Detection of bacterial pathogens in environmental samples using DNA microarrays

Polymerase chain reaction (PCR) is an important tool for pathogen detection, but historically, it has not been possible to accurately identify PCR products without sequencing, Southern blots, or dot-blots. Microarrays can be coupled with PCR where they serve as a set of parallel dot-blots to enhance product detection and identification. Microarrays are composed of many discretely located probes on a solid substrate such as glass. Each probe is composed of a sequence that is complimentary to a pathogen-specific gene sequence. PCR is used to amplify one or more genes and the products are then hybridized to the array to identify species-specific polymorphism within one or more genes. We illustrate this type of array using 16S rDNA probes suitable for distinguishing between several salmonid pathogens. We also describe the use of microarrays for direct detection of either RNA or DNA without the aid of PCR, although the sensitivity of these systems currently limits their application for pathogen detection. Finally, microarrays can also be used to "fingerprint" bacterial isolates and they can be used to identify diagnostic markers suitable for developing new PCR-based detection assays. We illustrate this type of array for subtyping an important food-borne pathogen, Listeria monocytogenes.     

Horizontal gene transfer: sustaining pathogenicity and optimizing host–pathogen interactions

Successful host–pathogen interactions require the presence, maintenance and expression of gene cassettes called 'pathogenicity islands' (PAIs) and 'metabolic islands' (MAIs) in the respective pathogen. The products of these genes confer on the pathogen the means to recognize their host(s) and to efficiently evade host defences in order to colonize, propagate within the host and eventually disseminate from the host. Virulence effectors secreted by type III and type IV secretion systems, among others, play vital roles in sustaining pathogenicity and optimizing host–pathogen interactions. Complete genome sequences of plant pathogenic bacteria have revealed the presence of PAIs and MAIs. The genes of these islands possess mosaic structures with regions displaying differences in nucleotide composition and codon usage in relation to adjacent genome structures, features that are highly suggestive of their acquisition from a foreign donor. These donors can be other bacteria, as well as lower members of the Archaea and Eukarya. Genes that have moved from the domains Archaea and Eukarya to the domain Bacteria are true cases of horizontal gene transfer. They represent interdomain genetic transfer. Genetic exchange between distinct members of the domain Bacteria, however, represents lateral gene transfer, an intradomain event. Both horizontal and lateral gene transfer events have been used to facilitate survival fitness of the pathogen.     

Antibody-mediated resistance against plant pathogens

Plant diseases have a significant impact on the yield and quality of crops. Many strategies have been developed to combat plant diseases, including the transfer of resistance genes to crops by conventional breeding. However, resistance genes can only be introgressed from sexually-compatible species, so breeders need alternative measures to introduce resistance traits from more distant sources. In this context, genetic engineering provides an opportunity to exploit diverse and novel forms of resistance, e.g. the use of recombinant antibodies targeting plant pathogens. Native antibodies, as a part of the vertebrate adaptive immune system, can bind to foreign antigens and eliminate them from the body. The ectopic expression of antibodies in plants can also interfere with pathogen activity to confer disease resistance. With sufficient knowledge of the pathogen life cycle, it is possible to counter any disease by designing expression constructs so that pathogen-specific antibodies accumulate at high levels in appropriate sub-cellular compartments. Although first developed to tackle plant viruses and still used predominantly for this purpose, antibodies have been targeted against a diverse range of pathogens as well as proteins involved in plant–pathogen interactions. Here we comprehensively review the development and implementation of antibody-mediated disease resistance in plants.     

DNA microarrays: new tools in the analysis of plant defence responses

Large-scale DNA sequencing is providing information on the number and organization of genes and genomes of plant species and their pathogens. The next phase is to identify gene functions and gene networks with key roles in compatible and incompatible plant–pathogen interactions. DNA microarrays can provide information on the expression patterns of thousands of genes in parallel. The application of this technology is already revealing new features of plant–pathogen interactions and will be a key tool for a wide range of experiments in molecular plant pathology.     

