Antimony resistance and gene expression in Leishmania: spotlight on molecular and proteomic aspects.

Leishmaniasis is a vector-borne parasitic disease caused by Leishmania parasites with a spectrum of clinical manifestations, ranging from skin lesions to severe visceral complications. Treatment of this infection has been extremely challenging with the concurrent emergence of drug resistance. The differential gene expression and the discrepancies in protein functions contribute to the appearance of 2 distinct phenotypes: resistant and sensitive, but the current diagnostic tools fail to differentiate between them. The identification of gene expression patterns and molecular mechanisms coupled with antimony (Sb) resistance can be leveraged to prompt diagnosis and select the most effective treatment methods. The present study attempts to use comparative expression of Sb resistance-associated genes in resistant and sensitive Leishmania, to disclose their relative abundance in clinical or in vitro selected isolates to gain an understanding of the molecular mechanisms of Sb response/resistance. Data suggest that the analysis of resistance gene expression would verify the Sb resistance or susceptibility only to a certain extent; however, none of the individual expression patterns of the studied genes was diagnostic as a biomarker of Sb response of Leishmania. The findings highlighted will be useful in bridging the knowledge gap and discovering innovative diagnostic tools and novel therapeutic targets.

Prevention of nodules and enhancement of antibody response to genetically engineered recombinant vaccine against Human Chorionic Gonadotropin (hCG) for contraception.

Objective: Human Chorionic Gonadotropin (hCG) plays a crucial role in embryo implantation and in maintenance of pregnancy. An immuno-contraceptive approach involves the use of a recombinant hCGß-LTB vaccine formulated with adjuvant Mycobacterium indicus pranii (MIP), to prevent pregnancy without disturbing ovulation, hormonal profiles, and menstrual cycles in women. The present work in mice was designed to address issues encountered in clinical trials conducted with hCGß-LTB vaccine, with focus on two primary concerns. Firstly, it aimed to determine the optimal vaccine dosage required to induce a high level of anti-hCG antibodies. Secondly, it aimed to assess the safety profile of the vaccine, specifically injection site reactions in the form of nodules, observed in some of the subjects.Methods and Results: Studies undertaken indicate that a 2 µg dose of the protein version of the vaccine, administered in mice through the intramuscular route, can induce high anti-hCG titres. Furthermore, administering a booster dose enhances the antibody response. Our findings suggest that the concentration and frequency of administration of the adjuvant MIP can also be reduced without compromising vaccine efficacy.Conclusion: The issue of nodule formation at the injection site can be mitigated either by administering the vaccine along with MIP intramuscularly or injecting hCG vaccine and MIP at separate intradermal sites. Thus, protein vaccine administered at a 2µg dose via the intramuscular route addresses both efficacy and safety concerns.

The Phase I/II clinical trials initiated with the recombinant hCG vaccine in women revealed inadequate antibody titres in all subjects, alongside the development of nodules at the injection sites in some participants. Studies were undertaken in mice to propose potential strategies for mitigating injection site reactions and enhancing the antibody response. It was concluded that the optimum dose of the protein version of the vaccine to get high antibody titres, is 2 µg administered intramuscularly while upholding safety standards.

NDK/NME proteins: a host-pathogen interface perspective towards therapeutics.

