O-antigen structural variation: mechanisms and possible roles in animal/plant-microbe interactions

Current data from bacterial pathogens of animals and from bacterial symbionts of plants support some of the more general proposed functions for lipopolysaccharides (LPS) and underline the importance of LPS structural versatility and adaptability. Most of the structural heterogeneity of LPS molecules is found in the O-antigen polysaccharide. In this review, the role and mechanisms of this striking flexibility in molecular structure of the O-antigen in bacterial pathogens and symbionts are illustrated by some recent findings. The variation in O-antigen that gives rise to an enormous structural diversity of O-antigens lies in the sugar composition and the linkages between monosaccharides. The chemical composition and structure of the O-antigen is strain-specific (interstrain LPS heterogeneity) but can also vary within one bacterial strain (intrastrain LPS heterogeneity). Both LPS heterogeneities can be achieved through variations at different levels. First of all, O-polysaccharides can be modified non-stoichiometrically with sugar moieties, such as glucosyl and fucosyl residues. The addition of non-carbohydrate substituents, i.e. acetyl or methyl groups, to the O-antigen can also occur with regularity, but in most cases these modifications are again non-stoichiometric. Understanding LPS structural variation in bacterial pathogens is important because several studies have indicated that the composition or size of the O-antigen might be a reliable indicator of virulence potential and that these important features often differ within the same bacterial strain. In general, O-antigen modifications seem to play an important role at several (at least two) stages of the infection process, including the colonization (adherence) step and the ability to bypass or overcome host defense mechanisms. There are many reports of modifications of O-antigen in bacterial pathogens, resulting either from altered gene expression, from lysogenic conversion or from lateral gene transfer followed by recombination. In most cases, the mechanisms underlying these changes have not been resolved. However, in recent studies some progress in understanding has been made. Changes in O-antigen structure mediated by lateral gene transfer, O-antigen conversion and phase variation, including fucosylation, glucosylation, acetylation and changes in O-antigen size, will be discussed. In addition to the observed LPS heterogeneity in bacterial pathogens, the structure of LPS is also altered in bacterial symbionts in response to signals from the plant during symbiosis. It appears to be part of a molecular communication between bacterium and host plant. Experiments ex planta suggest that the bacterium in the rhizosphere prepares its LPS for its roles in symbiosis by refining the LPS structure in response to seed and root compounds and the lower pH at the root surface. Moreover, modifications in LPS induced by conditions associated with infection are another indication that specific structures are important. Also during the differentiation from bacterium to bacteroid, the LPS of Rhizobium undergoes changes in the composition of the O-antigen, presumably in response to the change of environment. Recent findings suggest that, during symbiotic bacteroid development, reduced oxygen tension induces structural modifications in LPS that cause a switch from predominantly hydrophilic to predominantly hydrophobic molecular forms. However, the genetic mechanisms by which the LPS epitope changes are regulated remain unclear. Finally, the possible roles of O-antigen variations in symbiosis will be discussed.     

Phase and antigenic variation mediated by genome modifications

Phase and antigenic variation is used by several bacterial species to generate intra-population diversity that increases bacterial fitness and is important in niche adaptation, or to escape host defences. By this adaptive process, bacteria undergo frequent and usually reversible phenotypic changes resulting from genetic or epigenetic alterations at specific genetic loci. Phase variation or phenotypic switch allows the expression of a given phenotype to be switched ON or OFF. Antigenic variation refers to the expression of a number of alternative forms of an antigen on the cell surface, and at a molecular level, shares common features with phase variation mechanisms. This review will focus on phase and antigenic variation mechanisms implying genome modifications, with an emphasis on the diversity of phenotypes regulated by these mechanisms, and the ecological relevance of variant appearance within a given population.     

The role of phenotypic variation in rhizosphere Pseudomonas bacteria

Colony phase variation is a regulatory mechanism at the DNA level which usually results in high frequency, reversible switches between colonies with a different phenotype. A number of molecular mechanisms underlying phase variation are known: slipped-strand mispairing, genomic rearrangements, spontaneous mutations and epigenetic mechanisms such as differential methylation. Most examples of phenotypic variation or phase variation have been described in the context of host-pathogen interactions as mechanisms allowing pathogens to evade host immune responses. Recent reports indicate that phase variation is also relevant in competitive root colonization and biological control of phytopathogens. Many rhizospere Pseudomonas species show phenotypic variation, based on spontaneous mutation of the gacA and gacS genes. These morphological variants do not express secondary metabolites and have improved growth characteristics. The latter could contribute to efficient root colonization and success in competition, especially since (as shown for one strain) these variants were observed to revert to their wild-type form. The observation that these variants are present in rhizosphere-competent Pseudomonas bacteria suggests the existence of a conserved strategy to increase their success in the rhizosphere.     

