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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

A distinct difference is observed in immunoreactive protein pattern of pathogenic and nonpathogenic promastigote proteins: Protein extracts (~100 μg) of pathogenic (a and b) and nonpathogenic (c and d) promastigotes were analyzed by two-dimensional polyacrylamide gel electrophoresis and western blotted with virulent antisera (a and d) and avirulent sera (b and c) at 1:20 dilution. The low molecular weight proteins are enclosed with rectangle with arrows
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Figure 3: A distinct difference is observed in immunoreactive protein pattern of pathogenic and nonpathogenic promastigote proteins: Protein extracts (~100 μg) of pathogenic (a and b) and nonpathogenic (c and d) promastigotes were analyzed by two-dimensional polyacrylamide gel electrophoresis and western blotted with virulent antisera (a and d) and avirulent sera (b and c) at 1:20 dilution. The low molecular weight proteins are enclosed with rectangle with arrows

Mentions: Initially, we attempted to analyze the total parasite proteins isolated from both pathogenic and nonpathogenic population by SDS-PAGE followed by silver staining. To compare between two protein profiles by this way was not successful because of the very “congested” protein pattern generated by the sensitive silver staining method. In order to get more distinguishable protein pattern, whole parasite proteins were analyzed by two-dimensional gel electrophoresis (2D-PAGE). Unlike combined isoelectrophoresis followed by SDS-PAGE,[12] we used nonequilibrium pH gradient electrophoresis (NEPHGE) because it is less time consuming and separates proteins based on their isoelectric point.[10] The optimum resolution was obtained when proteins were run at 1000 VH in a mixture of ampholines with pH 3.5–10.0 and 4.0–6.0 at 1:2 ratio [Supplement Figure 1a and b]. To obtain total parasite protein profile, promastigotes were incubated with 35S-methionine for 4 h and extracts were analyzed by NEPHGE followed by SDS-PAGE. We observed around 50 major and several 100 minor clearly distinguishable protein spots in each gel [Figure 1a and b]. Though the background of these radioactive gels was very high, but we could still find few differences in the protein profile of pathogenic and nonpathogenic promastigotes. For example, if we consider the relative position of the peptide “a” and “b,” in Figure 1a and b, then we observed those peptides were present only in pathogenic extract. Similarly, few minor spots present in Figure 1b (indicated by arrow), appeared to be missing in pathogenic extract. Therefore, Figure 1a and b indicated for the 1st time that expression of proteins in two forms of promastigotes is not exactly identical. Next, we wanted to compare immunoreactive protein profile of both promastigotes because those protein may contribute directly or indirectly to the pathogenesis. Promastigote proteins were analyzed by 2D-PAGE followed by western blotting using antiserum raised against virulent (virulent serum) and avirulent AG83 promastigote extracts. At first, we western blotted pathogenic promastigote extract with virulent and avirulent serum and observed a significant difference between two protein profiles [Figure 2a and b]. Few proteins (marked by arrow) were expressed in pathogenic promastigotes were recognized by virulent serum. However, the most significant difference was observed in the case of a group of 3–4 LMW protein located inside the square. Two proteins (indicated by arrow inside the square) in this group were absolutely recognized by virulent serum but not by the avirulent serum. Other two minor proteins located at the edge of the square were also not recognized by avirulent serum. It, therefore, appeared that those LMW proteins are either not present in avirulent extract, or they expressed with altered antigenicity. To explore that possibility, we analyzed the nonpathogenic promastigote extract by 2D-PAGE-immunoblot experiments using the virulent and avirulent serum. We observed that avirulent serum recognized few high molecular weight proteins more strongly and few extra protein spots than virulent serum recognized in the same extract. Interestingly, the two proteins (indicated by arrow inside the box) very weakly detected by the serum and one of the two proteins remained undetected [Figure 2c]. These LMW proteins in nonpathogenic parasite extracts were not detected by the virulent serum [Figure 2d]. Above result, therefore, indicated that a group of LMW proteins were highly expressed in pathogenic promastigotes and their expression is downregulated in nonpathogenic counterpart perhaps with altered anitigenicity. All proteins detected in Western blot experiments by antiserum were of parasite origin and did not cross-react with proteins either from spleen tissue of mice or from serum [Supplement Figure 2a and b].


