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Pathogenicity of Leishmania donovani is associated with the high expression of a group low molecular weight proteins.

Mitra P - Trop Parasitol (2015 Jul-Dec)

Bottom Line: It has been proposed that the loss of pathogenicity is associated with the gradual disappearance of selective parasite proteins.We observed a gradual and significant downregulation of expression of a group of low molecular weight proteins (LMW, molecular weight 20-30 kDa) which are associated with loss of pathogenicity.Therefore, our data strongly support the hypothesis that the loss of pathogenicity of L. donovani is associated with a change in antigenic profile, but not due the selective deletion of pathogenic clones.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Queensland, Woolloongabba, QLD 4122, Australia.

ABSTRACT

Background: With few exceptions, members of the Leishmania donovani complex such as L. donovani, L. infantum and L. chagashi are the etiological agents of visceral leishmaniasis or kala-azar. Promastigotes of Leishmania spp. lose their Pathogenicity; the ability to establish infection in a susceptible host, after prolonged culture. The molecular basis of this evolution of pathogenic to nonpathogenic culture has not been very well understood. It has been proposed that the loss of pathogenicity is associated with the gradual disappearance of selective parasite proteins. An alternative hypothesis is that during prolonged culture, the pathogenic clonal population of the parasite is deleted from the mixed population due to their selection pressure. This clonal deletion is proposed to be responsible for the emergence of the nonpathogenic population.

Study methodology and results: We have a done a series of two-dimensional polyacrylamide gel electrophoresis followed by western blot experiments to study the antigenic profile of few L. donovani isolates of Indian origin. We observed a gradual and significant downregulation of expression of a group of low molecular weight proteins (LMW, molecular weight 20-30 kDa) which are associated with loss of pathogenicity. These proteins are recognized only by antiserum raised against the whole cell extract of one of the pathogenic Indian L. donovani isolates, Ag83, and remained undetected by antiserum raised against the nonpathogenic AG83 isolates. These LMW proteins were also present in the nonpathogenic extract in very low levels and remained undetected by the virulent serum, indicating a phenomenon of simultaneous downregulation of the expression and altered immunogenicity. LMW proteins were universally expressed in all early passage Indian isolate we tested and also detected in two clones obtained from pathogenic parasite culture. The antigenic patterns of none of the eight clones obtained from nonpathogenic culture were not exactly similar with the pathogenic clones.

Conclusion: Therefore, our data strongly support the hypothesis that the loss of pathogenicity of L. donovani is associated with a change in antigenic profile, but not due the selective deletion of pathogenic clones.

No MeSH data available.


Related in: MedlinePlus

The expression of low molecular weight promastigote proteins are downregulated during prolong culture. AG83 promastigotes were harvested after one (a), four (b), seven (c), and nine (d) months in culture after transformation from amastigotes. Proteins (100 μg) from harvested parasite stocks were analyzed by two-dimensional polyacrylamide gel electrophoresis followed by immunoblotting with virulent aniserum. Low molecular weight proteins are marked by arrows inside the box
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Figure 5: The expression of low molecular weight promastigote proteins are downregulated during prolong culture. AG83 promastigotes were harvested after one (a), four (b), seven (c), and nine (d) months in culture after transformation from amastigotes. Proteins (100 μg) from harvested parasite stocks were analyzed by two-dimensional polyacrylamide gel electrophoresis followed by immunoblotting with virulent aniserum. Low molecular weight proteins are marked by arrows inside the box

Mentions: From Figure 2, it is clear that we identified the high-level expression of a group of LMW protein which is differentially expressed in pathogenic promastigotes. We were more interested to know whether this high expression has any association with the ‘age’ of the parasite culture. AG83 promastigotes were maintained in a culture for more than 9 months, and parasite cultures were harvested with an interval of few months and extracts were analyzed as 2D-PAGE followed by immunoblotting experiments with virulent antiserum. From Figure 3a-d, it is clear that besides other minor changes in the protein profiles, the most significant difference was observed in the group of 3–4 LMW proteins. The level of those proteins remained nearly same in promastigotes maintained in the culture up to 4 months. After 7 months, the expression of all but one was significantly reduces, and only two of them could be detected in the extracts, when parasites were maintained in continuous culture for 9 months. It was observed that for AG83 promastigotes, pathogenicity started to decline when parasites were in continuous culture around 5–6 months (tested by BALB/c mice infection followed by measuring the spleen size after few months interval). After 9 months, we found that the infectivity of the parasite was 5–6-fold reduced (measured by the spleen size) in comparison to the very early passage parasite. Therefore, we conclude that both differential expression and altered composition of immunoreactive proteins are associated with the loss of pathogenicity of this L. donovani isolate. The LMW proteins were not detected in the amastigote extracts (data not shown), indicating they are specifically expressed in promastigotes. To check the promastigote specific expression, amastigotes were isolated from the spleen of the infected BALB/c mice and incubated in vitro at 22°C for transformation. We observed under our conditions, the transformation took 48 h. Therefore, parasite extracts were isolated and analyzed SDS-PAGE followed by immunoblotting by pathogenic and nonpathogenic serum. Four protein bands in between ~20 and 30 kDa, detected by pathogenic serum [Figure 4, left panel] but not by the nonpathogenic serum [Figure 4 right panel], corresponded well with our previous 2D-PAGE-immunoblot data showing the high-level expression of LMW proteins in pathogenic extracts. However, we are intrigued by the fact that amastigote extracts or “0” h extract had a very poor recognition by those two antiserums.


