Infectious Disorders - Drug Targets (Formerly Current Drug Targets - Infectious Disorders) - Volume 6, Issue 3, 2006

Microbiologists have realized that it is unlikely that all virulence determinants of a human pathogen could be identified simply by studying the pathogen in the laboratory since it is technically impossible to determine and mimic all of the complex and changing environmental stimuli that occur at the site of an infection. This shortcoming hampers our complete understanding of the virulence mechanisms employed by human pathogens, which is reflected in the relatively small number of effective vaccines that are currently available to combat the myriad of infections that afflict mankind. To overcome this problem, a number of investigators have designed methods to identify genes of pathogens that are specifically expressed during infection. The advent of in vivo expression technology (IVET) in 1993 triggered a concerted effort in various fields of microbiology to seek for differentially-expressed and/or in vivo induced genes. These genes were originally proposed as likely targets for the development of new vaccine, diagnostic and antibiotherapy strategies in the medical field. In counterpart, other non-human systems quickly followed the trend and included the development of methods particularly suited to study plant and animal pathogens in vivo. This Special Edition of Infectious Disorders-Drug Targets reviews some of the latest and outstanding developments accomplished using a number of methods that focus on differentially expressed genes to further our understanding of molecular microbial pathogenesis. A plethora of reviews have exhaustively covered many aspects of the novel techniques that led to the identification of numerous differentially expressed microbial genes. The present overview focuses on those methods that show substantial promise for-or have already led to-novel approaches for diagnosing, preventing or treating microbial diseases. In the first two papers, methods based on in vivo screens and selection will be presented. Jackson and Giddens (Development and Application of In Vivo Expression Technology (IVET) for Analysing Microbial Gene Expression in Complex Environments) will describe IVET and its various spin-offs as tools for analyzing microbial gene expression in complex environments and providing new targets for biotechnological development. Bossé et al. (High-Throughput Identification of Conditionally Essential Genes in Bacteria: From STM to TSM) will next depict the latest development obtained with signature-tagged mutagenesis (STM) and transposon screen by microarray (TSM), which combine the negative-selection principle of STM with the genome-wide screening strength of DNA microarrays. The next three papers will focus on transcriptomic approaches that have been used to dissect hostpathogen interactions. A review on the recent and ongoing developments obtained with bacterial microarrays will first be presented by Chen (DNA Microarrays - An Armory for Combating Infectious Diseases in the New Century). Besides describing exciting progress in microarray technology applied to the study of microbial pathogenesis, drug response, vaccine development and disease agent identification, Chen will address certain issues and challenges in the analysis, management and interpretation of microarray data. Kronstad (Serial Analysis of Gene Expression in Eukaryotic Pathogens) will then present SAGE as an alternative technique to microarrays to obtain information on transcript abundance and differential RNA expression, particularly with eukaryotic systems such as Saccharomyces cerevisiae and Caenorhabditis elegans. Finally, Mans et al. (Microarray Analysis of Human Epithelial Cell Responses to Bacterial Interaction) will present how the host transcriptional responses have been recently used to infer the function of virulence determinants of bacterial pathogens that are interacting with the epithelial mucosa during disease. 206 Infectious Disorders - Drug Targets 2006, Vol. 6, No. 3 Editorial In the last two papers, particular emphasis will be granted to the use of proteomic approaches leading to understanding of microbial pathogenesis and development of new vaccine, diagnostic and antibiotherapy tools. Lamont et al. (Mass Spectrometry-Based Proteomics and Its Application to Studies of Porphyromonas gingivalis Invasion and Pathogenicity) will depict the recent advances in proteomic methods based on multidimensional capillary HPLC and tandem mass spectrometry, which allow the acquisition of comprehensive protein expression datasets. These datasets are comparable with spotted cDNA arrays in terms of coverage and quantitative precision. Finally, Handfield and Hillman (In Vivo Induced Antigen Technology (IVIAT) and Change Mediated Antigen Technology (CMAT)) will review recent applications of IVIAT and CMAT to various human and plant pathogens.

