Microarray in parasitic infections.

Modern biology and genomic sciences are rooted in parasitic disease research. Genome sequencing efforts have provided a wealth of new biological information that promises to have a major impact on our understanding of parasites. Microarrays provide one of the major high-throughput platforms by which this information can be exploited in the laboratory. Many excellent reviews and technique articles have recently been published on applying microarrays to organisms for which fully annotated genomes are at hand. However, many parasitologists work on organisms whose genomes have been only partially sequenced. This review is mainly focused on how to use microarray in these situations.

INTRODUCTION

Array technology was in use as early as the 1980s but did not come into prominence until the mid 1990s when complementary DNA (cDNA) microarrays emerged as an exciting new biomolecular tool capable of probing the entire transcriptome of the cell. Today, microarrays are widespread in genomic research and have a diverse range of applications in biology and medicine. A few recent applications include microbe identification,[1] tumor classification,[2] evaluation of the host cell response to pathogens, and analysis of the endocrine system.[3] Microarrays opened up an entire new world to researchers. No longer is one restricted to studying a unique single aspect of the host/pathogen relationship; instead one can explore a genome-wide view of this complex interaction. This comprehensive and hypothesis-free analysis method is helping scientists discover and understand disease pathways, and ultimately develop better methods of detection, treatment, and prevention.

Data generated from whole-genome microarray studies are richer and deeper than ever before. Data from a single array experiment—whether gene expression or DNA analysis—can often be used for a number of different studies that otherwise would have required the compilation of data from numerous independent experiments. For instance, the same expression data from an infected host could be used to understand the mechanism of virulence, and might also be used to identify a unique host-response signature for pathogen and disease identification purposes. Moreover, arrays are being designed to simultaneously monitor whole-genome host and pathogen gene expression, providing a complete view of the progression of an infectious disease state—how a pathogen responds to its host and the host to its pathogen. The flexibility of the Affymetrix GeneChip® microarray analysis allows a single array and a single experiment to encompass different types of infectious disease studies.

The most recent generation of microarrays allows scientists to readily perform DNA sequence analysis, often providing the ability to sequence complete genomes in a single experiment. Whereas conventional sequencing methods typically require extensive resources (time, cost, and labor), the individual scientists can now use GeneChip CustomSeq® Arrays, for example, to resequence up to 300 kb of a genome within 48 hours with minimal amplification of the genomic target. By comparing sequences from different strains, scientists can now identify important genetic variations, leading to improved typing systems, vaccine candidates, and pathogen detection and identification methods. This application note describes the impact of GeneChip resequencing, genotyping, and expression arrays in all areas of infectious disease research, from understanding pathogenesis to developing better therapeutics and diagnostics.[4]

What to do with microarrays?

The most common use of microarrays is to examine differences in transcript abundance as a function of any of several variables. These can include time in a given physiological condition, developmental stage, drug treatment, population density, infection, strain, etc. Importantly, it should be remembered that, for most experiments, microarrays are not the last experiment performed but, like genetic screens, they serve as a springboard to unravelling complex molecular and cellular pathways. The two big questions for researchers are: What type of experiments can be done and how does one go from having a mass of data on a large number of genes to understanding the biology of the system as a whole?[5]

Types of microarray

Microarrays can be divided into various types based upon characteristics such as the nature of the probe, the solid-surface support used, and the specific method used for probe addressing and/or target detection. Here, we review the methodologies of printed and in situ-synthesized microarrays, high-density bead arrays, and electronic and suspension bead microarrays. In all of these approaches, the probe refers to the DNA sequence bound to the solid-surface support in the microarray, whereas the target is the “unknown” sequence of interest. In general terms, probes are synthesized and immobilized as discrete features, or spots. Each feature contains millions of identical probes. The target is fluorescently labeled and then hybridized to the probe microarray. A successful hybridization event between the labeled target and the immobilized probe will result in an increase of fluorescence intensity over a background level, which can be measured using a fluorescent scanner.[6]

Printed microarrays

These were the first microarrays utilized in research laboratories and are so called because of the “printing” or spotting of the probes onto the microarray surface, which is most commonly a glass microscope slide. Glass slides are an attractive medium for microarrays because they are economical; are stable throughout high temperatures and stringent washes; are nonporous, allowing for efficient kinetics during hybridization; and have minimal background fluorescence.