Pathogenicity of Leishmania donovani is associated with the high expression of a group low molecular weight proteins.

Mitra P - Trop Parasitol (2015 Jul-Dec)

The expression of low molecular weight promastigote proteins are downregulated during prolong culture. AG83 promastigotes were harvested after one (a), four (b), seven (c), and nine (d) months in culture after transformation from amastigotes. Proteins (100 μg) from harvested parasite stocks were analyzed by two-dimensional polyacrylamide gel electrophoresis followed by immunoblotting with virulent aniserum. Low molecular weight proteins are marked by arrows inside the box
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4557149&req=5

Figure 5: The expression of low molecular weight promastigote proteins are downregulated during prolong culture. AG83 promastigotes were harvested after one (a), four (b), seven (c), and nine (d) months in culture after transformation from amastigotes. Proteins (100 μg) from harvested parasite stocks were analyzed by two-dimensional polyacrylamide gel electrophoresis followed by immunoblotting with virulent aniserum. Low molecular weight proteins are marked by arrows inside the box
Mentions: From Figure 2, it is clear that we identified the high-level expression of a group of LMW protein which is differentially expressed in pathogenic promastigotes. We were more interested to know whether this high expression has any association with the ‘age’ of the parasite culture. AG83 promastigotes were maintained in a culture for more than 9 months, and parasite cultures were harvested with an interval of few months and extracts were analyzed as 2D-PAGE followed by immunoblotting experiments with virulent antiserum. From Figure 3a-d, it is clear that besides other minor changes in the protein profiles, the most significant difference was observed in the group of 3–4 LMW proteins. The level of those proteins remained nearly same in promastigotes maintained in the culture up to 4 months. After 7 months, the expression of all but one was significantly reduces, and only two of them could be detected in the extracts, when parasites were maintained in continuous culture for 9 months. It was observed that for AG83 promastigotes, pathogenicity started to decline when parasites were in continuous culture around 5–6 months (tested by BALB/c mice infection followed by measuring the spleen size after few months interval). After 9 months, we found that the infectivity of the parasite was 5–6-fold reduced (measured by the spleen size) in comparison to the very early passage parasite. Therefore, we conclude that both differential expression and altered composition of immunoreactive proteins are associated with the loss of pathogenicity of this L. donovani isolate. The LMW proteins were not detected in the amastigote extracts (data not shown), indicating they are specifically expressed in promastigotes. To check the promastigote specific expression, amastigotes were isolated from the spleen of the infected BALB/c mice and incubated in vitro at 22°C for transformation. We observed under our conditions, the transformation took 48 h. Therefore, parasite extracts were isolated and analyzed SDS-PAGE followed by immunoblotting by pathogenic and nonpathogenic serum. Four protein bands in between ~20 and 30 kDa, detected by pathogenic serum [Figure 4, left panel] but not by the nonpathogenic serum [Figure 4 right panel], corresponded well with our previous 2D-PAGE-immunoblot data showing the high-level expression of LMW proteins in pathogenic extracts. However, we are intrigued by the fact that amastigote extracts or “0” h extract had a very poor recognition by those two antiserums.

Bottom Line: It has been proposed that the loss of pathogenicity is associated with the gradual disappearance of selective parasite proteins.We observed a gradual and significant downregulation of expression of a group of low molecular weight proteins (LMW, molecular weight 20-30 kDa) which are associated with loss of pathogenicity.Therefore, our data strongly support the hypothesis that the loss of pathogenicity of L. donovani is associated with a change in antigenic profile, but not due the selective deletion of pathogenic clones.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Queensland, Woolloongabba, QLD 4122, Australia.

ABSTRACT

Background: With few exceptions, members of the Leishmania donovani complex such as L. donovani, L. infantum and L. chagashi are the etiological agents of visceral leishmaniasis or kala-azar. Promastigotes of Leishmania spp. lose their Pathogenicity; the ability to establish infection in a susceptible host, after prolonged culture. The molecular basis of this evolution of pathogenic to nonpathogenic culture has not been very well understood. It has been proposed that the loss of pathogenicity is associated with the gradual disappearance of selective parasite proteins. An alternative hypothesis is that during prolonged culture, the pathogenic clonal population of the parasite is deleted from the mixed population due to their selection pressure. This clonal deletion is proposed to be responsible for the emergence of the nonpathogenic population.

Study methodology and results: We have a done a series of two-dimensional polyacrylamide gel electrophoresis followed by western blot experiments to study the antigenic profile of few L. donovani isolates of Indian origin. We observed a gradual and significant downregulation of expression of a group of low molecular weight proteins (LMW, molecular weight 20-30 kDa) which are associated with loss of pathogenicity. These proteins are recognized only by antiserum raised against the whole cell extract of one of the pathogenic Indian L. donovani isolates, Ag83, and remained undetected by antiserum raised against the nonpathogenic AG83 isolates. These LMW proteins were also present in the nonpathogenic extract in very low levels and remained undetected by the virulent serum, indicating a phenomenon of simultaneous downregulation of the expression and altered immunogenicity. LMW proteins were universally expressed in all early passage Indian isolate we tested and also detected in two clones obtained from pathogenic parasite culture. The antigenic patterns of none of the eight clones obtained from nonpathogenic culture were not exactly similar with the pathogenic clones.

Conclusion: Therefore, our data strongly support the hypothesis that the loss of pathogenicity of L. donovani is associated with a change in antigenic profile, but not due the selective deletion of pathogenic clones.

No MeSH data available.


Related in: MedlinePlus