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Immunization with an autotransporter protein of Orientia tsutsugamushi provides protective immunity against scrub typhus.

Ha NY, Sharma P, Kim G, Kim Y, Min CK, Choi MS, Kim IS, Cho NH - PLoS Negl Trop Dis (2015)

Bottom Line: Despite the wide range of preventative approaches that have been attempted in the past 70 years, all have failed to develop an effective prophylactic vaccine.Currently, the selection of the proper antigens is one of the critical barriers to generating cross-protective immunity against antigenically-variable strains of O. tsutsugamushi.Our findings demonstrate that ScaA functions as a bacterial adhesion factor, and anti-ScaA antibody significantly neutralizes bacterial infection of host cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, Republic of Korea; Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea.

ABSTRACT

Background: Scrub typhus is an acute febrile disease caused by Orientia tsutsugamushi infection. Recently, the rapid increase of scrub typhus incidence in several countries within the endemic region has become a serious public health issue. Despite the wide range of preventative approaches that have been attempted in the past 70 years, all have failed to develop an effective prophylactic vaccine. Currently, the selection of the proper antigens is one of the critical barriers to generating cross-protective immunity against antigenically-variable strains of O. tsutsugamushi.

Methodology/principal findings: We examined the potential role of ScaA protein, an autotransporter protein of O. tsutsugamushi, in bacterial pathogenesis and evaluated the protective attributes of ScaA immunization in lethal O. tsutsugamushi infection in mice. Our findings demonstrate that ScaA functions as a bacterial adhesion factor, and anti-ScaA antibody significantly neutralizes bacterial infection of host cells. In addition, immunization with ScaA not only provides protective immunity against lethal challenges with the homologous strain, but also confers significant protection against heterologous strains when combined with TSA56, a major outer membrane protein of O. tsutsugamushi.

Conclusions/significance: Immunization of ScaA proteins provides protective immunity in mice when challenged with the homologous strain and significantly enhanced protective immunity against infection with heterologous strains. To our knowledge, this is the most promising result of scrub typhus vaccination trials against infection of heterologous strains in mouse models thus far.

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Related in: MedlinePlus

Adhesion function of ScaA.(A) HeLa cells were incubated with fluorescent microbeads coated with GST or GST-ScaA (ScaA) for 1 h, washed extensively, and fixed. Cell-bound microbeads (green) were visualized by fluorescence microscopy after staining of cell nuclei (blue). Scale bars, 10 μm. (B) Relative binding of the microbeads coated with GST (dotted line) or GST-ScaA (thick line) to HeLa cells was quantified directly using fluorescence-activated cell sorter (FACS) analysis. The gray histogram represents unbound cells (cells not incubated with microbeads). (C) Immunofluorescence microscopy using an anti-ScaA antibody revealed the presence of ScaA on the surface of the recombinant E. coli (lower panels). Preimmune serum did not detect the recombinant protein (upper panels). Scale bars, 5 μm. (D) E. coli transformed with the pET28a vector or with pScaA was induced with IPTG and incubated with HeLa cells. After being washed to remove adherent bacteria, the cells were fixed, permeabilized, and stained with an anti-E. coli antibody (green) and ToPro-3 for nuclear staining (blue). Scale bars, 10 μm. (E) CFU-based quantification of adherent E. coli transformed with the vector or pScaA was performed. The results are presented as percentages of adherent bacteria relative to the total bacterial input. Data are representative of three independent assays for each of the host cells. **, p < 0.01. (F) Inclusion of anti-ScaA serum in the medium (α-ScaA) significantly inhibited adhesion of E. coli expressing ScaA into host cells. After addition of anti-ScaA or preimmune serum into infection media, CFU-based quantification of adherent E. coli transformed with the vector or pScaA was performed. **, p < 0.01.
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pntd.0003585.g002: Adhesion function of ScaA.(A) HeLa cells were incubated with fluorescent microbeads coated with GST or GST-ScaA (ScaA) for 1 h, washed extensively, and fixed. Cell-bound microbeads (green) were visualized by fluorescence microscopy after staining of cell nuclei (blue). Scale bars, 10 μm. (B) Relative binding of the microbeads coated with GST (dotted line) or GST-ScaA (thick line) to HeLa cells was quantified directly using fluorescence-activated cell sorter (FACS) analysis. The gray histogram represents unbound cells (cells not incubated with microbeads). (C) Immunofluorescence microscopy using an anti-ScaA antibody revealed the presence of ScaA on the surface of the recombinant E. coli (lower panels). Preimmune serum did not detect the recombinant protein (upper panels). Scale bars, 5 μm. (D) E. coli transformed with the pET28a vector or with pScaA was induced with IPTG and incubated with HeLa cells. After being washed to remove adherent bacteria, the cells were fixed, permeabilized, and stained with an anti-E. coli antibody (green) and ToPro-3 for nuclear staining (blue). Scale bars, 10 μm. (E) CFU-based quantification of adherent E. coli transformed with the vector or pScaA was performed. The results are presented as percentages of adherent bacteria relative to the total bacterial input. Data are representative of three independent assays for each of the host cells. **, p < 0.01. (F) Inclusion of anti-ScaA serum in the medium (α-ScaA) significantly inhibited adhesion of E. coli expressing ScaA into host cells. After addition of anti-ScaA or preimmune serum into infection media, CFU-based quantification of adherent E. coli transformed with the vector or pScaA was performed. **, p < 0.01.

