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Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut

BACKGROUND: In vector-borne diseases such as leishmaniasis, the sand fly midgut is considered to be an important site for vector-parasite interaction. Digestive enzymes including serine peptidases such as trypsin and chymotrypsin, which are secreted in the midgut are one of the obstacles for Leishma...

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Autores principales: Verma, Sudha, Das, Sushmita, Mandal, Abhishek, Ansari, Md Yousuf, Kumari, Sujata, Mansuri, Rani, Kumar, Ajay, Singh, Ruby, Saini, Savita, Abhishek, Kumar, Kumar, Vijay, Sahoo, Ganesh Chandra, Das, Pradeep
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481909/
https://www.ncbi.nlm.nih.gov/pubmed/28645315
http://dx.doi.org/10.1186/s13071-017-2239-9
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author Verma, Sudha
Das, Sushmita
Mandal, Abhishek
Ansari, Md Yousuf
Kumari, Sujata
Mansuri, Rani
Kumar, Ajay
Singh, Ruby
Saini, Savita
Abhishek, Kumar
Kumar, Vijay
Sahoo, Ganesh Chandra
Das, Pradeep
author_facet Verma, Sudha
Das, Sushmita
Mandal, Abhishek
Ansari, Md Yousuf
Kumari, Sujata
Mansuri, Rani
Kumar, Ajay
Singh, Ruby
Saini, Savita
Abhishek, Kumar
Kumar, Vijay
Sahoo, Ganesh Chandra
Das, Pradeep
author_sort Verma, Sudha
collection PubMed
description BACKGROUND: In vector-borne diseases such as leishmaniasis, the sand fly midgut is considered to be an important site for vector-parasite interaction. Digestive enzymes including serine peptidases such as trypsin and chymotrypsin, which are secreted in the midgut are one of the obstacles for Leishmania in establishing a successful infection. The presence of some natural inhibitors of serine peptidases (ISPs) has recently been reported in Leishmania. In the present study, we deciphered the role of these ISPs in the survival of Leishmania donovani in the hostile sand fly midgut environment. METHODS: In silico and co-immunoprecipitation studies were performed to observe the interaction of L. donovani ISPs with trypsin and chymotrypsin. Zymography and in vitro enzyme assays were carried out to observe the inhibitory effect of purified recombinant ISPs of L. donovani (rLdISPs) on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of ISPs in the amastigote to promastigote transition stages were studied by semi-quantitative RT-PCR and Western blot. The role of LdISP on the survival of ISP overexpressed (OE) and ISP knocked down (KD) Leishmania parasites inside the sand fly gut was investigated by in vitro and in vivo cell viability assays. RESULTS: We identified two ecotin-like genes in L. donovani, LdISP1 and LdISP2. In silico and co-immunoprecipitation results clearly suggest a strong interaction of LdISP molecules with trypsin and chymotrypsin. Zymography and in vitro enzyme assay confirmed the inhibitory effect of rLdISP on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of LdISP2 was found to be strongly associated with the amastigote to promastigote phase transition. The activities of the digestive enzymes were found to be significantly reduced in the infected sand flies when compared to uninfected. To our knowledge, our study is the first report showing the possible reduction of chymotrypsin activity in L. donovani infected sand flies compared to uninfected. Interestingly, during the early transition stage, substantial killing was observed in ISP2 knocked down (ISP2KD) parasites compared to wild type (WT), whereas ISP1 knocked down (ISP1KD) parasites remained viable. Therefore, our study clearly indicates that LdISP2 is a more effective inhibitor of serine peptidases than LdISP1. CONCLUSION: Our results suggest that the lack of ISP2 is detrimental to the parasites during the early transition from amastigotes to promastigotes. Moreover, the results of the present study demonstrated for the first time that LdISP2 has an important role in the inhibition of peptidases and promoting L. donovani survival inside the Phlebotomus argentipes midgut. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-017-2239-9) contains supplementary material, which is available to authorized users.
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spelling pubmed-54819092017-06-23 Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut Verma, Sudha Das, Sushmita Mandal, Abhishek Ansari, Md Yousuf Kumari, Sujata Mansuri, Rani Kumar, Ajay Singh, Ruby Saini, Savita Abhishek, Kumar Kumar, Vijay Sahoo, Ganesh Chandra Das, Pradeep Parasit Vectors Research BACKGROUND: In vector-borne diseases such as leishmaniasis, the sand fly midgut is considered to be an important site for vector-parasite interaction. Digestive enzymes including serine peptidases such as trypsin and chymotrypsin, which are secreted in the midgut are one of the obstacles for Leishmania in establishing a successful infection. The presence of some natural inhibitors of serine peptidases (ISPs) has recently been reported in Leishmania. In the present study, we deciphered the role of these ISPs in the survival of Leishmania donovani in the hostile sand fly midgut environment. METHODS: In silico and co-immunoprecipitation studies were performed to observe the interaction of L. donovani ISPs with trypsin and chymotrypsin. Zymography and in vitro enzyme assays were carried out to observe the inhibitory effect of purified recombinant ISPs of L. donovani (rLdISPs) on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of ISPs in the amastigote to promastigote transition stages were studied by semi-quantitative RT-PCR and Western blot. The role of LdISP on the survival of ISP overexpressed (OE) and ISP knocked down (KD) Leishmania parasites inside the sand fly gut was investigated by in vitro and in vivo cell viability assays. RESULTS: We identified two ecotin-like genes in L. donovani, LdISP1 and LdISP2. In silico and co-immunoprecipitation results clearly suggest a strong interaction of LdISP molecules with trypsin and chymotrypsin. Zymography and in vitro enzyme assay confirmed the inhibitory effect of rLdISP on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of LdISP2 was found to be strongly associated with the amastigote to promastigote phase transition. The activities of the digestive enzymes were found to be significantly reduced in the infected sand flies when compared to uninfected. To our knowledge, our study is the first report showing the possible reduction of chymotrypsin activity in L. donovani infected sand flies compared to uninfected. Interestingly, during the early transition stage, substantial killing was observed in ISP2 knocked down (ISP2KD) parasites compared to wild type (WT), whereas ISP1 knocked down (ISP1KD) parasites remained viable. Therefore, our study clearly indicates that LdISP2 is a more effective inhibitor of serine peptidases than LdISP1. CONCLUSION: Our results suggest that the lack of ISP2 is detrimental to the parasites during the early transition from amastigotes to promastigotes. Moreover, the results of the present study demonstrated for the first time that LdISP2 has an important role in the inhibition of peptidases and promoting L. donovani survival inside the Phlebotomus argentipes midgut. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-017-2239-9) contains supplementary material, which is available to authorized users. BioMed Central 2017-06-23 /pmc/articles/PMC5481909/ /pubmed/28645315 http://dx.doi.org/10.1186/s13071-017-2239-9 Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Verma, Sudha
Das, Sushmita
Mandal, Abhishek
Ansari, Md Yousuf
Kumari, Sujata
Mansuri, Rani
Kumar, Ajay
Singh, Ruby
Saini, Savita
Abhishek, Kumar
Kumar, Vijay
Sahoo, Ganesh Chandra
Das, Pradeep
Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut
title Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut
title_full Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut
title_fullStr Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut
title_full_unstemmed Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut
title_short Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut
title_sort role of inhibitors of serine peptidases in protecting leishmania donovani against the hydrolytic peptidases of sand fly midgut
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481909/
https://www.ncbi.nlm.nih.gov/pubmed/28645315
http://dx.doi.org/10.1186/s13071-017-2239-9
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