Genomic markers for parasitic infections


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Genomic markers for parasitic infections

  2. 2. AGENDA Introduction Tools for Gene detection and analysis . Tools For Molecular Epidemiology of Parasites Functional Genomic Study Application of Genomic Studies in Parasitology Conclusion. 2
  3. 3. Introduction Parasites have kept many secrets from the researchers who have sought to eradicate them over past decades.Little is known about them: Evade drugs, Escape the immune system, Regulate switching between genes involved in immune evasion, and Orchestrate development. 3
  4. 4. IntroductionProblems with Study of Parasites: Lack of reliable culture system (Difficult to keep and breed) Shortage of material for biochemical studies; Lack of traditional genetic methods to study gene functions. They parasitize hosts that are not ideal experimental subjects. 4
  5. 5. Introduction Human helminths are much more complex organisms (e.g., they are diploid, have reproductive and other organs, nervous systems, etc.) compared to Protozoans There are many more species of them They belong to two completely unrelated phyla (Platyhelminthes and Nematoda) No cell lines are available The developmental cycles cannot be completed in vitro Their developmental cycles are not only dissimilar to those of Protozoans, but they are also generally 5 dissimilar to each other.
  6. 6. Introduction The availability of parasite genome sequences and related genome-based tools have provided substantial opportunities to overcome these problems to a large extent. 6
  7. 7. Introduction A genome is all of a living things genetic material The genome is divided into chromosomes, chromosomes contain genes, and genes are made of DNA. 7
  8. 8. Introduction Gene and chromosome. Coined in 1920 by Hans Winkler. The complete set of genes in an organism : GENOME. Frederick Sanger in 1977 introduced the ―dideoxy‖ chain termination method to sequence DNA molecules. 8
  9. 9. IntroductionHuman Parasites Complete sequence: Timeline2001: Encephalitozoon cuniculi2002: P.falciparum2004: Cryptosporidium parvum and hominis2005: E. histolytica, Trypanosoma brucei, cruzi; L.major2007: Brugia malayi, Giardia lamblia, Trichomonas vaginalis2008: P.vivax, P. knowelsi2009: Schistosoma mansoni, and S. japonicum2011: Ascaris suumOther parasites: Toxoplasma gondii, Leishmania braziliensis, L. infantum, L.mexicana,(Partial/ Draft). 9
  10. 10. Introduction The study of Genomic Markers is expected to lead to major technological advances: New Diagnostics New therapeutics, and Vaccine development, Understanding of disease mechanisms, Elucidation of Host–parasite interactions, Insight into transmission biology. 10
  11. 11. Tools For Gene Detection and AnalysisI. Amplification Techniques: PCR Real Time PCR NASBA LAMP LCR2. Hybridization Techniques: FISH Microarrays3. Biosensors Liposome Based Lateral Flow Biosensor Matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI TOF) 11
  12. 12. Tools For Gene AnalysisUsed for MOLECULAR EPIDEMIOLOGY OF PARASITES Multi Locus Enzyme Elecrophoresis (MLEE) Amplified Fragment Length Polymorphism (AFLP) Random Amplified Polymorphic DNA (RAPD) PCR-Restriction Fragment Length Polymorphism (RFLP) Kinetoplastid minicircle DNA (kDNA) RFLP for Leishmania Gene sequencing and Sequencing of ribosomal loci Multi locus Microsatellite Typing (MLMT) Genome wide Single Nucleotide Polymorphism (SNP) Single Strand Conformation Polymorphism (SSCP). 12
  13. 13. Real Time PCRThe primary advantages of real-time PCR over conventional PCR : It provides high-throughput analysis in a closed-tube format (no post-PCR handling is required) It can be used for quantitation over a broad dynamic range It can be used to differentiate DNA fragments by analyzing the melting curve of DNA. Simple, fast, closed, and automated amplification system responsible for decreasing the risk of cross- contamination. 13
  14. 14. Real Time PCR Various Fluorescent chemistries are being used1. TaqMan Probe: One of the most widely used chemistries Assay design is simple and assays are robust. However, Taqman probes do not confirm that the correct fragment has been amplified (other than by running a gel). 14
  15. 15. Real Time PCR2. Minor-groove binder(MGB) Eclipse probes3. Molecular beacons4. Fluorescence- (or Forster) resonance-energy- transfer (FRET)-based assays5. Intercalating Dyes: SYBR Green, BEBO, LC Green: SYBR Green is the most cost-efficient chemistry and, thus, are the most popular option among researchers.15
  16. 16. Applications of RT-PCR Detection of Cryptosporidium, Leishmania, Trypanosoma, Giardia, Toxoplasma Diagnosis, animal models, drug efficacy and vectorial capacity for Leishmania Monitoring antimalarial therapy Species discrimination and SNP analysis (FRET assay) Discrimination between species and genotypes of Cryptosporidium 16
