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Article ; Online: GRASP negatively regulates the secretion of the virulence factor gp63 in Leishmania.

Kumar, Kamal / Basak, Rituparna / Rai, Aakansha / Mukhopadhyay, Amitabha

2024 Volume 121, Issue 5, Page(s) 1063–1078

Abstract: Metalloprotease-gp63 is a virulence factor secreted by Leishmania. However, secretory pathway in Leishmania is not well defined. Here, we cloned and expressed the GRASP homolog from Leishmania. We found that Leishmania expresses one GRASP homolog of 58 ... ...

Abstract	Metalloprotease-gp63 is a virulence factor secreted by Leishmania. However, secretory pathway in Leishmania is not well defined. Here, we cloned and expressed the GRASP homolog from Leishmania. We found that Leishmania expresses one GRASP homolog of 58 kDa protein (LdGRASP) which localizes in LdRab1- and LPG2-positive Golgi compartment in Leishmania. LdGRASP was found to bind with COPII complex, LdARF1, LdRab1 and LdRab11 indicating its role in ER and Golgi transport in Leishmania. To determine the function of LdGRASP, we generated LdGRASP knockout parasites using CRISPR-Cas9. We found fragmentation of Golgi in Ld:GRASPKO parasites. Our results showed enhanced transport of non-GPI-anchored gp63 to the cell surface leading to higher secretion of this form of gp63 in Ld:GRASPKO parasites in comparison to Ld:WT cells. In contrast, we found that transport of GPI-anchored gp63 to the cell surface is blocked in Ld:GRASPKO parasites and thereby inhibits its secretion. The overexpression of dominant-negative mutant of LdRab1 or LdSar1 in Ld:GRASPKO parasites significantly blocked the secretion of non-GPI-anchored gp63. Interestingly, we found that survival of transgenic parasites overexpressing Ld:GRASP-GFP is significantly compromised in macrophages in comparison to Ld:WT and Ld:GRASPKO parasites. These results demonstrated that LdGRASP differentially regulates Ldgp63 secretory pathway in Leishmania.
MeSH term(s)	Virulence Factors/metabolism ; Virulence Factors/genetics ; Protozoan Proteins/metabolism ; Protozoan Proteins/genetics ; Metalloendopeptidases/metabolism ; Metalloendopeptidases/genetics ; Golgi Apparatus/metabolism ; Endoplasmic Reticulum/metabolism ; Macrophages/parasitology ; Macrophages/metabolism ; Animals ; Leishmania/metabolism ; Leishmania/genetics ; Protein Transport ; CRISPR-Cas Systems ; Golgi Matrix Proteins/metabolism ; Golgi Matrix Proteins/genetics
Language	English
Publishing date	2024-04-01
Publishing country	England
Document type	Journal Article
ZDB-ID	619315-8
ISSN	1365-2958 ; 0950-382X
ISSN (online)	1365-2958
ISSN	0950-382X
DOI	10.1111/mmi.15255
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Article ; Online: Unconventional role of Rab4 in the secretory pathway in Leishmania.

Ansari, Irshad / Singh, Amir Kumar / Kapoor, Anjali / Mukhopadhyay, Amitabha

Biochimica et biophysica acta. Molecular cell research

2024 Volume 1871, Issue 4, Page(s) 119687

Abstract: Leishmania donovani is an auxotroph for heme. Parasite acquires heme by clathrin-mediated endocytosis of hemoglobin by specific receptor. However, the regulation of receptor recycling pathway is not known in Leishmania. Here, we have cloned, expressed ... ...