Establishing the mechanisms by which microbes interact with their environment, including eukaryotic hosts, is a major challenge that is essential for the economic utilisation of microbes and their products. Techniques for determining global gene expression profiles of microbes, such as microarray analyses, are often hampered by methodological restraints, particularly the recovery of bacterial transcripts (RNA) from complex mixtures and rapid degradation of RNA. A pioneering technology that avoids this problem is In Vivo Expression Technology (IVET). IVET is a ‘promotertrapping’ methodology that can be used to capture nearly all bacterial promoters (genes) upregulated during a microbeenvironment interaction. IVET is especially useful because there is virtually no limit to the type of environment used (examples to date include soil, oomycete, a host plant or animal) to select for active microbial promoters. Furthermore, IVET provides a powerful method to identify genes that are often overlooked during genomic annotation, and has proven to be a flexible technology that can provide even more information than identification of gene expression profiles. A derivative of IVET, termed resolvase-IVET (RIVET), can be used to provide spatio-temporal information about environment-specific gene expression. More recently, niche-specific genes captured during an IVET screen have been exploited to identify the regulatory mechanisms controlling their expression. Overall, IVET and its various spin-offs have proven to be a valuable and robust set of tools for analysing microbial gene expression in complex environments and providing new targets for biotechnological development.

Signature-tagged mutagenesis (STM) provided the first widely applicable high-throughput method for detecting conditionally essential genes in bacteria by using negative selection to screen large pools of transposon (Tn) mutants. STM requires no prior knowledge of the bacterium's genome sequence, and has been used to study a large number of Gram-positive and Gram-negative species, greatly expanding the repertoires of known virulence factors for these organisms. Originally, hybridization of radiolabelled probes to colony or dot blots was used to detect differences in populations of tagged mutants before and after growth under a selective condition. Modifications of the tag detection method involving polymerase chain reaction (PCR) amplification and visualisation by gel electrophoresis have been developed and can be automated through the use of robotics. Genetic footprinting is another negative selection technique that uses PCR amplification to detect loss of mutants from a pool. Unlike PCR-STM, this technique allows direct amplification of Tn-flanking sequences. However, it requires the bacterium's whole genome sequence in order to design specific primers for every gene of interest. More recently, a number of techniques have been described that combine the negative-selection principle of STM and genetic footprinting with the genome-wide screening power of DNA microarrays. These techniques, although also requiring whole genome sequences, use either a form of linker-mediated or semi-random PCR to amplify and label Tn-flanking regions for hybridization to microarrays. The superior sensitivity microarray detection allows greater numbers of mutants to be screened per pool, as well as determination of the coverage/distribution of insertions in the library prior to screening, two significant advantages over STM.

DNA microarrays are high-throughput platforms that take advantage of the vast amount of sequence information and allow scientists to perform gene expression profiling or genotyping studies on a “global” or “genomewide” scale. The global monitoring of gene expression in hosts and pathogens, either separately or interactively, has given us systemic views of the disease mechanisms. Ongoing improvements in DNA sequencing and microarray technologies continue to open up new opportunities for better understanding and developing more effective approaches in diagnosis, treatments, and preventions of infectious diseases. This review focuses on the latest developments and applications of the DNA microarray technologies designed for studying pathogens in the areas of pathogenesis, host-pathogen interaction, drug response, vaccine development, and disease agent identification. Issues and challenges in the analysis, management and interpretation of microarray data are also addressed.

The tag-based method of serial analysis of gene expression (SAGE) has been used to measure mRNA abundance and differential expression in a variety of organisms including several parasites and fungal pathogens. SAGE is based on the collection of short sequence tags as a measure of transcript abundance and the method provides an alternative, and in some instances, complementary approach to array-based methods of measuring differential gene expression. These methods are being used to improve our molecular understanding of the pathogenesis of eukaryotic microbes and SAGE in particular presents valuable opportunities for gene discovery and genome annotation. For eukaryotic pathogens, the SAGE method has been employed for the parasites Plasmodium falciparum, Toxoplasma gondii and Giardia lamblia, as well as fungal pathogens of plants (Magnaporthe grisea, Blumeria graminis, Ustilago maydis) and humans (Cryptococcus neoformans, Coccidiodes posadasii, Trichophyton rubrum). The accumulating information promises to speed the identification of key pathogen functions for virulence and proliferation in the host with the hope that some of these will represent important targets for drug and vaccine development.

