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H. Steven Seifert - One of the best experts on this subject based on the ideXlab platform.
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PacBio Amplicon Sequencing Method To Measure Pilin Antigenic Variation Frequencies of Neisseria gonorrhoeae.
mSphere, 2019Co-Authors: Egon A. Ozer, Lauren L. Prister, Shaohui Yin, Billy H. Ward, Stanimir S. Ivanov, H. Steven SeifertAbstract:ABSTRACT Gene diversification is a common mechanism pathogens use to alter surface structures to aid in immune avoidance. Neisseria gonorrhoeae uses a gene conversion-based diversification system to alter the primary sequence of the gene encoding the major subunit of the pilus, pilE. Antigenic Variation occurs when one of the nonexpressed 19 silent copies donates part of its DNA sequence to pilE. We have developed a method using Pacific Biosciences (PacBio) amplicon sequencing and custom software to determine pilin Antigenic Variation frequencies. The program analyzes 37 variable regions across the strain FA1090 1-81-S2 pilE gene and can be modified to determine sequence Variation from other starting pilE sequences or other diversity generation systems. Using this method, we measured pilin Antigenic Variation frequencies for various derivatives of strain FA1090 and showed we can also analyze pilin Antigenic Variation frequencies during macrophage infection. IMPORTANCE Diversity generation systems are used by many unicellular organism to provide subpopulations of cell with different properties that are available when needed. We have developed a method using the PacBio DNA sequencing technology and a custom computer program to analyze the pilin Antigenic Variation system of the organism that is the sole cause of the sexually transmitted infection, gonorrhea.
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Analysis of Pilin Antigenic Variation in Neisseria meningitidis by Next-Generation Sequencing.
Journal of bacteriology, 2018Co-Authors: H. Steven SeifertAbstract:Many pathogenic microbes evade host immune surveillance by varying the surface antigens, a process termed Antigenic Variation. While the process of pilin Antigenic Variation has been extensively studied in the human pathogen Neisseria gonorrhoeae (gonococcus [Gc]), relatively few studies of pilin Antigenic Variation have been conducted with Neisseria meningitidis (meningococcus [Mc]). Mc is usually a commensal organism that colonizes the human nasopharynx, but when it translocates to the bloodstream or meninges, it results in the severe and often deadly meningococcal disease. The type IV pili of Mc isolates play a critical role in host surface adherence, and its major pilin component (PilE) can undergo Antigenic Variation. In this study, Roche 454 pyrosequencing was used to examine the pilin Antigenic Variation of Mc strain 8013, as well as 8013 recA, recX, recQ, rep, and recJ mutants, Gc orthologues which have been shown to play a role in pilin Antigenic Variation. This study confirms that the Mc recA, rep, and recJ genes are essential for pilin Antigenic Variation. While the Mc recQ and recX gene products contribute to normal frequencies of Antigenic Variation, the loss of these factors does not alter the types of pilin variants produced. Overall, this study shows that the mechanisms of pilin Antigenic Variation are conserved between Gc and Mc.IMPORTANCE Antigenic Variation is a strategy used by many pathogens to escape host immune surveillance and establish persistent infections. This study successfully applies next-generation sequencing to study pilin Antigenic Variation in the human pathogen Neisseria meningitidis This assay provides an affordable and efficient solution for quantifying Antigenic Variation frequency in mutant strains and for defining the recombination products of the process. We determined that there is a nonuniformity of silent donor copies used during meningococcus Antigenic Variation, and by the analysis of selected mutants deficient for specific recombination pathways, we show for the first time that the processes are conserved between N. meningitidis and Neisseria gonorrhoeae.
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Antigenic Variation in Bacterial Pathogens.
Microbiology spectrum, 2016Co-Authors: Guy H. Palmer, Troy Bankhead, H. Steven SeifertAbstract:Antigenic Variation is a strategy used by a broad diversity of microbial pathogens to persist within the mammalian host. Whereas viruses make use of a minimal proofreading capacity combined with large amounts of progeny to use random mutation for variant generation, Antigenically variant bacteria have evolved mechanisms which use a stable genome, which aids in protecting the fitness of the progeny. Here, three well-characterized and highly Antigenically variant bacterial pathogens are discussed: Anaplasma, Borrelia, and Neisseria. These three pathogens display a variety of mechanisms used to create the structural and Antigenic Variation needed for immune escape and long-term persistence. Intrahost Antigenic Variation is the focus; however, the role of these immune escape mechanisms at the population level is also presented.
