Ashok K Gupta

Heat Shock Protein 90 as a Drug Target against Protozoan Infections BIOCHEMICAL CHARACTERIZATION OF HSP90 FROM PLASMODIUM FALCIPARUM AND TRYPANOSOMA EVANSI AND EVALUATION OF ITS INHIBITOR AS A CANDIDATE DRUG

Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s; its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90 ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90 multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases.

Proteomics of Trypanosoma evansi Infection in Rodents

Background Trypanosoma evansi infections, commonly called ‘surra’, cause significant economic losses to livestock industry. While this infection is mainly restricted to large animals such as camels, donkeys and equines, recent reports indicate their ability to infect humans. There are no World Animal Health Organization (WAHO) prescribed diagnostic tests or vaccines available against this disease and the available drugs show significant toxicity. There is an urgent need to develop improved methods of diagnosis and control measures for this disease. Unlike its related human parasites T. brucei and T. cruzi whose genomes have been fully sequenced T. evansi genome sequence remains unavailable and very little efforts are being made to develop improved methods of prevention, diagnosis and treatment. With a view to identify potential diagnostic markers and drug targets we have studied the clinical proteome of T. evansi infection using mass spectrometry (MS). Methodology/Principal Findings Using shot-gun proteomic approach involving nano-lc Quadrupole Time Of Flight (QTOF) mass spectrometry we have identified over 160 proteins expressed by T. evansi in mice infected with camel isolate. Homology driven searches for protein identification from MS/MS data led to most of the matches arising from related Trypanosoma species. Proteins identified belonged to various functional categories including metabolic enzymes; DNA metabolism; transcription; translation as well as cell-cell communication and signal transduction. TCA cycle enzymes were strikingly missing, possibly suggesting their low abundances. The clinical proteome revealed the presence of known and potential drug targets such as oligopeptidases, kinases, cysteine proteases and more. Conclusions/Significance Previous proteomic studies on Trypanosomal infections, including human parasites T. brucei and T. cruzi, have been carried out from lab grown cultures. For T. evansi infection this is indeed the first ever proteomic study reported thus far. In addition to providing a glimpse into the biology of this neglected disease, our study is the first step towards identification of diagnostic biomarkers, novel drug targets as well as potential vaccine candidates to fight against T. evansi infections.

Genetic diversity and bottleneck studies in the Marwari horse breed

Genetic diversity within the Marwari breed of horses was evaluated using 26 different microsatellite pairs with 48 DNA samples from unrelated horses. This molecular characterisation was undertaken to evaluate the problem of genetic bottlenecks also, if any, in this breed. The estimated mean (± s.e.) allelic diversity was 5.9 (± 2.24), with a total of 133 alleles. A high level of genetic variability within this breed was observed in terms of high values of mean (±s.e.) effective number of alleles (3.3 ± 1.27), observed heterozygosity (0.5306 ± 0.22), expected Levene’s heterozygosity (0.6612 ± 0.15), expected Nei’s heterozygosity (0.6535 ± 0.14), and polymorphism information content (0.6120 ± 0.03). Low values of Wright’s fixation index, FIS (0.2433 ± 0.05) indicated low levels of inbreeding. This basic study indicated the existence of substantial genetic diversity in the Marwari horse population. No significant genotypic linkage disequilibrium was detected across the population, suggesting no evidence of linkage between loci. A normal ‘L’ shaped distribution of mode-shift test, non-significant heterozygote excess on the basis of different models, as revealed from Sign, Standardized differences and Wilcoxon sign rank tests as well as non-significantM ratio value suggested that there was no recent bottleneck in the existing Marwari breed population, which is important information for equine breeders. This study also revealed that the Marwari breed can be differentiated from some other exotic breeds of horses on the basis of three microsatellite primers.

Development of EMA-2 recombinant antigen based enzyme-linked immunosorbent assay for seroprevalence studies of Theileria equi infection in Indian equine population

Equine piroplasmosis is a tick-transmitted protozoan disease caused by Theileria equi and/or Babesia caballi. In the present study, we expressed a 53kDa protein from the truncated EMA-2 gene of T. equi (Indian strain) and developed EMA-2ELISA using this expressed protein. This ELISA is able to detect T. equi-specific antibodies in experimentally infected animals as early as 9 days post-infection. The assay developed was validated with the OIE recommended competitive ELISA (cELISA) on 120 serum samples and significant agreement (kappa=0.93) was observed between results of both the ELISAs which indicates suitability of EMA-2ELISA for use in sero-diagnosis. Diagnostic sensitivity and specificity of EMA-2ELISA - as compared with cELISA - were 0.97 and 0.96, respectively. Analysis of 5651 equine serum samples - collected during 2007-2012 from 12 states of India representing eight agro-climatic zones - by EMA-2ELISA revealed 32.65% seroprevalence of T. equi in India. In conclusion, the EMA-2ELISA developed using the T. equi EMA-2 recombinant protein as antigen for detecting T. equi-specific antibodies has good diagnostic potential for sero-epidemiological surveys.