The probe spots, or features, can be applied by either noncontact or contact printing. A noncontact printer uses the same technology as computer printers (i.e., bubble jet or inkjet) to expel small droplets of probe solution onto the glass slide. In contact printing, each print pin directly applies the probe solution onto the microarray surface. The result in both cases is the application of a few nanoliters of probe solution per spot to create an array of 100- to 150-m features. During the printing process, it is imperative to control for cross-contamination and printing consistency to preserve the integrity of the microarray and subsequent hybridization data. Due to the relatively large size of the features, printed microarrays are of lower density (10,000 to 30,000 features) than in situ-synthesized microarrays and high-density bead arrays (discussed below) but offer considerably more features than either electronic microarrays or suspension bead arrays.

In situ-synthesized oligonucleotide microarrays

In situ-synthesized arrays are extremely-high-density microarrays that use oligonucleotide probes, of which GeneChips (Affymetrix, Santa Clara, CA) are the most widely known. Unlike the printed oligonucleotide arrays described above, the oligonucleotide probes are synthesized directly on the surface of the microarray, which is typically a 1.2-cm2 quartz wafer. Because in situ-synthesized probes are typically short (20 to 25 bp), multiple probes per target are included to improve sensitivity, specificity, and statistical accuracy. Classically, 11 probes are used per 600 bases being examined.[7] The use of probe sets further increases the specificity. A probe set includes one perfect-match probe and one mismatch probe that contains a 1-bp difference in the middle position of the probe (i.e., position 13 of a 25-bp probe). Each member of the probe set is located in a separate feature, which allows the mismatch probe to act as a negative control to identify possible nonspecific cross-hybridization events. Recent advances in GeneChips include the use of longer probes, the design of arrays that interrogate across entire genes or exons, and the implementation of multiple independent and nonoverlapping perfect-match probes in lieu of classic probe sets. The microarray surface is chemically protected from a nucleotide addition until unprotected by light.

When the array surface is exposed to UV light, reactive nucleotides modified with a photolabile protecting group can be added to growing oligonucleotide chains. To target specific nucleotides to exact probe sites, photolithographic masks are used. Each photolithographic mask has a defined pattern of windows, which acts as a filter to either transmit or block UV light from specific features on the chemically protected microarray surface. Areas of the microarray surface in which UV light has been blocked will remain protected from the addition of nucleotides, whereas areas exposed to light will be deprotected, and specific nucleotides can be added. The pattern of windows in each mask directs the order of nucleotide addition. In-situ probe synthesis is therefore accomplished through the cycling of masking, light exposure, and the addition of either adenine (A), cytosine (C), thymine (T), or guanine (G) bases to the growing oligonucleotide.[7]

High-density bead arrays

Similar to the printed and in situ-hybridized microarrays, Bead Arrays (Illumina, San Diego, CA) provide a patterned substrate for the high-density detection of target nucleic acids. However, instead of glass slides or silicon wafers as direct substrates, Bead Arrays rely on 3 μm silica beads that randomly self-assemble onto one of two available substrates: The Sentrix Array Matrix (SAM) or the Sentrix Bead Chip.[8] The SAM contains 96 1.4-mm fiber-optic bundles. Each bundle is an individual array consisting of 50,000 5-μm light-conducting fibers, each of which is chemically etched to create a microwell for a single bead.[8] In the universal Bead Array, up to 1,536 bead types (each with a unique capture sequence) assemble onto each fiber bundle, resulting in ~30 beads of each type in the array.[9] Each SAM allows the analysis of 96 independent samples. The Bead-Chip can be used to assay 1 to 16 samples at a time on a silicon slide that has been processed by micro-electro-mechanical systems technology to provide microwells for individual beads.[10] Bead Chips are more appropriate for very high-density applications such as whole-genome genotyping, which requires 105 to 106 features for determining genome-wide single nucleotide polymorphisms (SNP) (Infinium assay; Illumina).[11]