Comparative phylogenomics of pathogenic bacteria by microarray analysis

DNA microarrays represent a powerful technology that enables whole-scale comparison of bacterial genomes. This, coupled with new methods to model DNA microarray data, is facilitating the development of robust comparative phylogenomics analyses. Such studies have dramatically increased our ability to differentiate between bacteria, highlighting previously undetected genetic differences and population structures and providing new insight into virulence and evolution of bacterial pathogens. Recent results from such studies have generated insights into the evolution of bacterial pathogens, the levels of diversity and plasticity in the genome of a species, as well as the differences in virulence amongst pathogenic bacteria.     

Bacterial endophytes and their interactions with hosts

Recent molecular studies on endophytic bacterial diversity have revealed a large richness of species. Endophytes promote plant growth and yield, suppress pathogens, may help to remove contaminants, solubilize phosphate, or contribute assimilable nitrogen to plants. Some endophytes are seedborne, but others have mechanisms to colonize the plants that are being studied. Bacterial mutants unable to produce secreted proteins are impaired in the colonization process. Plant genes expressed in the presence of endophytes provide clues as to the effects of endophytes in plants. Molecular analysis showed that plant defense responses limit bacterial populations inside plants. Some human pathogens, such as Salmonella spp., have been found as endophytes, and these bacteria are not removed by disinfection procedures that eliminate superficially occurring bacteria. Delivery of endophytes to the environment or agricultural fields should be carefully evaluated to avoid introducing pathogens.     

Type III effector proteins: doppelgangers of bacterial virulence

Bacterial pathogens have co-evolved with their hosts in their ongoing quest for advantage in the resulting interaction. These intimate associations have resulted in remarkable adaptations of prokaryotic virulence proteins and their eukaryotic molecular targets. An important strategy used by microbial pathogens of animals to manipulate host cellular functions is structural mimicry of eukaryotic proteins. Recent evidence demonstrates that plant pathogens also use structural mimicry of host factors as a virulence strategy. Nearly all virulence proteins from phytopathogenic bacteria have eluded functional annotation on the basis of primary amino-acid sequence. Recent efforts to determine their three-dimensional structures are, however, revealing important clues about the mechanisms of bacterial virulence in plants.     

Bacterial elicitation and evasion of plant innate immunity

Recent research on plant responses to bacterial attack has identified extracellular and intracellular host receptors that recognize conserved pathogen-associated molecular patterns and more specialized virulence proteins, respectively. These findings have shed light on our understanding of the molecular mechanisms by which bacteria elicit host defences and how pathogens have evolved to evade or suppress these defences.     

From genotype to phenotype: buffering mechanisms and the storage of genetic information

DNA sequence variation is abundant in wild populations. While molecular biologists use genetically homogeneous strains of model organisms to avoid this variation, evolutionary biologists embrace genetic variation as the material of evolution since heritable differences in fitness drive evolutionary change. Yet, the relationship between the phenotypic variation affecting fitness and the genotypic variation producing it is complex. Genetic buffering mechanisms modify this relationship by concealing the effects of genetic and environmental variation on phenotype. Genetic buffering allows the build-up and storage of genetic variation in phenotypically normal populations. When buffering breaks down, thresholds governing the expression of previously silent variation are crossed. At these thresholds, phenotypic differences suddenly appear and are available for selection. Thus, buffering mechanisms modulate evolution and regulate a balance between evolutionary stasis and change. Recent work provides a glimpse of the molecular details governing some types of genetic buffering.     

