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Differential Immune Response against Recombinant Leishmania donovani Peroxidoxin 1 and Peroxidoxin 2 Proteins in BALB/c Mice.

Daifalla NS, Bayih AG, Gedamu L - J Immunol Res (2015)

Bottom Line: We also evaluated the effect of coadministration of TLR agonists (CpG ODN and GLA-SE) on the antigen-specific immune response.Immunization with recombinant LdPxn1 alone induced a predominantly Th2 type immune response that is associated with the production of high level of IgG1 and no IgG2a isotype while rLdPxn2 resulted in a mixed Th1/Th2 response characterized by the production of antigen-specific IgG2a in addition to IgG1 isotype.TLR agonists also enhanced a more polarized Th 1 type immune response against rLdPxn2.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Calgary, Room 374, 2500 University Drive NW, Calgary, AB, Canada T2N 1N4 ; The Forsyth Institute, Cambridge, MA 02142, USA.

ABSTRACT
We assessed the immune response against recombinant proteins of two related, albeit functionally different, peroxidoxins from Leishmania donovani: peroxidoxin 1 (LdPxn1) and peroxidoxin 2 (LdPxn2) in BALB/c mice. We also evaluated the effect of coadministration of TLR agonists (CpG ODN and GLA-SE) on the antigen-specific immune response. Immunization with recombinant LdPxn1 alone induced a predominantly Th2 type immune response that is associated with the production of high level of IgG1 and no IgG2a isotype while rLdPxn2 resulted in a mixed Th1/Th2 response characterized by the production of antigen-specific IgG2a in addition to IgG1 isotype. Antigen-stimulated spleen cells from mice that were immunized with rLdPxn1 produced low level of IL-10 and IL-4 and no IFN-γ, whereas cells from mice immunized with rLdPxn2 secreted high level of IFN-γ, low IL-4, and no IL-10. Coadministration of CpG ODN or GLA-SE with rLdPxn1 skewed the immune response towards a Th 1 type as indicated by robust production of IgG2a isotype. Furthermore, the presence of TLR agonists together with rLdPxn1 antigen enhanced the production of IFN-γ and to a lesser extent of IL-10. TLR agonists also enhanced a more polarized Th 1 type immune response against rLdPxn2.

No MeSH data available.


(a) Sequence comparison of Leishmania donovani Pxn1 and Pxn2. Alignment of amino acid sequence depicts the high homology between LdPxn1 and LdPxn2. Highlighted areas show positions of mismatch. LdPxn2 possesses extra 9 amino acids at the carboxy terminus (underlined) that are missing from LdPxn1. (b) SDS-PAGE and western blot of rLdPxn1 and rLdPxn2 proteins. One microgram per lane of rLdPxn1 (lane 1) and rLdPxn2 (lane 2) was separated on a 12% SDS-PAGE and stained with Coomassie blue, top. The separated samples were transferred to Hybond-P membrane and were probed with pooled sera from mice immunized with the respective recombinant protein, bottom. Molecular weight in kDa is shown on the left.
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fig1: (a) Sequence comparison of Leishmania donovani Pxn1 and Pxn2. Alignment of amino acid sequence depicts the high homology between LdPxn1 and LdPxn2. Highlighted areas show positions of mismatch. LdPxn2 possesses extra 9 amino acids at the carboxy terminus (underlined) that are missing from LdPxn1. (b) SDS-PAGE and western blot of rLdPxn1 and rLdPxn2 proteins. One microgram per lane of rLdPxn1 (lane 1) and rLdPxn2 (lane 2) was separated on a 12% SDS-PAGE and stained with Coomassie blue, top. The separated samples were transferred to Hybond-P membrane and were probed with pooled sera from mice immunized with the respective recombinant protein, bottom. Molecular weight in kDa is shown on the left.

Mentions: One of the evasive mechanisms used by Leishmania parasites to bypass the microbicidal effect of free radicals produced by macrophages is the expression of antioxidant enzymes known as peroxidoxins. These enzymes are conserved and highly abundant proteins in almost all living organisms which suggest essential function in oxidative homeostasis. It has been shown that peroxidoxins from different organisms including Leishmania are important in the protection of these organisms against oxidative stress [14–16]. We isolated and characterized three peroxidoxins as part of a multigene family from L. donovani complex: Pxn1, Pxn2, and Pxn3 [14, 17]. Both Pxn1 and Pxn2 are cytosolic whereas Pxn3 is predicted to be glycosomal. A fourth mitochondrial peroxidoxin, Pxn4, has also been identified in L. donovani [18]. In addition to the common localization of Pxn1 and Pxn2 in the cytoplasm, the two proteins have 89.4% homology. The difference between these two proteins is brought about by an extra 9 amino acids at the carboxy terminus of Pxn2 plus few nucleotide mismatches along the entire sequence [14, 17] (Figure 1(a)). Despite the high similarity between LdPxn1 and LdPxn2 at the amino acid level, there are striking differences between the proteins encoded by the two genes. Unlike LdPxn1, which is upregulated during the amastigote stage, LdPxn2 is expressed at high levels during the promastigote stage and the expression declines towards the amastigote stage. In addition, while recombinant LdPxn1 protein has been shown to detoxify various free radicals including ROS and RNS, LdPxn2 can only detoxify H2O2 [14].