Tolerance to oxidative stress is associated with both oxidative stress response and inherent growth in a fungal wheat pathogen

Reactive oxygen species are toxic byproducts of aerobic respiration that are also important in mediating a diversity of cellular functions. Reactive oxygen species form an important component of plant defenses to inhibit microbial pathogens during pathogen–plant interactions. Tolerance to oxidative stress is likely to make a significant contribution to the viability and pathogenicity of plant pathogens, but the complex network of oxidative stress responses hinders identification of the genes contributing to this trait. Here, we employed a forward genetic approach to investigate the genetic architecture of oxidative stress tolerance in the fungal wheat pathogen Zymoseptoria tritici. We used quantitative trait locus (QTL) mapping of growth and melanization under axenic conditions in two cross-populations to identify genomic regions associated with tolerance to oxidative stress. We found that QTLs associated with growth under oxidative stress as well as inherent growth can affect oxidative stress tolerance, and we identified two uncharacterized genes in a major QTL associated with this trait. Our data suggest that melanization does not affect tolerance to oxidative stress, which differs from what was found for animal pathogens. This study provides a whole-genome perspective on the genetic basis of oxidative stress tolerance in a plant pathogen.     

Genetics-squared: combining host and pathogen genetics in the analysis of innate immunity and bacterial virulence

The interaction of bacterial pathogens with their hosts’ innate immune systems can be extremely complex and is often difficult to disentangle experimentally. Using mouse models of bacterial infections, several laboratories have successfully applied genetic approaches to identify novel host genes required for innate immune defense. In addition, a variety of creative bacterial genetic schemes have been developed to identify key bacterial genes involved in triggering or evading host immunity. In cases where both the host and pathogen are amenable to genetic manipulation, a combination of host and pathogen genetic approaches can be used. Focusing on bacterial infections of mice, this review summarizes the benefits and limitations of applying genetic analysis to the study of host–pathogen interactions. In particular, we consider how prokaryotic and eukaryotic genetic strategies can be combined, or “squared,” to yield new insights in host–pathogen biology.     

Mathematical and computational approaches can complement experimental studies of host–pathogen interactions

In addition to traditional and novel experimental approaches to study host–pathogen interactions, mathematical and computer modelling have recently been applied to address open questions in this area. These modelling tools not only offer an additional avenue for exploring disease dynamics at multiple biological scales, but also complement and extend knowledge gained via experimental tools. In this review, we outline four examples where modelling has complemented current experimental techniques in a way that can or has already pushed our knowledge of host–pathogen dynamics forward. Two of the modelling approaches presented go hand in hand with articles in this issue exploring fluorescence resonance energy transfer and two-photon intravital microscopy. Two others explore virtual or ' in silico ' deletion and depletion as well as a new method to understand and guide studies in genetic epidemiology. In each of these examples, the complementary nature of modelling and experiment is discussed. We further note that multi-scale modelling may allow us to integrate information across length (molecular, cellular, tissue, organism, population) and time (e.g. seconds to lifetimes). In sum, when combined, these compatible approaches offer new opportunities for understanding host–pathogen interactions.     

Comparative pathogenesis of Mycobacterium marinum and Mycobacterium tuberculosis

A thorough understanding of Mycobacterium tuberculosis pathogenesis in humans has been elusive in part because of imperfect surrogate laboratory hosts, each with its own idiosyncrasies. Mycobacterium marinum is the closest genetic relative of the M. tuberculosis complex and is a natural pathogen of ectotherms. In this review, we present evidence that the similar genetic programmes of M. marinum and M. tuberculosis and the corresponding host immune responses reveal a conserved skeleton of Mycobacterium host–pathogen interactions. While both species have made niche-specific refinements, an essential framework has persisted. We highlight genetic comparisons of the two organisms and studies of M. marinum in the developing zebrafish. By pairing M. marinum with the simplified immune system of zebrafish embryos, many of the defining mechanisms of mycobacterial pathogenesis can be distilled and investigated in a tractable host/pathogen pair.     

