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.     

Gut Microbiota of the Tick Vector Ixodes scapularis Modulate Colonization of the Lyme Disease Spirochete

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Dual Nuclease and Helicase Activities of Helicobacter pylori AddAB Are Required for DNA Repair, Recombination, and Mouse Infectivity

Helicobacter pylori infection of the human stomach is associated with disease-causing inflammation that elicits DNA damage in both bacterial and host cells. Bacteria must repair their DNA to persist. The H. pylori AddAB helicase-exonuclease is required for DNA repair and efficient stomach colonization. To dissect the role of each activity in DNA repair and infectivity, we altered the AddA and AddB nuclease (NUC) domains and the AddA helicase (HEL) domain by site-directed mutagenesis. Extracts of Escherichia coli expressing H. pylori addA^NUCB or addAB^NUC mutants unwound DNA but had approximately half of the exonuclease activity of wild-type AddAB; the addA^NUCB^NUC double mutant lacked detectable nuclease activity but retained helicase activity. Extracts with AddA^HELB lacked detectable helicase and nuclease activity. H. pylori with the single nuclease domain mutations were somewhat less sensitive to the DNA-damaging agent ciprofloxacin than the corresponding deletion mutant, suggesting that residual nuclease activity promotes limited DNA repair. The addA^NUC and addA^HEL mutants colonized the stomach less efficiently than the wild type; addB^NUC showed partial attenuation. E. coli ΔrecBCD expressing H. pylori addAB was recombination-deficient unless H. pylori recA was also expressed, suggesting a species-specific interaction between AddAB and RecA and also that H. pylori AddAB participates in both DNA repair and recombination. These results support a role for both the AddAB nuclease and helicase in DNA repair and promoting infectivity.Infection of the stomach with Helicobacter pylori causes a variety of diseases including gastritis, peptic ulcers, and gastric cancer (1). A central feature of the pathology of these conditions is the establishment of a chronic inflammatory response that acts both on the host and the infecting bacteria (2). Both epithelial (3, 4) and lymphoid (5, 6) cells in the gastric mucosa of infected individuals release DNA-damaging agents that can introduce double-stranded (ds)² breaks into the bacterial chromosome (7). The ds breaks must be repaired for the bacteria to survive and establish chronic colonization of the stomach. Homologous recombination is required for the faithful repair of DNA damage and bacterial survival. Alteration of the expression of one of a series of cell surface proteins on H. pylori occurs by an apparent gene conversion of babA, the frequency of which is reduced in repair-deficient strains (8, 9). This change in the cell surface, which may allow H. pylori to evade the host immune response, is a second means by which recombination can promote efficient colonization of the stomach by H. pylori.The initiation or presynaptic steps of recombination at dsDNA breaks in most bacteria involves the coordinated action of nuclease and helicase activities provided by one of two multisubunit enzymes, the AddAB and RecBCD enzymes (10). Escherichia coli recBCD null mutants have reduced cell viability, are hypersensitive to DNA-damaging agents, and are homologous recombination-deficient (11–14). Similarly, H. pylori addA and addB null mutants are hypersensitive to DNA-damaging agents, have reduced frequencies of babA gene conversion, and colonize the stomach of mice less efficiently than wild-type strains (8).The activities of RecBCD enzyme from E. coli (15–19) and AddAB from H. pylori (8) or Bacillus subtilis (20–23) indicate some common general features of the presynaptic steps of DNA repair. In the case of E. coli, repair begins when the RecBCD enzyme binds to a dsDNA end and unwinds the DNA using its ATP-dependent helicase activities (17, 24). Single-stranded (ss) DNA produced during unwinding, with or without accompanying nuclease, is coated with RecA protein (16, 25). This recombinogenic substrate engages in strand exchange with a homologous intact duplex to form a joint molecule. Joint molecules are thought to be converted into intact, recombinant DNA either by replication or by cutting and ligation of exchanged strands (26).Although the AddAB and RecBCD enzymes appear to play similar roles in promoting recombination and DNA repair, they differ in several ways. RecBCD is a heterotrimer, composed of one copy of the RecB, RecC, and RecD gene products (27), whereas AddAB has two subunits, encoded by the addA and addB genes (21, 28). The enzyme subunit(s) responsible for helicase activity can be inferred from the presence of conserved protein domains or the activity of purified proteins. AddA, RecB, and RecD are superfamily I helicases with six highly conserved helicase motifs, including the conserved Walker A box found in many enzymes that bind ATP (29–32). A Walker A box is defined by the consensus sequence (G/A)XXGXGKT (X is any amino acid (29). RecBCD enzymes in which the conserved Lys in this motif is changed to Gln have a reduced affinity for ATP binding (33, 34) and altered helicase activity (17, 35–37).A nuclease domain with the conserved amino acid sequence LDYK is found in RecB, AddA, AddB, and many other nucleases (38). The conserved Asp plays a role in Mg²⁺ binding at the active site; Mg²⁺ is required for nuclease activity (39). The recB1080 mutation, which changes codon 1080 from the conserved Asp in this motif to Ala, eliminates nuclease activity (39).We have recently shown that addA and addB deletion mutants are hypersensitive to DNA-damaging agents and impaired in colonization of the mouse stomach compared with wild-type strains (8). To determine the roles of the individual helicase and nuclease activities of H. pylori AddAB in DNA repair and infectivity, we used site-directed mutagenesis to inactivate the conserved nuclease domains of addA and addB and the conserved ATPase (helicase) domain of AddA. Here, we report that loss of the AddAB helicase is sufficient to impair H. pylori DNA repair and infectivity and, when the genes are expressed in E. coli, homologous recombination. AddAB retains partial activity in biochemical and genetic assays when either of the two nuclease domains is inactivated but loses all detectable nuclease activity when both domains are inactivated. Remarkably, H. pylori AddAB can produce recombinants in E. coli only in the presence of H. pylori RecA, suggesting a species-specific interaction in which AddAB facilitates the production of ssDNA-coated with RecA protein. Our results show that both the helicase and nuclease activities are required for the biological roles of H. pylori AddAB.     