Abstract	Leishmania donovani is an auxotroph for heme. Parasite acquires heme by clathrin-mediated endocytosis of hemoglobin by specific receptor. However, the regulation of receptor recycling pathway is not known in Leishmania. Here, we have cloned, expressed and characterized the Rab4 homologue from L. donovani. We have found that LdRab4 localizes in both early endosomes and Golgi in L. donovani. To understand the role of LdRab4 in L. donovani, we have generated transgenic parasites overexpressing GFP-LdRab4:WT, GFP-LdRab4:Q67L, and GFP-LdRab4:S22N. Our results have shown that overexpression of GFP-LdRab4:Q67L or GFP-LdRab4:S22N does not alter the cell surface localization of hemoglobin receptor in L. donovani. Surprisingly, we have found that overexpression of GFP-LdRab4:S22N significantly blocks the transport of Ldgp63 to the cell surface whereas the trafficking of Ldgp63 is induced to the cell surface in GFP-LdRab4:WT and GFP-LdRab4:Q67L overexpressing parasites. Consequently, we have found significant inhibition of gp63 secretion by GFP-LdRab4:S22N overexpressing parasites whereas secretion of Ldgp63 is enhanced in GFP-LdRab4:WT and GFP-LdRab4:Q67L overexpressing parasites in comparison to untransfected control parasites. Moreover, we have found that survival of transgenic parasites overexpressing GFP-LdRab4:S22N is severely compromised in macrophages in comparison to GFP-LdRab4:WT and GFP-LdRab4:Q67L expressing parasites. These results demonstrated that LdRab4 unconventionally regulates the secretory pathway in L. donovani.
MeSH term(s)	Animals ; Secretory Pathway ; Leishmania donovani/genetics ; Animals, Genetically Modified/metabolism ; Carrier Proteins/metabolism ; Hemoglobins/metabolism ; Heme/metabolism
Chemical Substances	Carrier Proteins ; Hemoglobins ; Heme (42VZT0U6YR)
Language	English
Publishing date	2024-02-10
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	60-7
ISSN	1879-2596 ; 1879-260X ; 1872-8006 ; 1879-2642 ; 1879-2618 ; 1879-2650 ; 0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 1570-9639 ; 0925-4439 ; 1874-9399
ISSN (online)	1879-2596 ; 1879-260X ; 1872-8006 ; 1879-2642 ; 1879-2618 ; 1879-2650
ISSN	0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 1570-9639 ; 0925-4439 ; 1874-9399
DOI	10.1016/j.bbamcr.2024.119687
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Article ; Online: Leishmania highjack host lipid body for its proliferation in macrophages by overexpressing host Rab18 and TRAPPC9 by downregulating miR-1914-3p expression.

Sood, Chandni / Verma, Jitender Kumar / Basak, Rituparna / Kapoor, Anjali / Gupta, Swarnima / Mukhopadhyay, Amitabha

PLoS pathogens

2024 Volume 20, Issue 2, Page(s) e1012024

Abstract: Lipids stored in lipid-bodies (LBs) in host cells are potential sources of fatty acids for pathogens. However, the mechanism of recruitment of LBs from the host cells by pathogens to acquire fatty acids is not known. Here, we have found that Leishmania ... ...

Abstract	Lipids stored in lipid-bodies (LBs) in host cells are potential sources of fatty acids for pathogens. However, the mechanism of recruitment of LBs from the host cells by pathogens to acquire fatty acids is not known. Here, we have found that Leishmania specifically upregulates the expression of host Rab18 and its GEF, TRAPPC9 by downregulating the expression of miR-1914-3p by reducing the level of Dicer in macrophages via their metalloprotease gp63. Our results also show that miR-1914-3p negatively regulates the expression of Rab18 and its GEF in cells. Subsequently, Leishmania containing parasitophorous vacuoles (Ld-PVs) recruit and retain host Rab18 and TRAPPC9. Leishmania infection also induces LB biogenesis in host cells and recruits LBs on Ld-PVs and acquires FLC12-labeled fatty acids from LBs. Moreover, overexpression of miR-1914-3p in macrophages significantly inhibits the recruitment of LBs and thereby suppresses the multiplication of parasites in macrophages as parasites are unable to acquire fatty acids. These results demonstrate a novel mechanism how Leishmania acquire fatty acids from LBs for their growth in macrophages.
MeSH term(s)	Leishmania ; Lipid Droplets/metabolism ; Macrophages/metabolism ; MicroRNAs/genetics ; MicroRNAs/metabolism ; Fatty Acids/metabolism ; Cell Proliferation
Chemical Substances	MicroRNAs ; Fatty Acids
Language	English
Publishing date	2024-02-27
Publishing country	United States
Document type	Journal Article
ZDB-ID	2205412-1
ISSN	1553-7374 ; 1553-7374
ISSN (online)	1553-7374
ISSN	1553-7374
DOI	10.1371/journal.ppat.1012024
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Article: Hemoglobin Endocytosis and Intracellular Trafficking: A Novel Way of Heme Acquisition by