Genetic characterization of Indian Spiti horses

India has a rich biodiversity of equines in the form of six distinct indigenous horse (Equus caballus) breeds, namely Kathiawari, Marwari, Spiti, Zanskari, Bhutia and Manipuri, in addition to indigenous donkeys and wild asses (Yadav et al. 2001). These horse breeds are well adapted to different agroclimatic regions and possess certain unique characteristics. Spiti horses are slow moving, sure-footed, and are found mainly in the hilly areas of the Himalayan range, mostly in the districts of Lahaul-Spiti and Kinnaur in the state of Himachal Pradesh, India. Spiti horses not only thrive under such harsh conditions but are also used as pack animals. Unfortunately, the population numbers of this breed are decreasing at an alarming rate and it is thought to be close to extinction. Consequently, there is an urgent need to characterize this valuable breed to set priorities for its conservation. Molecular characterization is an essential prerequisite to developing an effective and meaningful conservation programme for Spiti horses. Several DNA-based technologies have been developed in the last decade to type polymorphic loci, including random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), variable number of tandem repeats (VNTRs), single-strand conformational polymorphism (SSCP), and methods that make use of polymorphisms of short tandem repeats called microsatellites. Among the array of molecular markers, microsatellites are considered especially suitable for biodiversity evaluation, owing to their codominant inheritance, high heterozygosity, ease and reliability of scoring, ubiquitous presence throughout the genome, and high degree of polymorphism (Takezaki and Nei 1996). The aim of the present work was to assess genetic variation in the Spiti breed of Indian horses, using 25 microsatellite markers that had been observed in earlier studies to be polymorphic in exotic breeds of horses (Ellegren et al. 1992; van Haeringen et al. 1994, 1998; Ewen and Matthews 1995; Eggleston-Stott et al. 1996, 1997; Meyer et al. 1997; Swinburne et al. 1997, 2000; Coogle and Bailey 1998; Lindgren et al. 1999; Kakoi et al. 1999).

Microsatellite markers based genetic diversity and bottleneck studies in Zanskari pony

Genetic diversity in Zanskari pony breed was evaluated at 48 microsatellite loci using fifty adult, healthy and unrelated animals. Allele frequency data was used to detect genetic diversity and bottleneck. The estimated average number of alleles (±s.e.) was 8.5208±2.5010 with a total of 409 alleles. A high level of genetic diversity within this breed was observed in terms of number of alleles, observed heterozygosity (0.6763±0.1704), expected Levens heterozygosity (0.7724±0.795), expected Neis heterozygosity (0.7644±0.0787) and polymorphism information content (>0.5). In-breeding coefficient (F(is)) was 0.115±0.0209, suggesting moderately high in-breeding in Zanskari breed. Although analysis of bottleneck revealed no bottleneck in recent past but population of Zanskari ponies has decreased drastically and only a few thousand pure-bred animals are left. The information is useful for proposing effective population management strategies for future.

Molecular Variability in Different Indian Isolates of Equine Herpesvirus-1

Three abortigenic Indian isolates of equine herpesvirus-1 (EHV-1) (Tohana, Hisar and Bikaner), along with two exotic abortigenic isolates (AB4 and V592) and another EHV-1 isolate (Jind) obtained from a case of perinatal foal mortality, were studied for variability. For this purpose, PCR and restriction endonuclease (RE) digestion techniques were used simultaneously as a DNA fingerprinting system. Nine different regions of EHV-1 virus were amplified by PCR using primer pairs specific for the regions and the products obtained from these regions were subsequently subjected to various restriction endonucleases to further assess the variability in the number of RE sites as well as in their positions. No difference was observed in all the four abortigenic isolates in terms of the size of different PCR products amplified by all the nine primer pairs, except for primer pairs ‘E’ and ‘C’. PCR products obtained with primer pair E revealed that Tohana and Bikaner isolates were most similar while Hisar isolate was like V592 isolate. However, the PCR product obtained from Jind isolate had a size between the PCR products of Hisar and Tohan/Bikaner isolates. The primer pair ‘C’ used to amplify the region between 1151 to 3679 in ‘Gene 1,2,3’ clearly differentiated the EHV-1 isolate obtained from a case of perinatal foal mortality from isolates obtained from abortion cases. This primer pair needs to be exploited more extensively for use as a potential marker for differentiating the EHV-1 isolates, mainly the abortion cases from perinatal foal mortality ones. Restriction endonuclease studies done with PCR product of all the isolates with various primer pairs did not reveal any changes in the position or number of RE sites present in the products amplified, indicating no variation in different RE sites within the amplified PCR products. However, this study clarified that all the Indian isolates belonged to the IP group of EHV-1.

