Microbial genome analysis: insights into virulence, host adaptation and evolution

Genome analysis of microbial pathogens has provided unique insights into their virulence, host adaptation and evolution. Common themes have emerged, including lateral gene transfer among enteric pathogens, genome decay among obligate intracellular pathogens and antigenic variation among mucosal pathogens. The advent of post-genomic approaches and the sequencing of the human genome will enable scientists to investigate the complex and dynamic interplay between host and pathogen. This wealth of information will catalyse the development of new intervention strategies to reduce the burden of microbial-related disease.     

An assessment of opportunities to dissect host genetic variation in resistance to infectious diseases in livestock

This paper reviews the evidence for host genetic variation in resistance to infectious diseases for a wide variety of diseases of economic importance in poultry, cattle, pig, sheep and Atlantic salmon. Further, it develops a method of ranking each disease in terms of its overall impact, and combines this ranking with published evidence for host genetic variation and information on the current state of genomic tools in each host species. The outcome is an overall ranking of the amenability of each disease to genomic studies that dissect host genetic variation in resistance. Six disease-based assessment criteria were defined: industry concern, economic impact, public concern, threat to food safety or zoonotic potential, impact on animal welfare and threat to international trade barriers. For each category, a subjective score was assigned to each disease according to the relative strength of evidence, impact, concern or threat posed by that particular disease, and the scores were summed across categories. Evidence for host genetic variation in resistance was determined from available published data, including breed comparison, heritability studies, quantitative trait loci (QTL) studies, evidence of candidate genes with significant effects, data on pathogen sequence and on host gene expression analyses. In total, 16 poultry diseases, 13 cattle diseases, nine pig diseases, 11 sheep diseases and three Atlantic salmon diseases were assessed. The top-ranking diseases or pathogens, i.e. those most amenable to studies dissecting host genetic variation, were Salmonella in poultry, bovine mastitis, Marek's disease and coccidiosis, both in poultry. The top-ranking diseases or pathogens in pigs, sheep and Atlantic salmon were Escherichia coli, mastitis and infectious pancreatic necrosis, respectively. These rankings summarise the current state of knowledge for each disease and broadly, although not entirely, reflect current international research efforts. They will alter as more information becomes available and as genome tools become more sophisticated for each species. It is suggested that this approach could be used to rank diseases from other perspectives as well, e.g. in terms of disease control strategies.     

Genome hyperevolution and the success of a parasite

The strategy of antigenic variation is to present a constantly changing population phenotype that enhances parasite transmission, through evasion of immunity arising within, or existing between, host animals. Trypanosome antigenic variation occurs through spontaneous switching among members of a silent archive of many hundreds of variant surface glycoprotein (VSG) antigen genes. As with such contingency systems in other pathogens, switching appears to be triggered through inherently unstable DNA sequences. The archive occupies subtelomeres, a genome partition that promotes hypermutagenesis and, through telomere position effects, singular expression of VSG. Trypanosome antigenic variation is augmented greatly by the formation of mosaic genes from segments of pseudo-VSG, an example of implicit genetic information. Hypermutation occurs apparently evenly across the whole archive, without direct selection on individual VSG, demonstrating second-order selection of the underlying mechanisms. Coordination of antigenic variation, and thereby transmission, occurs through networking of trypanosome traits expressed at different scales from molecules to host populations.     

The effects of symmetry on the dynamics of antigenic variation

In the studies of dynamics of pathogens and their interactions with a host immune system, an important role is played by the structure of antigenic variants associated with a pathogen. Using the example of a model of antigenic variation in malaria, we show how many of the observed dynamical regimes can be explained in terms of the symmetry of interactions between different antigenic variants. The results of this analysis are quite generic, and have wider implications for understanding the dynamics of immune escape of other parasites, as well as for the dynamics of multi-strain diseases.     

