The Vacuolar ATPase (V-ATPase) is a proton pump responsible for controlling the intracellular and extracellular pH of cells. The structure of V-ATPase has been highly conserved among all eukaryotic cells and is involved in diverse functions across species. V-ATPase is best known for its acidification of endosomes and lysosomes and is also important for luminal acidification of specialized cells. Several reports have suggested the involvement of V-ATPase in maintaining an alkaline intracellular and acidic extracellular pH thereby aiding in proliferation and metastasis of cancer cells respectively. Increased expression of V-ATPase and relocation to the plasma membrane aids in cancer modulates key tumorigenic cell processes like autophagy, Warburg effect, immunomoduation, drug resistance and most importantly cancer cell signaling. In this review, we discuss the direct role of V-ATPase in acidification and indirect regulation of signaling pathways, particularly Notch Signaling.

Diagnostic parameters for visceral leishmaniasis (VL), a potentially fatal parasitic disease caused by Leishmania donovani, have been redefined in the last decade with the development of serological and molecular tests, though a definitive diagnosis still banks on the century-old parasitological methods in many areas. Recombinant antigens have improved performance of serodiagnostic methods. Serology-based tests, rk39 antigen dipstick, and direct agglutination test commonly employed in the field are highly sensitive methods, however, fail to distinguish past infections. Molecular approaches have become increasingly relevant due to remarkable sensitivity, specificity, and flexibility in choice of samples. Quantitative polymerase chain reaction is a highly sensitive and specific tool used in referral labs for detection/assessment of parasite load in VL patients and subsequently in monitoring treatment response to antileishmanial agents. The method displays potential to provide threshold for distinguishing asymptomatics in endemic areas. Currently, improvement in VL diagnostics is required for successful decentralized (point-of-care) testing in field conditions and to detect VL-HIV co-infection. Techniques such as loop-mediated isothermal amplification offer a reliable molecular diagnostic method for field application. The diagnosis based on bioanalytics/biosensors promise frontiers for point-of-care VL detection after adequate standardization. This review summarizes the recent developments in VL diagnostics, drawing attention towards the need for standardization of the diagnostics across the affected regions.

BackgroundWith widespread resistance to antimonials in Visceral Leishmaniasis (VL) in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy. Surveillance of MIL susceptibility in natural populations of Leishmania donovani is vital to preserve it and support the VL elimination program.Methodology and Principal FindingsWe measured in vitro susceptibility towards MIL and paromomycin (PMM) in L. donovani isolated from VL and PKDL, pre- and post-treatment cases, using an amastigote-macrophage model. MIL susceptibility of post-treatment isolates from cured VL cases (n = 13, mean IC50±SD = 2.43±1.44 µM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC50 = 4.72±1.99 µM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC50 = 1.86±0.75 µM). In PKDL, post-treatment isolates (n = 3, mean IC50 = 16.13±2.64 µM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC50 = 8.63±0.94 µM). Overall, PKDL isolates (n = 8, mean IC50 = 11.45±4.19 µM) exhibited significantly higher tolerance (p<0.0001) to MIL than VL isolates (n = 22, mean IC50 = 2.58±1.58 µM). Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes previously reported in parasites with experimentally induced MIL resistance were not present in the clinical isolates. Further, the mRNA expression profile of these genes was comparable in the pre- and post-treatment isolates. Parasite isolates from VL and PKDL cases were uniformly susceptible to PMM with respective mean IC50 = 7.05±2.24 µM and 6.18±1.51 µM.ConclusionThe in vitro susceptibility of VL isolates remained unchanged at the end of MIL treatment; however, isolates from relapsed VL and PKDL cases had lower susceptibility than the pre-treatment isolates. PKDL isolates were more tolerant towards MIL in comparison with VL isolates. All parasite isolates were uniformly susceptible to PMM. Mutations in the LdMT and LdRos3 genes as well as changes in the expression of these genes previously correlated with experimental resistance to MIL could not be verified for the field isolates.