Unlike the known locations of printed and in situ-hybridized microarray features, the beads in Bead Arrays randomly assort to their final location on the array. Thus, the bead location must be mapped, which is accomplished by a decoding process.[12] This “decoding” process is in contrast to the use of internal dyes for “encoding” the Luminex microspheres discussed in “Suspension Bead Arrays” below. Each bead has ~700,000 copies of a unique capture oligonucleotide covalently attached to it, which serves as the bead's identifier.

Electronic microarrays

The printed and in situ-synthesized microarrays and Bead Arrays described above rely on passive transport for the hybridization of nucleic acids. In contrast, electronic microarrays utilize active hybridization via electric fields to control nucleic acid transport. Microelectronic cartridges (NanoChip 400; Nanogen, San Diego, CA) use complementary metal oxide semiconductor technology for the electronic addressing of nucleic acids.[13] Each NanoChip cartridge has 12 connectors that control 400 individual test sites. Negatively charged nucleic acids are transported to specific sites or features, when a positive current is applied to one or more test sites on the microarray. The surface of the microarray contains streptavidin, which allows for the formation of streptavidin-biotin bonds once electronically addressed biotinylated probes reach their targeted location. The positive current is then removed from the active features, and new test sites can be activated by the targeted application of a positive current. Once the probes have been hybridized at discrete features, the microarray is ready for the application of fluorescently labeled target DNA. Typically, target DNA passively hybridizes with the immobilized probes on the microarray but can also be concentrated electronically. Although addressing the capture probe down first is the most commonly used format, amplicon-down and sandwich assays have also been utilized. Regardless of the addressing format used, if hybridization occurs between the probe and the target DNA, fluorescent reporters will be present at the positive test, which will be detected when the electronic microarray is scanned and analyzed.

Suspension bead arrays

In contrast to the two-dimensional, or planar arrays discussed above, suspension bead arrays are essentially three-dimensional arrays based on the use of microscopic polystyrene spheres (microspheres or beads) as the solid support and flow cytometry for bead and target detection. Furthermore, they are distinct from the high-density Illumina Bead Arrays discussed above, in which the beads are immobilized on fiberoptic strands or silicon slides. Suspension bead-based assays were initially described in 1977 and focused on the detection of antigens and antibodies.[14] Multiplexing was first achieved by using different-sized microsphere sets for the simultaneous detection of multiple antibodies. Currently, more robust multiplexing is accomplished using different microsphere sets based on color. Red (658-nm emission) and infrared (712-nm emission) fluorochromes are used at various concentrations to fill 5.6-μm microspheres. Each bead of the 100-microsphere set has a distinct red-to-infrared ratio, and therefore, each bead has a unique spectral address. Microspheres with a specific spectral address coupled to a specific probe are equivalent to a feature in a planar microarray. Once multiple individual microspheres have been coupled to separate specific probes, a mixture of microspheres (in theory, up to 100) can be used to interrogate extracted and amplified nucleic acids. The subsequent detection of a fluorescent reporter that indicates probe-target DNA hybridization is accomplished using a bench-top flow cytometer. A single-file microsphere suspension passes by two lasers. A 635-nm laser excites the red and infrared fluorochromes impregnated in the microspheres, which allows the classification of the bead and therefore the identity of the probe-target being analyzed. A 532-nm laser excites reporter fluorochromes such as R-phycoerythrin and Alexa 532 to quantify any hybridization that occurs on the microsphere.[14]