  17. 17. Loop Mediated Isothermal Amplification(LAMP) LAMP is a method of nucleic acid amplification with extremely high sensitivity and specificity to discriminate single nucleotide differences Four primers specially designed to recognize six different sequences on the target gene . Amplification occurs only when all primers bind, thus forming a product. DNA amplification can be achieved using simple incubators (water bath or block heater) because of isothermal conditions 17
  18. 18. LAMP Can amplify a few copies of genetic material to 109 in less than an hour Large amounts of white magnesium pyrophosphate are precipitated from DNA amplification . The turbidity caused by this reaction is proportional to the amount of DNA synthesized. As a consequence, it is possible to evaluate the reaction in real time by measuring the turbidity or, more importantly, by visualization by the naked eye. The simple requirements make LAMP easily available for small laboratories, especially in rural endemic areas 18
  19. 19. LAMP Has been used to detect several parasitic diseases, including the human parasites Cryptosporidium, Entamoeba histolytica, Plasmodium, Trypanosoma, Taenia, Schistosoma, Fasciola hepatica and Fasciola gigantica, and Toxoplasma gondii, Could detect the miracidium after the first day of exposure in snails, the intermediate hosts of Schistosoma Mosquitoes carrying the parasites Plasmodium and Dirofilaria immitis have been identified 19
  20. 20. Ligase Chain Reaction (LCR) DNA ligases are highly specific and do not tolerate base mismatches (unlike DNA polymerases). This makes LCR superior to PCR for the detection of SNPs, which has been its chief application Products of the LCR are detected in real-time by using either FRET probes as LCR primers or primers that are designed to form molecular beacons once ligated. 20
  21. 21. LUMINEX Luminex is a bead-based xMAP technology (multianalyte profiling), a system that combines flow cytometry, fluorescent microspheres (beads), lasers and digital signal processing Capable of simultaneously measuring up to 100 different analytes in a single sample Luminex was able to distinguish the species C. hominis and C. parvum in 143 DNA extractions, using oligonucleotide-specific probes for the ML-2 regions (microsatellite region-2 )of each species, without the need for DNA sequencing. 21
  22. 22. Hybridization Techniques: 1. FISH Uses fluorescently-labeled probes [either DNA or peptide nucleic acid (PNA)] that hybridize to complementary nucleic acid targets in whole cells to enable the direct detection of organisms in complex communities PNA probes are pseudopeptides that hybridize to complementary nucleic acid targets (DNA and RNA) with better specificity and stability than DNA probes PNA probes are expensive to construct. 22
  23. 23. FISH Applying FISH to whole cells gives information about microbial identity, cell morphology, abundance and spatial distribution of individual target species To study the partitioning and chromosome composition of nuclei in Giardia and to detect arthropozoonotic species and genotypes of Cryptosporidium and Giardia PNA probes have been used for the direct FISH detection of African trypanosomes 23
  24. 24. 2. MICROARRAYS DNA microarrays provide a powerful tool for the parallel analysis of multiple genes and gene transcript Microarrays are arrays of either cDNAs or oligonucleotides that are either spotted onto a glass microscope slide or synthesized on a silicone chip. DNA or mRNA extracted from cells or tissues is labeled with specific fluorescent molecules and hybridized to the microarray DNA. 24
  25. 25. Microarrays To improve sensitivity, samples are often pre- amplified by PCR The resulting image of fluorescent spots is visualized in a confocal scanner and digitized for quantitative analysis Has been used to detect and discriminate between a range of parasites, including different species and genotypes of Entamoeba, Giardia and Cryptosporidium in a single assay. 25
  26. 26. MicroarraysThe main application of microarrays has been to study gene expression in hosts To study differential gene expression in different lifecycle stages of T. brucei Gender-associated gene expression in Schistosoma japonicum Gene expression during asexual development of T. gondii Gene expression for Plasmodium in mosquitoes. 26
  27. 27. BIOSENSORS A biosensor is an analytical device which converts a biological response into an electrical signal Sandwich hybridization is used to capture target nucleic acid and the reporter liposome, which contains a dye. Following capture and washing, the liposomes are lyzed using detergent, which produces a signal. 27
  28. 28. (Monis et al 2005. Trends in Parasitology 21:28 340-46)
  29. 29. MALDI-TOF Matrix-assisted laser desorption–ionization time-of- flight mass spectrometry Based on the detection of diagnostic proteins; but a database that contains the mass information of the diagnostic biomarkers is needed to use MALDI -TOF as an identification tool Require expensive, specialized equipment and/or the establishment of appropriate sample databases. Has been used for the study of Cryptosporidium. 29
