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.

Decline in Hepatitis C Virus (HCV) Incidence in Men Who Have Sex With Men Living With Human Immunodeficiency Virus: Progress to HCV Microelimination in the United Kingdom?

BACKGROUND: Modeling of the London hepatitis C virus (HCV) epidemic in men who have sex with men (MSM) and are living with human immunodeficiency virus (HIV) suggested that early access to direct-acting antiviral (DAA) treatment may reduce incidence. With high rates of linkage to care, microelimination of HCV within MSM living with HIV may be realistic ahead of 2030 World Health Organization targets. We examined trends in HCV incidence in the pre- and post-DAA eras for MSM living with HIV in London and Brighton, United Kingdom. METHODS: A retrospective cohort study was conducted at 5 HIV clinics in London and Brighton between 2013 and 2018. Each site reported all acute HCV episodes during the study period. Treatment timing data were collected. Incidence rates and reinfection proportion were calculated. RESULTS: A total of.378 acute HCV infections were identified, comprising 292 first infections and 86 reinfections. Incidence rates of acute HCV in MSM living with HIV peaked at 14.57/1000 person-years of follow-up (PYFU; 95% confidence interval [CI], 10.95-18.20) in 2015. Rates fell to 4.63/1000 PYFU (95% CI, 2.60 to 6.67) by 2018. Time from diagnosis to starting treatment declined from 29.8 (2013) to 3.7 months (2018). CONCLUSIONS: We observed a 78% reduction in the incidence of first HCV episode and a 68% reduction in overall HCV incidence since the epidemic peak in 2015, which coincides with wider access to DAAs in England. Further interventions to reduce transmission, including earlier access to treatment and for reinfection, are likely needed for microelimination to be achieved in this population.

Functional characterization of the LdNAGD gene in Leishmania donovani.

The N-acetyl glucosamine catabolic pathway has been well established as a critically essential pathway for the survival and pathogenesis of several intracellular pathogens. The intracellular form of Leishmania donovani resides inside the parasitophorous vacuole of macrophages. Recent studies have shown that amino sugars, such as N-acetyl glucosamine, are released from the turnover of host macromolecules, such as glycosaminoglycans, glycoproteins, and proteoglycans, inside the parasitophorous vacuole. Three enzymes, hexokinase (Hxk), N-acetyl glucosamine-6-phosphate deacetylase (NAGD) and glucosamine-6-phosphate deaminase (GND), are sequentially involved in the catabolism of GlcNAc. The Leishmania donovani genome encodes all enzymes of the GlcNAc catabolic pathway. Here, we investigated the role of the GlcNAc catabolic pathway in the proliferation and survival of L. donovani by characterizing the NAGD gene of this pathway. Recombinant LdNAGD displayed deacetylation activity and was localized inside the glycosomes. LdNAGD gene deletion impaired GlcNAc catabolism and was indispensable for the viability of L. donovani in media containing GlcNAc as the sole carbon source. Furthermore, these Δnagd cells showed attenuated virulence in THP-1 cells and a significantly reduced proliferation rate compared to wild type (WT) cells inside THP-1 cells. Our data suggested that LdNAGD is important for the intracellular proliferation of L. donovani and may represent a potential drug target.

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.

Author Correction: Micro RNAs upregulated in Vitiligo skin play an important role in its aetiopathogenesis by altering TRP1 expression and keratinocyte-melanocytes cross-talk.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Micro RNAs upregulated in Vitiligo skin play an important role in its aetiopathogenesis by altering TRP1 expression and keratinocyte-melanocytes cross-talk.

Translation of genes is regulated by many factors including microRNAs (miRNAs). miRNA profiling of lesional and non-lesional epidermal RNA from 18 vitiligo patients revealed significant upregulation of 29 miRNAs in the lesional epidermis, of which 6 miRNAs were transfected in normal human epidermal keratinocytes (NHEKs) to study their downstream effects using quantitative proteomics. Many proteins involved in oxidative stress, Vesicle trafficking, Cellular apoptosis, Mitochondrial proteins and Keratins were regulated after miRNA transfections in the keratinocytes. However, tyrosinase related protein-1 (TRP1/TYRP1), a melanogenesis protein, was consistently downregulated in NHEKs by all the six miRNAs tested, which was quite intriguing. TRP1 was also downregulated in lesional epidermis compared with non-lesional epidermis. Since melanocytes synthesize and transfer melanosomes to the surrounding keratinocytes, we hypothesized that downregulation of TRP1 in NHEKs may have a role in melanosome transfer, which was confirmed by our co-culture experiments. Downregulation of TRP1 in keratinocytes negatively affected the melanosome transfer from melanocytes to keratinocytes resulting in melanin accumulation which may be leading to melanin induced cytotoxicity in melanocytes. Regulation of key processes involved in aetiopathogenesis of vitiligo along with TRP1 suggests that miRNAs act in an integrated manner which may be detrimental for the loss of melanocytes in vitiligo.