Nuclear Markers Reveal Predominantly North to South Gene Flow in Ixodes scapularis,the Tick Vector of the Lyme Disease Spirochete

Ixodes scapularis, the tick vector of the Lyme disease spirochete, is distributed over most of the eastern United States, but >80% of all Lyme disease cases occur in the northeast. The role that genetic differences between northern and southern tick populations play in explaining this disparate distribution of Lyme disease cases is unclear. The present study was conducted with 1,155 SNP markers in eight nuclear genes; the 16S mitochondrial gene was examined for comparison with earlier studies. We examined 350 I. scapularis from 7 states covering a representative area of the species. A demographic analysis using Bayesian Extended Skyline Analysis suggested that I. scapularis populations in Mississippi and Georgia began expanding 500,000 years ago, those in Florida and North Carolina 200,000 years ago and those from Maryland and New Jersey only during the past 50,000 years with an accompanying bottleneck. Wisconsin populations only began expanding in the last 20,000 years. Analysis of current migration patterns suggests large amounts of gene flow in northern collections and equally high rates of gene flow among southern collections. In contrast there is restricted and unidirectional gene flow between northern and southern collections, mostly occurring from northern into southern populations. Northern populations are characterized by nymphs that quest above the leaf litter, are easy to collect by flagging, frequently feed on mammals such as rodents and shrews, commonly attach to people, and about 25% of which are infected with B. burgdorferi. If there is a genetic basis for these behaviors, then the patterns detected in this study are of concern because they suggest that northern I. scapularis populations with a greater ability to vector B. burgdorferi to humans are expanding south.     

Dbp6p Is an Essential Putative ATP-Dependent RNA Helicase Required for 60S-Ribosomal-Subunit Assembly in Saccharomyces cerevisiae

A previously uncharacterized Saccharomyces cerevisiae open reading frame, YNR038W, was analyzed in the context of the European Functional Analysis Network. YNR038W encodes a putative ATP-dependent RNA helicase of the DEAD-box protein family and was therefore named DBP6 (DEAD-box protein 6). Dbp6p is essential for cell viability. In vivo depletion of Dbp6p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase labeling of pre-rRNA and steady-state analysis of pre-rRNA and mature rRNA by Northern hybridization and primer extension show that Dbp6p depletion leads to decreased production of the 27S and 7S precursors, resulting in a depletion of the mature 25S and 5.8S rRNAs. Furthermore, hemagglutinin epitope-tagged Dbp6p is detected exclusively within the nucleolus. We propose that Dbp6p is required for the proper assembly of preribosomal particles during the biogenesis of 60S ribosomal subunits, probably by acting as an rRNA helicase.     

