Publicação
Role of secreted aspartyl proteases in Candida albicans virulence, host immune response and immunoprotection in murine disseminated candidiasis
| Resumo: | The polymorphic yeast Candida albicans is an important opportunistic human pathogen and the most common causative agent of fungal invasive infections. Host physical barriers and immune system integrity are crucial factors in controlling the establishment of Candida infections. However, the high adaptability of C. albicans to different host niches is also a determinant factor. The host-fungus interplay is dynamic and the balance between fungal elimination and tissue damage depends on both the host response and how the pathogen reacts to immune effector molecules and cells. The murine model of hematogenously disseminated C. albicans infection is widely used to ascertain strain virulence and host-fungus interactions. However, distinct C. albicans strains may present distinct virulence phenotypes and elicit quantitatively and qualitatively different immune responses. Hence, results obtained with one strain cannot be taken as representative of the whole species. Moreover, most studies focused on C. albicans internalization and killing by phagocytes, rather than on the overall immune response. Here, a comparative analysis of the early immune response, host survival and kidney fungal burden was done in mice infected intravenously with three C. albicans strains with different attributed virulence, SC5314, ATCC 90028 and ATCC 32354. Strain SC5314 was the most virulent and elicited a more marked inflammatory response, with higher neutrophil recruitment. In contrast, ATCC 32354 presented the lowest virulence and stimulated less markedly the innate immune response than the other strains. These results provide additional evidence on the association between C. albicans virulence and the early immune response, which may be useful in delineating vaccination or immunotherapeutic strategies against disseminated candidiasis. Secretion of C. albicans hydrolytic enzymes during infection is a virulence attribute that aids adhesion to and invasion of host tissues, and immune evasion. Among these enzymes, secreted aspartyl proteases (Sap) encoded by a 10-member gene family (SAP1 to SAP10) have been particularly studied. Several members of the Sap family were claimed to play a significant role in the progression of candidiasis established by the hematogenous or intraperitoneal routes. This assumption was based on the observed attenuated virulence of sap-null mutant strains. However, the exclusive contribution of SAP genes to their attenuated phenotype was not unequivocally confirmed, as the Ura status of several mutant strains could also have contributed to the attenuation. In this study, the importance of SAP1 to SAP6 in murine models of hematogenously disseminated candidiasis and C. albicans peritonitis was reassessed by using sap-null mutant strains not affected in their URA3 gene expression. In systemic candidiasis established by intravenous infection, SAT1-flipping constructed Δsap123 and Δsap456 mutants did not present a significant reduction in virulence contrasting the attenuated virulence found in equivalent Ura-blaster mutants. Using the newly assessed mutant strains, the median survival time of BALB/c mice infected with the Δsap123 strain was similar to that of wild-type (WT) SC5314-infected mice, while those infected with mutant strains lacking SAP5 showed slightly extended survival times. Nevertheless, WT and Δsap456 strains were equally able to invade mice kidneys. Likewise, SAP4 to SAP6 deficiency had no noticeable impact on the immune response elicited in the spleen and kidneys of C. albicans-infected mice. These results suggest that Sap1 to Sap6 do not play a significant role in C. albicans virulence in the murine model of hematogenously disseminated candidiasis. Contrastingly, in the murine model of C. albicans peritonitis, Δsap456-infected mice presented lower kidney fungal burden than WT- or Δsap123-infected mice. WT-infected mice presented higher proportions of T regulatory cells (Foxp3+) in the spleen and mesenteric lymph nodes than non-infected or sap-null mutant-infected counterparts. In addition, CD4+CD25+ T cells of WT-infected mice were the most effective in suppressing the proliferative response of CD4+CD25- T cells whereas those of Δsap456-infected mice were the least suppressive. Furthermore, CD4+ T cells of WT-infected mice were the ones producing the highest levels of IL-10. Interestingly, Δsap456-infected mice presented less Foxp3+ cells in kidney lesions than the other fungal challenged mice. Altogether, these results implicate Sap4 to Sap6 in the host immune response to C. albicans peritonitis, providing additional evidence for the role of these enzymes in this infection model. To circumvent host immune defenses, C. albicans developed