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Histoplasma capsulatum yeast phase-specific protein Yps3p induces Toll-like receptor 2 signaling.

Aravalli RN, Hu S, Woods JP, Lokensgard JR - J Neuroinflammation (2008)

Bottom Line: Our data show that H. capsulatum Yps3p induced TLR2 signaling in wild-type microglia, but not in microglia isolated from TLR2 KO mice, confirming that Yps3p is a ligand for TLR2.Furthermore, Yps3p-induced TLR2 signaling was suppressed by vaccinia virus-encoded TLR inhibitors.This is the first demonstration of a fungal protein serving as a TLR ligand and mediating signaling in primary brain cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota Medical School, Minneapolis, Minnesota, USA. arava001@umn.edu

ABSTRACT
Histoplasma capsulatum is a common cause of fungal infection in certain geographic areas, and although most infections are asymptomatic, it is capable of causing histoplasmosis, a disseminated, life-threatening disease, especially in immunocompromised individuals. A deeper understanding of this host-pathogen interaction is needed to develop novel therapeutic strategies to counter lethal infection. Although several lines of evidence suggest that this fungus is neurotropic in HIV patients, little is known about the immunobiology of Histoplasma infection in the central nervous system [CNS]. The goal of the present study was to understand the innate neuroimmune mechanisms that recognize H. capsulatum during the initial stages of infection. Using a 293T stable cell line expressing murine Toll-like receptor 2 [TLR2], we show here that TLR2 recognizes H. capsulatum cell wall protein Yps3p and induces the activation of NF-kappaB. In further experiments, we tested the ability of Yps3p to induce signaling from TLR2 in primary microglial cells, the resident brain macrophages of the CNS. Our data show that H. capsulatum Yps3p induced TLR2 signaling in wild-type microglia, but not in microglia isolated from TLR2 KO mice, confirming that Yps3p is a ligand for TLR2. Furthermore, Yps3p-induced TLR2 signaling was suppressed by vaccinia virus-encoded TLR inhibitors. This is the first demonstration of a fungal protein serving as a TLR ligand and mediating signaling in primary brain cells.

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Inhibition of TLR2-mediated, Yps3p-induced NF-κB activation in 293T-mTLR2 cells by VV proteins. Plasmids carrying the open-reading frames of A46R, A52R, K1L, or N1L were co-transfected along with pNiFty2-Luc, cells were incubated overnight at 37°C and treated with Yps3p for 5 h. Cells were then harvested and the amount of luciferase produced was quantified using bright glow substrate. Data are presented as mean ± SD of triplicate samples and are representative of three independent experiments. Statistical analysis was performed by student's t test. *P < 0.05; **P < 0.01.
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Figure 3: Inhibition of TLR2-mediated, Yps3p-induced NF-κB activation in 293T-mTLR2 cells by VV proteins. Plasmids carrying the open-reading frames of A46R, A52R, K1L, or N1L were co-transfected along with pNiFty2-Luc, cells were incubated overnight at 37°C and treated with Yps3p for 5 h. Cells were then harvested and the amount of luciferase produced was quantified using bright glow substrate. Data are presented as mean ± SD of triplicate samples and are representative of three independent experiments. Statistical analysis was performed by student's t test. *P < 0.05; **P < 0.01.

Mentions: To further confirm the role of TLR2 in responding to H. capsulatum, we next attempted to inhibit this signaling pathway using four vaccinia virus([VV) proteins. Among these viral proteins, A46R inhibits signaling from MyD88, the cytoplasmic adaptor of TLR2 [41], and A52R interacts with and blocks the activity of two downstream molecules IRAK2 and TRAF6 along the TLR2 pathway [41,42]; whereas N1L and K1L prevent the release of NF-κB from its inhibitor IκBα [43,44] (Fig. 1). ORFs of each of these VV proteins were cloned into the pORF5 vector under the control of a composite binary promoter comprised of the elongation factor 1α (EF-1α) and the eukaryotic initiation factor 4g (eIF-4g). 1 μg of each pORF5-VV plasmid was co-transfected into 293T-mTLR2 cells together with 1 μg of pNiFty2-Luc. Following overnight incubation at 37°C, the cells were treated with Yps3p for 6 h, harvested and the expression levels of luciferase in the transfected cells were measured using a luciferase assay. The data show that NF-κB activation was severely impaired in cells expressing each of these viral proteins when compared to cells expressing pNiFty2-Luc alone, a result which demonstrates that all four viral proteins were able to inhibit Yps3p-induced TLR2 signaling in 293T-mTLR2 cells (Fig. 3). This result not only confirmed Yps3p mediation of TLR2 signaling but also showed that it can be inhibited at different levels (at the receptor as well as downstream) along the TLR2 signaling pathway.