No effective vaccine is available for any parasitic disease. The treatment to those is solely dependent on chemotherapy, which is always threatened due to development of drug resistance in bugs. This warrants identification of new drug targets. Here, we discuss Nucleoside diphosphate kinases (NDKs) of pathogens that alter host's intra and extracellular environment, as novel drug targets to simultaneously tackle multiple pathogens. NDKs having diverse functions, are highly conserved among prokaryotes and eukaryotes (the mammal NDKs are called NMEs [non-metastatic enzymes]). However, NDKs and NMEs have been separately analysed in the past for their structure and functions. The role of NDKs of pathogen in modulation of inflammation, phagocytosis, apoptosis, and ROS generation in host is known. Conversely, its combined contribution in host-pathogen interaction has not been studied yet. Through the sequence and domain analysis, we found that NDKs can be classified in two groups. One group comprised NMEs 1-4 and few NDKs of select essential protozoan parasites and the bacterium Mycobacterium tuberculosis. The other group included NME7 and the other NDKs of those parasites, posing challenges in the development of drugs specifically targeting pathogen NDKs, without affecting NME7. However, common drugs targeting group 2 NDKs of pathogens can be designed, as NME7 of group 2 is expressed only in ciliated host cells. This review thus analyses comparatively for the first time the structures and functions of human NMEs and pathogen NDKs and predicts the possibilities of NDKs as drug targets. In addition, pathogen NDKs have been now provided a nomenclature in alignment with the NMEs of humans.

Genome-wide analysis reveals allelic variation and chromosome copy number variation in paromomycin-resistant Leishmania donovani.

In the absence of adequate diagnosis and treatment, leishmaniasis remains a major public health concern on a global scale. Drug resistance remains a key obstacle in controlling and eliminating visceral leishmaniasis. The therapeutic gap due to lack of target-specific medicine and vaccine can be minimized by obtaining parasite's genomic information. This study compared whole-genome sequence of paromomycin-resistant parasite (K133PMM) developed through in vitro adaptation and selection with sensitive Leishmania clinical isolate (K133WT). We found a large number of upstream and intergenic gene variations in K133PMM. There were 259 single nucleotide polymorphisms (SNPs), 187 insertion-deletion (InDels), and 546 copy number variations (CNVs) identified. Most of the genomic variations were found in the gene's upstream and non-coding regions. Ploidy estimation revealed chromosome 5 in tetrasomy and 6, 9, and 12 in trisomy, uniquely in K133PMM. These contain the genes for protein degradation, parasite motility, autophagy, cell cycle maintenance, and drug efflux membrane transporters. Furthermore, we also observed reduction in ploidy of chromosomes 15, 20, and 23, in the resistant parasite containing mostly the genes for hypothetical proteins and membrane transporters. We chronicled correlated genomic conversion and aneuploidy in parasites and hypothesize that this led to rapid evolutionary changes in response to drug induced pressure, which causes them to become resistant.

A novel signal sequence negative multimeric glycosomal protein required for cell cycle progression of Leishmania donovani parasites.

Expression of the intracellular form amastigote specific genes in the Leishmania donovani parasite plays a major role in parasite replication in the macrophage. In the current work, we have characterized a novel hypothetical gene, Ld30b that is specifically transcribed in the intracellular stage of the parasite. The recombinant Ld30b protein exists as a pentamer in solution as identified by native-PAGE and size exclusion gel chromatography. Structural analysis using circular dichroism and molecular modeling indicate that Ld30b belongs to family of cAMP-dependent protein kinase type I-alpha regulatory subunit. Co-localization immunofluorescence microscopy and western blot analyses (using anti-Ld30b antibody and anti-hypoxanthine-guanine phosphoribosyl transferase, a glycosome marker) on the isolated parasite glycosome organelle fractions show that Ld30b is localized in glycosome, though lacked a glycosome targeting PTS1/2 signal in the protein sequence. Episomal expression of Ld30b in the parasite caused the arrest of promastigotes and amastigotes growth in vitro. Cell cycle analysis using flow cytometry indicates that these parasites are arrested in 'sub G0/G1' phase of the cell cycle. Single allele knockout of Ld30b in the parasite similarly attenuated its growth by accumulation of cells in the S phase of cell cycle, thus confirming the probable importance of appropriate level of protein in the cells. Studying such intracellular stage expressing genes might unravel novel regulatory pathways for the development of drugs or vaccine candidates against leishmaniasis.

A Leishmania-specific gene upregulated at the amastigote stage is crucial for parasite survival.

