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Identification of differentially expressed proteins from Leishmania amazonensis associated with the loss of virulence of the parasites.

Magalhães RD, Duarte MC, Mattos EC, Martins VT, Lage PS, Chávez-Fumagalli MA, Lage DP, Menezes-Souza D, Régis WC, Manso Alves MJ, Soto M, Tavares CA, Nagen RA, Coelho EA - PLoS Negl Trop Dis (2014)

Bottom Line: In addition, the proteins that presented a significant decrease or increase in their protein expression content were identified applying a proteomic approach.It is interesting to note that six proteins, considered hypothetical in Leishmania, showed a significant decrease in their expression and were also identified.The identified proteins that presented a significant decrease in their expression during cultivation, including the hypothetical, may also be related to this loss of parasites' infectivity, and applied in future studies, including vaccine candidates and/or immunotherapeutic targets against leishmaniasis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

ABSTRACT

Background: The present study analyzed whether or not the in vitro cultivation for long periods of time of pre-isolated Leishmania amazonensis from lesions of chronically infected BALB/c mice was able to interfere in the parasites' infectivity using in vivo and in vitro experiments. In addition, the proteins that presented a significant decrease or increase in their protein expression content were identified applying a proteomic approach.

Methodology/principal findings: Parasites were cultured in vitro for 150 days. Aliquots were collected on the day 0 of culture (R0), as well as after ten (R10; 50 days of culture), twenty (R20; 100 days of culture), and thirty (R30; 150 days of culture) passages, and were used to analyze the parasites' in vitro and in vivo infectivity, as well as to perform the proteomic approach. Approximately 837, 967, 935, and 872 spots were found in 2-DE gels prepared from R0, R10, R20, and R30 samples, respectively. A total of 37 spots presented a significant decrease in their intensity of expression, whereas a significant increase in protein content during cultivation could be observed for 19 proteins (both cases >2.0 folds). Some of these identified proteins can be described, such as diagnosis and/or vaccine candidates, while others are involved in the infectivity of Leishmania. It is interesting to note that six proteins, considered hypothetical in Leishmania, showed a significant decrease in their expression and were also identified.

Conclusions/significance: The present study contributes to the understanding that the cultivation of parasites over long periods of time may well be related to the possible loss of infectivity of L. amazonensis. The identified proteins that presented a significant decrease in their expression during cultivation, including the hypothetical, may also be related to this loss of parasites' infectivity, and applied in future studies, including vaccine candidates and/or immunotherapeutic targets against leishmaniasis.

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Immunoblotting validation of some proteins in Leishmania amazonensis.Representative immunoblotting of some proteins that presented a significant decrease or increase in their expression content between R0 and R30 passages, using promastigote and amastigotes-like forms of L. amazonensis, are shown here. For each protein [α-tubulin, in A; paraflagellar rod protein 1D, in B; glucose-regulated protein 78 (GRP78) in C, and heat shock protein 83 (HSP83), in D], this image presents one example of correspondent 2-DE spot of promastigote form obtained from R0 or R30 passages. The antibodies used to validate each spot are described in the material and methods section. Asterisks represent the comparison between the expression of the protein in the R0 condition in relation to the R30 sample in each parasite stage, applying the Student's t-test (P<0.05), and the numbers represent the relative variation of each protein in comparison to R0 of each parasite stage. All experiments were performed in triplicate.
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pntd-0002764-g003: Immunoblotting validation of some proteins in Leishmania amazonensis.Representative immunoblotting of some proteins that presented a significant decrease or increase in their expression content between R0 and R30 passages, using promastigote and amastigotes-like forms of L. amazonensis, are shown here. For each protein [α-tubulin, in A; paraflagellar rod protein 1D, in B; glucose-regulated protein 78 (GRP78) in C, and heat shock protein 83 (HSP83), in D], this image presents one example of correspondent 2-DE spot of promastigote form obtained from R0 or R30 passages. The antibodies used to validate each spot are described in the material and methods section. Asterisks represent the comparison between the expression of the protein in the R0 condition in relation to the R30 sample in each parasite stage, applying the Student's t-test (P<0.05), and the numbers represent the relative variation of each protein in comparison to R0 of each parasite stage. All experiments were performed in triplicate.

Mentions: Some of the identified proteins that presented a significant increase or decrease in their contents in the axenic cultures were used to validate the results found in this study. In this context, two of them presenting a significant decrease in their expression content, namely α-tubulin and paraflagellar rod protein 1D, and two of them, which presented an increase in their expression, namely HSP83 and GRP78, were used in the Western blot experiments (Figure 3). When promastigote extracts were employed, the selected proteins showed a variation that runs in line with the results obtained in the 2-DE gels. In addition, the decrease in the level of α-tubulin and paraflagellar rod protein 1D detected in the promastigote forms are also maintained when the R30 forms are axenically derived into the amastigote stage of the parasite.


