Molecular Mechanism and Diagnosis of Leishmaniasis

Health Planning &Resources Development

Investigators

Abstract

Leishmania parasite causes human disease with clinical symptoms ranging from cutaneous lesions to fatal visceral disease. The parasite poses serious public health risk worldwide either due to its pathogenesis or by blood transmission. There are no vaccines available for Leishmaniasis and suffers from the lack of availability of sensitive diagnostic methods. Further, parasite drug resistance makes it difficult to treat this disease. The research in my laboratory is focused on to identify and characterize genes that are essential for growth and differentiation of Leishmania because of their importance in parasite virulence. In addition, we also have focused our attention in modulating the secretion of the parasite proteins because number of such proteins has been implicated as virulent factors. We have identified several such genes and have modulated their expression in Leishmania by molecular manipulations such as gene-knockout, antisense and negative dominant expression of mutant forms of such genes. The goal of these studies is to develop an attenuated parasite, which can be tested as vaccine candidates. We also are developing DNA microarray for Leishmania genome, which could be useful to screen for multiple genes that are essential for drug resistance, and parasite differentiation using RNA from such parasites. Diagnosis of Leishmania is difficult in endemic areas because of the lack of availability of sensitive detection methods. We have developed PCR-based detection methods using parasite mitochondrial DNA (k-DNA) as a target because it is abundant in parasite. In addition, we are developing an oligonucleotide based microarray chip for Leishmania and other blood borne pathogens, which could be used for the rapid diagnosis of such agents in the blood. Molecular Mechanism of Leishmaniasis. Leishmania parasite causes human disease (Leishmaniasis) with clinical symptoms ranging from self healing cutaneous lesions to fatal visceral infection. The lack of understanding of the mechanism by which Leishmania parasite causes disease poses a serious public health risk worldwide and in particular for U.S. military personnel their families and tourists either living or traveling in endemic areas. As a first step towards understanding the molecular mechanism of Leishmania pathogenesis, we have began to analyze the processes that are involved in parasite life cycle in transformation from an avirulent to virulent form and to target such processes to control growth of the parasite. The role of unique structural and functional features of organelles, like the cytoskeleton , basal body in cell growth of Leishmania, is still obscure. In order to identify genes that control growth, we have isolated for the first time a gene encoding for centrin from L. donovani. Centrin is calcium binding cytoskeletal protein essential for centrosome duplication or segregation. The levels of centrin mRNA and protein were high during the exponential growth of the parasite in culture and declined to a low level in the stationary phase. Expression of N-terminal deleted centrin in the parasite significantly reduces its growth rate and it was found that significantly more cells are arrested in the G2/M stage than in control cells. Centrin mutant parasites were shown to be highly susceptible to killing by the human macrophages suggesting growth arrested centrin mutants have an attenuated phenotype. Further, these studies indicate that centrin may have a functional role in Leishmania growth. Currently studies are underway to modulate the expression of centrin gene product by disrupting the gene in the parasite which would allow us to further attenuate the parasite growth and to generate a vaccine candidate. Pathogen Chip for Detection of Bioterrorism Agents in Blood. We will target at least seven BT agents identified as "Category A" by the Centers for Disease Control. The objectives are: (1) To evaluate existing PCR-based nucleic acid technology that rapidly detects BT pathogens to determine the feasibility of adapting them for the screening of blood. (2) To select PCR primers that will amplify diagnostic gene sequences from the pathogens. (3) To optimize methods for amplification of pathogen sequences from spiked blood samples. (4) To select oligonucleotide sequences corresponding to BT agent genes and print them on a microarray for hybridization to labeled, PCR amplified BT pathogen sequences and to sequences from closely related organisms that will be used as models to test the detection method. (5) To optimize methods for printing the oligonucleotides on glass slides. (6) To optimize methods for hybridization of the labeled PCR products to the microarray so that multiple suspected pathogens could be identified with one standard procedure. Description of Research: Selection of pathogens. The first rapid PCR detection techniques and prototype microarray are designed to detect agents identified by the CDC to have the greatest danger for use in bioterrorism (Bacillus anthracis, Yersinia pestis, Francisella tularensis, Venezuelan Equine Encephalitis Virus, Hemorrhagic viruses-Ebola and Marburg, and Variola major virus). We are focusing particular attention on pathogens with potential to contaminate the blood supply. We have selected a model organism related to each pathogen that can be used to test the system without the requirement for biosafety level 4 (BSL4) isolation procedures. Selection of amplicons. For each pathogen, one region of nucleic acid sequence has been selected for PCR amplification (the amplicon). The region has been selected primarily by searching the literature to identify nucleic acid sequences that have been amplified successfully with known primers (17 to 30 nucleotides in length). Amplicons have been selected that can discriminate the bioterror pathogens from other organisms and that have demonstrated sensitivity to less than 1000 organisms per milliler. Some of the targets already identified are the protective antigen (PA) gene, lethal factor (LF) gene, and edema factor (EF) gene for Bacillus anthracis; the plasminogen activator (PA) gene for Yersinia pestis, and the tul4 gene of