Vlp Genes Research Articles

Multiple and Diverse vsp and vlp Sequences in Borrelia miyamotoi, a Hard Tick-Borne Zoonotic Pathogen.

Based on chromosome sequences, the human pathogen Borrelia miyamotoi phylogenetically clusters with species that cause relapsing fever. But atypically for relapsing fever agents, B. miyamotoi is transmitted not by soft ticks but by hard ticks, which also are vectors of Lyme disease Borrelia species. To further assess the relationships of B. miyamotoi to species that cause relapsing fever, I investigated extrachromosomal sequences of a North American strain with specific attention on plasmid-borne vsp and vlp genes, which are the underpinnings of antigenic variation during relapsing fever. For a hybrid approach to achieve assemblies that spanned more than one of the paralogous vsp and vlp genes, a database of short-reads from next-generation sequencing was supplemented with long-reads obtained with real-time DNA sequencing from single polymerase molecules. This yielded three contigs of 31, 16, and 11 kb, which each contained multiple and diverse sequences that were homologous to vsp and vlp genes of the relapsing fever agent B. hermsii. Two plasmid fragments had coding sequences for plasmid partition proteins that differed from each other from paralogous proteins for the megaplasmid and a small plasmid of B. miyamotoi. One of 4 vsp genes, vsp1, was present at two loci, one of which was downstream of a candiate prokaryotic promoter. A limited RNA-seq analysis of a population growing in the blood of mice indicated that of the 4 different vsp genes vsp1 was the one that was expressed. The findings indicate that B. miyamotoi has at least four types of plasmids, two or more of which bear vsp and vlp gene sequences that are as numerous and diverse as those of relapsing fever Borrelia. The database and insights from these findings provide a foundation for further investigations of the immune responses to this pathogen and of the capability of B. miyamotoi for antigenic variation.

Antigenic variation with a twist – the Borrelia story

A common mechanism of immune evasion in pathogenic bacteria and protozoa is antigenic variation, in which genetic or epigenetic changes result in rapid, sequential shifts in a surface-exposed antigen. In this issue of Molecular Microbiology, Dai et al. provide the most complete description to date of the vlp/vsp antigenic variation system of the relapsing fever spirochaete, Borrelia hermsii. This elaborate, plasmid-encoded system involves an expression site that can acquire either variable large protein (vlp) or variable small protein (vsp) surface lipoprotein genes from 59 different archival copies. The archival vlp and vsp genes are arranged in clusters on at least five different plasmids. Gene conversion occurs through recombination events at upstream homology sequences (UHS) found in each gene copy, and at downstream homology sequences (DHS) found periodically among the vlp/vsp archival genes. Previous studies have shown that antigenic variation in relapsing fever Borrelia not only permits the evasion of host antibody responses, but can also result in changes in neurotropism and other pathogenic properties. The vlsE antigenic variation locus of Lyme disease spirochaetes, although similar in sequence to the relapsing fever vlp genes, has evolved a completely different antigenic variation mechanism involving segmental recombination from a contiguous array of vls silent cassettes. These two systems thus appear to represent divergence from a common precursor followed by functional convergence to create two distinct antigenic variation processes.

Characterisation of silent and active genes for a variable large protein of Borrelia recurrentis.

BackgroundWe report the characterisation of the variable large protein (vlp) gene expressed by clinical isolate A1 of Borrelia recurrentis; the agent of the life-threatening disease louse-borne relapsing fever.MethodsThe major vlp protein of this isolate was characterised and a DNA probe created. Use of this together with standard molecular methods was used to determine the location of the vlp1B. recurrentis A1 gene in both this and other isolates.ResultsThis isolate was found to carry silent and expressed copies of the vlp1B. recurrentis A1 gene on plasmids of 54 kbp and 24 kbp respectively, whereas a different isolate, A17, had only the silent vlp1B. recurrentis A17 on a 54 kbp plasmid. Silent and expressed vlp1 have identical mature protein coding regions but have different 5' regions, both containing different potential lipoprotein leader sequences. Only one form of vlp1 is transcribed in the A1 isolate of B. recurrentis, yet both 5' upstream sequences of this vlp1 gene possess features of bacterial promoters.ConclusionTaken together these results suggest that antigenic variation in B. recurrentis may result from recombination of variable large and small protein genes at the junction between lipoprotein leader sequence and mature protein coding region. However, this hypothetical model needs to be validated by further identification of expressed and silent variant protein genes in other B. recurrentis isolates.