人源札幌病毒抗原多样性及遗传进化研究进展

人源札幌病毒(human sapovirus, HuSaV)是全球范围内引起散发性急性胃肠炎和相关疫情的重要病原,尤其对婴幼儿及免疫缺陷患者等高危人群存有致死的危险,人源札幌病毒具有丰富的抗原和遗传多样性,其抗原多样性及免疫原性主要位于P2亚结构域,并且衣壳蛋白免疫原性是人源札幌病毒疫苗研发的理论基础。由于人源札幌病毒可以耐受高衣壳突变而不失去病毒功能使它得以迅速进化,其在宿主体内进化过程中存在连续的氨基酸突变,且突变主要在VP1的P结构域内积累,少见于非结构蛋白和VP2中。序列和结构的改变使得人源札幌病毒逃脱先前存在的群体免疫,有必要进一步探索人源札幌病毒的免疫逃逸机制及其拮抗宿主的免疫应答。因此,本文针对人源札幌病毒在基因组特征、抗原多样性特点、遗传进化机制等领域的研究进展进行了系统综述,并对未来研究中亟待解决的科学问题进行了展望。     

Shared themes of antigenic variation and virulence in bacterial, protozoal, and fungal infections.

Pathogenic microbes have evolved highly sophisticated mechanisms for colonizing host tissues and evading or deflecting assault by the immune response. The ability of these microbes to avoid clearance prolongs infection, thereby promoting their long-term survival within individual hosts and, through transmission, between hosts. Many pathogens are capable of extensive antigenic changes in the face of the multiple constitutive and dynamic components of host immune defenses. As a result, highly diverse populations that have widely different virulence properties can arise from a single infecting organism (clone). In this review, we consider the molecular and genetic features of antigenic variation and corresponding host-parasite interactions of different pathogenic bacterial, fungal, and protozoan microorganisms. The host and microbial molecules involved in these interactions often determine the adhesive, invasive, and antigenic properties of the infecting organisms and can dramatically affect the virulence and pathobiology of individual infections. Pathogens capable of such antigenic variation exhibit mechanisms of rapid mutability in confined chromosomal regions containing specialized genes designated contingency genes. The mechanisms of hypermutability of contingency genes are common to a variety of bacterial and eukaryotic pathogens and include promoter alterations, reading-frame shifts, gene conversion events, genomic rearrangements, and point mutations.     

How protozoan parasites evade the immune response

Protozoan pathogens such as Plasmodium, Leishmania, Trypanosoma and Entamoeba are responsible for several of the most widespread and lethal human diseases. Their successful survival depends mainly on evading the host immune system by, for example, penetrating and multiplying within cells, varying their surface antigens, eliminating their protein coat, and modulating the host immune response. Immunosuppression is sometimes caused directly by parasite products and sometimes involves antigenic mimicry, which often appears in association with parasitic diseases. However, one of the most sophisticated mechanisms of evasion is the selective activation of a subset of T helper cells.     

Why are parasite contingency genes often associated with telomeres?

Contingency genes are common in pathogenic microbes and enable, through pre-emptive mutational events, rapid, clonal switches in phenotype that are conducive to survival and proliferation in hosts. Antigenic variation, which is a highly successful survival strategy employed by eubacterial and eukaryotic pathogens, involves large repertoires of distinct contingency genes that are expressed differentially, enabling evasion of host acquired immunity. Most, but not all, antigenic variation systems make extensive use of subtelomeres. Study of model systems has shown that subtelomeres have unusual properties, including reversible silencing of genes mediated by proteins binding to the telomere, and engagement in ectopic recombination with other subtelomeres. There is a general theory that subtelomeric location confers a capacity for gene diversification through such recombination, although experimental evidence is that there is no increased mitotic recombination at such loci and that sequence homogenisation occurs. Possible benefits of subtelomeric location for pathogen contingency systems are reversible gene silencing, which could contribute to systems for gene switching and mutually exclusive expression, and ectopic recombination, leading to gene family diversification. We examine, in several antigenic variation systems, what possible benefits apply.     

Intracellular pathogens go extreme: genome evolution in the Rickettsiales

The Rickettsiales, a genetically diverse group of the alpha-Proteobacteria, include major mammalian pathogens, such as the agents of epidemic typhus, scrub typhus, ehrlichioses and heartwater disease. Sequenced genomes of this bacterial order have provided exciting insights into reductive genome evolution, antigenic variation and host cell manipulation. Recent results suggest that human pathogens emerged relatively late in the evolution of the Rickettsiales. Surprisingly, there is no association between pathogenicity and the acquisition of novel virulence genes. Here, we explore the genomic differences between members of the Rickettsiales and ask what are the changes that enable infectious agents to emerge from seemingly harmless bacteria.     