Ansari, Irshad / Basak, Rituparna / Mukhopadhyay, Amitabha

Pathogens (Basel, Switzerland)

2022 Volume 11, Issue 5

Abstract: ... ...

Abstract	Leishmania
Language	English
Publishing date	2022-05-16
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2695572-6
ISSN	2076-0817
ISSN	2076-0817
DOI	10.3390/pathogens11050585
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Article ; Online: Planning for a Multi-Well Artificial Recharge Field in Kuwait with the Help of Numerical Modeling.

Mukhopadhyay, Amitabha / Akber, Adnan / Al-Saaidi, Mashal / Aladwani, Abdullah

Ground water

2022 Volume 60, Issue 5, Page(s) 685–698

Abstract: Kuwait, an arid country, wants to have a reserve of water for emergency use and has planned to create an underground reserve of water through multi-well artificial recharge at Kabd area. Numerical modeling of different recharge-recovery scenarios was ... ...

Abstract	Kuwait, an arid country, wants to have a reserve of water for emergency use and has planned to create an underground reserve of water through multi-well artificial recharge at Kabd area. Numerical modeling of different recharge-recovery scenarios was carried out to chalk out an optimum strategy for implementation of the project. These scenario runs suggested that apart from the aquifer parameters and the quality of the groundwater and the recharge water, the well spacing and well construction, orientation of the field with respect to the prevailing hydraulic gradient, and the injection and the pumping schedule would determine the overall recovery efficiency of the setup. For the selected site, following strategies were found to have positive impact on meeting the goals of the creation of a reserve of water for use in an emergency: (1) during the reserve creation stage, simultaneous recharge and pumping through alternate wells; (2) the compensation of the water lost from the reserve created due to the flow down the hydraulic gradient during the waiting period; and (3) the orientation of the long axis of the field perpendicular to the regional hydraulic gradient. The adoption of cyclic injection and recovery option for the creation of the reserve eliminates the need of the first two steps but calls for several cycles of injection followed by recovery and adequate and consistent supply of water for injection over that period. An optimum field design and two alternative recharge options have been suggested based on the above observations.
MeSH term(s)	Environmental Monitoring/methods ; Groundwater ; Kuwait ; Water ; Water Wells
Chemical Substances	Water (059QF0KO0R)
Language	English
Publishing date	2022-01-10
Publishing country	United States
Document type	Case Reports ; Research Support, Non-U.S. Gov't
ZDB-ID	246212-6
ISSN	1745-6584 ; 0017-467X
ISSN (online)	1745-6584
ISSN	0017-467X
DOI	10.1111/gwat.13166
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Article: Planning for a Multi‐Well Artificial Recharge Field in Kuwait with the Help of Numerical Modeling

Mukhopadhyay, Amitabha / Akber, Adnan / Al‐Saaidi, Mashal / Aladwani, Abdullah

Ground water. 2022 Sept., v. 60, no. 5

2022

Abstract: Kuwait, an arid country, wants to have a reserve of water for emergency use and has planned to create an underground reserve of water through multi‐well artificial recharge at Kabd area. Numerical modeling of different recharge–recovery scenarios was ... ...