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Microbial Antigenic Variation mediated by homologous DNA recombination
FEMS microbiology reviews, 2012Co-Authors: Cornelis Vink, Gloria Rudenko, H. Steven SeifertAbstract:Pathogenic microorganisms employ numerous molecular strategies in order to delay or circumvent recognition by the immune system of their host. One of the most widely used strategies of immune evasion is Antigenic Variation, in which immunogenic molecules expressed on the surface of a microorganism are continuously modified. As a consequence, the host is forced to constantly adapt its humoral immune response against this pathogen. An Antigenic change thus provides the microorganism with an opportunity to persist and/or replicate within the host (population) for an extended period of time or to effectively infect a previously infected host. In most cases, Antigenic Variation is caused by genetic processes that lead to the modification of the amino acid sequence of a particular antigen or to alterations in the expression of biosynthesis genes that induce changes in the expression of a variant antigen. Here, we will review Antigenic Variation systems that rely on homologous DNA recombination and that are found in a wide range of cellular, human pathogens, including bacteria (such as Neisseria spp., Borrelia spp., Treponema pallidum, and Mycoplasma spp.), fungi (such as Pneumocystis carinii) and parasites (such as the African trypanosome Trypanosoma brucei). Specifically, the various DNA recombination-based Antigenic Variation systems will be discussed with a focus on the employed mechanisms of recombination, the DNA substrates, and the enzymatic machinery involved.
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Focusing homologous recombination: pilin Antigenic Variation in the pathogenic Neisseria
Molecular microbiology, 2011Co-Authors: Laty A Cahoon, H. Steven SeifertAbstract:Some pathogenic microbes utilize homologous recombination to generate Antigenic variability in targets of immune surveillance. These specialized systems rely on the cellular recombination machinery to catalyse dedicated, high-frequency reactions that provide extensive diversity in the genes encoding surface antigens. A description of the specific mechanisms that allow unusually high rates of recombination without deleterious effects on the genome in the well-characterized pilin Antigenic Variation systems of Neisseria gonorrhoeae and Neisseria meningitidis is presented. We will also draw parallels to selected bacterial and eukaryotic Antigenic Variation systems, and suggest the most pressing unanswered questions related to understanding these important processes.
Richard Mcculloch - One of the best experts on this subject based on the ideXlab platform.
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emerging challenges in understanding trypanosome Antigenic Variation
Emerging topics in life sciences, 2017Co-Authors: Richard Mcculloch, Christina A Cobbold, Luisa M Figueiredo, Andrew P Jackson, Liam J Morrison, Monica R Mugnier, Nina Papavasiliou, Achim Schnaufer, Keith R MatthewsAbstract:Many pathogens evade host immunity by periodically changing the proteins they express on their surface — a phenomenon termed Antigenic Variation. An extreme form of Antigenic Variation, based around switching the composition of a variant surface glycoprotein (VSG) coat, is exhibited by the African trypanosome Trypanosoma brucei, which causes human disease. The molecular details of VSG switching in T. brucei have been extensively studied over the last three decades, revealing in increasing detail the machinery and mechanisms by which VSG expression is controlled and altered. However, several key components of the models of T. brucei Antigenic Variation that have emerged have been challenged through recent discoveries. These discoveries include new appreciation of the importance of gene mosaics in generating huge levels of new VSG variants, the contributions of parasite development and body compartmentation in the host to the infection dynamics and, finally, potential differences in the strategies of Antigenic Variation and host infection used by the crucial livestock trypanosomes T. congolense and T. vivax. This review will discuss all these observations, which raise questions regarding how secure the existing models of trypanosome Antigenic Variation are. In addition, we will discuss the importance of continued mathematical modelling to understand the purpose of this widespread immune survival process.
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DNA Recombination Strategies During Antigenic Variation in the African Trypanosome.