Mentions: Recently, several studies reported that rickettsial Sca proteins mediate bacterial adherence to and/or invasion into mammalian host cells [32,45]. Therefore, we examined whether the O. tsutsugamushi ScaA protein could function as a virulence factor for bacterial adhesion and/or invasion. First, we performed a bead-binding assay using fluorescent microbeads (1 μm in diameter) covalently conjugated to either purified GST or GST-ScaA. Incubation of HeLa cells with GST-ScaA-conjugated beads resulted in marked binding to the host cells, even after extensive washing. The control beads linked to GST alone interacted only weakly with the HeLa cells (Fig. 2A) [32]. The interaction of the fluorescent beads with the host cells was quantified using flow cytometry (Fig. 2B). After fixation, the mean fluorescence intensity (MFI) of the HeLa cells incubated with beads conjugated to GST-ScaA dramatically increased (MFI = 50.1) compared to that of cells incubated with beads conjugated to GST (MFI = 13.6) or that of untreated cells [32].


Immunization with an autotransporter protein of Orientia tsutsugamushi provides protective immunity against scrub typhus.

Ha NY, Sharma P, Kim G, Kim Y, Min CK, Choi MS, Kim IS, Cho NH - PLoS Negl Trop Dis (2015)

Adhesion function of ScaA.(A) HeLa cells were incubated with fluorescent microbeads coated with GST or GST-ScaA (ScaA) for 1 h, washed extensively, and fixed. Cell-bound microbeads (green) were visualized by fluorescence microscopy after staining of cell nuclei (blue). Scale bars, 10 μm. (B) Relative binding of the microbeads coated with GST (dotted line) or GST-ScaA (thick line) to HeLa cells was quantified directly using fluorescence-activated cell sorter (FACS) analysis. The gray histogram represents unbound cells (cells not incubated with microbeads). (C) Immunofluorescence microscopy using an anti-ScaA antibody revealed the presence of ScaA on the surface of the recombinant E. coli (lower panels). Preimmune serum did not detect the recombinant protein (upper panels). Scale bars, 5 μm. (D) E. coli transformed with the pET28a vector or with pScaA was induced with IPTG and incubated with HeLa cells. After being washed to remove adherent bacteria, the cells were fixed, permeabilized, and stained with an anti-E. coli antibody (green) and ToPro-3 for nuclear staining (blue). Scale bars, 10 μm. (E) CFU-based quantification of adherent E. coli transformed with the vector or pScaA was performed. The results are presented as percentages of adherent bacteria relative to the total bacterial input. Data are representative of three independent assays for each of the host cells. **, p < 0.01. (F) Inclusion of anti-ScaA serum in the medium (α-ScaA) significantly inhibited adhesion of E. coli expressing ScaA into host cells. After addition of anti-ScaA or preimmune serum into infection media, CFU-based quantification of adherent E. coli transformed with the vector or pScaA was performed. **, p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359152&req=5