Microarray fabrication and experimental design

There are three major types of spotted arrays, defined by the nature of DNA arrayed: cDNA, genomic DNA, or open reading frame-specific oligonucleotides.[15] Oligonucleotide arrays are best suited to situations where the gene boundaries, including their exon/intron organization, are known. Genomic DNA arrays require that introns and intergenic regions are not too common, whereas cDNA arrays have neither of these limitations. The advantages of genomic and cDNA microarrays are that they can be produced from libraries (cDNA or small, random fragments of genomic DNA) in which all inserts are cloned into a common vector such as pBluescript. As a result, only one pair of primers are required to amplify the inserts, which makes the generation of these microarrays relatively routine and inexpensive, albeit laborious. The quality of the resulting microarrays is dependent largely on the quality of the libraries used, including overall complexity and the length of the inserts. For example, non-normalized cDNA libraries will result in some genes being represented multiple times (an advantage for giving high confidence to the data) whereas others, corresponding to rare transcripts, will be completely absent. Another limitation of cDNA and genomic microarrays is that they are unable to discriminate between splice variants or gene family members that are closely related, whereas genomic libraries could contain segments that span two or more genes.

For organisms such as Plasmodium falciparum, whose genomes have been sequenced and annotated, long oligonucleotides (70 mers) can be prepared and spotted directly onto the microarrays.[1617] Although more expensive than the other types, oligonucleotide microarrays have several advantages. First, oligonucleotide microarrays permit distinction between genes that are differentially spliced and/or are members of highly conserved gene families. Second, oligonucleotide microarrays allow discrimination between sense and antisense transcripts through hybridization with strand-specific probes generated by labeling either first or second strand cDNA. Third, oligonucleotide microarrays can be used to reveal polymorphisms although achieving this level of specificity can be technically challenging. Fourth, the use of multiple, independent oligonucleotides for a given gene can increase the confidence in the result. Finally, spotting oligonucleotides directly onto microarrays avoids the laborious and error-prone step of polymerase chain reaction (PCR) amplification and amplicon purification.

Reproducibility of the microarray

Like other techniques, reproducibility is partly a function of the quality of the reagents used. Thus, the DNA to be spotted, as well as the glass on which the microarray will be fabricated, must be meticulously prepared and carefully processed to minimize the level of background fluorescence.[18] Another common concern is having confidence that a spot on the microarray corresponds to the gene assigned to it. This problem was acutely highlighted when researchers using human cDNA microarrays commonly found mistakes in the annotation of the UNIGENE expressed sequence tag (EST) clones used to make the microarrays. The source of this error is still not clear, but most probably arose out of the difficulty of assembling, handling and managing a very large number of clones.[19] Thus, it is advisable to spot onto the microarray two or more independent clones for each gene. In addition, one should validate a microarray by directly sequencing random samples of the spotted material and checking the “register” of the array using probes for specific genes. Finally, for any gene of special interest, it is prudent to expand the data by independent means, such as northern blot or real-time reverse transcriptase PCR, before investing significant effort into its further analysis. Such methods have their own pitfalls and are not necessarily more reliable than well-performed microarray experiments; however, they can provide additional, valuable data (e.g. they can reveal alternative splicing that might not be detected on all arrays).

Parasite development and life cycle

Transcriptional profiling of the changes in gene expression during development offers a new tool in the parasitologist's arsenal to unravel how a parasite develops. The transcriptional profiles of P. falciparum grown in red blood cells revealed that glycolytic and other metabolic enzymes are coordinately upregulated when the parasites are undergoing high rates of growth. By contrast, late schizonts that are ready to undergo egress display reduced global transcriptional levels except for a subset of genes whose transcripts encode proteins involved in signaling.[20] Time-course analysis of gene expression following exposure to some developmental trigger (e.g. nutrient starvation or drug treatment) can identify early-response genes that are probably involved in initiating developmental changes as well as late-response genes that are likely to dictate the unique metabolic and physical properties of that developmental stage. For example, at a late stage during the transition of Toxoplasma gondii tachyzoites to bradyzoites, a large number of genes encoding metabolic enzymes and bradyzoite secretory antigens were upregulated.[21] However, these data also highlight a major limitation of creating microarrays from a cDNA library. Because these microarrays were prepared from a mature bradyzoite cDNA library,[22] spots representing some of the “early-response” genes might not have been present.