Francisella tularensis to name a few. The quantitative PCR (qPCR) or "Taqman" approach will require a pair of primers that are 18-22 nucleotides in length, 40% to 60% GC content, melting temperature of 55 to 60oC, primers to be within 2oC melting temperature of each other, 1 to 2 nucleotide GC clamp, and amplicon size to be less than 200 base pairs in length. QPCR primers and probe have been synthesized for Bacillus anthracis. Selection of probes. For each amplicon, the qPCR method requires a fluorogenic probe that is 7 to 10oC higher than the primers' melting temperature, having no 5' terminal G residue, and its site to be less than 30 nucleotides from the corresponding strand primer. For the microarray, three internal oligonucleotides have been selected for each amplicon, 65-70 nucleotides in length, with melting temperatures 68-74?C. These probes have been searched against the Genbank database to insure they represent unique sequences. Oligonucleotide synthesis. Primers, probes and amplicons have been designed with the intent to minimize dimers, hairpins, long stretches of single base repeats, false priming sites; and to be able to multiplex the detection assay. Selected primer sequences were synthesized unmodified. Each probe was synthesized with an amino modification at the 5' end for covalently linking to an aldehyde coated glass slide. All oligos were column purified after synthesis to remove partial synthesis products. System development. The selected primers and probes will be combined into detection systems by two different approaches. I. Quantitative PCR. To achieve the shortest time for development of a usable test for the detection of BT agent nucleic acid in blood, primers and probes will be adapted for quantitative PCR. PCR will be performed on known amounts of template DNA or pathogen spiked into blood samples in a thermocycler. PCR products will be visualized on agarose gels. Sample preparation, cycling time and reaction buffer composition will be adjusted to reach maximum amplification. Subsequently, qPCR will be performed with the probes mentioned above in a Cephied Smartcycler (available at CBER). This device is equipped to measure fluorescence of up to 4 labeled probes. The sample preparation, reaction conditions and probe sequence will again be optimized to achieve the earliest possible cycle threshold (Ct) and satisfactory assay robustness (AR) with the minimum amount of pathogen in the sample. The earliest Ct and a good AR indicate maximum sensitivity measuring the correct target generated fluorescence distinct from background fluorescence. II. Microarrays. To achieve a more comprehensive, multiplex detection method, a microarray will be developed utilizing the same amplicons described above. A. Printing. The microarray probes for Leishmania, T. brucei, Hepatitis B Virus (HBV) and HCV as model organisms have been suspended in printing buffer in 384 well plates and spotted onto aldehyde coated glass slides with a robotic arrayer. Quality control oligos of "alien" sequence have been printed at the edges of each block. B. Sample preparation and amplification. Initial testing of PCR primers has been done with DNA samples and model organisms spiked into water. Detection of PCR products on an agarose gel stained with ethidium bromide was successful. Extraction procedures from whole blood samples will be optimized. Once successful PCR amplification was demonstrated, primer extension thermocyling (PET) reactions was performed to incorporate fluorescent label into single strand DNA for hybridization to the complementary strand probes on the microarray. Fluorescent-labeled DNA complementary to the quality control oligos was synthesized in similar fashion. C. Hybridization. Labeled PET products from the spiked samples and the quality control oligo were mixed with hybridization solution, and added to the microarray under a cover slip. The time (1-24 hours) and temperature (25?C-65?C) of incubation was optimized empirically. After hybridization, microarrays were washed to remove nonannealed DNA and dried for imaging. D. Microarray imaging. The hybridized microarrays were scanned and the image analyzed with the scanner software. The quality control spots were used to align the array grid over the image so that the identity of pathogen spots that show hybridization signal could be identified. Control of Leishmaniasis by Program Cell Death. Programmed cell death (PCD) is an essential part of cell biology and is thought to have evolved not only to regulate growth and development in multicellular organisms. However, recent studies, which showed the existence of PCD in unicellular organisms, have postulated a functional role of PCD in the biology of unicellular organisms. It has been postulated that in order to promote and maintain clonality within the population, the Trypanosomatids must have developed an altruistic mechanism to control growth. We wanted to explore whether PCD exists in Leishmania and if so can antiLeishmanial drugs induce it. This will provide an opportunity to develop future antiLeishmanial drugs. We have demonstrated some features characterizing programmed cell death (PCD) in the unicellular protozoan parasite Leishmania donovani, the causative agent of visceral Leishmaniasis. We report that PCD is initiated in stationary phase cultures of promastigotes and both in actively growing cultures of axenic amastigotes and promastigotes upon treatment with anti Leishmanial drugs (Pentostam and amphotericin B). However, the two cell types respond to antiLeishmanial drugs differently. The features of PCD in Leishmania donovani promastigotes and amastigotes are nuclear condensation, nicked DNA in the nucleus, DNA ladder formation, increased plasma membrane permeability, decrease in mitchondrial membrane poential, and induction of casapase-like activity. Therefore, these studies provide for the first time the presence of programmed cell death in Leishmania and the basis for understanding the mechanism of Leishmania pathogenesis. Currently we are characterizing the factors involved in the cell death program with an aim to exploit it for the control of parasite growth. This project incorporates FY2002 projects 1Z01BP005009-07, 1Z01BP005010-07, and 1Z01BP005021-01.