Abstract	Kuwait, an arid country, wants to have a reserve of water for emergency use and has planned to create an underground reserve of water through multi‐well artificial recharge at Kabd area. Numerical modeling of different recharge–recovery scenarios was carried out to chalk out an optimum strategy for implementation of the project. These scenario runs suggested that apart from the aquifer parameters and the quality of the groundwater and the recharge water, the well spacing and well construction, orientation of the field with respect to the prevailing hydraulic gradient, and the injection and the pumping schedule would determine the overall recovery efficiency of the setup. For the selected site, following strategies were found to have positive impact on meeting the goals of the creation of a reserve of water for use in an emergency: (1) during the reserve creation stage, simultaneous recharge and pumping through alternate wells; (2) the compensation of the water lost from the reserve created due to the flow down the hydraulic gradient during the waiting period; and (3) the orientation of the long axis of the field perpendicular to the regional hydraulic gradient. The adoption of cyclic injection and recovery option for the creation of the reserve eliminates the need of the first two steps but calls for several cycles of injection followed by recovery and adequate and consistent supply of water for injection over that period. An optimum field design and two alternative recharge options have been suggested based on the above observations.
Keywords	aquifers ; groundwater ; groundwater recharge ; Kuwait
Language	English
Dates of publication	2022-09
Size	p. 685-698.
Publishing place	Blackwell Publishing Ltd
Document type	Article
Note	Case Reports
ZDB-ID	246212-6
ISSN	1745-6584 ; 0017-467X
ISSN (online)	1745-6584
ISSN	0017-467X
DOI	10.1111/gwat.13166
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: GTPase Sar1 regulates the trafficking and secretion of the virulence factor gp63 in

Parashar, Smriti / Mukhopadhyay, Amitabha

The Journal of biological chemistry

2017 Volume 292, Issue 29, Page(s) 12111–12125

Abstract: Metalloprotease gp63 ( ...

Abstract	Metalloprotease gp63 (
MeSH term(s)	Amino Acid Substitution ; COP-Coated Vesicles/enzymology ; COP-Coated Vesicles/metabolism ; Cell Line, Tumor ; Cytosol/enzymology ; Cytosol/metabolism ; GTP Phosphohydrolases/chemistry ; GTP Phosphohydrolases/genetics ; GTP Phosphohydrolases/metabolism ; Humans ; Intracellular Membranes/enzymology ; Intracellular Membranes/metabolism ; Leishmania donovani/cytology ; Leishmania donovani/genetics ; Leishmania donovani/growth & development ; Leishmania donovani/metabolism ; Luminescent Proteins/chemistry ; Luminescent Proteins/genetics ; Luminescent Proteins/metabolism ; Macrophages/cytology ; Macrophages/metabolism ; Macrophages/parasitology ; Metalloendopeptidases/chemistry ; Metalloendopeptidases/genetics ; Metalloendopeptidases/metabolism ; Mutagenesis, Site-Directed ; Mutation ; Organisms, Genetically Modified ; Peptide Fragments/chemistry ; Peptide Fragments/genetics ; Peptide Fragments/metabolism ; Protein Interaction Domains and Motifs ; Protein Transport ; Protozoan Proteins/chemistry ; Protozoan Proteins/genetics ; Protozoan Proteins/metabolism ; Recombinant Fusion Proteins/chemistry ; Recombinant Fusion Proteins/metabolism ; Recombinant Proteins/chemistry ; Recombinant Proteins/metabolism ; Vesicular Transport Proteins/chemistry ; Vesicular Transport Proteins/genetics ; Vesicular Transport Proteins/metabolism ; Virulence Factors/chemistry ; Virulence Factors/genetics ; Virulence Factors/metabolism
Chemical Substances	Luminescent Proteins ; Peptide Fragments ; Protozoan Proteins ; Recombinant Fusion Proteins ; Recombinant Proteins ; Vesicular Transport Proteins ; Virulence Factors ; Metalloendopeptidases (EC 3.4.24.-) ; glycoprotein gp63, Leishmania (EC 3.4.24.-) ; GTP Phosphohydrolases (EC 3.6.1.-)
Language	English
Publishing date	2017-06-02
Publishing country	United States
Document type	Journal Article
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M117.784033
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Article ; Online: Trained immunity of alveolar macrophages enhances injury resolution via KLF4-MERTK-mediated efferocytosis.