One of the greatest challenges to this type of study involves obtaining pure preparations of the developmental stage of interest and generating sufficient quantities of RNA. Large scale, in vitro culture methods and the ability to selectively isolate parasites based on stage-specific molecular markers and/or physical properties can often overcome these challenges. However, the number of manipulations which the parasites undergo between the time of reaching the stage of interest and RNA preparation must be kept to a minimum to avoid inducing changes in gene expression as a result of those manipulations.

Host responses to infection

Most of the above discussion has focused on microarrays comprising parasite sequences. Of course, the essence of parasitology is the interaction between host and pathogen, and arrays consisting of host gene sequences are the perfect complement to the parasite gene arrays. For example, transcriptional profiling of the changes that occur within the host cell during infection, growth, latency, and killing can provide key insights into how the parasite establishes and maintains infection. Reduced to its simplest level, the three major goals of examining how the host responds to infection are elucidating: (1) how does it recognize that it has been infected with a specific parasite and initiate an appropriate immune response?; (2) how does the parasite stimulate these responses?; and (3) do the changes in host transcript abundance favor the host or the parasite? For intimately associated pathogens, the use of infected host material brings with it a special problem, however ensuring that the signal detected is not due to cross-hybridization between parasite transcripts and the host DNA spotted on the microarray. For some highly conserved genes, the sequences present in the parasite and host genomes could be so similar that each hybridizes to the other (e.g. some ribosomal protein genes).[23] In practice, however, such genes are relatively rare and the specificity of the hybridization is sufficient that the problem arises only in very few cases. These rare instances are easily identified in pilot experiments in which pure parasite genomic or cDNA is used as a probe on the host arrays and vice versa.

Recognition by the innate immune system of an infectious agent is a critical step in mounting an immune response against infection. Several groups have categorized the response of dendritic cells, macrophages, and other innate immune cells to pathogenic organisms as well as to specific activators of the innate immune system such as lipopolysaccharide, double-stranded RNA, CpG methylated DNA (in vertebrate systems, cytosines found immediately 50 of guanosines are frequently methylated, depending on whether the given gene is being transcribed or not) and mannan.[23] In many cases, these stimuli modulated common sets of genes that include those encoding chemokines, cytokines and other stress-related proteins. However, each stimulus did modulate its own unique subset of genes, suggesting that the innate immune system discriminates between infectious agents. In addition, some pathogens fail to trigger at least a subset of the common response genes. For example, the response of infected fibroblasts to two intracellular pathogens, T. gondii and Trypanosoma cruzi, varies dramatically, with T. gondii stimulating a rapid and classic proinflammatory response and T. Cruzi inducing much less change and in a very different repertoire of genes.[24]

In terms of the biology of the system, host genes modulated during infection represent three functionally distinct classes: (1) genes that improve the survival of the host (“pro-host”); (2) genes that promote the parasite's growth (“pro-parasite”); and (3) genes incidentally regulated as a consequence of modulating the first two classes (“bystander”). Of course, pro-host genes that ameliorate extreme virulence can serve a crucial role for the effective transmission of any pathogen but, for the purposes of this discussion, we will consider only the short-term consequences of the interactions between a given host and given pathogen within it.

Many pro-host genes (e.g. those encoding interferon-γ, tumor necrosis factor-α and other cytokines) have been previously identified, and microarrays have proven useful in the en masse characterization of their activation by numerous pathogens.[2526] Many more such genes, however, are likely to be identified by these sorts of analysis. On the pro-parasite side, very few genes have so far been definitively identified, but it can safely be assumed that the parasites have evolved to co-opt the host machinery to their own purposes and that changes in gene expression are likely to be one way to accomplish this. For example, intracellular parasites such as T. gondii, Theileria parva and Leishmania sp. are likely to upregulate anti-apoptotic genes as well as host metabolic genes necessary to satisfy their auxotrophies.