Host-pathogen interactions are inherently complex and dynamic. The recent use of human microarrays has been invaluable to monitor the effects of various bacterial and viral pathogens upon host cell gene expression programs. This methodology has allowed the host response transcriptome of several cell lines to be studied on a global scale. To this point, the great majority of reports have focused on the response of immune cells, including macrophages and dendritic cells. These studies revealed that the immune response to microbial pathogens is tailored to different microbial challenges. Conversely, the paradigm for epithelial cells has-until recently-held that the epithelium mostly served as a relatively passive physical barrier to infection. It is now generally accepted that the epithelial barrier contributes more actively to signaling events in the immune response. In light of this shift, this review will compare transcriptional profiling data from studies that involved host-pathogen interactions occurring with epithelial cells. Experiments that defined both a common core response, as well as pathogen-specific host responses will be discussed. This review will also summarize the contributions that transcriptional profiling analysis has made to our understanding of bacterial physio-pathogensis of infection. This will include a discussion of how host transcriptional responses can be used to infer the function of virulence determinants from bacterial pathogens interacting with epithelial mucosa. In particular, we will expand upon the lessons that have been learned from gastro-intestinal and oral pathogens, as well as from members of the commensal flora.

Porphyromonas gingivalis is a Gram-negative anaerobe that populates the subgingival crevice of the mouth. It is known to undergo a transition from its commensal status in healthy individuals to a highly invasive intracellular pathogen in human patients suffering from periodontal disease, where it is often the dominant species of pathogenic bacteria. The application of mass spectrometry-based proteomics to the study of P. gingivalis interactions with model host cell systems, invasion and pathogenicity is reviewed. These studies have evolved from qualitative identifications of small numbers of secreted proteins, using traditional gel-based methods, to quantitative whole cell proteomic studies using multiple dimension capillary HPLC coupled with linear ion trap mass spectrometry. It has become possible to generate a differential readout of protein expression change over the entire P. gingivalis proteome, in a manner analogous to whole genome mRNA arrays. Different strategies have been employed for generating protein level expression ratios from mass spectrometry data, including stable isotope metabolic labeling and most recently, spectral counting methods. A global view of changes in protein modification status remains elusive due to the limitations of existing computational tools for database searching and data mining. Such a view would be desirable for purposes of making global assessments of changes in gene regulation in response to host interactions during the course of adhesion, invasion and internalization. With a complete data matrix consisting of changes in transcription, protein abundance and protein modification during the course of invasion, the search for new protein drug targets would benefit from a more comprehensive understanding of these processes than what could be achieved prior to the advent of systems biology.

In this chapter, an overview of in vivo induced antigen technology (IVIAT) and change mediated antigen technology (CMAT) will be presented, including a discussion of the advantages and limitations of these methods. Over fifteen different microbial pathogens have been or are known to be currently studied with these methods. Salient data obtained from the application of IVIAT and/or CMAT to a selection of human and plant pathogens will be summarized. This includes recent reports on Streptococcus pyogenes (Group A) in neurological disorders and invasive diseases, Xylella fastidiosa in Pierce's disease, Xanthomonas campestris in bean blight, Salmonella enterica serovar typhi in typhoid fever and Leishmania spp. related infections. Special emphasis will be given to those targets that have been further investigated for the development of novel vaccine, diagnostic and/or antibiotherapy strategies. This encompasses a new point-of-care serological diagnostic test for chronic periodontal diseases. Finally, Mycobacterium tuberculosis in vivo induced products will be described as providing a rational basis for differentiating subjects with primary, dormant or secondary tuberculosis infections, from control subjects who have or did not have prior vaccination with BCG.