp, K. Trulzsch and J. Heesemann (2004). Transcriptional responses of murine macrophages to infection with Yersinia enterocolitica. Cell Microbiol 6(4): 377-90.