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

Mitra P - Trop Parasitol (2015 Jul-Dec)

A distinct difference is observed in immunoreactive protein pattern of pathogenic and nonpathogenic promastigote proteins: Protein extracts (~100 μg) of pathogenic (a and b) and nonpathogenic (c and d) promastigotes were analyzed by two-dimensional polyacrylamide gel electrophoresis and western blotted with virulent antisera (a and d) and avirulent sera (b and c) at 1:20 dilution. The low molecular weight proteins are enclosed with rectangle with arrows
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: A distinct difference is observed in immunoreactive protein pattern of pathogenic and nonpathogenic promastigote proteins: Protein extracts (~100 μg) of pathogenic (a and b) and nonpathogenic (c and d) promastigotes were analyzed by two-dimensional polyacrylamide gel electrophoresis and western blotted with virulent antisera (a and d) and avirulent sera (b and c) at 1:20 dilution. The low molecular weight proteins are enclosed with rectangle with arrows
Mentions: Initially, we attempted to analyze the total parasite proteins isolated from both pathogenic and nonpathogenic population by SDS-PAGE followed by silver staining. To compare between two protein profiles by this way was not successful because of the very “congested” protein pattern generated by the sensitive silver staining method. In order to get more distinguishable protein pattern, whole parasite proteins were analyzed by two-dimensional gel electrophoresis (2D-PAGE). Unlike combined isoelectrophoresis followed by SDS-PAGE,[12] we used nonequilibrium pH gradient electrophoresis (NEPHGE) because it is less time consuming and separates proteins based on their isoelectric point.[10] The optimum resolution was obtained when proteins were run at 1000 VH in a mixture of ampholines with pH 3.5–10.0 and 4.0–6.0 at 1:2 ratio [Supplement Figure 1a and b]. To obtain total parasite protein profile, promastigotes were incubated with 35S-methionine for 4 h and extracts were analyzed by NEPHGE followed by SDS-PAGE. We observed around 50 major and several 100 minor clearly distinguishable protein spots in each gel [Figure 1a and b]. Though the background of these radioactive gels was very high, but we could still find few differences in the protein profile of pathogenic and nonpathogenic promastigotes. For example, if we consider the relative position of the peptide “a” and “b,” in Figure 1a and b, then we observed those peptides were present only in pathogenic extract. Similarly, few minor spots present in Figure 1b (indicated by arrow), appeared to be missing in pathogenic extract. Therefore, Figure 1a and b indicated for the 1st time that expression of proteins in two forms of promastigotes is not exactly identical. Next, we wanted to compare immunoreactive protein profile of both promastigotes because those protein may contribute directly or indirectly to the pathogenesis. Promastigote proteins were analyzed by 2D-PAGE followed by western blotting using antiserum raised against virulent (virulent serum) and avirulent AG83 promastigote extracts. At first, we western blotted pathogenic promastigote extract with virulent and avirulent serum and observed a significant difference between two protein profiles [Figure 2a and b]. Few proteins (marked by arrow) were expressed in pathogenic promastigotes were recognized by virulent serum. However, the most significant difference was observed in the case of a group of 3–4 LMW protein located inside the square. Two proteins (indicated by arrow inside the square) in this group were absolutely recognized by virulent serum but not by the avirulent serum. Other two minor proteins located at the edge of the square were also not recognized by avirulent serum. It, therefore, appeared that those LMW proteins are either not present in avirulent extract, or they expressed with altered antigenicity. To explore that possibility, we analyzed the nonpathogenic promastigote extract by 2D-PAGE-immunoblot experiments using the virulent and avirulent serum. We observed that avirulent serum recognized few high molecular weight proteins more strongly and few extra protein spots than virulent serum recognized in the same extract. Interestingly, the two proteins (indicated by arrow inside the box) very weakly detected by the serum and one of the two proteins remained undetected [Figure 2c]. These LMW proteins in nonpathogenic parasite extracts were not detected by the virulent serum [Figure 2d]. Above result, therefore, indicated that a group of LMW proteins were highly expressed in pathogenic promastigotes and their expression is downregulated in nonpathogenic counterpart perhaps with altered anitigenicity. All proteins detected in Western blot experiments by antiserum were of parasite origin and did not cross-react with proteins either from spleen tissue of mice or from serum [Supplement Figure 2a and b].

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