multiple evasion mechanisms. Among these, production of Saps has been particularly highlighted due to their ability to degrade and/or inactivate diverse host immune effector molecules. The mammalian pattern recognition receptor galectin-3 (Gal-3) was shown to specifically bind β-1,2 mannosides on C. albicans cell wall. This interaction directly induces fungal cell death and also promotes pro-inflammatory cytokine TNF-α production by host cells. In this study, we assessed whether this lectin could be degraded by native Sap2 or recombinant Sap1 to Sap3 isoenzymes. All C. albicans Saps tested degraded and inactivated the host receptor Gal-3. This may constitute a fungal strategy to control and evade host immune mechanisms dependent on Gal-3. Degradation of this protein might thus generate a protective microenvironment of reduced Gal-3 activity, which may facilitate C. albicans survival in the host. Previous reports have shown that mice immunized with Sap2 were protected against mucosal or peritoneal C. albicans infection. Here, we extended these studies and evaluated the suitability of C. albicans recombinant Sap2 (rSap2) as protective immunogen for vaccination against hematogenously disseminated candidiasis. Four different immunogenic preparations were tested, respectively using Alum, Imiquimod, Freund’s or CpG plus Alum as adjuvants. Moreover, as the hypha-associated isoenzyme Sap5 is preferentially expressed during systemic candidiasis, it was also evaluated as target antigen for Candida vaccination, together with Alum or Imiquimod. All these approaches failed in protecting the immunized mice from fungal infection. This suggests that the C. albicans enzymes Sap2 and Sap5, despite their potential role in virulence, do not appear to be suitable target proteins in immunopreventive strategies against hematogenously disseminated candidiasis. In summary, results presented in this thesis provide additional evidence for the differential involvement of Saps in distinct C. albicans infection models. Moreover, these results, by showing that host immune response to C. albicans is affected by lack of SAP expression, are in support of a previously hypothesized immunomodulatory role for Saps. Finally, the lack of host immune protection against hematogenously disseminated candidiasis by means of Sap immunization reinforces the limited role of these proteins in this type of infection. |
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| Autores principais: | Correia, Isabel Alexandra Duarte Ferreira Lopes |
| Ano: | 2012 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | The polymorphic yeast Candida albicans is an important opportunistic human pathogen and the most common causative agent of fungal invasive infections. Host physical barriers and immune system integrity are crucial factors in controlling the establishment of Candida infections. However, the high adaptability of C. albicans to different host niches is also a determinant factor. The host-fungus interplay is dynamic and the balance between fungal elimination and tissue damage depends on both the host response and how the pathogen reacts to immune effector molecules and cells. The murine model of hematogenously disseminated C. albicans infection is widely used to ascertain strain virulence and host-fungus interactions. However, distinct C. albicans strains may present distinct virulence phenotypes and elicit quantitatively and qualitatively different immune responses. Hence, results obtained with one strain cannot be taken as representative of the whole species. Moreover, most studies focused on C. albicans internalization and killing by phagocytes, rather than on the overall immune response. Here, a comparative analysis of the early immune response, host survival and kidney fungal burden was done in mice infected intravenously with three C. albicans strains with different attributed virulence, SC5314, ATCC 90028 and ATCC 32354. Strain SC5314 was the most virulent and elicited a more marked inflammatory response, with higher neutrophil recruitment. In contrast, ATCC 32354 presented the lowest virulence and stimulated less markedly the innate immune response than the other strains. These results provide additional evidence on the association between C. albicans virulence and the early immune response, which may be useful in delineating vaccination or immunotherapeutic strategies against disseminated candidiasis. Secretion of C. albicans hydrolytic enzymes during infection is a virulence attribute that aids adhesion to and invasion of host tissues, and immune evasion. Among these enzymes, secreted aspartyl proteases (Sap) encoded by a 10-member gene family (SAP1 to SAP10) have been particularly studied. Several members of the Sap family were claimed to play a significant role in the progression of candidiasis established by the hematogenous or intraperitoneal