Microbiology spectrum, 2015Co-Authors: Richard Mcculloch, Liam J Morrison, James P J HallAbstract:Survival of the African trypanosome in its mammalian hosts has led to the evolution of Antigenic Variation, a process for evasion of adaptive immunity that has independently evolved in many other viral, bacterial and eukaryotic pathogens. The essential features of trypanosome Antigenic Variation have been understood for many years and comprise a dense, protective Variant Surface Glycoprotein (VSG) coat, which can be changed by recombination-based and transcription-based processes that focus on telomeric VSG gene transcription sites. However, it is only recently that the scale of this process has been truly appreciated. Genome sequencing of Trypanosoma brucei has revealed a massive archive of >1000 VSG genes, the huge majority of which are functionally impaired but are used to generate far greater numbers of VSG coats through segmental gene conversion. This chapter will discuss the implications of such VSG diversity for immune evasion by Antigenic Variation, and will consider how this expressed diversity can arise, drawing on a growing body of work that has begun to examine the proteins and sequences through which VSG switching is catalyzed. Most studies of trypanosome Antigenic Variation have focused on T. brucei, the causative agent of human sleeping sickness. Other work has begun to look at Antigenic Variation in animal-infective trypanosomes, and we will compare the findings that are emerging, as well as consider how Antigenic Variation relates to the dynamics of host-trypanosome interaction.
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Antigenic Variation in African trypanosomes: the importance of chromosomal and nuclear context in VSG expression control
Cellular Microbiology, 2013Co-Authors: Lucy Glover, Richard Mcculloch, Sam Alsford, Sebastian Hutchinson, Mark Field, David HornAbstract:African trypanosomes are lethal human and animal parasites that use Antigenic Variation for evasion of host adaptive immunity. To facilitate Antigenic Variation, trypanosomes dedicate approximately one third of their nuclear genome, including many minichromosomes, and possibly all sub-telomeres, to variant surface glycoprotein (VSG) genes and associated sequences. Antigenic Variation requires transcription of a single VSG by RNA polymerase I (Pol-I), with silencing of other VSGs, and periodic switching of the expressed gene, typically via DNA recombination with duplicative translocation of a new VSG to the active site. Thus, telomeric location, epigenetic controls and monoallelic transcription by Pol-I at an extranucleolar site are prominent features of VSGs and their expression, with telomeres, chromatin structure and nuclear organization all making vitally important contributions to monoallelic VSG expression control and switching. We discuss VSG transcription, recombination and replication control within this chromosomal and sub-nuclear context.
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Antigenic Variation in African trypanosomes: monitoring progress
Trends in parasitology, 2004Co-Authors: Richard MccullochAbstract:Antigenic Variation is central to the success of African trypanosomes and other eukaryotic, bacterial and viral pathogens. Our understanding of the control and execution of this immune evasion strategy in trypanosomes is incomplete, despite the molecular basis of Antigenic Variation being first described over 20 years ago. Recent research progress in this field is highlighted here and some of the unresolved questions raised.
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Antigenic Variation in trypanosomes: enhanced phenotypic Variation in a eukaryotic parasite.