Differential Immune Response against Recombinant Leishmania donovani Peroxidoxin 1 and Peroxidoxin 2 Proteins in BALB/c Mice.

Daifalla NS, Bayih AG, Gedamu L - J Immunol Res (2015)

(a) Sequence comparison of Leishmania donovani Pxn1 and Pxn2. Alignment of amino acid sequence depicts the high homology between LdPxn1 and LdPxn2. Highlighted areas show positions of mismatch. LdPxn2 possesses extra 9 amino acids at the carboxy terminus (underlined) that are missing from LdPxn1. (b) SDS-PAGE and western blot of rLdPxn1 and rLdPxn2 proteins. One microgram per lane of rLdPxn1 (lane 1) and rLdPxn2 (lane 2) was separated on a 12% SDS-PAGE and stained with Coomassie blue, top. The separated samples were transferred to Hybond-P membrane and were probed with pooled sera from mice immunized with the respective recombinant protein, bottom. Molecular weight in kDa is shown on the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: (a) Sequence comparison of Leishmania donovani Pxn1 and Pxn2. Alignment of amino acid sequence depicts the high homology between LdPxn1 and LdPxn2. Highlighted areas show positions of mismatch. LdPxn2 possesses extra 9 amino acids at the carboxy terminus (underlined) that are missing from LdPxn1. (b) SDS-PAGE and western blot of rLdPxn1 and rLdPxn2 proteins. One microgram per lane of rLdPxn1 (lane 1) and rLdPxn2 (lane 2) was separated on a 12% SDS-PAGE and stained with Coomassie blue, top. The separated samples were transferred to Hybond-P membrane and were probed with pooled sera from mice immunized with the respective recombinant protein, bottom. Molecular weight in kDa is shown on the left.
Mentions: One of the evasive mechanisms used by Leishmania parasites to bypass the microbicidal effect of free radicals produced by macrophages is the expression of antioxidant enzymes known as peroxidoxins. These enzymes are conserved and highly abundant proteins in almost all living organisms which suggest essential function in oxidative homeostasis. It has been shown that peroxidoxins from different organisms including Leishmania are important in the protection of these organisms against oxidative stress [14–16]. We isolated and characterized three peroxidoxins as part of a multigene family from L. donovani complex: Pxn1, Pxn2, and Pxn3 [14, 17]. Both Pxn1 and Pxn2 are cytosolic whereas Pxn3 is predicted to be glycosomal. A fourth mitochondrial peroxidoxin, Pxn4, has also been identified in L. donovani [18]. In addition to the common localization of Pxn1 and Pxn2 in the cytoplasm, the two proteins have 89.4% homology. The difference between these two proteins is brought about by an extra 9 amino acids at the carboxy terminus of Pxn2 plus few nucleotide mismatches along the entire sequence [14, 17] (Figure 1(a)). Despite the high similarity between LdPxn1 and LdPxn2 at the amino acid level, there are striking differences between the proteins encoded by the two genes. Unlike LdPxn1, which is upregulated during the amastigote stage, LdPxn2 is expressed at high levels during the promastigote stage and the expression declines towards the amastigote stage. In addition, while recombinant LdPxn1 protein has been shown to detoxify various free radicals including ROS and RNS, LdPxn2 can only detoxify H2O2 [14].

Bottom Line: We also evaluated the effect of coadministration of TLR agonists (CpG ODN and GLA-SE) on the antigen-specific immune response.Immunization with recombinant LdPxn1 alone induced a predominantly Th2 type immune response that is associated with the production of high level of IgG1 and no IgG2a isotype while rLdPxn2 resulted in a mixed Th1/Th2 response characterized by the production of antigen-specific IgG2a in addition to IgG1 isotype.TLR agonists also enhanced a more polarized Th 1 type immune response against rLdPxn2.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Calgary, Room 374, 2500 University Drive NW, Calgary, AB, Canada T2N 1N4 ; The Forsyth Institute, Cambridge, MA 02142, USA.

ABSTRACT
We assessed the immune response against recombinant proteins of two related, albeit functionally different, peroxidoxins from Leishmania donovani: peroxidoxin 1 (LdPxn1) and peroxidoxin 2 (LdPxn2) in BALB/c mice. We also evaluated the effect of coadministration of TLR agonists (CpG ODN and GLA-SE) on the antigen-specific immune response. Immunization with recombinant LdPxn1 alone induced a predominantly Th2 type immune response that is associated with the production of high level of IgG1 and no IgG2a isotype while rLdPxn2 resulted in a mixed Th1/Th2 response characterized by the production of antigen-specific IgG2a in addition to IgG1 isotype. Antigen-stimulated spleen cells from mice that were immunized with rLdPxn1 produced low level of IL-10 and IL-4 and no IFN-γ, whereas cells from mice immunized with rLdPxn2 secreted high level of IFN-γ, low IL-4, and no IL-10. Coadministration of CpG ODN or GLA-SE with rLdPxn1 skewed the immune response towards a Th 1 type as indicated by robust production of IgG2a isotype. Furthermore, the presence of TLR agonists together with rLdPxn1 antigen enhanced the production of IFN-γ and to a lesser extent of IL-10. TLR agonists also enhanced a more polarized Th 1 type immune response against rLdPxn2.

No MeSH data available.