Variable VlsE Is Critical for Host Reinfection by the Lyme Disease Spirochete

Many pathogens make use of antigenic variation as a way to evade the host immune response. A key mechanism for immune evasion and persistent infection by the Lyme disease spirochete, Borrelia burgdorferi, is antigenic variation of the VlsE surface protein. Recombination results in changes in the VlsE surface protein that prevent recognition by VlsE-specific antibodies in the infected host. Despite the presence of a substantial number of additional proteins residing on the bacterial surface, VlsE is the only known antigen that exhibits ongoing variation of its surface epitopes. This suggests that B. burgdorferi may utilize a VlsE-mediated system for immune avoidance of its surface antigens. To address this, the requirement of VlsE for host reinfection by the Lyme disease pathogen was investigated. Host-adapted wild type and VlsE mutant spirochetes were used to reinfect immunocompetent mice that had naturally cleared an infection with a VlsE-deficient clone. Our results demonstrate that variable VlsE is necessary for reinfection by B. burgdorferi, and this ability is directly related to evasion of the host antibody response. Moreover, the data presented here raise the possibility that VlsE prevents recognition of B. burgdorferi surface antigens from host antibodies. Overall, our findings represent a significant advance in our knowledge of immune evasion by B. burgdorferi, and provide insight to the possible mechanisms involved in VlsE-mediated immune avoidance.     

Trafficking of STEVOR to the Maurer's clefts in Plasmodium falciparum-infected erythrocytes

The human malarial parasite Plasmodium falciparum exports proteins to destinations within its host erythrocyte, including cytosol, surface and membranous profiles of parasite origin termed Maurer's clefts. Although several of these exported proteins are determinants of pathology and virulence, the mechanisms and trafficking signals underpinning protein export are largely uncharacterized-particularly for exported transmembrane proteins. Here, we have investigated the signals mediating trafficking of STEVOR, a family of transmembrane proteins located at the Maurer's clefts and believed to play a role in antigenic variation. Our data show that, apart from a signal sequence, a minimum of two addition signals are required. This includes a host cell targeting signal for export to the host erythrocyte and a transmembrane domain for final sorting to Maurer's clefts. Biochemical studies indicate that STEVOR traverses the secretory pathway as an integral membrane protein. Our data suggest general principles for transport of transmembrane proteins to the Maurer's clefts and provide new insights into protein sorting and trafficking processes in P. falciparum.     

Molecular microbiology and pathogenesis of Helicobacter and Campylobacter updated: a meeting report of the 11th conference on Campylobacter,Helicobacter and related organisms

The genome analysis of the gastrointestinal pathogens Helicobacter and Campylobacter has stimulated a wealth of new research activities, which are presented every 2 years at the international conferences on Campylobacter, Helicobacter and Related Organisms (CHRO). Both organisms represent excellent models for the identification of new molecular mechanisms involved in pathogenesis, host response and physiological adaptation in course of acute and chronic infectious diseases. The investigation of their global distribution, pronounced genetic and antigenic diversity as well as the molecular mechanisms allowing long-term persistence in hostile and unusual microbial habitats, is a challenge for scientists of many different disciplines world-wide. With a focus on the molecular microbiology aspects, this review summarizes recent trends in Helicobacter and Campylobacter research by highlighting selected presentations at the 11th CHRO conference. The topics include the discovery of new virulence factors, functional analysis of protein secretion systems, host signalling pathways, adaptation to stress conditions, global gene regulation, and genetic variability.     

Co-evolution and exploitation of host cell signaling pathways by bacterial pathogens

Bacterial pathogens have evolved by combinations of gene acquisition, deletion, and modification, which increases their fitness. Additionally, bacteria are able to evolve in "quantum leaps" via the ability to promiscuously acquire new genes. Many bacterial pathogens - especially Gram-negative enteric pathogens - have evolved mechanisms by which to subvert signal transduction pathways of eukaryotic cells by expressing genes that mimic or regulate host protein factors involved in a variety of signaling cascades. This results in the ability to cause diseases ranging from tumor formation in plants to gastroenteritis and bubonic plague. Here, we present recent advances on mechanisms of bacterial pathogen evolution, including specific signaling cascades targeted by their virulence genes with an emphasis on the ubiquitin modification system, Rho GTPase regulators, cytoskeletal modulators, and host innate immunity. We also comment briefly on evolution of host defense mechanisms in place that limit disease caused by bacterial pathogens.     