Genetic approaches to dissect plant nonhost resistance mechanisms

Nonhost resistance (NHR) refers to the immunity of most tested genotypes of a plant species to most tested variants of a pathogen species. Thus, NHR is broad spectrum and durable in nature and constitutes a major safety barrier against invasion of a myriad of potentially pathogenic microbes in any plants including domesticated crops. Genetic study of NHR is generally more difficult compared to host resistance mainly because NHR is genetically more complicated and often lacks intraspecific polymorphisms. Nevertheless, substantial progress has been made towards the understanding of the molecular basis of NHR in the past two decades using various approaches. Not surprisingly, molecular mechanisms of NHR revealed so far encompasses pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity. In this review, we briefly discuss the inherent difficulty in genetic studies of NHR and summarize the main approaches that have been taken to identify genes contributing to NHR. We also discuss new enabling strategies for dissecting multilayered NHR in model plants with a focus on NHR against filamentous pathogens, especially biotrophic pathogens such as powdery mildew and rust fungi.     

Host-pathogen interplay and the evolution of bacterial effectors

Many bacterial pathogens require a type III secretion system (T3SS) and suite of type III secreted effectors (T3SEs) to successfully colonize their hosts, extract nutrients and consequently cause disease. T3SEs, in particular, are key components of the bacterial arsenal, as they function directly inside the host to disrupt or suppress critical components of the defence network. The development of host defence and surveillance systems imposes intense selective pressures on these bacterial virulence factors, resulting in a host–pathogen co-evolutionary arms race. This arms race leaves its genetic signature in the pattern and structure of natural genetic variation found in T3SEs, thereby permitting us to infer the specific evolutionary processes and pressures driving these interactions. In this review, we summarize our current knowledge of T3SS-mediated host–pathogen co-evolution. We examine the evolution of the T3SS and the T3SEs that traverse it, in both plant and animal pathosystems, and discuss the processes that maintain these important pathogenicity determinants within pathogen populations. We go on to examine the possible origins of T3SEs, the mechanisms that give rise to new T3SEs and the processes that underlie their evolution.     

Genetic diversity in cytokines associated with immune variation and resistance to multiple pathogens in a natural rodent population

Pathogens are believed to drive genetic diversity at host loci involved in immunity to infectious disease. To date, studies exploring the genetic basis of pathogen resistance in the wild have focussed almost exclusively on genes of the Major Histocompatibility Complex (MHC); the role of genetic variation elsewhere in the genome as a basis for variation in pathogen resistance has rarely been explored in natural populations. Cytokines are signalling molecules with a role in many immunological and physiological processes. Here we use a natural population of field voles (Microtus agrestis) to examine how genetic diversity at a suite of cytokine and other immune loci impacts the immune response phenotype and resistance to several endemic pathogen species. By using linear models to first control for a range of non-genetic factors, we demonstrate strong effects of genetic variation at cytokine loci both on host immunological parameters and on resistance to multiple pathogens. These effects were primarily localized to three cytokine genes (Interleukin 1 beta (Il1b), Il2, and Il12b), rather than to other cytokines tested, or to membrane-bound, non-cytokine immune loci. The observed genetic effects were as great as for other intrinsic factors such as sex and body weight. Our results demonstrate that genetic diversity at cytokine loci is a novel and important source of individual variation in immune function and pathogen resistance in natural populations. The products of these loci are therefore likely to affect interactions between pathogens and help determine survival and reproductive success in natural populations. Our study also highlights the utility of wild rodents as a model of ecological immunology, to better understand the causes and consequences of variation in immune function in natural populations including humans.     