Advances in parasitology, 2001Co-Authors: J. David Barry, Richard MccullochAbstract:Abstract African trypanosomes are unicellular, eukaryotic parasites that live extracellularly in a wide range of mammals, including humans. They have a surface coat, composed of variant surface glycoprotein (VSG), which probably is essential and acts as a defence against general innate immunity and against acquired immunity directed at invariant surface antigens. In effect, the VSG is the only antigen that the host can target, and each trypanosome expresses only one VSG. To counter specific antibodies againts the VSG, trypanosomes periodically undergo Antigenic Variation, the change to expression of another VSG. Antigenic Variation belongs to the general survival strategy of enhanced phenotypic Variation, where a subset of ‘contigency’ genes of viruses, bacteria and parasites hypermutate, allowing adaptation to hostile or changing environments. A fundamental feature of Antigenic Variation is its link with the population dynamics of trypanosomes within the single host. Antigenic variants appear hierachically within the mammalian host, with a mixture of order and randomness. The underlying mechanisms of this are not understood, although differential VSG gene activation may play a prominent part. Trypanosome Antigenic Variation has evolved a second arm in which the infective metalcyclic population in the tsetse fly expresses a defined mixture of VSGs, although again each trypanosome expresses a single VSG. Differential VSG expression enhances transmission to new hosts, in the case of blood-stream trypanosomes by prolonging infection, and in the metacyclic population by generating diversity that may counter existing partial immunity in reservoir hosts. Antigenic Variation employs a huge repertoire of VSG genes. Only one is expressed at a time bloodstream trypanosomes, as a result of transcription being restricted to a set of about 20 bloodstream expression sites ( BES s), which are at chromosome telomeres. Only one BES is active at a time, probably through transcriptional elongation being inhibited in the silent BES s. Although transcriptional switching between BES s can effect a VSG switch, the most prolific switch route involves homologous recombination of deoxyribonucleic acid, usually by the copying of a silent gene into a BES . Hierarchical expression of VSGs may be dictated in part by the different types of locus occupied by VSG genes. The VSG genes expressed int the metacyclic population also occupy telomeric sites, which appear to be derived from BES s but have a simpler structure. Their differential expression is achieved by random transcriptional activation; the detailed story requires direct study of the metacylic stage itself. Available evidence suggests that the VSG originated as a surface receptor, and it can be proposed that a number of selective events have contributed to the evolution of the complex, multisystem phenomenon that Antigenic Variation has become.
J. David Barry - One of the best experts on this subject based on the ideXlab platform.
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Mosaic VSGs and the scale of Trypanosoma brucei Antigenic Variation.
PLoS pathogens, 2013Co-Authors: James P J Hall, Huanhuan Wang, J. David BarryAbstract:A main determinant of prolonged Trypanosoma brucei infection and transmission and success of the parasite is the interplay between host acquired immunity and Antigenic Variation of the parasite variant surface glycoprotein (VSG) coat. About 0.1% of trypanosome divisions produce a switch to a different VSG through differential expression of an archive of hundreds of silent VSG genes and pseudogenes, but the patterns and extent of the trypanosome diversity phenotype, particularly in chronic infection, are unclear. We applied longitudinal VSG cDNA sequencing to estimate variant richness and test whether pseudogenes contribute to Antigenic Variation. We show that individual growth peaks can contain at least 15 distinct variants, are estimated computationally to comprise many more, and that Antigenically distinct ‘mosaic’ VSGs arise from segmental gene conversion between donor VSG genes or pseudogenes. The potential for trypanosome Antigenic Variation is probably much greater than VSG archive size; mosaic VSGs are core to Antigenic Variation and chronic infection.
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Probabilistic order in Antigenic Variation of Trypanosoma brucei.
International journal for parasitology, 2005Co-Authors: Liam J Morrison, Phelix A.o. Majiwa, Andrew F. Read, J. David BarryAbstract:Antigenic Variation in African trypanosomes displays a degree of order that is usually described as 'semi-predictable' but which has not been analysed in statistical detail. It has been proposed that, during switching, the variable antigen type (VAT) being inactivated can influence which VAT is subsequently activated. Antigenic Variation proceeds by the differential activation of members of the large archive of distinct variable surface glycoprotein (VSG) genes. The most popular model for ordered expression of VATs invokes differential activation probabilities for individual VSG genes, dictated in part by which of the four types of genetic locus they occupy. We have shown, in pilot experiments in cattle, correlation between the timing of appearance of VSG-specific mRNA and of lytic antibodies corresponding to seven VSGs encoded by single-copy genes. We have then determined the times of appearance of VAT-specific antibodies, as a measure of appearance of the VATs, in a statistically significant number of mouse infections (n=22). There is a surprisingly high degree of order in temporal appearance of the VATs, indicating that Antigenic Variation proceeds through order in the probability of activation of each VAT. In addition, for the few examples of each available, the locus type inhabited by the silent 'donor' VSG plays a significant role in determination of order. We have analysed in detail previously published data on VATs appearing in first relapse peaks, and find that the variant being switched off does not influence which one is being switched on. This differs from what has been reported for Plasmodium falciparum var Antigenic Variation. All these features of trypanosome Antigenic Variation can be explained by a one-step model in which, following an initial deactivation event, the switch process and the imposition of order early in infection arise from the inherent activation probabilities of the specific VSG being switched on.
