Contribution of Toll-like receptors 2 and 4 in an oral Yersinia enterocolitica mouse infection model

doi:10.1078/1438-4221-00277

International Journal of Medical Microbiology

Volume 293, Issue 5, 2003, Pages 341-348

https://doi.org/10.1078/1438-4221-00277 Get rights and content

Abstract

A characteristic of the three human-pathogenic Yersinia spp. (the plague agent Y. pestis and the enteropathogenic Y. pseudotuberculosis and Y. enterocolitica) is the expression of the virulence (V)-antigen (LcrV). LcrV is a released multifunctional protein which is involved in contact-induced secretion of Yersinia antihost proteins and in evasion of the host innate immune response. Recently, we reported that recombinant LcrV signals in a CD14- and TLR2-dependent fashion leading to immunosuppression by interleukin-10 (IL-10) induction. The impact of this immunosuppressive effect for Yersinia pathogenesis was underlined by the observation that IL-10- and TLR2-deficient mice were found to be less susceptible to Y. enterocolitica infection than isogenic C57BL/6 wild-type animals. In the present study, we show that Y. enterocolitica leads to higher IL-10 and lower TNF-α levels in spleens from infected C57BL/6 wild-type mice than in those from TLR2-deficient mice. By comparing Y. enterocolitica infection in TLR2-, TLR4-, and TLR2/TLR4-deficient mice, we found that TLR2 is more important in yersiniosis than TLR4. Strikingly and in contrast to the results obtained in TLR2-deficient mice of C57BL/6 background, TLR2-deficient mice of C3H genetic background were more susceptible to an oral Y. enterocolitica infection than wild-type C3H mice. To our knowledge, this is the first report on a divergent influence of a TLR-deficiency on infection outcome in mice of different genetic backgrounds.

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Cited by (23)

