Leishmania highjack host lipid body for its proliferation in macrophages by overexpressing host Rab18 and TRAPPC9 by downregulating miR-1914-3p expression.

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.

Virus-Like Particles of SARS-CoV-2 as Virus Surrogates: Morphology, Immunogenicity, and Internalization in Neuronal Cells.

The engineering of virus-like particles (VLPs) is a viable strategy for the development of vaccines and for the identification of therapeutic targets without using live viruses. Here, we report the generation and characterization of quadruple-antigen SARS-CoV-2 VLPs. VLPs were generated by transient transfection of two expression cassettes in adherent HEK293T cells─one cassette containing Mpro for processing of three structural proteins (M, E, and N), and the second cassette expressing the Spike protein. Further characterization revealed that the VLPs retain close morphological and antigenic similarity with the native virus and also bind strongly to the SARS-CoV-2 receptor hACE-2 in an in vitro binding assay. Interestingly, the VLPs were found to internalize into U87-MG cells through cholesterol-rich domains in a dynamin-dependent process. Finally, our results showed that mice immunized with VLPs induce robust humoral and cellular immune responses mediated by enhanced levels of IL-4, IL-17, and IFNγ. Taken together, our results demonstrate that VLPs mimic the native virus and induce a strong immune response, indicating the possible use of these particles as an alternative vaccine candidate against SARS-CoV-2. VLPs can also be effective in mapping the initial stages of virus entry and screening inhibitors.

Rab5b function is essential to acquire heme from hemoglobin endocytosis for survival of Leishmania.

Previously, we showed that Rab5a and Rab5b differentially regulate fluid-phase and receptor-mediated endocytosis in Leishmania, respectively. To unequivocally demonstrate the role of Rab5b in hemoglobin endocytosis in Leishmania, we generated null-mutants of Rab5b parasites by sequentially replacing both copies of LdRab5b with the hygromycin and neomycin resistance gene cassettes. LdRab5b-/- null-mutant parasite was confirmed by qPCR analysis of genomic DNA using LdRab5b specific primers. LdRab5b-/- cells showed severe growth defect indicating essential function of LdRab5b in parasite. To characterize the role of Rab5b in Hb endocytosis in parasites, LdRab5b-/- cells were rescued by exogenous addition of hemin in growth medium. Our results showed that LdRab5b-/- cells are relatively smaller in size. Ultrastructural analysis revealed the presence of relatively enlarged flagellar pocket and bigger intracellular vesicles in these cells in comparison to control cells. Both promastigotes and amastigotes of Rab5b null-mutant parasites were unable to internalize Hb but fluid phase endocytosis of different markers was not affected. However, complementation of LdRab5b:WT in LdRab5b-/- cells (LdRab5b-/-:pRab5b:WT) rescued Hb internalization in these cells. Interestingly, LdRab5b-/- cells showed significantly less Hb-receptor on cell surface in comparison to control cells indicating a block in HbR trafficking. Finally, we showed that LdRab5b-/- parasites can infect the macrophages but are unable to survive after 96 h of infection in comparison to control cells. However, supplementation of hemin in the growth medium significantly rescued LdRab5b-/-Leishmania survival in macrophage indicating that LdRab5b function is essential for the acquisition of heme from internalized Hb for the survival of Leishmania.

Identification and characterization of the hemoglobin-binding domain of hemoglobin receptor in Leishmania.

Leishmania internalize hemoglobin (Hb) via a specific receptor (HbR) for their survival. To identify the Hb-binding domain of HbR, we cloned and expressed several truncated proteins of HbR and determined their ability to bind Hb. Our findings reveal that 90% of Hb-binding activity is retained in HbR41-80 in comparison with HbR1-471 . We synthesized a 40 amino acid peptide (SSEKMKQLTMYMIHEMVEGLEGRPSTVRMLPSFVYTSDPA) corresponding to HbR41-80 and found that it specifically binds Hb. Subsequently, we found that the HbR41-80 peptide completely blocks Hb uptake in both promastigote and amastigote forms of Leishmania and, thereby, inhibits the growth of the parasite. These results demonstrate that HbR41-80 is the Hb-binding domain of HbR, which might be used as a potential therapeutic agent to inhibit the growth of Leishmania.

A trimeric metazoan Rab7 GEF complex is crucial for endocytosis and scavenger function.

Endosome biogenesis in eukaryotic cells is critical for nutrient uptake and plasma membrane integrity. Early endosomes initially contain Rab5, which is replaced by Rab7 on late endosomes prior to their fusion with lysosomes. Recruitment of Rab7 to endosomes requires the Mon1-Ccz1 guanine-nucleotide-exchange factor (GEF). Here, we show that full function of the Drosophila Mon1-Ccz1 complex requires a third stoichiometric subunit, termed Bulli (encoded by CG8270). Bulli localises to Rab7-positive endosomes, in agreement with its function in the GEF complex. Using Drosophila nephrocytes as a model system, we observe that absence of Bulli results in (i) reduced endocytosis, (ii) Rab5 accumulation within non-acidified enlarged endosomes, (iii) defective Rab7 localisation and (iv) impaired endosomal maturation. Moreover, longevity of animals lacking bulli is affected. Both the Mon1-Ccz1 dimer and a Bulli-containing trimer display Rab7 GEF activity. In summary, this suggests a key role for Bulli in the Rab5 to Rab7 transition during endosomal maturation rather than a direct influence on the GEF activity of Mon1-Ccz1.

Leishmania donovani resides in modified early endosomes by upregulating Rab5a expression via the downregulation of miR-494.

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.

Rab5 Isoforms Specifically Regulate Different Modes of Endocytosis in Leishmania.

Differential functions of Rab5 isoforms in endocytosis are not well characterized. Here, we cloned, expressed, and characterized Rab5a and Rab5b from Leishmania and found that both of them are localized in the early endosome. To understand the role of LdRab5 isoforms in different modes of endocytosis in Leishmania, we generated transgenic parasites overexpressing LdRab5a, LdRab5b, or their dominant-positive (LdRab5a:Q93L and LdRab5b:Q80L) or dominant-negative mutants (LdRab5a:N146I and LdRab5b:N133I). Using LdRab5a or its mutants overexpressing parasites, we found that LdRab5a specifically regulates the fluid-phase endocytosis of horseradish peroxidase and also specifically induced the transport of dextran-Texas Red to the lysosomes. In contrast, cells overexpressing LdRab5b or its mutants showed that LdRab5b explicitly controls receptor-mediated endocytosis of hemoglobin, and overexpression of LdRab5b:WT enhanced the transport of internalized Hb to the lysosomes in comparison with control cells. To unequivocally demonstrate the role of Rab5 isoforms in endocytosis in Leishmania, we tried to generate null-mutants of LdRab5a and LdRab5b parasites, but both were lethal indicating their essential functions in parasites. Therefore, we used heterozygous LdRab5a(+/-) and LdRab5b(+/-) cells. LdRab5a(+/-) Leishmania showed 50% inhibition of HRP uptake, but hemoglobin endocytosis was uninterrupted. In contrast, about 50% inhibition of Hb endocytosis was observed in LdRab5b(+/-) cells without any significant effect on HRP uptake. Finally, we tried to identify putative LdRab5a and LdRab5b effectors. We found that LdRab5b interacts with clathrin heavy chain and hemoglobin receptor. However, LdRab5a failed to interact with the clathrin heavy chain, and interaction with hemoglobin receptor was significantly less. Thus, our results showed that LdRab5a and LdRab5b differentially regulate fluid phase and receptor-mediated endocytosis in Leishmania.