Tumor associated vacuolar H+-ATPases (V-ATPases) are multi-subunit proton pumps that acidify tumor microenvironment, thereby promoting tumor invasion. Subunit ‘a’ of its V0 domain is the major pH sensing unit that additionally controls sub-cellular targeting of V-ATPase and exists in four different isoforms. Our study reports an elevated expression of the V-ATPase-V0a2 isoform in ovarian cancer(OVCA) tissues and cell lines(A2780, SKOV-3 and TOV-112D). Among all V0’a’ isoforms, V0a2 exhibited abundant expression on OVCA cell surface while normal ovarian epithelia did not. Sub-cellular distribution of V-ATPase-V0a2 confirmed its localization on plasma-membrane, where it was also co-associated with cortactin, an F-actin stabilizing protein at leading edges of cancer cells. Additionally, V0a2 was also localized in early and late endosomal compartments that are sites for modulations of several signaling pathways in cancer. Targeted inhibition of V-ATPase-V0a2 suppressed matrix metalloproteinase activity(MMP-9 & MMP-2) in OVCA cells. In conclusion, V-ATPase-V0a2 isoform is abundantly expressed on ovarian tumor cell surface in association with invasion assembly related proteins and plays critical role in tumor invasion by modulating the activity of matrix-degrading proteases. This study highlights for the first time, the importance of V-ATPase-V0a2 isoform as a distinct biomarker and possible therapeutic target for treatment of ovarian carcinoma.

Indian Leishmania donovani isolates (n ‫؍‬ 19) from regional zones representing various levels of antimony resistance displayed significantly (P < 0.01) correlated results with respect to in vitro susceptibility to the antileishmanial drugs sodium antimony gluconate, amphotericin B, and Miltefosine, raising the possibility of cross-resistance mechanisms operating in the field isolates. The results of gene expression analysis of LdMT and LdRos3 were suggestive of alternate mechanisms of Miltefosine susceptibility in the isolates.A high (Ͼ60%) proportion of non-antimony-responsive cases of Kala azar in India and the anthroponotic mode of transmission of the parasite causing the disease increase the chances of the generation and spreading of drug-resistant parasites (15, 17). The second-line antileishmanials amphotericin B (AmB) and Miltefosine (MIL) are highly effective for treatment of antimony-resistant patients but are of limited utility because of adverse reactions and high cost. A recent report of unresponsiveness to Ambisome in Sudanese patients of VL is worrisome and indicates the emergence of AmB-resistant parasites (9). Preliminary data from a phase IV trial with MIL suggested a doubling of the relapse rate, indicating lower drug efficacy than in phase II and III trials and providing a warning about the emergence of resistance (3,18,19).Earlier studies using isolates from responsive and nonresponsive patients indicated that resistance to antimonials is an intrinsic property of the parasite (4,8,15,16). Antimony resistance varies among zones representing differing levels of endemicity, emphasizing the acquired nature of resistance in the region (15). Sodium antimony gluconate (SAG)-resistant isolates exhibited cross-resistance to AmB and MIL, with HSP83 and a calpain-related protein being implicated in resistance by modulating drug-induced programmed cell death (21). Since the use of MIL for VL treatment has been introduced only recently, resistance has not yet been reported in the field; however, a wide range of 50% effective doses (ED 50 ) of MIL has been observed for parasite isolates from Nepal and Peru (23). The results of earlier studies revealed a role in MIL uptake and susceptibility for the LdMT-LdRos3-dependent flippase machinery at the plasma membrane (10-12). The present study was aimed at (i) evaluating the in vitro natural susceptibility of field isolates of Leishmania donovani to SAG, AmB, and MIL and (ii) correlating MIL susceptibility with the mRNA expression of LdMT and LdRos3 to explore their role in MIL resistance and potential as markers of MIL resistance in field isolates.The present study considered 19 L. donovani isolates from VL patients representing regional zones with various degrees of disease endemicity. In vitro susceptibility of parasites from SAG-treated patients (responsive and nonresponsive) and AmB-treated patients (all responded to treatment, and no clinical resistance was observed) was studied. Informed consent based on the guidelines of the Ethical Committee, Safdarjun...

