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Alterations of protein expression in conditions of copper-deprivation for Paracoccidioides lutzii in the presence of extracellular matrix components.

de Oliveira HC, da Silva Jde F, Matsumoto MT, Marcos CM, Peres da Silva R, Moraes da Silva RA, Labate MT, Labate CA, Fusco Almeida AM, Mendes Giannini MJ - BMC Microbiol. (2014)

Bottom Line: A significant increase in binding to all ECM components was observed when the fungus was cultured without Cu; which might be related to some adhesins expression.A proteomic assay was developed and revealed 39 proteins expressed that are involved in processes such as virulence, protein synthesis, metabolism, energy, transcription, transport, stress response and the cell cycle when the fungus was interacting with the ECM components.The up-regulated expression of two important adhesins, enolase and 14-3-3, was observed at the fungal cell wall during the interaction with the ECM components, indicating the role of these proteins in the Paracoccidioides-host interaction.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, UNESP - Univ Estadual Paulista, Laboratório de Micologia Clinica, Rodovia Araraquara-Jaú, Km 1, Araraquara, SP, Brazil. giannini@fcfar.unesp.br.

ABSTRACT

Background: Paracoccidioides spp is a fungi genus and the agent of paracoccidioidomycosis. The strategies of infection used by these pathogens involve the expression of proteins related to adaptation to the host, particularly regarding the uptake of micronutrients. This study analyzed the adhesion of Paracoccidioides lutzii during conditions of copper (Cu) and iron (Fe) deprivation, while also evaluating the proteins expressed in conditions of Cu depletion in the presence of four extracellular matrix (ECM) components (laminin, fibronectin and types I and IV collagen).

Results: We cultured the P. lutzii in a chemically defined media without Cu and Fe. The fungus was then placed in contact with different ECM components and adhesion was evaluated. A significant increase in binding to all ECM components was observed when the fungus was cultured without Cu; which might be related to some adhesins expression. A proteomic assay was developed and revealed 39 proteins expressed that are involved in processes such as virulence, protein synthesis, metabolism, energy, transcription, transport, stress response and the cell cycle when the fungus was interacting with the ECM components. The up-regulated expression of two important adhesins, enolase and 14-3-3, was observed at the fungal cell wall during the interaction with the ECM components, indicating the role of these proteins in the Paracoccidioides-host interaction.

Conclusions: This study is important for determining prospective proteins that may be involved in the interaction of Paracoccidioides with a host. Understanding the adaptive response to different growth conditions, elucidating the processes of adhesion and cell invasion, and identifying the proteins that are differentially expressed during the fungus-host interaction may help elucidate mechanisms used for survival and growth of Paracoccidioides in various human tissues.

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Quantitative analyses of expressed A) enolase and B) 14-3-3 proteins during the interaction with ECM components.
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Fig5: Quantitative analyses of expressed A) enolase and B) 14-3-3 proteins during the interaction with ECM components.

Mentions: Real-time PCR analysis revealed higher expression levels of two adhesins, enolase and 14-3-3, under conditions of Cu depletion and in the presence of the different ECM components. Thus, we explored the subcellular location of these proteins using anti-enolase and anti-14-3-3 polyclonal antibodies in combination with immunoelectron microscopy (IEM) to confirm the differential expression of these proteins during the interaction of P. lutzii with the different ECM components (Figures 3 and 4). P. lutzii yeast cells with and without Cu in contact with all the ECM components were processed by post embedding with gold particles. The immunocytochemistry assays revealed a ubiquitous distribution of the gold particles in all conditions, but we observed that, when the fungus was grown under simulated infection conditions, there was an increased expression of enolase and 14-3-3 (indicated with arrows in Figures 3 and 4) in the fungal cell wall. A quantitative analysis was developed by counting the number of expressed enolase and 14-3-3 proteins in the fungal cell wall and a real increase of protein expression occurred in the cell wall during its interaction with all ECM components (Figure 5). These results showed that these proteins, when interacting with the host structures, are recruited to the cell wall and may be used during this interaction, as shown by da Silva et al. [26] and Marcos et al. [27].Figure 3