Source-sink dynamics of virulence evolution

To understand the evolution of genetic diversity within species--bacterial and others--we must dissect the first steps of genetic adaptation to novel habitats, particularly habitats that are suboptimal for sustained growth where there is strong selection for adaptive changes. Here, we present the view that bacterial human pathogens represent an excellent model for understanding the molecular mechanisms of the adaptation of a species to alternative habitats. In particular, bacterial pathogens allow us to develop analytical methods to detect genetic adaptation using an evolutionary 'source-sink' model, with which the evolution of bacterial pathogens can be seen from the angle of continuous switching between permanent (source) and transient (sink) habitats. The source-sink model provides a conceptual framework for understanding the population dynamics and molecular mechanisms of virulence evolution.     

Lessons from signature-tagged mutagenesis on the infectious mechanisms of pathogenic bacteria

Studies on the genetic basis of bacterial pathogenicity have been undertaken for almost 30 years, but the development of new genetic tools in the past 10 years has considerably increased the number of identified virulence factors. Signature-tagged mutagenesis (STM) is one of the most powerful general genetic approaches, initially developed by David Holden and colleagues in 1995, which has now led to the identification of hundreds of new genes requested for virulence in a broad range of bacterial pathogens. We have chosen to present in this review, the most recent and/or most significant contributions to the understanding of the molecular mechanisms of bacterial pathogenicity among over 40 STM screens published to date. We will first briefly review the principle of the method and its major technical limitations. Then, selected studies will be discussed where genes implicated in various aspects of the infectious process have been identified (including tropism for specific host and/or particular tissues, interactions with host cells, mechanisms of survival and persistence within the host, and the crossing of the blood brain barrier). The examples chosen will cover intracellular as well as extracellular Gram-negative and Gram-positive pathogens.     

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.     

病原菌毒力岛研究进展

毒力岛作为基因组岛的一种亚类,是细菌染色体上具有特定结构和功能特征的可移动基因大片段,经基因水平转移（转导、接合或转化）获得,可使细菌基因组进化在短期内发生“量的飞跃”,直接或间接增强细菌的生态适应性,与病原菌的致病性密切相关。毒力岛存在于多种动植物病原细菌中,对于细菌的毒力变异、遗传进化甚至新病原亚种形成有重要意义。简要综述了病原菌毒力岛的研究进展,介绍了毒力岛的结构、功能特征及其在病原菌进化中作用。     

Whole genome plasticity in pathogenic bacteria

The exploitation of bacterial genome sequences has so far provided a wealth of new general information about the genetic diversity of bacteria, such as that of many pathogens. Comparative genomics uncovered many genome variations in closely related bacteria and revealed basic principles involved in bacterial diversification, improving our knowledge of the evolution of bacterial pathogens. A correlation between metabolic versatility and genome size has become evident. The degenerated life styles of obligate intracellular pathogens correlate with significantly reduced genome sizes, a phenomenon that has been termed "evolution by reduction". These mechanisms can permanently alter bacterial genotypes and result in adaptation to their environment by genome optimization. In this review, we summarize the recent results of genome-wide approaches to studying the genetic diversity of pathogenic bacteria that indicate that the acquisition of DNA and the loss of genetic information are two important mechanisms that contribute to strain-specific differences in genome content.     

Bacterial strategies to overcome insect defences

Recent genetic and molecular analyses have revealed how several strategies enable bacteria to persist and overcome insect immune defences. Genetic and genomic tools that can be used with Drosophila melanogaster have enabled the characterization of the pathways that are used by insects to detect bacterial invaders and combat infection. Conservation of bacterial virulence factors and insect immune repertoires indicates that there are common strategies of host invasion and pathogen eradication. Long-term interactions of bacteria with insects might ensure efficient dissemination of pathogens to other hosts, including humans.     

Genetic control of susceptibility to bacterial infections in mouse models

Historically, the laboratory mouse (Mus musculus) has been the experimental model of choice to study pathophysiology of infection with bacterial pathogens, including natural and acquired host defence mechanisms. Inbred mouse strains differ significantly in their degree of susceptibility to infection with various human pathogens such as Mycobacterium, Salmonella, Legionella and many others. Segregation analyses and linkage studies have indicated that some of these differences are under simple genetic control whereas others behave as complex traits. Major advances in genome technologies have greatly facilitated positional cloning of single gene effects. Thus, a number of genes playing a key role in initial susceptibility, progression and outcome of infection have been uncovered and the functional characterization of the encoded proteins has provided new insight into the molecular basis of antimicrobial defences of polymorphonuclear leukocytes, macrophages, as well as T and B lymphocytes. The multigenic control of susceptibility to infection with certain human pathogens is beginning to be characterized by quantitative trait locus mapping in genome wide scans. This review summarizes recent progress on the mapping, cloning and characterization of genes and proteins that affect susceptibility to infection with major intracellular bacterial pathogens.     