The morphological and biochemical alterations between the two life stages of Leishmania are governed by stage-regulated expression of several genes. Amastigote-specific genes are believed to have a role in the survival and replication of the parasite in the hostile environment of the mammalian host. Previously, we have reported the upregulated expression of A1 gene (LdA1) at the amastigote stage at RNA level. In the present study, we have further characterized LdA1, in order to understand its role in Leishmania. Sequence homology search revealed that LdA1 is unique to the Leishmania genus. Sequence- and structure-level functional annotations predicted the involvement of LdA1 in a range of biological processes critical for survival of the parasites. Western blot confirmed the upregulated expression of LdA1 at the protein level at the amastigote stage. Overexpression of LdA1 in Leishmania did not affect its growth, phenotype, or infectivity. Attempts to generate null mutants of LdA1 by homologous recombination were not successful. Repeated inability to create null mutants of LdA1 was suggestive of gene essentiality. Mutant parasites with a single allele deletion of LdA1 (LdA1+/-) showed reduction in motility, size, and growth rate at both the life stages in vitro, which was restored following gene add-back by episomal expression of LdA1 in mutant parasites. Although LdA1+/- parasites were able to infect macrophages ex vivo, their capacity to survive inside macrophages was reduced significantly (P < 0.01) beyond 72 h of infection. In conclusion, LdA1 is a Leishmania-specific gene having upregulated expression at the amastigote stage and is important for survival of Leishmania parasite.

Leishmania donovani-specific Ub-related modifier-1: an early endosome-associated ubiquitin-like conjugation in Leishmania donovani.

Protein modification by ubiquitin (Ub) and Ub-like molecules (Ubls) is a diverse biological process that regulates the activity of the target proteins. Systematic studies of Ubls in trypanosomatids like Leishmania, the causative organism of potentially fatal visceral leishmaniasis, would yield a better understanding of the disease pathogenesis and identify novel therapeutic targets. The present study is the first to characterize Leishmania donovani-specific Ub-related modifier-1 (LdUrm1) and the associated conjugation pathway. Based on homology modeling, LdUrm1 was found to possess a ß-grasp fold and a C-terminal di-glycine motif unique to Ub/Ubls, essential for its conjugation to the target proteins. We identified LdUba4 as the E1 enzyme for LdUrm1 and demonstrated its energy-dependent enzymatic activity. LdUrm1 was immunolocalized anteriorly near the flagellar reservoir, while LdUba4 was cytoplasmic, both in promastigotes and axenic amastigotes. Expression of nonconjugatable LdUrm1 in L. donovani resulted in depleted parasite growth suggesting its role in the pathogenesis. By mass spectrometry, we identified Rab5, a known mediator of early endosome regulated hemoglobin endocytosis in Leishmania, as a target of LdUrm1. Our data suggest that LdUrm1 conjugation pathway may have a role in early endosome-mediated heme uptake in Leishmania that may be explored as a drug target.

Genetic diversity of Leishmania donovani that causes cutaneous leishmaniasis in Sri Lanka: a cross sectional study with regional comparisons.

BACKGROUND: Leishmania donovani is the etiological agent of visceral leishmaniasis (VL) in the Indian subcontinent. However, it is also known to cause cutaneous leishmaniasis (CL) in Sri Lanka. Sri Lankan L. donovani differs from other L. donovani strains, both at the molecular and biochemical level. To investigate the different species or strain-specific differences of L. donovani in Sri Lanka we evaluated sequence variation of the kinetoplastid DNA (kDNA). METHODS: Parasites isolated from skin lesions of 34 CL patients and bone marrow aspirates from 4 VL patients were genotyped using the kDNA minicircle PCR analysis. A total of 301 minicircle sequences that included sequences from Sri Lanka, India, Nepal and six reference species of Leishmania were analyzed. RESULTS: Haplotype diversity of Sri Lankan isolates were high (H d = 0.757) with strong inter-geographical genetic differentiation (F ST > 0.25). In this study, L. donovani isolates clustered according to their geographic origin, while Sri Lankan isolates formed a separate cluster and were clearly distinct from other Leishmania species. Within the Sri Lankan group, there were three distinct sub-clusters formed, from CL patients who responded to standard antimony therapy, CL patients who responded poorly to antimony therapy and from VL patients. There was no specific clustering of sequences based on geographical origin within Sri Lanka. CONCLUSION: This study reveals high levels of haplotype diversity of L. donovani in Sri Lanka with a distinct genetic association with clinically relevant phenotypic characteristics. The use of genetic tools to identify clinically relevant features of Leishmania parasites has important therapeutic implications for leishmaniasis.