Identification of differentially expressed proteins from Leishmania amazonensis associated with the loss of virulence of the parasites.

Magalhães RD, Duarte MC, Mattos EC, Martins VT, Lage PS, Chávez-Fumagalli MA, Lage DP, Menezes-Souza D, Régis WC, Manso Alves MJ, Soto M, Tavares CA, Nagen RA, Coelho EA - PLoS Negl Trop Dis (2014)

Immunoblotting validation of some proteins in Leishmania amazonensis.Representative immunoblotting of some proteins that presented a significant decrease or increase in their expression content between R0 and R30 passages, using promastigote and amastigotes-like forms of L. amazonensis, are shown here. For each protein [α-tubulin, in A; paraflagellar rod protein 1D, in B; glucose-regulated protein 78 (GRP78) in C, and heat shock protein 83 (HSP83), in D], this image presents one example of correspondent 2-DE spot of promastigote form obtained from R0 or R30 passages. The antibodies used to validate each spot are described in the material and methods section. Asterisks represent the comparison between the expression of the protein in the R0 condition in relation to the R30 sample in each parasite stage, applying the Student's t-test (P<0.05), and the numbers represent the relative variation of each protein in comparison to R0 of each parasite stage. All experiments were performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974679&req=5

pntd-0002764-g003: Immunoblotting validation of some proteins in Leishmania amazonensis.Representative immunoblotting of some proteins that presented a significant decrease or increase in their expression content between R0 and R30 passages, using promastigote and amastigotes-like forms of L. amazonensis, are shown here. For each protein [α-tubulin, in A; paraflagellar rod protein 1D, in B; glucose-regulated protein 78 (GRP78) in C, and heat shock protein 83 (HSP83), in D], this image presents one example of correspondent 2-DE spot of promastigote form obtained from R0 or R30 passages. The antibodies used to validate each spot are described in the material and methods section. Asterisks represent the comparison between the expression of the protein in the R0 condition in relation to the R30 sample in each parasite stage, applying the Student's t-test (P<0.05), and the numbers represent the relative variation of each protein in comparison to R0 of each parasite stage. All experiments were performed in triplicate.
Mentions: Some of the identified proteins that presented a significant increase or decrease in their contents in the axenic cultures were used to validate the results found in this study. In this context, two of them presenting a significant decrease in their expression content, namely α-tubulin and paraflagellar rod protein 1D, and two of them, which presented an increase in their expression, namely HSP83 and GRP78, were used in the Western blot experiments (Figure 3). When promastigote extracts were employed, the selected proteins showed a variation that runs in line with the results obtained in the 2-DE gels. In addition, the decrease in the level of α-tubulin and paraflagellar rod protein 1D detected in the promastigote forms are also maintained when the R30 forms are axenically derived into the amastigote stage of the parasite.

Bottom Line: In addition, the proteins that presented a significant decrease or increase in their protein expression content were identified applying a proteomic approach.It is interesting to note that six proteins, considered hypothetical in Leishmania, showed a significant decrease in their expression and were also identified.The identified proteins that presented a significant decrease in their expression during cultivation, including the hypothetical, may also be related to this loss of parasites' infectivity, and applied in future studies, including vaccine candidates and/or immunotherapeutic targets against leishmaniasis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

ABSTRACT

Background: The present study analyzed whether or not the in vitro cultivation for long periods of time of pre-isolated Leishmania amazonensis from lesions of chronically infected BALB/c mice was able to interfere in the parasites' infectivity using in vivo and in vitro experiments. In addition, the proteins that presented a significant decrease or increase in their protein expression content were identified applying a proteomic approach.

Methodology/principal findings: Parasites were cultured in vitro for 150 days. Aliquots were collected on the day 0 of culture (R0), as well as after ten (R10; 50 days of culture), twenty (R20; 100 days of culture), and thirty (R30; 150 days of culture) passages, and were used to analyze the parasites' in vitro and in vivo infectivity, as well as to perform the proteomic approach. Approximately 837, 967, 935, and 872 spots were found in 2-DE gels prepared from R0, R10, R20, and R30 samples, respectively. A total of 37 spots presented a significant decrease in their intensity of expression, whereas a significant increase in protein content during cultivation could be observed for 19 proteins (both cases >2.0 folds). Some of these identified proteins can be described, such as diagnosis and/or vaccine candidates, while others are involved in the infectivity of Leishmania. It is interesting to note that six proteins, considered hypothetical in Leishmania, showed a significant decrease in their expression and were also identified.

Conclusions/significance: The present study contributes to the understanding that the cultivation of parasites over long periods of time may well be related to the possible loss of infectivity of L. amazonensis. The identified proteins that presented a significant decrease in their expression during cultivation, including the hypothetical, may also be related to this loss of parasites' infectivity, and applied in future studies, including vaccine candidates and/or immunotherapeutic targets against leishmaniasis.

Show MeSH
Related in: MedlinePlus