Role of β1 integrins and bacterial adhesins for Yop injection into leukocytes in Yersinia enterocolitica systemic mouse infection
2016, International Journal of Medical Microbiology
Citation Excerpt :
To circumvent this problem and address whether YadA is required for Yop injection, we infected TLR2−/−xTLR4−/− mice with a C57BL/6 background with E40-pBla or ΔYadA-pBla for two days. TLR2−/−xTLR4−/− mice were chosen because they are highly susceptible to Ye infection (Sing et al., 2003). Under these conditions, we were able to detect a comparable bacterial load in the spleen after infection with both E40-pBla and ΔYadA-pBla (Fig. 4A) and could therefore analyze Yop injection.
Injection of Yersinia outer proteins (Yops) into host cells by a type III secretion system is an important immune evasion mechanism of Yersinia enterocolitica (Ye). In this process Ye invasin (Inv) binds directly while Yersinia adhesin A (YadA) binds indirectly via extracellular matrix (ECM) proteins to β1 integrins on host cells. Although leukocytes turned out to be an important target of Yop injection by Ye, it was unclear which Ye adhesins and which leukocyte receptors are required for Yop injection. To explain this, we investigated the role of YadA, Inv and β1 integrins for Yop injection into leukocytes and their impact on the course of systemic Ye infection in mice. Ex vivo infection experiments revealed that adhesion of Ye via Inv or YadA is sufficient to promote Yop injection into leukocytes as revealed by a β-lactamase reporter assay. Serum factors inhibit YadA- but not Inv-mediated Yop injection into B and T cells, shifting YadA-mediated Yop injection in the direction of neutrophils and other myeloid cells. Systemic Ye mouse infection experiments demonstrated that YadA is essential for Ye virulence and Yop injection into leukocytes, while Inv is dispensable for virulence and plays only a transient and minor role for Yop injection in the early phase of infection. Ye infection of mice with β1 integrin-depleted leukocytes demonstrated that β1 integrins are dispensable for YadA-mediated Yop injection into leukocytes, but contribute to Inv-mediated Yop injection. Despite reduced Yop injection into leukocytes, β1 integrin-deficient mice exhibited an increased susceptibility for Ye infection, suggesting an important role of β1 integrins in immune defense against Ye. This study demonstrates that Yop injection into leukocytes by Ye is largely mediated by YadA exploiting, as yet unknown, leukocyte receptors.
Amino acid and structural variability of Yersinia pestis LcrV protein
2010, Infection, Genetics and Evolution
The LcrV protein is a multifunctional virulence factor and protective antigen of the plague bacterium and is generally conserved between the epidemic strains of Yersinia pestis. We investigated the diversity in the LcrV sequences among non-epidemic Y. pestis strains which have a limited virulence in selected animal models and for humans. Sequencing of lcrV genes from 19 Y. pestis strains belonging to different phylogenetic groups (subspecies) showed that the LcrV proteins possess four major variable hotspots at positions 18, 72, 273, and 324–326. These major variations, together with other minor substitutions in amino acid sequences, allowed us to classify the LcrV alleles into five sequence types (A–E). We observed that the strains of different Y. pestis “subspecies” can have the same type of LcrV, including that conserved in epidemic strains, and different types of LcrV can exist within the same natural plague focus. Therefore, the phenomenon of “selective virulence” characteristic of the strains of the microtus biovar is unlikely to be the result of polymorphism of the V antigen. The LcrV polymorphisms were structurally analyzed by comparing the modeled structures of LcrV from all available strains. All changes except one occurred either in flexible regions or on the surface of the protein, but local chemical properties (i.e. those of a hydrophobic, hydrophilic, amphipathic, or charged nature) were conserved across all of the strains. Polymorphisms in flexible and surface regions are likely subject to less selective pressure, and have a limited impact on the structure. In contrast, the substitution of tryptophan at position 113 with either glutamic acid or glycine likely has a serious influence on the regional structure of the protein, and these mutations might have an effect on the function of LcrV. The polymorphisms at positions 18, 72 and 273 were accountable for differences in the oligomerization of LcrV.
The weak interaction of LcrV and TLR2 does not contribute to the virulence of Yersinia pestis
2007, Microbes and Infection
Citation Excerpt :
In Y. enterocolitica O:8 infections, we have shown that LcrVO:8 contributes to virulence by TLR2-mediated IL-10 induction [12] resulting in a higher Y. enterocolitica O:8 resistance of TLR2−/− mice from a C57BL/6 genetic background [10]. Moreover, Y. enterocolitica O:8-infected TLR2−/− mice exhibited lower levels of IL-10 and higher levels of protective TNF-α in spleens [11]. Therefore we asked if this mechanism of TLR2-dependent IL-10 induction via LcrV is also important in plague.
Yersinia pestis and the enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica share the virulence-antigen LcrV. Previously, using reverse genetics we have proven that LcrV contributes to the virulence of Y. enterocolitica serotype O:8 by inducing IL-10 via Toll-like receptor 2 (TLR2). However, both the ability of Y. pestis LcrV to activate TLR2 and a possible role of TLR2-dependent IL-10 induction by LcrV in Y. pestis are not yet known. To eliminate interference from additional protein sequences, we produced LcrVs without affinity tags from Y. pestis and from Y. enterocolitica O:8 (LcrVO:8). LcrVO:8 was much more potent in TLR2-activity than Y. pestis LcrV. To analyse the role of TLR2 in plague, we infected both wild-type and TLR2−/− mice subcutaneously with Y. pestis GB. While TLR2−/− mice exhibited lower blood levels of IL-10 (day 2 post-infection) and of the pro-inflammatory cytokines TNF-α, IFN-γ and MCP-1 (day 4) than wild-type mice, there was no significant difference in survival. The low TLR2-activity of Y. pestis LcrV and associated cytokine expression might explain why - in contrast to Y. enterocolitica O:8 infection - TLR2-deficient mice are not more resistant than wild-type mice in a bubonic plague model.
Cellular mechanisms of bacterial internalization counteracted by Yersinia
2005, International Review of Cytology
Citation Excerpt :
Yersinia triggers an inflammatory response that involves activation of NF‐κB and secretion of IL‐8 upon contact with host cells, which is similar to the response seen with pathogens like Helicobacter pylori, Salmonella typhimurium, and enteropathogenic Escherichia coli (Eaves‐Pyles et al., 1999; Fischer et al., 2001; Foryst‐Ludwig et al., 2004; Keates et al., 1997). Although several virulence plasmid‐encoded factors, such as YadA, LcrV (Sing et al., 2003), and YopB (Viboud et al., 2003), have been suggested to contribute to the induction caused by Yersinia, recent cDNA array data imply that invasin and lipopolysaccharides (LPSs) are the predominant factors causing upregulation of proinflammatory genes in both epithelial cells and macrophages (Bohn et al., 2004; Hoffmann et al., 2004). In experiments using invasin‐coated beads, it has been shown that invasin alone is sufficient to induce NF‐κB activation and IL‐8 synthesis (Grassl et al., 2003; Schulte et al., 2000).
Upon host‐cell contact, human pathogenic Yersinia species inject Yop virulence effectors into the host through a Type III secretion‐and‐translocation system. These virulence effectors cause a block in phagocytosis (YopE, YopT, YpkA, and YopH) and suppression of inflammatory mediators (YopJ). The Yops that block phagocytosis either interfere with the host cell actin regulation of Rho GTPases (YopE, YopT, and YpkA) or specifically and rapidly inactivate host proteins involved in signaling from the receptor to actin (YopH). The block in uptake has been shown to be activated following binding to Fc, Complement, and β1‐integrin receptors in virtually any kind of host cell. Thus, the use of Yersinia as a model system to study Yersinia–host cell interactions provides a good tool to explore signaling pathways involved in phagocytosis.
Yersinia V antigen induces both TLR homo- and heterotolerance in an IL-10-involving manner
2004, Cellular Immunology
Citation Excerpt :
TLRs have recently been identified as major pattern recognition molecules sensing highly conserved microbial structures and transducing signals into the host cell. In previous studies we have shown that rLcrV uses both CD14 and TLR2 for IL-10 or NF-κB induction in murine PPMs and CD14/TLR2-transfected human embryonic kidney cells, respectively [18,38,49]. Here we show that rLcrV-induced IL-10 production is also CD14/TLR2-dependent in the human monocytic cell line Mono-Mac-6 suggesting that exploitation of CD14/TLR2 by LcrV is important for immunomodulation both in murine and human macrophages.
The virulence antigen (LcrV) of pathogenic yersiniae “silences” macrophages against stimulation with the TLR2-agonist zymosan A in a CD14/TLR2-dependent fashion via IL-10 induction. This pathogenically important “silencing” resembles TLR tolerance phenomena; in these, pre-exposure to a primary tolerizing TLR-agonist renders macrophages unresponsive to stimulation with a secondary challenging TLR-agonist which may involve either the same (TLR homotolerance) or a different TLR (TLR heterotolerance) as the primary TLR-agonist. Here, we show that rLcrV induces TLR homo- and heterotolerance against TLR2- or TLR4-agonists both in human and murine macrophages, respectively. The underlying mechanism of LcrV-induced tolerance is most likely not due to changes in TLR2- or TLR4 expression, but involves LcrV-mediated IL-10 production, since LcrV-induced TLR homo- and heterotolerance is highly impaired in IL-10^−/− macrophages. Moreover, the involvement of IL-10 in TLR tolerance induction seems to be a more general phenomenon as shown by experiments using different TLR-agonists in IL-10^−/− macrophages. Since LcrV acts as a secreted protein upon macrophages without requiring direct cell contact, as shown in transwell assays, we propose that yersiniae exploit IL-10-involving TLR tolerance mechanisms by the virulence factor LcrV.
Immunoglobulin for treating bacterial infections: One more mechanism of action
2019, Antibodies