Characterization of cross-protection by genetically modified live-attenuated Leishmania donovani parasites against Leishmania mexicana.

Previously, we showed that genetically modified live-attenuated Leishmania donovani parasite cell lines (LdCen(-/-) and Ldp27(-/-)) induce a strong cellular immunity and provide protection against visceral leishmaniasis in mice. In this study, we explored the mechanism of cross-protection against cutaneous lesion-causing Leishmania mexicana. Upon challenge with wild-type L. mexicana, mice immunized either for short or long periods showed significant protection. Immunohistochemical analysis of ears from immunized/challenged mice exhibited significant influx of macrophages, as well as cells expressing MHC class II and inducible NO synthase, suggesting an induction of potent host-protective proinflammatory responses. In contrast, substantial inhibition of IL-10, IL-4, and IL-13 expression and the absence of degranulated mast cells and less influx of eosinophils within the ears of immunized/challenged mice suggested a controlled anti-inflammatory response. L. mexicana Ag-stimulated lymph node cell culture from the immunized/challenged mice revealed induction of IFN-γ secretion by the CD4 and CD8 T cells compared with non-immunized/challenged mice. We also observed suppression of Th2 cytokines in the culture supernatants of immunized/challenged lymph nodes compared with non-immunized/challenged mice. Adoptively transferred total T cells from immunized mice conferred strong protection in recipient mice against L. mexicana infection, suggesting that attenuated L. donovani can provide protection against heterologous L. mexicana parasites by induction of a strong T cell response. Furthermore, bone marrow-derived dendritic cells infected with LdCen(-/-) and Ldp27(-/-) parasites were capable of inducing a strong proinflammatory response leading to the proliferation of Th1 cells. These studies demonstrate the potential of live-attenuated L. donovani parasites as pan-Leishmania species vaccines.

Genetically Modified Live Attenuated Leishmania donovani Parasites Induce Innate Immunity through Classical Activation of Macrophages That Direct the Th1 Response in Mice.

Visceral leishmaniasis (VL) causes significant mortality and there is no effective vaccine. Previously, we have shown that genetically modified Leishmania donovani parasites, here described as live attenuated parasites, induce a host protective adaptive immune response in various animal models. In this study, we demonstrate an innate immune response upon infection with live attenuated parasites in macrophages from BALB/c mice both in vitro and in vivo. In vitro infection of macrophages with live attenuated parasites (compared to that with wild-type [WT] L. donovani parasites) induced significantly higher production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-12 [IL-12], gamma interferon [IFN-γ], and IL-6), chemokines (monocyte chemoattractant protein 1/CCL-2, macrophage inflammatory protein 1α/CCL-3, and IP-10), reactive oxygen species (ROS), and nitric oxide, while concomitantly reducing anti-inflammatory cytokine IL-10 and arginase-1 activities, suggesting a dominant classically activated/M1 macrophage response. The classically activated response in turn helps in presenting antigen to T cells, as observed with robust CD4(+) T cell activation in vitro. Similarly, parasitized splenic macrophages from live attenuated parasite-infected mice also demonstrated induction of an M1 macrophage phenotype, indicated by upregulation of IL-1ß, TNF-α, IL-12, and inducible nitric oxide synthase 2 and downregulation of genes associated with the M2 phenotype, i.e., the IL-10, YM1, Arg-1, and MRC-1 genes, compared to WT L. donovani-infected mice. Furthermore, an ex vivo antigen presentation assay showed macrophages from live attenuated parasite-infected mice induced higher IFN-γ and IL-2 but significantly less IL-10 production by ovalbumin-specific CD4(+) T cells, resulting in proliferation of Th1 cells. These data suggest that infection with live attenuated parasites promotes a state of classical activation (M1 dominant) in macrophages that leads to the generation of protective Th1 responses in BALB/c mice.