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Antigenic Variation in trypanosomes: enhanced phenotypic Variation in a eukaryotic parasite.
Advances in parasitology, 2001Co-Authors: J. David Barry, Richard MccullochAbstract:Abstract African trypanosomes are unicellular, eukaryotic parasites that live extracellularly in a wide range of mammals, including humans. They have a surface coat, composed of variant surface glycoprotein (VSG), which probably is essential and acts as a defence against general innate immunity and against acquired immunity directed at invariant surface antigens. In effect, the VSG is the only antigen that the host can target, and each trypanosome expresses only one VSG. To counter specific antibodies againts the VSG, trypanosomes periodically undergo Antigenic Variation, the change to expression of another VSG. Antigenic Variation belongs to the general survival strategy of enhanced phenotypic Variation, where a subset of ‘contigency’ genes of viruses, bacteria and parasites hypermutate, allowing adaptation to hostile or changing environments. A fundamental feature of Antigenic Variation is its link with the population dynamics of trypanosomes within the single host. Antigenic variants appear hierachically within the mammalian host, with a mixture of order and randomness. The underlying mechanisms of this are not understood, although differential VSG gene activation may play a prominent part. Trypanosome Antigenic Variation has evolved a second arm in which the infective metalcyclic population in the tsetse fly expresses a defined mixture of VSGs, although again each trypanosome expresses a single VSG. Differential VSG expression enhances transmission to new hosts, in the case of blood-stream trypanosomes by prolonging infection, and in the metacyclic population by generating diversity that may counter existing partial immunity in reservoir hosts. Antigenic Variation employs a huge repertoire of VSG genes. Only one is expressed at a time bloodstream trypanosomes, as a result of transcription being restricted to a set of about 20 bloodstream expression sites ( BES s), which are at chromosome telomeres. Only one BES is active at a time, probably through transcriptional elongation being inhibited in the silent BES s. Although transcriptional switching between BES s can effect a VSG switch, the most prolific switch route involves homologous recombination of deoxyribonucleic acid, usually by the copying of a silent gene into a BES . Hierarchical expression of VSGs may be dictated in part by the different types of locus occupied by VSG genes. The VSG genes expressed int the metacyclic population also occupy telomeric sites, which appear to be derived from BES s but have a simpler structure. Their differential expression is achieved by random transcriptional activation; the detailed story requires direct study of the metacylic stage itself. Available evidence suggests that the VSG originated as a surface receptor, and it can be proposed that a number of selective events have contributed to the evolution of the complex, multisystem phenomenon that Antigenic Variation has become.
Alan G. Barbour - One of the best experts on this subject based on the ideXlab platform.
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Antigenic Variation in Vector-Borne Pathogens
Emerging Infectious Diseases, 2000Co-Authors: Alan G. Barbour, Blanca I. RestrepoAbstract:Several pathogens of humans and domestic animals depend on hematophagous arthropods to transmit them from one vertebrate reservoir host to another and maintain them in an environment. These pathogens use Antigenic Variation to prolong their circulation in the blood and thus increase the likelihood of transmission. By convergent evolution, bacterial and protozoal vector-borne pathogens have acquired similar genetic mechanisms for successful Antigenic Variation. Borrelia spp. and Anaplasma marginale (among bacteria) and African trypanosomes, Plasmodium falciparum, and Babesia bovis (among parasites) are examples of pathogens using these mechanisms. Antigenic Variation poses a challenge in the development of vaccines against vectorborne pathogens.
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Antigenic Variation in lyme disease borreliae by promiscuous recombination of vmp like sequence cassettes
Cell, 1997Co-Authors: Jing Ren Zhang, Alan G. Barbour, John M Hardham, Steven J. NorrisAbstract:We have identified and characterized an elaborate genetic system in the Lyme disease spirochete Borrelia burgdorferi that promotes extensive Antigenic Variation of a surface-exposed lipoprotein, VlsE. A 28 kb linear plasmid of B. burgdorferi B31 (lp28-1) was found to contain a vmp-like sequence (vls) locus that closely resembles the variable major protein (vmp) system for Antigenic Variation of relapsing fever organisms. Portions of several of the 15 nonexpressed (silent) vls cassette sequences located upstream of vlsE recombined into the central vlsE cassette region during infection of C3H/HeN mice, resulting in Antigenic Variation of the expressed lipoprotein. This combinatorial Variation could potentially produce millions of Antigenic variants in the mammalian host.