  30. 30. TOOLS FOR MOLECULAREPIDEMIOLOGY: 1. MLEE Non DNA Based Based on phenotypic polymorphism dependent on protein structures which in turn is based on structural gene sequences. It can detect polymorphism only within the coding region of the gene, not the mutations in the promoter region or introns. A set of 10-20 genes is analysed to generate meaningful data. Extensively used for Leishmania, Trypanosoma, P. falciparum 30
  31. 31. 2. Amplified Fragment LengthPolymorphism (AFLP) Allows detection of a DNA polymorphism without prior information on the sequence. Employs PCR to selectively amplify the groups of restriction fragments of totally digested DNA. Polymorphisms are identified based on the presence or absence of DNA fragments by polyacrylamide gel analysis 31
  32. 32. AFLPThe advantages of this technique are The ability to search an entire genome for polymorphisms, The reproducibility of the method, and The possibility of being used against parasites about which there is no prior genetic information Highly efficient because of the possibility of analyzing a large number of bands simultaneously, with extensive coverage of the genome 32
  33. 33. AFLP: Uses To differentiate isolates of C. parvum into two distinct genotypes Has revealed high genetic variability among the genome species of Leishmania major, L. tropica and L. donovani, which was sufficient to distinguish between Cutaneous and Visceral Leishmaniasis. 33
  34. 34. 3. Random Amplified Polymorphic DNA(RAPD ) Extensively used for description of strains in epidemiological studies RAPD is a very simple, fast, and inexpensive technique which makes survey of parasite genome easier. Does not require either prior knowledge of the DNA sequence or DNA hybridization Has been used to delineate strains of microorganisms. 34
  35. 35. RAPD Highly efficient in the differentiation of amplification profiles, as well as its ability to distinguish polymorphisms between helminthic parasites. Used to map genes for the characterization of species, to stimulate the genetic variability and determine the genetic structure of populations of different microorganisms. It also reveals polymorphisms in the noncoding regions of the genome. 35
  36. 36. RAPD: Uses Species differentiation of Leishmania, To study polymorphisms of parasites such as Plasmodium and Trypanosoma Differentiation of endemic strains of Wuchereria bancrofti. 36
  37. 37. 4. RFLP This reaction is based on the digestion of the PCR products by restriction enzymes or endonucleases. These enzymes cleave DNA into fragments of certain sizes, whose analysis on agarose or polyacrylamide gel results in different patterns of fragment sizes, enabling the identification . Diversity of Cryptosporidium spp Diagnosis of species and genotypes of Toxoplasma gondii. 37
  38. 38. 5. Multi locus Microsatellite Typing (MLMT) Microsatellites are short DNA sequences (about 300 base pairs) composed of tandem repeats of one to six nucleotides, with approximately one hundred repeats. Microsatellites are abundant in eukaryotic genomes and can mutate rapidly by loss or gain of repeat units. They show frequent polymorphism, co-dominant inheritance, high reproducibility and high resolution, require simple typing methods, and can be detected by PCR . 38
  39. 39. MLMT Microsatellite markers have been developed only for some parasitic nematodes such as species of Trichostrongyloid . Strain Typing of L.donovani The low popularity of these genetic markers is due to the high number of microsatellites, which cause technical difficulties in isolating parasites by PCR. 39
  40. 40. Genome wide Single NucleotidePolymorphism (SNP) SNP is a change in a single nucleotide in one sequence relative to another caused by nucleotide mutation, gene transfer, or intragenic recombination. Variation in a single base in the genetic code between different individuals of the same species can be detected. SNP variation occurs when a single nucleotide, such as an A, is replaced by one of the other three nucleotide—C, G, or T. e.g. AAGGTTA to ATGGTTA 40
  41. 41. Single Strand Conformation Polymorhism(SSCP) It is electrophoretic separation of single-stranded nucleic acids based on subtle differences in sequence (often a single base pair) which results in a different secondary structure and a measurable difference in mobility through a gel. Like RFLP, SSCPs are allelic variants of inherited, genetic traits that can be used as genetic markers Unlike RFLP analysis, however, SSCP analysis can detect DNA polymorphisms and mutations at multiple places in DNA fragments. 41
  42. 42. Functional Genomic Study Some of the available genomes are incomplete, poorly annotated, or not annotated at all. There are almost no tools available with which gene function can be tested directly. Without annotation and functional genomic tools, the sequence data are not truly useful.Annotation: a note added by way of explanation or commentary mentioning what the gene does (its function) to make sense. 42