Chakraborty, Sreeparna / Singh, Abhalaxmi / Wang, Li / Wang, Xinge / Sanborn, Mark A / Ye, Zijing / Maienschein-Cline, Mark / Mukhopadhyay, Amitabha / Ganesh, Balaji B / Malik, Asrar B / Rehman, Jalees

The Journal of experimental medicine

2023 Volume 220, Issue 11

Abstract: Recent studies suggest that training of innate immune cells such as tissue-resident macrophages by repeated noxious stimuli can heighten host defense responses. However, it remains unclear whether trained immunity of tissue-resident macrophages also ... ...

Abstract	Recent studies suggest that training of innate immune cells such as tissue-resident macrophages by repeated noxious stimuli can heighten host defense responses. However, it remains unclear whether trained immunity of tissue-resident macrophages also enhances injury resolution to counterbalance the heightened inflammatory responses. Here, we studied lung-resident alveolar macrophages (AMs) prechallenged with either the bacterial endotoxin or with Pseudomonas aeruginosa and observed that these trained AMs showed greater resilience to pathogen-induced cell death. Transcriptomic analysis and functional assays showed greater capacity of trained AMs for efferocytosis of cellular debris and injury resolution. Single-cell high-dimensional mass cytometry analysis and lineage tracing demonstrated that training induces an expansion of a MERTKhiMarcohiCD163+F4/80low lung-resident AM subset with a proresolving phenotype. Reprogrammed AMs upregulated expression of the efferocytosis receptor MERTK mediated by the transcription factor KLF4. Adoptive transfer of these trained AMs restricted inflammatory lung injury in recipient mice exposed to lethal P. aeruginosa. Thus, our study has identified a subset of tissue-resident trained macrophages that prevent hyperinflammation and restore tissue homeostasis following repeated pathogen challenges.
MeSH term(s)	Animals ; Mice ; Adoptive Transfer ; c-Mer Tyrosine Kinase/genetics ; Macrophages, Alveolar ; Phagocytosis ; Trained Immunity
Chemical Substances	c-Mer Tyrosine Kinase (EC 2.7.10.1) ; Mertk protein, mouse (EC 2.7.10.1) ; Klf4 protein, mouse
Language	English
Publishing date	2023-08-24
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	218343-2
ISSN	1540-9538 ; 0022-1007
ISSN (online)	1540-9538
ISSN	0022-1007
DOI	10.1084/jem.20221388
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Article ; Online: Ubiquitin ligase CHFR mediated degradation of VE-cadherin through ubiquitylation disrupts endothelial adherens junctions.

Tiruppathi, Chinnaswamy / Wang, Dong-Mei / Ansari, Mohammad Owais / Bano, Shabana / Tsukasaki, Yoshikazu / Mukhopadhyay, Amitabha / Joshi, Jagdish C / Loch, Christian / Niessen, Hans W M / Malik, Asrar B

Nature communications

2023 Volume 14, Issue 1, Page(s) 6582

Abstract: Vascular endothelial cadherin (VE-cadherin) expressed at endothelial adherens junctions (AJs) is vital for vascular integrity and endothelial homeostasis. Here we identify the requirement of the ubiquitin E3-ligase CHFR as a key mechanism of ... ...