routes. This assumption was based on the observed attenuated virulence of sap-null mutant strains. However, the exclusive contribution of SAP genes to their attenuated phenotype was not unequivocally confirmed, as the Ura status of several mutant strains could also have contributed to the attenuation. In this study, the importance of SAP1 to SAP6 in murine models of hematogenously disseminated candidiasis and C. albicans peritonitis was reassessed by using sap-null mutant strains not affected in their URA3 gene expression. In systemic candidiasis established by intravenous infection, SAT1-flipping constructed Δsap123 and Δsap456 mutants did not present a significant reduction in virulence contrasting the attenuated virulence found in equivalent Ura-blaster mutants. Using the newly assessed mutant strains, the median survival time of BALB/c mice infected with the Δsap123 strain was similar to that of wild-type (WT) SC5314-infected mice, while those infected with mutant strains lacking SAP5 showed slightly extended survival times. Nevertheless, WT and Δsap456 strains were equally able to invade mice kidneys. Likewise, SAP4 to SAP6 deficiency had no noticeable impact on the immune response elicited in the spleen and kidneys of C. albicans-infected mice. These results suggest that Sap1 to Sap6 do not play a significant role in C. albicans virulence in the murine model of hematogenously disseminated candidiasis. Contrastingly, in the murine model of C. albicans peritonitis, Δsap456-infected mice presented lower kidney fungal burden than WT- or Δsap123-infected mice. WT-infected mice presented higher proportions of T regulatory cells (Foxp3+) in the spleen and mesenteric lymph nodes than non-infected or sap-null mutant-infected counterparts. In addition, CD4+CD25+ T cells of WT-infected mice were the most effective in suppressing the proliferative response of CD4+CD25- T cells whereas those of Δsap456-infected mice were the least suppressive. Furthermore, CD4+ T cells of WT-infected mice were the ones producing the highest levels of IL-10. Interestingly, Δsap456-infected mice presented less Foxp3+ cells in kidney lesions than the other fungal challenged mice. Altogether, these results implicate Sap4 to Sap6 in the host immune response to C. albicans peritonitis, providing additional evidence for the role of these enzymes in this infection model. To circumvent host immune defenses, C. albicans developed multiple evasion mechanisms. Among these, production of Saps has been particularly highlighted due to their ability to degrade and/or inactivate diverse host immune effector molecules. The mammalian pattern recognition receptor galectin-3 (Gal-3) was shown to specifically bind β-1,2 mannosides on C. albicans cell wall. This interaction directly induces fungal cell death and also promotes pro-inflammatory cytokine TNF-α production by host cells. In this study, we assessed whether this lectin could be degraded by native Sap2 or recombinant Sap1 to Sap3 isoenzymes. All C. albicans Saps tested degraded and inactivated the host receptor Gal-3. This may constitute a fungal strategy to control and evade host immune mechanisms dependent on Gal-3. Degradation of this protein might thus generate a protective microenvironment of reduced Gal-3 activity, which may facilitate C. albicans survival in the host. Previous reports have shown that mice immunized with Sap2 were protected against mucosal or peritoneal C. albicans infection. Here, we extended these studies and evaluated the suitability of C. albicans recombinant Sap2 (rSap2) as protective immunogen for vaccination against hematogenously disseminated candidiasis. Four different immunogenic preparations were tested, respectively using Alum, Imiquimod, Freund’s or CpG plus Alum as adjuvants. Moreover, as the hypha-associated isoenzyme Sap5 is preferentially expressed during systemic candidiasis, it was also evaluated as target antigen for Candida vaccination, together with Alum or Imiquimod. All these approaches failed in protecting the immunized mice from fungal infection. This suggests that the C. albicans enzymes Sap2 and Sap5, despite their potential role in virulence, do not appear to be suitable target proteins in immunopreventive strategies against hematogenously disseminated candidiasis. In summary, results presented in this thesis provide additional evidence for the differential involvement of Saps in distinct C. albicans infection models. Moreover, these results, by showing that host immune response to C. albicans is affected by lack of SAP expression, are in support of a previously hypothesized immunomodulatory role for Saps. Finally, the lack of host immune protection against hematogenously disseminated candidiasis by means of Sap immunization reinforces the limited role of these proteins in this type of infection. |
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