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Antigenic Variation in Borrelial Diseases of Humans
Global Infectious Diseases, 1992Co-Authors: Alan G. BarbourAbstract:Relapsing fever and Lyme disease are human infections caused by different members of the spirochetal genus Borrelia (1). In both diseases a spirochetemia in the vertebrate is the source of infection for the arthropod. One presumes that a bacterium persisting in the blood of the vertebrate longer than another bacterium would have a selective advantage over its more short-lived siblings. It is not surprising, therefore, to find that one of the strategies used by borrelias to achieve this persistence in the host is Antigenic Variation.
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Antigenic Variation in Borrelia.
Research in microbiology, 1991Co-Authors: I. Saint Girons, Alan G. BarbourAbstract:Antigenic Variation was demonstrated for the agent of relapsing fever, Borrelia hermsii. The phenomenon is correlated with changes in major surface proteins called Vmp. The genes encoding these antigens are located on linear plasmids. Expression occurs by transposition of genes encoding Vmp to a telomeric expression site located on another linear plasmid. Activation of a vmp gene occurs by placing it downstream from a promoter. Resemblance to the Antigenic Variation of trypanosomes is discussed.
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Molecular biology of Antigenic Variation in Lyme borreliosis and relapsing fever: a comparative analysis.
Scandinavian journal of infectious diseases. Supplementum, 1991Co-Authors: Alan G. BarbourAbstract:Lyme borreliosis and relapsing fever are human diseases caused by different members of the genus Borrelia. Antigenic Variation has been a well-known feature of the pathogenesis of relapsing fever for decades. More recently it has been recognized that Borrelia burgdorferi, the agent of Lyme borreliosis, also can vary its surface antigens. In this review the biology and molecular biology of Antigenic Variation of the pathogens in these two disorders are compared.
Steven H Seifert - One of the best experts on this subject based on the ideXlab platform.
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an alternative dna structure is necessary for pilin Antigenic Variation in neisseria gonorrhoeae
Science, 2009Co-Authors: Laty A Cahoon, Steven H SeifertAbstract:Pathogens can utilize DNA recombination to promote Antigenic Variation of surface structures to avoid immune detection. We identified a cis-acting DNA sequence near the Antigenically variable pilin locus of the human pathogen, Neisseria gonorrhoeae. This 16 base pair G-rich sequence was required for pilin Antigenic Variation and formed a guanine quartet (G4) structure in vitro. Individual mutations that disrupted the structure also blocked pilin Antigenic Variation and prevented nicks required for recombination from occurring within the G4 region. A compound that binds and stabilizes G4 structures also inhibited pilin Antigenic Variation and prevented nicks from occurring on the G-rich strand. This site constitutes a recombination initiation sequence/structure that directs gene conversion to a specific chromosomal locus.
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differential roles of homologous recombination pathways in neisseria gonorrhoeae pilin Antigenic Variation dna transformation and dna repair
Molecular Microbiology, 1998Co-Authors: Ian J Mehr, Steven H SeifertAbstract:Neisseria gonorrhoeae (Gc) pili undergo Antigenic Variation when the amino acid sequence of the pilin protein is changed, aiding in immune avoidance and altering pilus expression. Pilin Antigenic Variation occurs by RecA-dependent unidirectional transfer of DNA sequences from a silent pilin locus to the expressed pilin gene through high-frequency recombination events that occur at limited regions of homology. We show that the Gc recQ and recO genes are essential for pilin Antigenic and phase Variation and DNA repair but are not involved in natural DNA transformation. This suggests that a RecF-like pathway of recombination exists in Gc. In addition, mutations in the Gc recB, recC or recD genes revealed that a Gc RecBCD pathway also exists and is involved in DNA transformation and DNA repair but not in pilin Antigenic Variation.