Prophage Induction Is Enhanced and Required for Renal Disease and Lethality in an EHEC Mouse Model

Enterohemorrhagic Escherichia coli (EHEC), particularly serotype O157:H7, causes hemorrhagic colitis, hemolytic uremic syndrome, and even death. In vitro studies showed that Shiga toxin 2 (Stx2), the primary virulence factor expressed by EDL933 (an O157:H7 strain), is encoded by the 933W prophage. And the bacterial subpopulation in which the 933W prophage is induced is the producer of Stx2. Using the germ-free mouse, we show the essential role 933W induction plays in the virulence of EDL933 infection. An EDL933 derivative with a single mutation in its 933W prophage, resulting specifically in that phage being uninducible, colonizes the intestines, but fails to cause any of the pathological changes seen with the parent strain. Hence, induction of the 933W prophage is the primary event leading to disease from EDL933 infection. We constructed a derivative of EDL933, SIVET, with a biosensor that specifically measures induction of the 933W prophage. Using this biosensor to measure 933W induction in germ-free mice, we found an increase three logs greater than was expected from in vitro results. Since the induced population produces and releases Stx2, this result indicates that an activity in the intestine increases Stx2 production.     

Arabidopsis ROOT INITIATION DEFECTIVE1, a DEAH-Box RNA Helicase Involved in Pre-mRNA Splicing,Is Essential for Plant Development

Pre-mRNA splicing is a critical process in gene expression in eukaryotic cells. A multitude of proteins are known to be involved in pre-mRNA splicing in plants; however, the physiological roles of only some of these have been examined. Here, we investigated the developmental roles of a pre-mRNA splicing factor by analyzing root initiation defective1-1 (rid1-1), an Arabidopsis thaliana mutant previously shown to have severe defects in hypocotyl dedifferentiation and de novo meristem formation in tissue culture under high-temperature conditions. Phenotypic analysis in planta indicated that RID1 is differentially required during development and has roles in processes such as meristem maintenance, leaf morphogenesis, and root morphogenesis. RID1 was identified as encoding a DEAH-box RNA helicase implicated in pre-mRNA splicing. Transient expression analysis using intron-containing reporter genes showed that pre-mRNA splicing efficiency was affected by the rid1 mutation, which supported the presumed function of RID1 in pre-mRNA splicing. Our results collectively suggest that robust levels of pre-mRNA splicing are critical for several specific aspects of plant development.     

HrpA, a DEAH-box RNA helicase, is involved in global gene regulation in the Lyme disease spirochete

Spirochetes causing Lyme borreliosis are obligate parasites that can only be found in a tick vector or a vertebrate host. The ability to survive in these two disparate environments requires up and downregulation of specific genes by regulatory circuits that remain largely obscure. In this work on the Lyme spirochete, B. burgdorferi, we show that a disruption of the hrpA gene, which encodes a putative RNA helicase, results in a complete loss in the ability of the spirochetes to infect mice by needle inoculation. Studies of protein expression in culture by 2D gels revealed a change in the expression of 33 proteins in hrpA clones relative to the wild-type parent. Quantitative characterization of protein expression by iTRAQ analysis revealed a total of 187 differentially regulated proteins in an hrpA background: 90 downregulated and 97 upregulated. Forty-two of the 90 downregulated and 65 of the 97 upregulated proteins are not regulated under any conditions by the previously reported regulators in B. burgdorferi (bosR, rrp2, rpoN, rpoS or rrp1). Downregulated and upregulated proteins also fell into distinct functional categories. We conclude that HrpA is part of a new and distinct global regulatory pathway in B. burgdorferi gene expression. Because an HrpA orthologue is present in many bacteria, its participation in global regulation in B. burgdorferi may have relevance in other bacterial species where its function remains obscure. We believe this to be the first report of a role for an RNA helicase in a global regulatory pathway in bacteria. This finding is particularly timely with the recent growth of the field of RNA regulation of gene expression and the ability of RNA helicases to modulate RNA structure and function.     

TbPIF5 Is a Trypanosoma brucei Mitochondrial DNA Helicase Involved in Processing of Minicircle Okazaki Fragments