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¹: Both authors contributed equally to this work.

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International Journal of Medical Microbiology

Abstract

References (27)

Toll-like receptors in the induction of the innate immune response

Nature

The tranquilizing injection of Yersinia proteins: a pathogen's strategy to resist host defence

Biol. Chem

Immune responses to Yersinia enterocolitica in susceptible BALB/c and resistant C57BL/6 mice: an essential role for gamma interferon

Infect. Immun

In vivo neutralization of tumor necrosis factor-alpha and interferon-gamma abrogates resistance to Yersinia enterocolitica infection in mice

Med. Microbiol. Immunol. Berl

IL-12 is essential for resistance against Yersinia enterocolitica by triggering IFN-gamma production in NK cells and CD4 + T cells

J. Immunol

Early gamma interferon mRNA expression is associated with resistance of mice against Yersinia enterocolitica

Infect. Immun

An antigen determining virulence in Pasteurella pestis

Nature

The virulence plasmid of Yersinia, an antihost genome

Microbiol. Mol. Biol. Rev

Toll-like receptor 2-deficient mice are highly susceptible to Streptococcus pneumoniae meningitis because of reduced bacterial clearing and enhanced inflammation

J. Infect. Dis

Virulence role of V antigen of Yersinia pestis at the bacterial surface

Infect. Immun

Yersinia enterocolitica infection in resistant and susceptible strains of mice

Infect. Immun

Polymerization of a single protein of the pathogen Yersinia enterocolitica into needles punctures eukaryotic cells

Proc. Natl. Acad. Sci. USA

LcrV is a channel size-determining component of the Yop effector translocon of Yersinia

Mol. Microbiol

Cited by (23)

Role of β1 integrins and bacterial adhesins for Yop injection into leukocytes in Yersinia enterocolitica systemic mouse infection

Amino acid and structural variability of Yersinia pestis LcrV protein

The weak interaction of LcrV and TLR2 does not contribute to the virulence of Yersinia pestis

Cellular mechanisms of bacterial internalization counteracted by Yersinia

Yersinia V antigen induces both TLR homo- and heterotolerance in an IL-10-involving manner

Immunoglobulin for treating bacterial infections: One more mechanism of action