Live attenuated Leishmania donovani p27 gene knockout parasites are nonpathogenic and elicit long-term protective immunity in BALB/c mice.

Leishmaniasis causes significant morbidity and mortality worldwide, and no vaccines against this disease are available. Previously, we had shown that the amastigote-specific protein p27 (Ldp27) is a component of an active cytochrome c oxidase complex in Leishmania donovani and that upon deletion of its gene the parasite had reduced virulence in vivo. In this study, we have shown that Ldp27(-/-) parasites do not survive beyond 20 wk in BALB/c mice and hence are safe as an immunogen. Upon virulent challenge, mice 12 wk postimmunization showed significantly lower parasite burden in the liver and spleen. When mice were challenged 20 wk postimmunization, a significant reduction in parasite burden was still noted, suggesting long-term protection by Ldp27(-/-) immunization. Immunization with Ldp27(-/-) induced both pro- and anti-inflammatory cytokine responses and activated splenocytes for enhanced leishmanicidal activity in association with NO production. Protection in both short- and long-term immunized mice after challenge with the wild-type parasite correlated with the stimulation of multifunctional Th1-type CD4 and CD8 T cells. Adoptive transfer of T cells from long-term immunized mice conferred protection against virulent challenge in naive recipient mice, suggesting involvement of memory T cell response in protection against Leishmania infection. Immunization of mice with Ldp27(-/-)also demonstrated cross-protection against Leishmania major and Leishmania braziliensis infection. Our data show that genetically modified live attenuated Ldp27(-/-) parasites are safe, induce protective immunity even in the absence of parasites, and can provide protection against homologous and heterologous Leishmania species.

Implications of co-infection of Leptomonas in visceral leishmaniasis in India.

Protozoan parasites Leishmania donovani (family: Trypanosomatidae) cause fatal visceral leishmaniasis (VL) and the infection relapses in apparently cured population as post kala-azar dermal leishmaniasis (PKDL) in the Indian subcontinent. In recent years co-infection of another Trypanosomatid parasite Leptomonas with L. donovani during VL/PKDL in this region has become prominent. The observation of clinically lesser-known insect parasite, Leptomonas in leishmaniasis is intriguing to researchers. The presence of Leishmania look alike Leptomonas in the cultures of clinical isolates of Leishmania has been worrisome to those, who prefer to work with pure Leishmania cultures for drug and vaccine development or immune response studies. The exact implications of such a co-habitation, which might lead to a delay in the diagnostics of VL and elevate mortality, need a thorough investigation. Also whether Leptomonas is involved in leishmaniasis manifestation needs to be ascertained. Thus we are currently witnessing a new paradigm of a parasitic co-infection in VL/PKDL cases in India and this review outlines various opportunities for further research in understanding such emerging co-infection.

Global Remodeling of Host Proteome in Response to Leishmania Infection.