Histoplasma capsulatum yeast phase-specific protein Yps3p induces Toll-like receptor 2 signaling.

Aravalli RN, Hu S, Woods JP, Lokensgard JR - J Neuroinflammation (2008)

Inhibition of TLR2-mediated, Yps3p-induced NF-κB activation in 293T-mTLR2 cells by VV proteins. Plasmids carrying the open-reading frames of A46R, A52R, K1L, or N1L were co-transfected along with pNiFty2-Luc, cells were incubated overnight at 37°C and treated with Yps3p for 5 h. Cells were then harvested and the amount of luciferase produced was quantified using bright glow substrate. Data are presented as mean ± SD of triplicate samples and are representative of three independent experiments. Statistical analysis was performed by student's t test. *P < 0.05; **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2474602&req=5

Figure 3: Inhibition of TLR2-mediated, Yps3p-induced NF-κB activation in 293T-mTLR2 cells by VV proteins. Plasmids carrying the open-reading frames of A46R, A52R, K1L, or N1L were co-transfected along with pNiFty2-Luc, cells were incubated overnight at 37°C and treated with Yps3p for 5 h. Cells were then harvested and the amount of luciferase produced was quantified using bright glow substrate. Data are presented as mean ± SD of triplicate samples and are representative of three independent experiments. Statistical analysis was performed by student's t test. *P < 0.05; **P < 0.01.
Mentions: To further confirm the role of TLR2 in responding to H. capsulatum, we next attempted to inhibit this signaling pathway using four vaccinia virus([VV) proteins. Among these viral proteins, A46R inhibits signaling from MyD88, the cytoplasmic adaptor of TLR2 [41], and A52R interacts with and blocks the activity of two downstream molecules IRAK2 and TRAF6 along the TLR2 pathway [41,42]; whereas N1L and K1L prevent the release of NF-κB from its inhibitor IκBα [43,44] (Fig. 1). ORFs of each of these VV proteins were cloned into the pORF5 vector under the control of a composite binary promoter comprised of the elongation factor 1α (EF-1α) and the eukaryotic initiation factor 4g (eIF-4g). 1 μg of each pORF5-VV plasmid was co-transfected into 293T-mTLR2 cells together with 1 μg of pNiFty2-Luc. Following overnight incubation at 37°C, the cells were treated with Yps3p for 6 h, harvested and the expression levels of luciferase in the transfected cells were measured using a luciferase assay. The data show that NF-κB activation was severely impaired in cells expressing each of these viral proteins when compared to cells expressing pNiFty2-Luc alone, a result which demonstrates that all four viral proteins were able to inhibit Yps3p-induced TLR2 signaling in 293T-mTLR2 cells (Fig. 3). This result not only confirmed Yps3p mediation of TLR2 signaling but also showed that it can be inhibited at different levels (at the receptor as well as downstream) along the TLR2 signaling pathway.

Bottom Line: Our data show that H. capsulatum Yps3p induced TLR2 signaling in wild-type microglia, but not in microglia isolated from TLR2 KO mice, confirming that Yps3p is a ligand for TLR2.Furthermore, Yps3p-induced TLR2 signaling was suppressed by vaccinia virus-encoded TLR inhibitors.This is the first demonstration of a fungal protein serving as a TLR ligand and mediating signaling in primary brain cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota Medical School, Minneapolis, Minnesota, USA. arava001@umn.edu

ABSTRACT
Histoplasma capsulatum is a common cause of fungal infection in certain geographic areas, and although most infections are asymptomatic, it is capable of causing histoplasmosis, a disseminated, life-threatening disease, especially in immunocompromised individuals. A deeper understanding of this host-pathogen interaction is needed to develop novel therapeutic strategies to counter lethal infection. Although several lines of evidence suggest that this fungus is neurotropic in HIV patients, little is known about the immunobiology of Histoplasma infection in the central nervous system [CNS]. The goal of the present study was to understand the innate neuroimmune mechanisms that recognize H. capsulatum during the initial stages of infection. Using a 293T stable cell line expressing murine Toll-like receptor 2 [TLR2], we show here that TLR2 recognizes H. capsulatum cell wall protein Yps3p and induces the activation of NF-kappaB. In further experiments, we tested the ability of Yps3p to induce signaling from TLR2 in primary microglial cells, the resident brain macrophages of the CNS. Our data show that H. capsulatum Yps3p induced TLR2 signaling in wild-type microglia, but not in microglia isolated from TLR2 KO mice, confirming that Yps3p is a ligand for TLR2. Furthermore, Yps3p-induced TLR2 signaling was suppressed by vaccinia virus-encoded TLR inhibitors. This is the first demonstration of a fungal protein serving as a TLR ligand and mediating signaling in primary brain cells.

Show MeSH
Related in: MedlinePlus