Alterations of protein expression in conditions of copper-deprivation for Paracoccidioides lutzii in the presence of extracellular matrix components.

de Oliveira HC, da Silva Jde F, Matsumoto MT, Marcos CM, Peres da Silva R, Moraes da Silva RA, Labate MT, Labate CA, Fusco Almeida AM, Mendes Giannini MJ - BMC Microbiol. (2014)

Quantitative analyses of expressed A) enolase and B) 14-3-3 proteins during the interaction with ECM components.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4302596&req=5

Fig5: Quantitative analyses of expressed A) enolase and B) 14-3-3 proteins during the interaction with ECM components.
Mentions: Real-time PCR analysis revealed higher expression levels of two adhesins, enolase and 14-3-3, under conditions of Cu depletion and in the presence of the different ECM components. Thus, we explored the subcellular location of these proteins using anti-enolase and anti-14-3-3 polyclonal antibodies in combination with immunoelectron microscopy (IEM) to confirm the differential expression of these proteins during the interaction of P. lutzii with the different ECM components (Figures 3 and 4). P. lutzii yeast cells with and without Cu in contact with all the ECM components were processed by post embedding with gold particles. The immunocytochemistry assays revealed a ubiquitous distribution of the gold particles in all conditions, but we observed that, when the fungus was grown under simulated infection conditions, there was an increased expression of enolase and 14-3-3 (indicated with arrows in Figures 3 and 4) in the fungal cell wall. A quantitative analysis was developed by counting the number of expressed enolase and 14-3-3 proteins in the fungal cell wall and a real increase of protein expression occurred in the cell wall during its interaction with all ECM components (Figure 5). These results showed that these proteins, when interacting with the host structures, are recruited to the cell wall and may be used during this interaction, as shown by da Silva et al. [26] and Marcos et al. [27].Figure 3

Bottom Line: A significant increase in binding to all ECM components was observed when the fungus was cultured without Cu; which might be related to some adhesins expression.A proteomic assay was developed and revealed 39 proteins expressed that are involved in processes such as virulence, protein synthesis, metabolism, energy, transcription, transport, stress response and the cell cycle when the fungus was interacting with the ECM components.The up-regulated expression of two important adhesins, enolase and 14-3-3, was observed at the fungal cell wall during the interaction with the ECM components, indicating the role of these proteins in the Paracoccidioides-host interaction.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, UNESP - Univ Estadual Paulista, Laboratório de Micologia Clinica, Rodovia Araraquara-Jaú, Km 1, Araraquara, SP, Brazil. giannini@fcfar.unesp.br.

ABSTRACT

Background: Paracoccidioides spp is a fungi genus and the agent of paracoccidioidomycosis. The strategies of infection used by these pathogens involve the expression of proteins related to adaptation to the host, particularly regarding the uptake of micronutrients. This study analyzed the adhesion of Paracoccidioides lutzii during conditions of copper (Cu) and iron (Fe) deprivation, while also evaluating the proteins expressed in conditions of Cu depletion in the presence of four extracellular matrix (ECM) components (laminin, fibronectin and types I and IV collagen).

Results: We cultured the P. lutzii in a chemically defined media without Cu and Fe. The fungus was then placed in contact with different ECM components and adhesion was evaluated. A significant increase in binding to all ECM components was observed when the fungus was cultured without Cu; which might be related to some adhesins expression. A proteomic assay was developed and revealed 39 proteins expressed that are involved in processes such as virulence, protein synthesis, metabolism, energy, transcription, transport, stress response and the cell cycle when the fungus was interacting with the ECM components. The up-regulated expression of two important adhesins, enolase and 14-3-3, was observed at the fungal cell wall during the interaction with the ECM components, indicating the role of these proteins in the Paracoccidioides-host interaction.

Conclusions: This study is important for determining prospective proteins that may be involved in the interaction of Paracoccidioides with a host. Understanding the adaptive response to different growth conditions, elucidating the processes of adhesion and cell invasion, and identifying the proteins that are differentially expressed during the fungus-host interaction may help elucidate mechanisms used for survival and growth of Paracoccidioides in various human tissues.

Show MeSH
Related in: MedlinePlus