The protozoan parasite Leishmania possesses an intrinsic ability to modulate a multitude of pathways in the host, toward aiding its own proliferation. In response, the host reprograms its cellular, immunological, and metabolic machinery to evade the parasite's lethal impact. Besides inducing various antioxidant signaling pathways to counter the elevated stress response proteins like heme oxygenase-1 (HO-1), Leishmania also attempts to delay host cell apoptosis by promoting anti-apoptotic proteins like Bcl-2. The downstream modulation of apoptotic proteins is regulated by effector pathways, including the PI3K/Akt survival pathway, the mitogen-activated protein kinases (MAPKs) signaling pathway, and STAT phosphorylation. In addition, Leishmania assists in its infection in a time-dependent manner by modulating the level of various proteins of autophagic machinery. Immune effector cells, such as mast cells and neutrophils, entrap and kill the pathogen by secreting various granular proteins. In contrast, the host macrophages exert their leishmanicidal effect by secreting various cytokines, such as IL-2, IL-12, etc. An interplay of various signaling pathways occurs in an organized network that is highly specific to both pathogen and host species. This Review analyzes the modulation of expression of proteins, including the cytokines, providing a realistic approach toward understanding the pathophysiology of disease and predicting some prominent markers for disease intervention and vaccine support strategies.

Biophysical Evidence for the Amyloid Formation of a Recombinant Rab2 Isoform of Leishmania donovani.

BACKGROUND: The most fatal form of Visceral leishmaniasis or kala-azar is caused by the intracellular protozoan parasite Leishmania donovani. The life cycle and the infection pathway of the parasite are regulated by the small GTPase family of Rab proteins. The involvement of Rab proteins in neurodegenerative amyloidosis is implicated in protein misfolding, secretion abnormalities and dysregulation. The inter and intra-cellular shuttlings of Rab proteins are proposed to be aggregation-prone. However, the biophysical unfolding and aggregation of protozoan Rab proteins is limited. Understanding the aggregation mechanisms of Rab protein will determine their physical impact on the disease pathogenesis and individual health. OBJECTIVE: This work investigates the acidic pH-induced unfolding and aggregation of a recombinant Rab2 protein from L. donovani (rLdRab2) using multi-spectroscopic probes. METHODS: The acidic unfolding of rLdRab2 induced at acidic pH is characterised by intrinsic fluorescence and ANS assay, while aggregation is determined by Thioflavin-T and 90° light scattering assay. Circular dichroism determined the secondary structure of monomers and aggregates. The aggregate morphology was imaged by transmission electron microscopy. RESULTS: rLdRab2 was modelled to be a Rab2 isoform with loose globular packing. The acidinduced unfolding of the protein is a plausible non-two-state process. At pH 2.0, a partially folded intermediate (PFI) state characterised by ~ 30 % structural loss and exposed hydrophobic core was found to accumulate. The PFI state slowly converted into well-developed protofibrils at high protein concentrations demonstrating its amyloidogenic nature. The native state of the protein was also observed to be aggregation-prone at high protein concentrations. However, it formed amorphous aggregation instead of fibrils. CONCLUSION: To our knowledge, this is the first study to report in vitro amyloid-like behaviour of Rab proteins in L donovani. This study provides a novel opportunity to understand the complete biophysical characteristics of Rab2 protein of the lower eukaryote, L. donovani.

Plumbagin accelerates serum albumin's amyloid aggregation kinetics and generates fibril polymorphism by inducing non-native ß-sheet structures.

The ligand-induced conformational switch of proteins has great significance in understanding the biophysics and biochemistry of their self-assembly. In this work, we have investigated the ability of plumbagin (PL), a hydroxynaphthoquinone compound found in the root of the medicinal plant Plumbago zeylanica, to modulate aggregation precursor state, aggregation kinetics and generate distinct fibril of human serum albumin (HSA). PL was found to moderately bind (binding constant Ka âˆ¼ 10-4 M-1)) to domain-II of HSA in the stoichiometric ratio of 1:1. We found that PL-HSA complex aggregation was accelerated as compared to that of HSA aggregation and it may be through an independent pathway. We also detected that fibril produced in the presence of PL is wider in diameter, contains a higher amount of ß-sheet (∼18%) and disordered (∼46%) structures, and is less stable. We concluded that the acceleration of aggregation reaction and generation of fibril polymorphism was mainly because of the higher extent of unfolding and high content of non-native ß-sheet structure in the aggregation precursor state of PL-HSA complex. This study offers opportunities to explore the ability of ligand binding to modulate aggregation reactions and generate polymorphic protein fibrils.