  43. 43. Functional Genomic Study Essential to resolve uncertainties in the molecular physiology of parasites; and To illuminate mechanisms of pathogenesis that may lead to development of new interventions to control and eliminate these parasites or the diseases. 43
  44. 44. Functional Genomic StudyTwo Categories1. Bioinformatic tools for sequence mining to generate hypotheses concerning likely biological function, and2. Experimental tools with which gene expression can be manipulated in the target organism (or, in the case of parasites, also the host) and the consequences of that manipulation for the biology of the parasite and its relationship with the host can be observed and measured a. RNA Interference b. Transgenesis 44
  45. 45. Functional Genomic Study: BioinformaticTools The first bioinformatic tools that are applied are generally genome-wide homologue searches, usually using variants of basic local alignment search tool (BLAST) to generate automatic annotations based on sequence homology. Very rough guide and at worst downright misleading In parasites the limited utility of a homology based approach is undermined further by the poor performance of gene-finding software in parasite genomic sequences. 45
  46. 46. Functional Genomic Study: Experimental Tools: 1.RNA Interfernce Most important functional genomic tool Gene expression is knocked down by exposing the parasites to gene-specific dsRNA or small interfering (si)RNA (gene silencing) Double-stranded RNA [dsRNA] triggers degradation of homologous mRNA transcripts Efficiency of RNAi in parasites varies between species Problems often arise with the efficiency, specificity and reproducibility of some methodologies, especially with nematode species. 46
  47. 47. RNA Interfernce To find out the parasite biology and the possibility of identifying and validating novel anthelmintic drug targets in B. malayi. In contrast to the situation in nematodes, RNAi in schistosomes seems to be more robust and reproducible and is currently being used by a number of groups to elucidate the function of some key proteins and pathways in these parasites: Haemoglobin digestion, Tegument formation, and the biological role of tegumental proteins These approaches have helped to identify vaccine/drug candidates, some of which are in various stages of clinical development. 47
  48. 48. 2. Transgenesis The converse of knock-down of expression is manipulation of expression by gene knock-in. It is a process by which gene function is inferred by studying the effects of transferring native or altered copies of genes into subject organisms. Either transient or heritable transgenesis of at least three species of human parasitic nematodes and trematodes (B.malayi, S.stercoralis, S. mansoni) and in at least one cestode, E. multilocularis has been attempted; apart from malaria parasites. 48
  49. 49. Functional Genomic Study Help in rational design of agents directed at defined molecular targets. Important in developing new drugs and identifying potential vaccine candidates for parasitic helminths and protozoans. 49
  50. 50. Genomic Studies: Applications and Uses.A. Parasite Systematics: Highly conserved coding regions e.g. Small Subunit rDNA and certain mitochondrial genes can help in discrimination above species level. Below the species level, relationships between genes sampled from different individuals are not hierarchical because homologous genes from the two parents combine in their offspring . 50
  51. 51. Genomic Studies: Applications and Uses Above the species level, different taxa are hierarchical because they are a consequence of speciation followed by long periods of reproductive isolation. Representation of phylogenetic relationships above the species level as networks rather than as strictly bifurcating trees is gaining interest. 51
  52. 52. Genomic Studies: Applications and UsesB. Parasite Diagnostics and Epidemiology Discrimination between species by molecular techniques like multiplex PCR, LAMP etc can be achieved by detecting the moderately conserved regions e.g. coding mitochondrial genes, internal transcribed spacer, rDNA, and other loci 52
  53. 53. Genomic Studies: Applications and Uses Molecular identification is particularly important when discriminating different parasites with morphologically identical life cycle stages, such as eggs or cysts, from faecal samples, or when attempting to match different life cycle stages of the same parasite from intermediate and definitive hosts In some endemic areas humans may be infected with more than one species of hookworm or of taenias , the eggs of which are identical. 53
  54. 54. Genomic Studies: Applications and UsesC. Intraspecies variation study: Finds extensive application in population genetics; breeding systems (e.g. cross vs. self fertilization); host specificity analysis; molecular epidemiology; conservation (e.g. predicting susceptibility to pathogens); and biosecurity (exotic and emerging pathogens). 54