Probably the greatest challenge for a biologist is determining the class (pro-host, pro-parasite or bystander) to which a gene belongs. It is obviously not feasible to examine each of the hundreds of modulated genes in detail, and so careful choices will need to be made based on some knowledge of the gene's function and, more importantly, the parasite's biology. Clues will come from a variety of sources, including, for example, the type of host cells that respond in a similar way, the timing of the induction and the response to virulent versus non-virulent strains. Ultimately, the most useful information will come from analysis of mutant hosts (or host cell lines) that produce more or less of the given gene's product. This knockout approach has been very powerful in analyzing the most obvious pro-host genes over the past two decades and will be crucial in the analysis of the less obvious genes identified from microarray experiments.

Dissecting how the parasite effects changes on the host cell will require identifying the parasite factors that modulate host cell transcription and determining the host transcriptional and signaling pathways influenced by infection. Reporter assays based upon transcripts and pathways identified in the microarray experiments can be utilized to facilitate identification of these factors. Finally, it should also be noted that, while the individual bystander genes might not be directly relevant, they could be invaluable in providing clues to pro-parasite genes or pro-parasite transcriptional and signaling pathways through providing a list of genes that respond to the stimulus.[5]

Vaccine development

One of the primary goals of infectious disease research is to prevent infection, by developing a vaccine that protects an individual from the disease in the first place. Historically, finding the right vaccine candidate has been a challenge since researchers typically focused on major surface proteins for potential vaccine candidates, while critical minor proteins would often escape discovery. Using GeneChip arrays, researchers can examine transcriptional activity of all genes of a pathogenic microorganism under in vivo conditions, allowing the identification of even rarely expressed, but potentially important, genes.

Discovering vaccine targets

For example, scientists at the University of Wurzburg, Germany analyzed the genome-wide expression of Neisseria meningitidis (meningococcus) to find surface proteins that were induced under in vivo conditions.[27] They analyzed gene expression during different stages of infection in N. meningitidis serogroup B, one of the serogroups that is responsible for the majority of meningococcal disease in industrialized countries. They were able to identify group-specific antigenic determinants, which served as a basis for rational, protein-based vaccine design against bacterial meningitis. Microarrays not only provide scientists with a quantitatively larger list of potential vaccine candidates, but expression profiles also provide a richer type of information that allows scientists to more quickly identify successful vaccine candidates. For example, scientists studying malaria have used a whole-genome GeneChip array to understand global gene activity during the different stages of the P. falciparum parasite life cycle that transfers from vector to host and back to vector.[28] This group found that most genes used in current vaccine trials are expressed at the same time and map to the same cluster. By looking at the uncharacterized genes from that “vaccine cluster,” the scientists anticipate finding additional vaccine candidates. They are now using follow-up approaches to confirm these potential targets as items of genuine vaccine interest.

CONCLUSION

Microarrays have the unprecedented potential to simultaneously detect and identify thousands of microbial genes, which provides another evolutionary technical advance in the field of clinical microbiology. Although, historically, microarrays have been used largely for gene expression studies, microarrays have gradually been applied in the detection and characterization of microbial pathogens, determination of antimicrobial resistance, typing of microbial pathogens, and monitoring of microbial infections by investigating host genomic expression and polymorphism profiles. Even with these major advances, the potential power behind microarray applications in clinical microbiology has yet to be fully realized. The flexibility and high-throughput nature of current microarray technology offers unprecedented opportunities for parasitic disease research. This technology has placed a completely new set of tools into the microbiologist's arsenal, and has created a novel method of experimental design. Whole genome analysis studies promise to rapidly accelerate our understanding of the host-pathogen interaction, and to allow major changes in clinical approaches to infectious disease detection, treatment, and prevention, not only in humans, but in animals and plants as well.