QbD-Assisted Development and Optimization of Doxycycline Hyclate- and Hydroxyapatite-Loaded Nanoparticles for Periodontal Delivery.

The current research aims to develop a carrier system for the delivery of a matrix metalloproteinase (MMP) inhibitor along with a bioceramic agent to the periodontal pocket. It is proposed that the present system, if given along with a systemic antibiotic, would be a fruitful approach for periodontitis amelioration. To fulfill the aforementioned objective, a doxycycline hyclate- and hydroxyapatite-adsorbed composite was prepared by a physical adsorption method and successfully loaded inside sodium alginate-chitosan nanoparticles and optimized based on particle size and drug content. Optimized formulation was then subjected to different evaluation parameters like encapsulation efficiency, hydroxyapatite content, ζ potential, surface morphology, in vitro drug release, cell line studies, and stability studies. For the optimized formulation, particle size, polydispersity index (PDI), entrapment efficiency, ζ potential, and drug content were found to be 336.50 nm, 0.23, 41.77%, -13.85 mV, and 14.00%, respectively. The surface morphology of the placebo and adsorbed composite-loaded nanoparticles as observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed the spherical shape and rough surface of the particles. In gingival crevicular fluid (GCF) 7.6, a sustained drug release profile was obtained up to 36 h. In vitro % viability studies performed on murine fibroblast cells (NIH3T3) and human periodontal ligament (hPDL) cell lines confirmed the proliferative nature of the formulation. Also, when subjected to stability studies for 4 weeks, particle size, PDI, and drug content did not vary considerably, thereby ensuring the stable nature of nanoparticles. Henceforth, sodium alginate-chitosan nanoparticles appeared to be a good carrier system for doxycycline hyclate and hydroxyapatite for periodontal therapy. If given along with a system antibiotic, the system will serve as a fruitful tool for infection-mediated periodontal regeneration and healing.

Manufacturing and preclinical toxicity of GLP grade gene deleted attenuated Leishmania donovani parasite vaccine.

Centrin1 gene deleted Leishmania donovani parasite (LdCen1-/-) was developed and extensively tested experimentally as an intracellular stage-specific attenuated and immunoprotective live parasite vaccine candidate ex vivo using human PBMCs and in vivo in animals. Here we report manufacturing and pre-clinical evaluation of current Good-Laboratory Practice (cGLP) grade LdCen1-/- parasites, as a prerequisite before proceeding with clinical trials. We screened three batches of LdCen1-/- parasites manufactured in bioreactors under cGLP conditions, for their consistency in genetic stability, attenuation, and safety. One such batch was preclinically tested using human PBMCs and animals (hamsters and dogs) for its safety and protective immunogenicity. The immunogenicity of the CGLP grade LdCen1-/- parasites was similar to one grown under laboratory conditions. The cGLP grade LdCen1-/- parasites were found to be safe and non-toxic in hamsters and dogs even at 3 times the anticipated vaccine dose. When PBMCs from healed visceral leishmaniasis (VL) cases were infected with cGLP LdCen1-/-, there was a significant increase in the stimulation of cytokines that contribute to protective responses against VL. This effect, measured by multiplex ELISA, was greater than that observed in PBMCs from healthy individuals. These results suggest that cGLP grade LdCen1-/- manufactured under cGMP complaint conditions can be suitable for future clinical trials.

Zooming in on common immune evasion mechanisms of pathogens in phagolysosomes: potential broad-spectrum therapeutic targets against infectious diseases.