  55. 55. Genomic Studies: Applications and Uses Genes are analyzed using tools like MLEE ( allozymes), RAPD, AFLP, PFGE, PCR-RFLP, pyrosequencing, mPCR, LAMP, and qPCR. The extent to which we recognize intraspecific groups of parasites will be a function of the extent to which intraspecific variation is structured among different hosts or among different geographic areas. 55
  56. 56. Genomic Studies: Applications and Uses In T. brucei, for example, three subspecies have historically been defined on the basis of geographic and host distribution, and the clinical course of disease. T. b. gambiense : Human parasite distributed through western and central Africa, causing chronic disease. T. b. rhodesiense : Human parasite distributed through eastern and southern Africa causing acute disease and T. b. brucei infects domestic and game animals, but not humans and is widely distributed throughout sub- Saharan Africa. 56
  57. 57. Genomic Studies: Applications and UsesD. Discrimination between individual isolates: The techniques of ―fingerprinting helps in tracking transmission of subgenotypes ; to find out sources of infection and risk factors and also the course of infection Done by using Mini/microsatellites, SSCP, and qPCR. 57
  58. 58. Genomic Studies: Applications and UsesE. Identifying phenotypic traits of clinical and epidemiological significance: Linkage studies between phenotype and genotype of a particular parasite are useful to determine the virulence, infectivity, and drug sensitivity Done by using genetic map; sequencing and/or RT- PCR of genes thought to be linked to phenotypic trait. Drug resistance in P. falciparum , virulence in T. gondii and resistance to malaria parasites in Anopheles gambiae have been studied . 58
  59. 59. Genomic Studies: Applications and Uses Useful to explore genetic changes involved in resistance to anti-parasitic drugs and understanding the potential mechanisms of drug resistance in human parasites, and can be expected to facilitate development of genetic markers to monitor and manage any future appearance and spread of drug resistance. 59
  60. 60. Application and Uses To understand how these parasite products act on immune responses Comparisons of parasite molecules with orthologues/paralogues from free-living relatives will strengthen efforts to decipher the strategies adopted by parasites to evade and subvert their host immune responses. This information can be exploited for development of drugs and vaccines against the parasites. 60
  61. 61. Application and Uses Treatment for helminthic infections, responsible for hundreds of thousands of deaths each year, depends almost exclusively on just two or three drugs: praziquantel, the benzimidazoles, and ivermectin Pharmacogenomics with the new helminth genomes represents a practicable route forward toward new drugs. For example, chemogenomics screening of the genome sequence of S. mansoni identified 20 parasite proteins for which potential drugs are available approved for other human ailments [schistosome thioredoxin glutathione reductase, auranofin (an anti-arthritis medication) was shown recently to exhibit potent anti-schistosomal activity]. 61
  62. 62. Conclusions Genomic studies of parasites should be done keeping the following points in focus: Highly conserved regions can reveal information about taxonomic relationships between species Moderately conserved regions can differentiate strains and closely related species; Moderately variable regions can reveal population genetic structure Highly variable regions enables tracking of particular isolates in a population; and ‗mapping‘of genetic markers can be used to find markers that correlate genotype to phenotype . 62
  63. 63. Conclusions Sequencing detects the highest level of variation down to changes in individual base pairs Techniques based on restriction-fragment-length polymorphisms only detect changes that affect restriction sites, but can be used to sample across the entire genome. 63
  64. 64. Conclusions The recent research landscape for parasites has been dominated by rapid progress in genome sequencing of several parasites of significance to human disease. Future genome-wide analyses will support efforts to elucidate the basic biology of parasites, including immune mediated and other host–parasite interactions that are relevant to parasitic diseases of humans. They will help to develop novel intervention strategies such as drugs and therapeutic or prophylactic vaccines, as well as to identify parasite biomarkers and devise improved diagnostics. 64
  65. 65. Conclusions As many of the parasitic infections are transmitted by arthropod vectors or involve intermediate hosts, a greater understanding of the interaction between vector/intermediate hosts and parasites is also important. The future may belong to Systems Biology approach to link high throughput molecular sciences such as genomics, proteomics and transcriptomics ; where individual streams of information can be processed as a whole, rather than remaining as an isolated descriptor of the action of individual components. 65
  66. 66. 66