Trypanosoma brucei''s mitochondrial genome, kinetoplast DNA (kDNA), is a giant network of catenated DNA rings. The network consists of a few thousand 1 kb minicircles and several dozen 23 kb maxicircles. Here we report that TbPIF5, one of T. brucei''s six mitochondrial proteins related to Saccharomyces cerevisiae mitochondrial DNA helicase ScPIF1, is involved in minicircle lagging strand synthesis. Like its yeast homolog, TbPIF5 is a 5′ to 3′ DNA helicase. Together with other enzymes thought to be involved in Okazaki fragment processing, TbPIF5 localizes in vivo to the antipodal sites flanking the kDNA. Minicircles in wild type cells replicate unidirectionally as theta-structures and are unusual in that Okazaki fragments are not joined until after the progeny minicircles have segregated. We now report that overexpression of TbPIF5 causes premature removal of RNA primers and joining of Okazaki fragments on theta structures. Further elongation of the lagging strand is blocked, but the leading strand is completed and the minicircle progeny, one with a truncated H strand (ranging from 0.1 to 1 kb), are segregated. The minicircles with a truncated H strand electrophorese on an agarose gel as a smear. This replication defect is associated with kinetoplast shrinkage and eventual slowing of cell growth. We propose that TbPIF5 unwinds RNA primers after lagging strand synthesis, thus facilitating processing of Okazaki fragments.     

The Putative RNA Helicase Dbp4p Is Required for Release of the U14 snoRNA from Preribosomes in Saccharomyces cerevisiae

Around 70 yeast snoRNAs guide rRNA modification, frequently forming base-paired interactions predicted to be very stable at physiological temperatures. Eighteen putative RNA helicases are required for ribosome synthesis, but their actual substrates were not known. We report that depletion of the DEAD box helicase Dbp4p dramatically increased cosedimentation of the snoRNAs U14 and snR41 with preribosomes. Cosedimentation was maintained after deproteinization by proteinase K, indicating that the snoRNAs remained base paired to the pre-rRNA. Affinity purification showed that U14 was strongly accumulated in early 90S preribosomes and depleted from later pre-40S complexes. U14 is required for pre-rRNA processing, and depletion of Dbp4p caused a very similar pre-rRNA processing defect, perhaps due to the reduced pool of free U14. Point mutations in helicase motifs I and III of Dbp4p blocked release of U14 from preribosomes. We conclude that the helicase activity of Dbp4p is required to unwind U14 and snR41 from the pre-rRNA.     

Population Structure of the Lyme Borreliosis Spirochete Borrelia burgdorferi in the Western Black-Legged Tick (Ixodes pacificus) in Northern California

Factors potentially contributing to the lower incidence of Lyme borreliosis (LB) in the far-western than in the northeastern United States include tick host-seeking behavior resulting in fewer human tick encounters, lower densities of Borrelia burgdorferi-infected vector ticks in peridomestic environments, and genetic variation among B. burgdorferi spirochetes to which humans are exposed. We determined the population structure of B. burgdorferi in over 200 infected nymphs of the primary bridging vector to humans, Ixodes pacificus, collected in Mendocino County, CA. This was accomplished by sequence typing the spirochete lipoprotein ospC and the 16S-23S rRNA intergenic spacer (IGS). Thirteen ospC alleles belonging to 12 genotypes were found in California, and the two most abundant, ospC genotypes H3 and E3, have not been detected in ticks in the Northeast. The most prevalent ospC and IGS biallelic profile in the population, found in about 22% of ticks, was a new B. burgdorferi strain defined by ospC genotype H3. Eight of the most common ospC genotypes in the northeastern United States, including genotypes I and K that are associated with disseminated human infections, were absent in Mendocino County nymphs. ospC H3 was associated with hardwood-dominated habitats where western gray squirrels, the reservoir host, are commonly infected with LB spirochetes. The differences in B. burgdorferi population structure in California ticks compared to the Northeast emphasize the need for a greater understanding of the genetic diversity of spirochetes infecting California LB patients.In the United States, Lyme borreliosis (LB) is the most commonly reported vector-borne illness and is caused by infection with the spirochete Borrelia burgdorferi (3, 9, 52). The signs and symptoms of LB can include a rash, erythema migrans, fever, fatigue, arthritis, carditis, and neurological manifestations (50, 51). The black-legged tick, Ixodes scapularis, and the western black-legged tick, Ixodes pacificus, are the primary vectors of B. burgdorferi to humans in the United States, with the former in the northeastern and north-central parts of the country and the latter in the Far West (9, 10). These ticks perpetuate enzootic transmission cycles together with a vertebrate reservoir host such as the white-footed mouse, Peromyscus leucopus, in the Northeast and Midwest (24, 35), or the western gray squirrel, Sciurus griseus, in California (31, 46).B. burgdorferi is a spirochete species with a largely clonal population structure (14, 16) comprising several different strains or lineages (8). The polymorphic ospC gene of B. burgdorferi encodes a surface lipoprotein that increases expression within the tick during blood feeding (47) and is required for initial infection of mammalian hosts (25, 55). To date, approximately 20 North American ospC genotypes have been described (40, 45, 49, 56). At least four, and possibly up to nine, of these genotypes are associated with B. burgdorferi invasiveness in humans (1, 15, 17, 49, 57). Restriction fragment length polymorphism (RFLP) and, subsequently, sequence analysis of the 16S-23S rRNA intergenic spacer (IGS) are used as molecular typing tools to investigate genotypic variation in B. burgdorferi (2, 36, 38, 44, 44, 57). The locus maintains a high level of variation between related species, and this variation reflects the heterogeneity found at the genomic level of the organism (37). The IGS and ospC loci appear to be linked (2, 8, 26, 45, 57), but the studies to date have not been representative of the full range of diversity of B. burgdorferi in North America.Previous studies in the northeastern and midwestern United States have utilized IGS and ospC genotyping to elucidate B. burgdorferi evolution, host strain specificity, vector-reservoir associations, and disease risk to humans. In California, only six ospC and five IGS genotypes have been described heretofore in samples from LB patients or I. pacificus ticks (40, 49, 56) compared to approximately 20 ospC and IGS genotypes identified in ticks, vertebrate hosts, or humans from the Northeast and Midwest (8, 40, 45, 49, 56). Here, we employ sequence analysis of both the ospC gene and IGS region to describe the population structure of B. burgdorferi in more than 200 infected I. pacificus nymphs from Mendocino County, CA, where the incidence of LB is among the highest in the state (11). Further, we compare the Mendocino County spirochete population to populations found in the Northeast.     