Elongated versions of Vlp surface lipoproteins protect Mycoplasma hyorhinis escape variants from growth-inhibiting host antibodies.

Variation in Vlp surface proteins of Mycoplasma hyorhinis was evaluated in terms of its role in determining susceptibility of organisms to growth inhibition by host antibodies (Abs). High-frequency switching of Vlp surface lipoproteins has been studied in isogenic lineages of M. hyorhinis SK76. In these lineages, the products of three genes, vlpA, vlpB, and vlpC, are subject to phase and size variation in vitro, which occur through distinct mutator elements that independently govern the expression of each vlp gene (promoter mutations) or the size of the vlp gene product (by intragenic expansion or contraction of a 3' region containing tandem repeats). Isogenic clonal variants of M. hyorhinis SK76 expressing distinct profiles of Vlp products were assessed for their susceptibility to complement-independent growth inhibition by serum Abs of swine experimentally infected with the arthritigenic SK76 strain. Invariably, variants expressing longer versions of VlpA, VlpB, or VlpC (each expressed individually) were completely resistant to host immune serum Abs, whereas variants expressing shorter allelic versions of each Vlp were susceptible. The target of growth-inhibiting Abs was not the Vlp products, since removal of anti-Vlp Abs had no effect on the inhibitory activity of the host immune serum on susceptible variants. Escape variant populations derived by propagating susceptible variants in an immune (versus control) host serum revealed a strong selection for the long-Vlp phenotype, irrespective of the identity of the Vlp expressed. Apparent mutational pathways of acquiring the protective phenotype included mutational switches to express long vlp genes that had been transcriptionally silent or switches to elongate expressed vlp genes. These results suggest that a major function of the Vlp system is to shield the wall-less mycoplasma surface from host Abs capable of binding vital (and as-yet-unidentified) surface antigens of this organism.

Mycoplasma hyorhinis vlp gene transcription: critical role in phase variation and expression of surface lipoproteins.

Mycoplasma hyorhinis contains clustered vlp genes encoding variable lipoproteins (Vlps), the major coat proteins and surface antigens of this wall-less prokaryotic pathogen. vlp genes are subject to discrete, high frequency mutations independently affecting the size or the expression of variant Vlp products. Change in Vlp size occurs by mutations altering the number of tandem intragenic repeats at the 3' end of each single-copy vlp gene. In this report, phase-variant Vlp expression is shown to result from altered vlp gene transcription. vlpA, vlpB and vlpC transcripts were monitored in a clonal lineage selected to display various Vlp phenotypes. Each vlp gene was expressed as a distinct transcript, which was subject to drastic ON/OFF switches associated with random insertion/ deletion mutations in a homopolymeric tract of adenine residues in the promoter region of all vlp genes. Unexpectedly, the level of vlp transcripts appeared to depend on the length of the corresponding genes in the ON configuration. Higher proportional levels of shorter vlp transcripts were shown to reflect a greater abundance of short Vlp lipoproteins present in L-[35S]-cysteine-labelled membrane protein preparations. The vlp cluster provides a heritable, and highly mutable, locus for the generation of surface diversity through random promoter mutations affecting the expression of genes, whose products also vary in length and abundance, by virtue of separate mutations in structural regions of the genes.

Increased structural and combinatorial diversity in an extended family of genes encoding Vlp surface proteins of Mycoplasma hyorhinis.