Pathogens as potential selective agents in the wild

Pathogens are considered a serious threat to which wild populations must adapt, most particularly under conditions of rapid environmental change. One way host adaptation has been studied is through genetic population structure at the major histocompatibility complex (MHC), a complex of adaptive genes involved in pathogen resistance in vertebrates. However, while associations between specific pathogens and MHC alleles or diversity have been documented from laboratory studies, the interaction between hosts and pathogens in the wild is more complex. As such, identifying selective agents and understanding underlying co-evolutionary mechanisms remains a major challenge. In this issue of Molecular Ecology , Evans & Neff (2009) characterized spatial and temporal variation in the bacterial parasite community infecting Chinook salmon ( Oncorhynchus tshawytscha ) fry from five populations in British Columbia, Canada. They used a 16S rDNA sequencing-based approach to examine the prevalence of bacterial infection in kidney and looked for associations with MHC class I and II genetic variability. The authors found a high diversity of bacteria infecting fry, albeit at low prevalence. It was reasoned that spatial variability in infection rate and bacterial community phylogenetic similarity found across populations may represent differential pathogen-mediated selection pressures. The study revealed some evidence of heterozygote advantage at MHC class II, but not class I, and preliminary associations between specific MHC alleles and bacterial infections were uncovered. This research adds an interesting perspective to the debate on host–pathogen co-evolutionary mechanisms and emphasizes the importance of considering the complexity of pathogen communities in studies of host local adaptation.     

In silico identification of potential therapeutic targets in the human pathogen Helicobacter pylori

Availability of genome sequences of pathogens has provided a tremendous amount of information that can be useful in drug target and vaccine target identification. One of the recently adopted strategies is based on a subtractive genomics approach, in which the subtraction dataset between the host and pathogen genome provides information for a set of genes that are likely to be essential to the pathogen but absent in the host. This approach has been used successfully in recent times to identify essential genes in Pseudomonas aeruginosa. We have used the same methodology to analyse the whole genome sequence of the human gastric pathogen Helicobacter pylori. Our analysis revealed that out of the 1590 coding sequences of the pathogen, 40 represent essential genes that have no human homolog. We have further analysed these 40 genes by the protein sequence databases to list some 10 genes whose products are possibly exposed on the pathogen surface. This preliminary work reported here identifies a small subset of the Helicobacter proteome that might be investigated further for identifying potential drug and vaccine targets in this pathogen.     

Fungal multilocus sequence typing--it's not just for bacteria

Multilocus sequence typing uses nucleotide sequence from several genes to identify individual microbial pathogens. The data obtained for multilocus sequence typing can be used to recognize fungal species and to determine if the fungi are purely clonal, or if they also recombine. Genetic regions with more polymorphisms and microsatellites might be used to recognize populations within species and are well suited to Bayesian methods of assigning unknown individuals to populations of origin. Knowledge of species, populations and reproductive mode can help answer questions common to all emerging diseases: is the disease due to the recent spread of a pathogen, to the emergence of a virulent strain of an existing pathogen, or to a change in the environment that promotes disease?     

Proteomic analysis of differentially expressed proteins in fungal elicitor-treated Arabidopsis cell cultures

Slow progress has been made in discovering plant genes governing the interaction of plant pathogens and their hosts using classical genetic approaches. Extensive studies employing DNA microarray techniques to identify global changes in gene expression during pathogen-host interaction have greatly enhanced discovery of genetic components regulating the plant defence response to pathogen attack. In this study, a complementary approach was used to identify changes in protein abundance during interaction of Arabidopsis cell cultures with a pathogen-derived elicitor. The soluble protein fractions were analysed by two-dimensional difference gel electrophoresis and proteins differentially expressed in response to treatment with fungal elicitor were identified via matrix-assisted laser desorption ionization-time of flight mass spectrometry. Elicitor responsive proteins included molecular chaperones, oxidative stress defence proteins, mitochondrial proteins, and enzymes of a diverse number of metabolic pathways. The findings, in combination with currently available microarray data, will form the basis of a filter to identify pivotal genes whose role in pathogen defence systems will require confirmation using gene knockout mutants.