Abstract	Vascular endothelial cadherin (VE-cadherin) expressed at endothelial adherens junctions (AJs) is vital for vascular integrity and endothelial homeostasis. Here we identify the requirement of the ubiquitin E3-ligase CHFR as a key mechanism of ubiquitylation-dependent degradation of VE-cadherin. CHFR was essential for disrupting the endothelium through control of the VE-cadherin protein expression at AJs. We observe augmented expression of VE-cadherin in endothelial cell (EC)-restricted Chfr knockout (Chfr
MeSH term(s)	Animals ; Mice ; Adherens Junctions/metabolism ; Ubiquitin/metabolism ; Ligases/metabolism ; Lipopolysaccharides/pharmacology ; Cadherins/genetics ; Cadherins/metabolism ; Endothelium/metabolism ; Ubiquitination ; Endothelium, Vascular/metabolism ; Cells, Cultured
Chemical Substances	cadherin 5 ; Ubiquitin ; Ligases (EC 6.-) ; Lipopolysaccharides ; Cadherins
Language	English
Publishing date	2023-10-18
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-023-42225-2
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Article ; Online: Leishmania donovani resides in modified early endosomes by upregulating Rab5a expression via the downregulation of miR-494.

Verma, Jitender Kumar / Rastogi, Ruchir / Mukhopadhyay, Amitabha

PLoS pathogens

2017 Volume 13, Issue 6, Page(s) e1006459

Abstract: Several intracellular pathogens arrest the phagosome maturation in the host cells to avoid transport to lysosomes. In contrast, the Leishmania containing parasitophorous vacuole (PV) is shown to recruit lysosomal markers and thus Leishmania is postulated ...

Abstract	Several intracellular pathogens arrest the phagosome maturation in the host cells to avoid transport to lysosomes. In contrast, the Leishmania containing parasitophorous vacuole (PV) is shown to recruit lysosomal markers and thus Leishmania is postulated to be residing in the phagolysosomes in macrophages. Here, we report that Leishmania donovani specifically upregulates the expression of Rab5a by degrading c-Jun via their metalloprotease gp63 to downregulate the expression of miR-494 in THP-1 differentiated human macrophages. Our results also show that miR-494 negatively regulates the expression of Rab5a in cells. Subsequently, L. donovani recruits and retains Rab5a and EEA1 on PV to reside in early endosomes and inhibits transport to lysosomes in human macrophages. Similarly, we have also observed that Leishmania PV also recruits Rab5a by upregulating its expression in human PBMC differentiated macrophages. However, the parasite modulates the endosome by recruiting Lamp1 and inactive pro-CathepsinD on PV via the overexpression of Rab5a in infected cells. Furthermore, siRNA knockdown of Rab5a or overexpression of miR-494 in human macrophages significantly inhibits the survival of the parasites. These results provide the first mechanistic insights of parasite-mediated remodeling of endo-lysosomal trafficking to reside in a specialized early endocytic compartment.
MeSH term(s)	Animals ; Down-Regulation ; Endosomes/microbiology ; Endosomes/parasitology ; Humans ; Leishmania donovani/physiology ; Leukocytes, Mononuclear/parasitology ; Lysosomes/metabolism ; Macrophages/parasitology ; MicroRNAs/genetics ; Phagosomes/microbiology ; Transcriptional Activation/genetics ; Up-Regulation ; Vacuoles/parasitology ; rab5 GTP-Binding Proteins/metabolism
Chemical Substances	MIRN494 microRNA, human ; MicroRNAs ; RAB5C protein, human (EC 3.6.1.-) ; rab5 GTP-Binding Proteins (EC 3.6.5.2)
Language	English
Publishing date	2017-06
Publishing country	United States
Document type	Journal Article
ZDB-ID	2205412-1
ISSN	1553-7374 ; 1553-7366
ISSN (online)	1553-7374
ISSN	1553-7366
DOI	10.1371/journal.ppat.1006459
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