Variable lipoproteins (Vlp) constitute the major coat protein of Mycoplasma hyorhinis. They are products of multiple, divergent, single-copy genes organized in a chromosomal cluster. Three genes, vlpA, vlpB, and vlpC, have been previously identified in clonal isolates of M. hyorhinis SK76. Each is linked to a characteristic promoter region containing a homopolymeric tract of adenine residues [poly(A) tract], subject to hypermutation, that transcriptionally controls phase variation of vlp genes and leads to combinatorial surface mosaics of distinct Vlp products. The size of the natural vlp gene repertoire is unknown but may critically determine the degree of structural and combinatorial diversity available in this species. In this study, the vlp repertoire of M. hyorhinis GDL-1 was characterized and shown to contain three additional genes, vlpD, vlpE, and vlpF, clustered with other known vlp genes in the order 5'-vlpD-vlpE-vlpF-IS-vlpA-IS-vlpB-vlpC+ ++-3', where IS represents copies of the IS1221 element of M. hyorhinis. The 5' boundary of this expanded family was identical to that of the more limited family 5'-vlpA-IS-vlpB-vlpC-3' previously described in a clonal isolate of strain SK76. A recombinant construct containing vlpD, vlpE, and vlpF expressed antigenically distinguishable products corresponding to each gene. These genes encode characteristic C-terminal repetitive regions that are subject to size variation by insertion or deletion of intragenic repeats but maintain an extended, charged structure. Each vlp gene also contained characteristic alternative open reading frames, which provide a potential reservoir of coding sequence for Vlp diversity, possibly recruited through insertion and/or deletion mutations. These findings demonstrate a vastly expanded potential for structural diversity and combinatorial display of surface mosaics on this organism and suggest that modulation of the vlp repertoire, possibly in conjunction with mobile elements, may determine the capacity for surface variation in natural populations and laboratory strains of this mycoplasma species.

Variation and Genetic Control of Surface Antigen Expression in Mycoplasmas: The VIp System of Mycoplasma hyorhinis

Surface antigenic diversity in the swine pathogen Mycoplasma hyorhinis is generated by random combinatorial expression and high-frequency phase variation of multiple, size-variant membrane surface lipoproteins (Vlps) which represent the major coat proteins of this wall-less procaryote. The distinctive structural basis for Vlp variation was revealed in a family of several related but divergent vlp genes. These occur in one cluster as single chromosomal copies, each encoding a conserved domain for membrane insertion and lipoprotein processing, and a divergent external domain that changes size by deletion or insertion of repetitive intragenic coding sequences while retaining a distinctive charge motif. Lack of detectable changes in restriction fragment patterns or DNA sequence of vlp structural genes during phase transitions between ON and OFF expression states ruled out long range genomic rearrangements and frameshift mutations as a means of controlling Vlp phase variation. However, highly homologous vlp promoter regions contain a homopolymeric tract of contiguous adenine residues [poly(A)] upstream of the transcriptional start site which is subject to frequent mutations altering its length. These mutations are the only sequence changes detected during phase transitions, and are highly correlated with the expression state of each vlp gene. This suggests a mechanism of transcriptional control regulating Vlp phase variation by critical changes within the poly(A) region affecting the spacing between the -10 and -35 hexamers or a putative regulator binding site. The multiple levels of structural and antigenic diversity embodied in the vlp gene family may provide essential adaptive capabilities for this wall-less microbial pathogen.

Molecular basis of Mycoplasma surface antigenic variation: a novel set of divergent genes undergo spontaneous mutation of periodic coding regions and 5′ regulatory sequences.

Antigenic diversity is generated in the wall-less pathogen Mycoplasma hyorhinis by combinatorial expression and phase variation of multiple, size-variant membrane surface lipoproteins (Vlps). The unusual structural basis for Vlp variation was revealed in a cluster of related but divergent vlp genes, vlpA, vlpB and vlpC, which occur as single chromosomal copies. These encode conserved N-terminal domains for membrane insertion and lipoprotein processing, but divergent external domains undergoing size variation by loss or gain of repetitive intragenic coding sequences while retaining a motif with distinctive charge distribution. Genetic analysis of phenotypically switched isogenic lineages representing ON or OFF expression states of Vlp products ruled out chromosomal rearrangement or frameshift mutations as mechanisms for Vlp phase variation. However, highly conserved vlp promoter regions contain a tract of contiguous A residues immediately upstream of the -10 box which is subject to frequent mutations altering its length in exact correspondence with the ON and OFF phase states of specific genes. This suggests a mechanism of transcriptional control regulating high frequency phase variation and random combinatorial expression of Vlps. The multiple levels of diversity embodied in the vlp gene cluster represents a novel adaptive capability particularly suited for this class of wall-less microbe.