pntd.0003585.g002: Adhesion function of ScaA.(A) HeLa cells were incubated with fluorescent microbeads coated with GST or GST-ScaA (ScaA) for 1 h, washed extensively, and fixed. Cell-bound microbeads (green) were visualized by fluorescence microscopy after staining of cell nuclei (blue). Scale bars, 10 μm. (B) Relative binding of the microbeads coated with GST (dotted line) or GST-ScaA (thick line) to HeLa cells was quantified directly using fluorescence-activated cell sorter (FACS) analysis. The gray histogram represents unbound cells (cells not incubated with microbeads). (C) Immunofluorescence microscopy using an anti-ScaA antibody revealed the presence of ScaA on the surface of the recombinant E. coli (lower panels). Preimmune serum did not detect the recombinant protein (upper panels). Scale bars, 5 μm. (D) E. coli transformed with the pET28a vector or with pScaA was induced with IPTG and incubated with HeLa cells. After being washed to remove adherent bacteria, the cells were fixed, permeabilized, and stained with an anti-E. coli antibody (green) and ToPro-3 for nuclear staining (blue). Scale bars, 10 μm. (E) CFU-based quantification of adherent E. coli transformed with the vector or pScaA was performed. The results are presented as percentages of adherent bacteria relative to the total bacterial input. Data are representative of three independent assays for each of the host cells. **, p < 0.01. (F) Inclusion of anti-ScaA serum in the medium (α-ScaA) significantly inhibited adhesion of E. coli expressing ScaA into host cells. After addition of anti-ScaA or preimmune serum into infection media, CFU-based quantification of adherent E. coli transformed with the vector or pScaA was performed. **, p < 0.01.
Mentions: Recently, several studies reported that rickettsial Sca proteins mediate bacterial adherence to and/or invasion into mammalian host cells [32,45]. Therefore, we examined whether the O. tsutsugamushi ScaA protein could function as a virulence factor for bacterial adhesion and/or invasion. First, we performed a bead-binding assay using fluorescent microbeads (1 μm in diameter) covalently conjugated to either purified GST or GST-ScaA. Incubation of HeLa cells with GST-ScaA-conjugated beads resulted in marked binding to the host cells, even after extensive washing. The control beads linked to GST alone interacted only weakly with the HeLa cells (Fig. 2A) [32]. The interaction of the fluorescent beads with the host cells was quantified using flow cytometry (Fig. 2B). After fixation, the mean fluorescence intensity (MFI) of the HeLa cells incubated with beads conjugated to GST-ScaA dramatically increased (MFI = 50.1) compared to that of cells incubated with beads conjugated to GST (MFI = 13.6) or that of untreated cells [32].

Bottom Line: Despite the wide range of preventative approaches that have been attempted in the past 70 years, all have failed to develop an effective prophylactic vaccine.Currently, the selection of the proper antigens is one of the critical barriers to generating cross-protective immunity against antigenically-variable strains of O. tsutsugamushi.Our findings demonstrate that ScaA functions as a bacterial adhesion factor, and anti-ScaA antibody significantly neutralizes bacterial infection of host cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, Republic of Korea; Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea.

ABSTRACT

Background: Scrub typhus is an acute febrile disease caused by Orientia tsutsugamushi infection. Recently, the rapid increase of scrub typhus incidence in several countries within the endemic region has become a serious public health issue. Despite the wide range of preventative approaches that have been attempted in the past 70 years, all have failed to develop an effective prophylactic vaccine. Currently, the selection of the proper antigens is one of the critical barriers to generating cross-protective immunity against antigenically-variable strains of O. tsutsugamushi.

Methodology/principal findings: We examined the potential role of ScaA protein, an autotransporter protein of O. tsutsugamushi, in bacterial pathogenesis and evaluated the protective attributes of ScaA immunization in lethal O. tsutsugamushi infection in mice. Our findings demonstrate that ScaA functions as a bacterial adhesion factor, and anti-ScaA antibody significantly neutralizes bacterial infection of host cells. In addition, immunization with ScaA not only provides protective immunity against lethal challenges with the homologous strain, but also confers significant protection against heterologous strains when combined with TSA56, a major outer membrane protein of O. tsutsugamushi.

Conclusions/significance: Immunization of ScaA proteins provides protective immunity in mice when challenged with the homologous strain and significantly enhanced protective immunity against infection with heterologous strains. To our knowledge, this is the most promising result of scrub typhus vaccination trials against infection of heterologous strains in mouse models thus far.

Show MeSH
Related in: MedlinePlus