Fission Yeast RecQ Helicase Rqh1 Is Required for the Maintenance of Circular Chromosomes

Protection of telomeres protein 1 (Pot1) binds to single-stranded telomere overhangs and protects chromosome ends. RecQ helicases regulate homologous recombination at multiple stages, including resection, strand displacement, and resolution. Fission yeast pot1 and RecQ helicase rqh1 double mutants are synthetically lethal, but the mechanism is not fully understood. Here, we show that the synthetic lethality of pot1Δ rqh1Δ double mutants is due to inappropriate homologous recombination, as it is suppressed by the deletion of rad51⁺. The expression of Rad51 in the pot1Δ rqh1Δ rad51Δ triple mutant, which has circular chromosomes, is lethal. Reduction of the expression of Rqh1 in a pot1 disruptant with circular chromosomes caused chromosome missegregation, and this defect was partially suppressed by the deletion of rad51⁺. Taken together, our results suggest that Rqh1 is required for the maintenance of circular chromosomes when homologous recombination is active. Crossovers between circular monomeric chromosomes generate dimers that cannot segregate properly in Escherichia coli. We propose that Rqh1 inhibits crossovers between circular monomeric chromosomes to suppress the generation of circular dimers.     

Inactivation of Genes for Antigenic Variation in the Relapsing Fever Spirochete Borrelia hermsii Reduces Infectivity in Mice and Transmission by Ticks

Borrelia hermsii, a causative agent of relapsing fever of humans in western North America, is maintained in enzootic cycles that include small mammals and the tick vector Ornithodoros hermsi. In mammals, the spirochetes repeatedly evade the host’s acquired immune response by undergoing antigenic variation of the variable major proteins (Vmps) produced on their outer surface. This mechanism prolongs spirochete circulation in blood, which increases the potential for acquisition by fast-feeding ticks and therefore perpetuation of the spirochete in nature. Antigenic variation also underlies the relapsing disease observed when humans are infected. However, most spirochetes switch off the bloodstream Vmp and produce a different outer surface protein, the variable tick protein (Vtp), during persistent infection in the tick salivary glands. Thus the production of Vmps in mammalian blood versus Vtp in ticks is a dominant feature of the spirochete’s alternating life cycle. We constructed two mutants, one which was unable to produce a Vmp and the other was unable to produce Vtp. The mutant lacking a Vmp constitutively produced Vtp, was attenuated in mice, produced lower cell densities in blood, and was unable to relapse in animals after its initial spirochetemia. This mutant also colonized ticks and was infectious by tick-bite, but remained attenuated compared to wild-type and reconstituted spirochetes. The mutant lacking Vtp also colonized ticks but produced neither Vtp nor a Vmp in tick salivary glands, which rendered the spirochete noninfectious by tick bite. Thus the ability of B. hermsii to produce Vmps prolonged its survival in blood, while the synthesis of Vtp was essential for mammalian infection by the bite of its tick vector.