In cancer cells, vacuolar ATPase (V-ATPase), a multi-subunit enzyme, is expressed on the plasma as well as vesicular membranes and critically influences metastatic behavior. The soluble, cleaved N-terminal domain of V-ATPase a2 isoform is associated with in vitro induction of tumorigenic characteristics in macrophages. This activity led us to further investigate its in vivo role in cancer progression by inhibition of a2 isoform (a2V) in tumor cells and the concomitant effect on tumor microenvironment in the mouse 4T-1 breast cancer model. Results showed that macrophages cocultivated with a2V knockdown (sh-a2) 4T-1 cells produce lower amounts of tumorigenic factors in vitro and have reduced ability to suppress T-cell activation and proliferation compared with control 4T-1 cells. Data analysis showed a delayed mammary tumor growth in Balb/c mice inoculated with sh-a2 4T-1 cells compared with control. The purified CD11b(+) macrophages from sh-a2 tumors showed a reduced expression of mannose receptor-1 (CD206), interleukin-10, transforming growth factor-β, arginase-1, matrix metalloproteinase and vascular endothelial growth factor. Flow cytometric analysis of tumor-infiltrated macrophages showed a significantly low number of F4/80(+)CD11c(+)CD206(+) macrophages in sh-a2 tumors compared with control. In sh-a2 tumors, most of the macrophages were F4/80(+)CD11c(+) (antitumor M1 macrophages) suggesting it to be the reason behind delayed tumor growth. Additionally, tumor-infiltrating macrophages from sh-a2 tumors showed a reduced expression of CD206 compared with control whereas CD11c expression was unaffected. These findings demonstrate that in the absence of a2V in tumor cells, the resident macrophage population in the tumor microenvironment is altered which affects in vivo tumor growth. We suggest that by involving the host immune system, tumor growth can be controlled through targeting of a2V on tumor cells.

Resistance to antimony is a major cause of failure to therapy in a large proportion of visceral leishmaniasis cases. Methods to distinguish resistant and sensitive parasite are urgently needed as the standard in vitro intracellular drug susceptibility assays are cumbersome and time consuming. Differential expression profiling studies have led to the identification of several antimony resistance-associated genes; however, their efficacy as a potential biomarker for monitoring antimony resistance remains imprecise. We analysed the expression of eight genes [antimony metabolism-associated genes - multidrug resistance protein A (MRPA), γ-glutamylcysteine synthetase (γ-GCS) and aquaporin-1 (AQP1) - and genes identified by proteome/transcriptome profiling—heat shock protein 83, mitogen-activated protein kinase 1 and histones H1, H2A and H4) in antimony-resistant (n=10) and antimony-sensitive (n=4) clinical isolates of Leishmania donovani by quantitative real-time PCR, in comparison with a lab-generated resistant and a standard sensitive isolate. We observed a significant differential expression of MRPA, histone H1 (p<0.01), γ-GCS, HSP83 (p<0.005) and histone H2A and H4 (p<0.0001) in a group of sodium antimony gluconate-resistant isolates compared to sensitive isolates. Preferential AQP1 expression was observed in all the sensitive isolates (p<0.0001). Overall, expression profile in field isolates for all the genes studied showed altered expression in majority of isolates, while in some, the expression was static. All the isolates showed a mosaic of expression pattern of the genes analysed indicating constellation of genes contributes towards the drug susceptibility of parasite. As none of the genes exhibit an absolute correlation with phenotype, targeted expression analysis of a set of genes should be considered as biomarker for distinguishing the antimony-resistant and antimony-sensitive parasite.

Macrophage polarization contributes to distinct human pathologies. In tumors, a polarized M2 phenotype called tumor-associated macrophages (TAMs) are associated with promotion of invasion and angiogenesis. In cancer cells, vacuolar ATPase (V-ATPase), a multi-subunit enzyme, is expressed on the plasma/vesicular membranes and critically influences the metastatic behavior. In addition, the soluble, cleaved N-terminal domain of a2 isoform of V-ATPase (a2NTD) is associated with in vitro induction of pro-tumorigenic properties in monocytes. This activity of a2 isoform of V-ATPase (a2V) caused us to investigate its role in cancer progression through the evaluation of the immunomodulatory properties of a2NTD. Here, we present direct evidence that surface expression of V-ATPase is associated with macrophage polarization in tumor tissue. Macrophages from BALB/c mice (peritoneal/bone marrow derived) were stimulated with recombinant a2NTD in both ex vivo and in vivo systems and evaluated for TAM characteristics. a2V was highly expressed in tumor tissues (breast and skin) as well as on the surface of tumor cell lines. The a2NTD-stimulated macrophages (a2MΦ) acquired TAM phenotype, which was characterized by elevated expression of mannose receptor-1, Arginase-1, interleukin-10 and transforming growth factor-β. a2MΦ also exhibited increased production of other tumorigenic factors including matrix metalloproteinase-9 and vascular endothelial growth factor. Further, a2MΦ were cocultured with mouse B-16F0 melanoma cells for their functional characterization. The coculture of these a2MΦ subsequently increased the invasion and angiogenesis of less invasive B-16F0 cells. When cocultured with naive T cells, a2MΦ significantly inhibited T-cell activation. The present data establish the role of V-ATPase in modulating a macrophage phenotype towards TAMs through the action of a2NTD, suggesting it to be a potential therapeutic target in cancer.