Genetic diversity, recombination and cryptic clades in Pseudomonas viridiflava infecting natural populations of Arabidopsis thaliana

Species-level genetic diversity and recombination in bacterial pathogens of wild plant populations have been nearly unexplored. Pseudomonas viridiflava is a common natural bacterial pathogen of Arabidopsis thaliana, for which pathogen defense genes and mechanisms are becoming increasing well known. The genetic variation contained within a worldwide sample of P. viridiflava collected from wild populations of A. thaliana was investigated using five genomic sequence fragments totaling 2.3 kb. Two distinct and deeply diverged clades were found within the P. viridiflava sample and in close proximity in multiple populations, each genetically diverse with synonymous variation as high as 9.3% in one of these clades. Within clades, there is evidence of frequent recombination within and between each sequenced locus and little geographic differentiation. Isolates from both clades were also found in a small sample of other herbaceous species in Midwest populations, indicating a possibly broad host range for P. viridiflava. The high levels of genetic variation and recombination together with a lack of geographic differentiation in this pathogen distinguish it from other bacterial plant pathogens for which intraspecific variation has been examined.     

A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256

Biofilm formation of Staphylococcus epidermidis on smooth polymer surfaces has been shown to be mediated by the ica operon. Upon activation of this operon, a polysaccharide intercellular adhesin (PIA) is synthesized that supports bacterial cell-to-cell contacts and triggers the production of thick, multilayered biofilms. Thus, the ica gene cluster represents a genetic determinant that significantly contributes to the virulence of specific Staphylococcus epidermidis strains. PIA synthesis has been reported recently to undergo a phase variation process. In this study, biofilm-forming Staphylococcus epidermidis strains and their PIA-negative phase variants were analysed genetically to investigate the molecular mechanisms of phase variation. We have characterized biofilm-negative variants by Southern hybridization with ica-specific probes, polymerase chain reaction and nucleotide sequencing. The data obtained in these analyses suggested that in approximately 30% of the variants the missing biofilm formation was due to the inactivation of either the icaA or the icaC gene by the insertion of the insertion sequence element IS256. Furthermore, it was shown that the transposition of IS256 into the ica operon is a reversible process. After repeated passages of the PIA-negative insertional mutants, the biofilm-forming phenotype could be restored. Nucleotide sequence analyses of the revertants confirmed the complete excision of IS256, including the initially duplicated 8 bp target sites. These results elucidate, for the first time, a molecular mechanism mediating phase variation in staphylcocci, and they demonstrate that a naturally occurring insertion sequence element is actively involved in the modulation of expression of a Staphylococcus virulence factor.     

Bistability and phase variation in Salmonella enterica

Cell-to-cell differences in bacterial gene expression can merely reflect the occurrence of noise. In certain cases, however, heterogeneous gene expression is a programmed event that results in bistable expression. If bistability is heritable, bacterial lineages are formed. When programmed bistability is reversible, the phenomenon is known as phase variation. In certain cases, bistability is controlled by genetic mechanisms (e. g., DNA rearrangement). In other cases, bistability has epigenetic origin. A robust epigenetic mechanism for the formation of bacterial lineages is the formation of heritable DNA methylation patterns. However, bistability can also arise upon propagation of gene expression patterns by feedback loops that are stable upon cell division. This review describes examples of bistability and phase variation in Salmonella enterica and discusses their adaptive value, sometimes in a speculative manner.     

Cut and move: protein machinery for DNA processing in bacterial conjugation

Conjugation is a paradigmatic example of horizontal or lateral gene transfer, whereby DNA is translocated between bacterial cells. It provides a route for the rapid acquisition of new genetic information. Increased antibiotic resistance among pathogens is a troubling consequence of this microbial capacity. DNA transfer across cell membranes requires a sophisticated molecular machinery that involves the participation of several proteins in DNA processing and replication, cell recruitment, and the transport of DNA and proteins from donor to recipient cells. Although bacterial conjugation was first reported in the 1940s, only now are we beginning to unravel the molecular mechanisms behind this process. In particular, structural biology is revealing the detailed molecular architecture of several of the pieces involved.