The intracellular viral, bacterial, or parasitic pathogens evade the host immune challenges to propagate and cause fatal diseases. The microbes overpower host immunity at various levels including during entry into host cells, phagosome formation, phagosome maturation, phagosome-lysosome fusion forming phagolysosomes, acidification of phagolysosomes, and at times after escape into the cytosol. Phagolysosome is the final organelle in the phagocyte with sophisticated mechanisms to degrade the pathogens. The immune evasion strategies by the pathogens include the arrest of host cell apoptosis, decrease in reactive oxygen species, the elevation of Th2 anti-inflammatory response, avoidance of autophagy and antigen cross-presentation pathways, and escape from phagolysosomal killing. Since the phagolysosome organelle in relation to infection/cure is seldom discussed in the literature, we summarize here the common host as well as pathogen targets manipulated or utilized by the pathogens established in phagosomes and phagolysosomes, to hijack the host immune system for their benefit. These common molecules or pathways can be broad-spectrum therapeutic targets for drug development for intervention against infectious diseases caused by different intracellular pathogens.

Identification of protein biomarkers of attenuation and immunogenicity of centrin or p27 gene deleted live vaccine candidates of Leishmania against visceral leishmaniasis.

Currently, no licensed vaccine is available for human visceral leishmaniasis (VL), a fatal disease caused by the protozoan parasite Leishmania donovani. Two of our live attenuated L. donovani vaccine candidates, either deleted for Centrin1 (LdCen1-/-) or p27 gene (Ldp27-/-), that display reduced growth in macrophages were studied to be safe, immunogenic and protective against VL in various animal models. This report involves the identification of differentially expressed proteins, their related pathways and its underlying mechanism in the intracellular stage of these parasites, using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) methods. Out of 50-60 proteins, found to be differentially expressed in these mutant parasites, 36 were found to be common in both the parasites. Such proteins mainly belong to the functional categories viz. metabolic enzymes, chaperones and stress proteins, proteins involved in translation, processing and transport and proteins involved in nucleic acid processing. Proteins known to be host protective, like Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cytochrome c, calreticulin and those responsible for inducing immune response, namely tubulins, DEAD box RNA helicases, HSP70 and tryparedoxin, have been detected to be modulated in these parasites. Such proteins could be predicted as biomarkers, with further scope of study for their role in growth attenuation. SIGNIFICANCE: This study aims at predicting proteomic biomarkers of Leishmania parasite growth attenuation, that have immunomodulatory role in the disease leishmaniasis. Advanced studies could be helpful in establishing the role of these identified proteins in parasitic virulence and to predict the host interaction at molecular level. Also, these proteins could be exploited as attenuation markers during the development of genetically modified live attenuated parasites as vaccine candidates. These could be cross validated in varied species of Leishmania and other tyrpanosomatids for similar response towards identifying them as universal biomarkers of attenuation.

Interaction of novel proteins, centrin4 and protein of centriole in Leishmania parasite and their effects on the parasite growth.

Centrins are cytoskeletal proteins associated with the centrosomes or basal bodies in the eukaryotes. We previously reported the involvement of Centrin 1-3 proteins in cell division in the protozoan parasites Leishmania donovani and Trypanosoma brucei. Centrin4 and 5, unique to such parasites, had never been characterized in Leishmania parasite. In the current study, we addressed the function of centrin4 (LdCen4) in Leishmania. By dominant-negative study, the episomal expression of C-terminal truncated LdCen4 in the parasite reduced the parasite growth. LdCen4 double allele gene deletion by either homologous recombination or CRISPR-Cas9 was not successful in L. donovani. However, CRISPR-Cas9-based deletion of the homologous gene was possible in L. mexicana, which attenuated the parasite growth in vitro, but not ex vivo in the macrophages. LdCen4 also interacts with endogenous and overexpressed LdPOC protein, a homolog of centrin reacting human POC (protein of centriole) in a calcium sensitive manner. LdCen4 and LdPOC binding has also been confirmed through in silico analysis by protein structural docking and validated by co-immunoprecipitation. By immunofluorescence studies, we found that both the proteins share a common localization at the basal bodies. Thus, for the first time, this article describes novel centrin4 and its binding protein in the protozoan parasites.