Gene conversion and concerted evolution in bacterial genomes

Gene conversion is defined as the non-reciprocal transfer of information between homologous sequences. Despite methodological problems to establish non-reciprocity, gene conversion has been demonstrated in a wide variety of bacteria. Besides examples of high-frequency reversion of mutations in repeated genes, gene conversion in bacterial genomes has been implicated in concerted evolution of multigene families. Gene conversion also has a prime importance in the generation of antigenic variation, an interesting mechanism whereby some bacterial pathogens are able to avoid the host immune system. In this review, we analyze examples of bacterial gene conversion (some of them spawned from the current genomic revolution), as well as the molecular models that explain gene conversion and its association with crossovers.     

Infectomics: genomics and proteomics of microbial infections

The completion of genomic sequences is the greatest triumph of molecular reductionism since the discovery of the DNA double helix in 1953. However, the utility of reductionism is becoming limited and holistic approaches, including theories and techniques, are desperately needed in the postgenomic era. In the field of infectious diseases there is an urgent need for global approaches that can efficiently, precisely and integratively study structural and functional genomics and proteomics of microbial infections (infectomics). The combination of new (e.g. DNA and protein microarrays) and traditional approaches (e.g. cloning, PCR, gene knockout and knockin, and antisense) will help overcome the challenges we are facing today. We assume that the global phenotypic changes (infectomes) in microbes and their host during infections are encoded by the genomes of microbial pathogens and their hosts, expressed in certain environmental conditions devoted to specific microbe-host interactions. Global drug responses (pharmacomes) in microbes and their host can be detected by genomic and proteomic approaches. Genome-wide approaches to genotyping and phenotyping or expression profiling will eventually lead to global dissection of microbial pathogenesis, efficient and rapid diagnosis of infectious diseases, and the development of novel strategies to control infections. The key fundamental issue of infectious diseases is how to globally and integratively understand the interactions between microbial pathogens and their hosts by using infectomics. In this review, we focus on the events that are considered important in infectomics. Electronic Publication     

Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks

Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.     

Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks

Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.     

Immune evasion strategies of trypanosomes: a review

Trypanosomes are digenetic protozoans that infect domestic and wild animals, as well as humans. They cause important medical and veterinary diseases, making them a major public health concern. There are many species of trypanosomes that infect virtually all vertebrate taxa. They typically cycle between insect or leech vectors and vertebrate hosts, and they undergo biochemical and morphological changes in the process. Trypanosomes have received much attention in the last 4 decades because of the diseases they cause and their remarkable armamentarium of immune evasion mechanisms. The completed genome sequences of trypanosomes have revealed an extensive array of molecules that contribute to various immune evasion mechanisms. The different species interact uniquely with their vertebrate hosts with a wide range of evasion strategies and some of the most fascinating immune evasion mechanisms, including antigenic variation that was first described in the trypanosomes. This review focuses on the variety of strategies that these parasites have evolved to evade or modulate immunity of endothermic and ectothermic vertebrates.     

DNA metabolism and genetic diversity in Trypanosomes

Trypanosomes are protozoan parasites that cause major diseases in humans and other animals. Trypanosoma brucei and Trypanosoma cruzi are the etiologic agents of African and American Trypanosomiasis, respectively. In spite of large amounts of information regarding various aspects of their biology, including the essentially complete sequences of their genomes, studies directed towards an understanding of mechanisms related to DNA metabolism have been very limited. Recent reports, however, describing genes involved with DNA recombination and repair in T. brucei and T. cruzi, indicated the importance of these processes in the generation of genetic variability, which is crucial to the success of these parasites. Here, we review these data and discuss how the DNA repair and recombination machineries may contribute to strikingly different strategies evolved by the two Trypanosomes to create genetic variability that is needed for survival in their hosts. In T. brucei, two genetic components are critical to the success of antigenic variation, a strategy that allows the parasite to evade the host immune system by periodically changing the expression of a group of variant surface glycoproteins (VSGs). One component is a mechanism that provides for the exclusive expression of a single VSG at any one time, and the second is a large repository of antigenically distinct VSGs. Work from various groups showing the importance of recombination reactions in T. brucei, primarily to move a silent VSG into an active VSG expression site, is discussed. T. cruzi does not use the strategy of antigenic variation for host immune evasion but counts on the extreme heterogeneity of their population for parasite adaptation to different hosts. We discuss recent evidence indicating the existence of major differences in the levels of genomic heterogeneity among T. cruzi strains, and suggest that metabolic changes in the mismatch repair pathway could be an important source of antigenic diversity found within the T. cruzi population.