Allergic fungal rhino sinusitis with granulomas: A new entity?

INTRODUCTION Allergic fungal rhino sinusitis (AFS) is classically described as allergic manifestation to the fungal antigen present in sinuses with no evidence of invasion. Granulomas in histopathology, suspicious of invasion, are occasionally observed in AFS and the disease in these patients behaves like invasive fungal sinusitis even without histologic evidence of invasion. We retrospectively studied AFS patients to analyze whether AFS should be continued to be designated as an allergic entity. MATERIAL AND METHODS AFS patients operated from January 2009 to July 2013 were retrospectively reviewed. Of the total 57 cases operated in last 4 years, nine showing presence of granuloma in histology were included in the AFS with granuloma Group (group 1) and the rest 48 were included in the AFS group (group 2). Both the groups were compared in terms of various parameters at presentation, treatment course and rate of recurrence. RESULTS Group 1 had significantly high rates of orbital erosion (P = .000), with positive association of skull base erosion (P = .092) and high rates of telecanthus (P = .000), diplopia (P = .000), proptosis (P = .161) and facial pain. Recurrent surgery was needed in 8 of 9 patients in the group 1 as compared to 1 of 48 patients group 2. CONCLUSION Granulomas suggests a more severe disease with a trend toward the invasive fungal sinusitis and alerts the clinician regarding the nature of progression. AFS seems to be a part of a continuous spectrum of fungal sinusitis rather than an allergic form as a distinct entity.

Mitochondrial DNA- a Tool for Phylogenetic and Biodiversity Search in Equines

It is imperative to assess the maternal lineage in order to achieve a broad picture of evolution, phylogenetic and genetic biodiversity within and among different breeds of livestock. In recent past, there has been a considerable advancement in sequencing of complete mammalian mtDNA molecules and their analysis. Most of the studies have focused on the mitochondrial D-loop region, the most variable part of mtDNA due to increased substitution rate than in the rest of the mtDNA genome which serves as a better genetic marker to assess the diversity. Mitochondrial DNA (mtDNA) possesses several favorable characteristics, including large quantity in the cell, small genome size, haploid, maternal inheritance with extremely low probability of paternal leakage, higher mutation rate than nuclear DNA, and amenable to change mainly through mutation rather than recombination. All these features make mtDNA a useful and one of the most frequently used markers in molecular systematic and has been widely employed to address questions of genetic diversity, population structure and population evolution of animals including equines. Many native breeds of horses as well as ponies were assessed for their genetic diversity and ancestry on the basis of studies on mitochondrial DNA to address the questions of evolution along with breed development and conservation.

Development of Lateral Flow Assay for Point-of-Care Diagnosis of Trypanosomosis in Equines

Abstract Trypanosomosis, commonly known as “Surra” in Indian subcontinent caused by Trypanosoma evansi, is a worldwide parasitic disease of wild and domestic animals. The disease is transmitted mechanically by biting and interrupted feeding of infected animals by hematophagous flies. The diagnostic methods available for this disease require high‐end instruments and skilled manpower and are time‐consuming. In the present investigation, we attempted an immunoassay based on gold nanoparticles for diagnosis of T. evansi infection in equines. Antibodies to horse IgG were raised in rabbits and conjugated to gold nanoparticles after purification with the protein‐A column. The soluble whole cell lysate antigen was prepared from purified T. evansi parasites and coated as test line on nitrocellulose membrane laminates, along with control line to detect anti‐Trypanosoma antibodies. The experimental and field serum samples were tested and evaluated to determine the sensitivity and specificity of the assay. The antigen‐specific antibodies were detected in experimental serum samples between 10 and 21 days post infection by the lateral flow assay (LFA). The LFA results showed 96.3% sensitivity and 93.9% specificity comparable with enzyme‐linked immunosorbent assay. The study suggests that the LFA can be used as a point‐of‐care diagnostic method in the field, which does not require any specialized instrument for the detection of T. evansi infection in animals. HighlightsA point‐of‐care diagnostic assay is developed for detection of Trypanosoma evansi infection in equines.The lateral flow assay successfully detected T. evansi–specific antibodies present in serum samples within 15 minutes.The lateral flow assay results showed 96.3% sensitivity and 93.9% specificity comparable with enzyme‐linked immunosorbent assay.